Mass Spectrometry in The Clinical Laboratory: Donald H. Chace

Chem. Rev.
2001, 101, 445−477 445
Mass Spectrometry in the Clinical Laboratory

Donald H. Chace*
Division of Bio-Analytical Chemistry and Mass Spectrometry, Neo Gen Screening, Inc., P.O. Box 219, Bridgeville, Pennsylvania 15017
Received April 4, 2000
Contents VI. Conclusions 470

A. Outlook of Clinical MS 471
I. Introduction 445
VII. Acknowledgments 472
A. Clinical Chemistry and MS 446 VIII. References 472
B. Laboratory Testing and Diagnosis of Disease 447
C. Scope of Review 447
II. Diagnostic Metabolites 448 I. Introduction
A. Metabolome Analyses 448 The utilization of mass spectrometers in clinical
B. Organic Acids 449 laboratories is undergoing considerable expansion at
1. GC/MS Applications 449 the outset of the 21st century.1,2 This expansion is
2. LC-MS Applications 452 largely due to extraordinary advances in mass spec-
C. Amino Acids 452 trometry (MS) developed in the previous decade.3 A
1. GC/MS Applications 453 clear vision of the importance of MS in medicine was
2. LC-MS Applications 453 provided at the 11th Sanibel Conference on Mass
3. Homocysteine 456 Spectrometry4 entitled “Mass Spectrometry in the
4. Thyroid Hormones 457 Clinical Diagnosis of Disease”. The consensus of the
D. Acylcarnitines and Acylglycines 458 conference was that MS is poised to take an increas-
ingly important role in clinical chemistry. MS is no
1. Inherited Disorders of Fatty Acid and 459
Organic Acid Metabolism longer the complex and laborious tool used exclu-
sively by experienced mass spectrometrists. It is an
2. Acylcarnitines 459
accessible, versatile, and powerful technology that is
3. Acylglycines and Other Fatty Acyl 461 best suited to solve research and analytical problems
Conjugates
in an extensive number of scientific disciplines.1 The
E. Bile Acids 461 rapid pace of developments in liquid chromatography
1. GC/MS 462 and mass spectrometry (LC-MS) has profoundly
2. LC-MS 462 influenced the potential number of applications of MS
F. Cholesterol and Steroids 462 in the clinical laboratory.5 Historically, most mass
1. GC/MS 463 spectrometric applications were restricted to the
2. LC-MS 463 analysis of small volatile molecules that were ana-
3. Time-of-Flight MS 463 lyzed, using gas chromatography and mass spectrom-
G. Biogenic Amines 464 etry (GC/MS). These methods required extensive
H. Other Classes of Small Biomarkers 464 sample preparation that included sample extraction
1. Lipids 464 and derivatization. The analysis of intact high mo-
lecular weight or extremely polar biomolecules was
2. Carbohydrates 464
either difficult or not possible in most laboratories
3. Trace Elements 464 just 15 yr ago. Today, new LC-MS applications
III. Diagnostic Proteins and Glycoproteins 465 provide solutions to the analysis of these biomarkers.
A. Proteome Analyses 465 Most compounds that characterize human biochem-
B. Hemoglobin 465 istry are extremely hydrophilic, ranging in size from
1. Proteolytic Fragment Analysis 466 small molecules with molecular masses generally less
2. Intact Globin Analysis 466 than 500 Da (e.g. amino acids, fatty acids, bile acids,
3. Glycohemoglobins 467 and steroids) to higher molecular weight molecules
C. Specific Disease Diagnostics Using MS 467 such as peptides and large biomolecules (e.g. proteins,
1. Protein Profiling 467 glycoproteins and oligonucleotides). The advent of
IV. Diagnostic Biopolymers 468 new ionization techniques such as electrospray ion-
ization (ESI) and matrix-assisted laser desorption
A. Genome Analyses 468
(MALDI) together with improvements in mass ana-
B. Current MS Applications 469 lyzers such as time-of-flight (ToF) and benchtop
V. Quantitative Analysis and Quality Assurance 469
A. Quantification in Clinical Chemistry 469 * Corresponding author phone: (412)220-2300; fax: (412)220-0784;
B. Quality Assurance 470 e-mail: [email protected].
10.1021/cr990077+ CCC: $36.00 © 2001 American Chemical Society

Published on Web 01/09/2001
446 Chemical Reviews, 2001, Vol. 101, No. 2 Chace
versatile, and robust analytical systems make mass

spectrometric applications ideal for solving routine
and complex clinical laboratory problems. However,
as pointed out by L. Bowers,4 the challenge for the
clinical scientist is to present complex MS data in
an easy to understand format. This format includes
interpretation of complex mass spectra and the
integration of these data with other laboratory re-
sults. The analytical chemist, who is not traditionally
trained in medicine, is challenged to provide some
form of interpretation of clinical data to a physician,
certified geneticist, or other health professional.
During this decade, chemists/mass spectrometrists
trained in the clinical sciences or clinical specialists
Dr. Donald Chace received his B.S. in Chemistry from Boston College in (physicians) trained in MS will provide expertise in
1981. He earned two graduate degrees from The George Washington a special area of MS defined as clinical mass spec-
University. In 1984, Dr. Chace received a M.S. in Forensic Science trometry.
specializing in forensic toxicology. In 1989, he received a Ph.D. in The most widely used application of MS in clinical
Pharmacology specializing in mass spectrometry and drug metabolism.
A new technique for the selective detection of isotopically enriched drugs chemistry is GC/MS.12-15 Due to analyte volatility
and their metabolites was developed using gas chromatography chemical and size limitations, GC/MS analyses are restricted
reaction interface mass spectrometry (GC−CRIMS). In 1989, he began to derivatizable compounds such as fatty acids,
his postdoctoral research at the University of Maryland School of Pharmacy organic acids, amino acids, monosaccharides, prosta-
investigating the use of diamine reagents to improve the analysis of glandins, bile acids, and steroids.16 The most widely
phosphate analgoues using thermospray MS. Dr. Chace joined the faculty
at Duke University as a Medical Research Assistant Professor in the
used GC/MS assay in clinical chemistry is the analy-
Department of Pediatrics in 1990. His research efforts included the sis of urine specimens from patients with suspected
application of tandem mass spectrometry for use in early identification of or known metabolic disturbances.17-19 The power of
metabolic disease in newborns. In 1997, he joined Neo Gen Screening, GC/MS in analyzing complex urinary profiles and
Inc. to continue the development of mass spectrometry applications in providing physicians with key evidence that assists
newborn screening and clinical chemistry. He is currently the Director of in the diagnosis of a disease has led to its “ac-
the Division of Bio-Analytical Chemistry and Mass Spectrometry. This
laboratory analyzes more than 250 000 blood samples obtained from ceptance” by clinical chemists, physicians, and other
newborns per year using tandem mass spectrometry for metabolic disease medical specialists.
screening. Historically, the use of MS was limited to univer-
sity and private specialty laboratories where the
tandem quadrupole (MS/MS), quadrupole ion trap expertise was available to use this complex technol-
(QIT), and the newest hybrid mass spectrometers, ogy and clinically interpret these results. Why has
e.g., quadrupole-time-of-flight (Q-ToF), have enabled the use and scope of MS recently increased in clinical
clinical chemists to consider analyzing these very laboratories as demonstrated by the recent surge in
polar and very large compounds.1,5-9 The number of the use of tandem mass spectrometry (desginated
potential clinical applications of MS is inestimable. here as MS/MS) throughout the world for newborn
screening of metabolic disease? The answer2 resides
A. Clinical Chemistry and MS in the fact that MS has become simple enough to be
Clinical10 is defined as having to do with the direct operated by inexperience users in a variety of scien-
treatment and observation of patients, as distin- tific disciplines. Although the machines have been
guished from experimental or laboratory study. The made easier to use and operation simplified, mass
application of chemistry and clinical science therefore spectrometers have retained, even dramatically im-
relates primarily to providing patient-derived chemi- proved, the power and flexibility demanded by expe-
cally relevant data to the physician. The physician rienced mass spectrometrist and analytical chemists.
uses data obtained from the clinical chemistry labo- We may be experiencing a “renaissance” of mass
ratory and other physical assessments to make a spectrometric based analyses in the clinical labora-
hypothesis (diagnosis) regarding a disease state. The tory. This reviewer would estimate2 that more than
number of clinical results that will be provided to a 1 000 000 newborn blood samples will be analyzed
physician that are based in MS will increase in this using LC-MS/MS applications in 2000. LC-MS ap-
decade. plications are replacing many of the older traditional
Bermes and Young11 state that the objective of a specialty clinical laboratory methods20,21 that used
clinical chemistry laboratory is “to perform qualita- immunological, fluorometric, and biological tech-
tive and quantitative analyses on body fluids such niques18 as demonstrated by the newborn screening
as blood, urine, cerebrospinal fluid ...” and further application of MS/MS for phenylketonuria.22-24 Will
state that “if the results are to be useful to the MS completely replace these traditional clinical
physician in the diagnosis and treatment of disease, chemistry analyzers during this decade? It is un-
the tests must be performed as accurately as possible. likely. However, the number and scope of mass
This [testing] requires the use of sound analytical spectrometric applications will certainly increase,
methods and good instrumentation”. Innovative and MS will be an indispensable tool in the clinical
sample preparation methods together with accurate, laboratory.
MS in the Clinical Laboratory Chemical Reviews, 2001, Vol. 101, No. 2 447
The chemical diversity of compounds that are and screening best espouses this concept.23 When MS
will probably be routinely analyzed using MS is methods are used correctly, with proper safeguards
extensive. MS-based clinical assays that provide and with communication of easily understood results
rapid, comprehensive, multicomponent analyses and to medical professionals, physicians will diagnose
maintain or improve sensitivity, selectivity, and disease more accurately and rapidly with a lower cost
accuracy25 will likely lead the growth of MS in clinical and higher benefit, thus improving the state-of-the-
laboratories. The use of stable isotopes in mass art of health care delivery. These facts do not neces-
spectrometric quantitative analyses will confer a sarily translate to increased use of MS in the clinical
technical advantage over other methodologies used laboratory. Some potential barriers to implementa-
in the clinical laboratory because of its inherent tion include reimbursement by health care payers,
accuracy and precision. Isotope dilution methods not the availability of significantly less expensive and
only improve precision and accuracy in many clinical easy-to-use albeit less accurate techniques such as
analyses but also potentially provide solutions for immunoassays, lack of physician acceptance or edu-
quality assurance, standardization, and interlabora- cation, poor assay availability, high costs at low
tory comparisons.26 LC-MS, MALDI-MS, and other volumes, and other negative influences that are
MS-based systems will be important for the mea- either economic or political.
surement of informative biomarkers such as protein
and gene fragments27 as GC/MS has proven to be a C. Scope of Review
powerful clinical tool for qualitative and quantitative
analysis of important small molecules. The number and type of clinically significant bio-
molecules and their analysis using MS are consider-
B. Laboratory Testing and Diagnosis of Disease able. These MS applications range from elements
such as iron and selenium to metabolites (such as
A physician presumes that the laboratory data phenylalanine and glucose) peptides, and proteins
provided to him/her are accurate and precise, vali- (such as insulin and hemoglobin) to large oligonucle-
dated, and subjected to rigorous quality assurance. otides (such as DNA and RNA fragments). Important
This presumption demands that the clinical chemist biomarkers may be compounds of endogenous origin
uses the most accurate methodologies available and produced by intermediary metabolism or xenobiotics
that a protocol is followed exactly. Each MS analysis produced by exogenous metabolism following drug
must be error-free because the information provided administration or environmental exposure. Clinical
affects an individual’s health and life. Obviously, the applications have also included biomedical research,
demands on the clinical mass spectrometrist are clinical toxicology,30,31 and other chemical disciplines
extraordinarily high. Every effort must be made to such as immunology, virology, bacteriology, and
prevent analytical inconsistencies, and methods re- oncology. It is impractical to cover every application
quire extensive validation.28 Furthermore, clinical of MS in clinical chemistry. Therefore, those applica-
assays must pass quality control and assurance tions that are currently practiced in clinical labora-
assessments before data can be used in clinical tories that will likely impact clinical analysis soon
practice.29 or that best illustrate the potential of mass spectro-
Raw MS results have no clinical significance to metric analysis will be emphasized. Referenced pub-
most physicians. Therefore, the clinical laboratory lications will primarily span 15 yr. Some older,
must develop extensive interpretation schemes that historically important or frequently cited papers32
clearly communicate pertinent laboratory testing will also be included, especially for applications using
information to a physician before this information can GC/MS. The clinical applications of MS will be
be useful to diagnose a disease in a patient. arranged in three basic groups: small metabolites
Expertise in the interpretation of mass spectro- such as organic acids, amino acids, fatty acids,
metric results does not reside in most routine clinical steroids and their conjugates;33 peptides, proteins,
laboratories. The effort required to implement an and glycoproteins;34,35 and oligonucleotides derived
interpretation and follow-up system for mass spec- from biopolymers (DNA, RNA). A brief discussion of
trometers is substantial. This is one reason that MS the quantitative aspects of MS, quality assurance,
has primarily resided in small reference laboratories and integration of MS in the clinical laboratory will
rather than large commercial clinical laboratories. In complete the review.
addition, information concerning false-positive and Of important note is the use of abbreviations and
false-negative rates provide reliability indicators to terminology. I have attempted to follow guidelines
the physician and should be provided with test written by O. David Sparkman in his text “Mass Spec
results. Methods with low false-positive rates enable Desk Reference”.7 The use of standardized terminol-
the clinician to respond more rapidly. Conversely, ogy is important for communication of meaning as
methods with high false-positive rates require time- seen in one recent example: MS/MS is now being
consuming repeat analysis to confirm a result. Labo- used by several newborn screening laboratories, and
ratory errors may result in a disease going undetec- there has been a recent trend among these users,
ted, delay a timely diagnosis, or raise health care many of whom are not classical chemists and mass
costs by requiring further tests or patient referrals. spectrometrists, to abbreviate tandem mass spec-
Clearly, the power of MS-based analysis is its inher- trometry as TMS. TMS is the abbreviation for trim-
ent accuracy. Precision will be the driving force for ethylsilyl, a type of chemical derivative used fre-
its use in the clinical laboratory. An Editorial by H. quently in the GC/MS analysis of organic acid
Levy that describes the use of MS/MS and newborn metabolites found in urine. Therefore, the currently
accepted abbreviations developed by groups such as

the American Society of Mass Spectrometry (ASMS)36
(MS/MS for tandem mass spectrometry), the Ameri-
can Chemical Society, and IUPAC should be used to
clearly communicate meaning. Users in other fields
should apply these standard terms rather than create
new ones.
II. Diagnostic Metabolites

A. Metabolome Analyses
Metabolome is a term used to characterize endog-
enous metabolites, i.e., those classes of compounds
that are products or substrates of endogenous me-
tabolism. In the clinical setting, the term metabolo-
mics complements the terms proteomics and genom-
ics. Metabolomics is more descriptive than the most
common method for describing the laboratory inves-
tigation of low molecular weight substances as “small
molecule analyses”. The metabolome is to small
organic molecules in cells and tissues as proteins and
oligonucleotides are to the proteome and genome,
respectively. Most mass spectrometric applications Figure 1. Simplified concept of the relationship between
that are in clinical use today are characterized by the phenotypes and genotypes in genetic disease. In a “normal”
analysis of these small organic molecules, which state, genes (genotype) code for proteins, which produce
includes the rapid analyses of multicomponent mix- metabolites and no disease expression. Abnormal genes
tures. (genotype) produce an abnormal phenotype if the gene is
expressed as an abnormal protein and the abnormal
Nearly 200 genetic disorders of intermediary me- protein alters metabolism. Alternatively, if the expressed
tabolism are characterized by a defect in a single protein is a functional protein, such as hemoglobin, then
enzyme.37 Many of these inherited genetic diseases the disease is characterized by an abnormal protein rather
produce an abnormal protein with reduced or absent than a metabolite. The relationships of gene to protein to
catalytic activity. If the defective enzyme plays a metabolite to normal disease-free state are indicated by
major role in the primary catabolic pathways of a solid arrows. In some cases, an abnormal gene may not be
expressed, an abnormal protein may not alter metabolism,
substrate, then a significant reduction in that sub- or an abnormal metabolite not produce toxicity. For these
strate’s metabolic rate will occur. Results of decreased examples, although an abnormal gene, protein, or metabo-
enzymatic activity may produce substrate A ac- lite concentration is present, no disease state is observed.
cumulation and product B deficiency when compared This process is represented as a dashed arrow to the
to normal. To maintain homeostasis, the excess column representing a normal, disease-free condition.
substrate A will be metabolized by an alternative
metabolic pathway. The product C of the alternative ations or vitamin supplements can be used to sub-
pathway, at high concentration, may produce toxicity, stantially reduce or prevent the most serious conse-
which includes cellular damage, impaired cellular quences in very many cases.
function or inhibition of other metabolic pathways. The analysis of small substrates and products of
Because the alternative metabolic pathway is gener- enzymes systems (lower than 1000 Da molecular
ally less efficient than the primary pathway, the mass) can provide good evidence for disease detection
substrate A may also accumulate in a metabolic because they often closely correlate with the disease
disorder. Substrate A, at very high concentrations, (i.e., they are substrates or products of the abnormal
can also be toxic. With regard to product B of an enzyme produced by a defective gene). The analysis
abnormal enzyme, a deficiency can also have adverse of larger proteins serving a functional role, i.e.,
metabolic consequence, especially if it participates in hemoglobin and oxygen delivery to tissues, are biom-
other anabolic or catabolic pathways. arkers that also directly relate to disease detection,
The clinical effects of abnormal metabolism include e.g., hemoglobin S and sickle cell anemia. The
organ malfunction or tissue damage, impaired de- analysis of functional proteins, like hemoglobin, is
velopment, delayed growth, mental retardation, physi- used in a manner similar to measurement of me-
cal disabilities, neurological disorders, cardiovascular tabolites in the diagnosis of metabolic disease. As
disease, and premature death.37 Identification and observed in Figure 1, an abnormal gene product does
quantitation of these abnormal metabolites are criti- not necessarily produce an abnormal protein (repre-
cally important to the recognition and confirmation sented as a dashed line from the abnormal column
of a disorder of endogenous metabolism. Early detec- to the column representing a normal, disease-free,
tion through newborn screening prior to symptoms expressed clinical condition). Likewise, an abnormal
or clinical diagnostic testing after the onset of disease protein does not necessarily adversely affect metabo-
symptoms permits pharmacological and/or dietary lism. The accuracy of disease detection is directly
intervention. Although the cures for inherited meta- related to the correlation between the causative agent
bolic diseases are limited at this time, dietary alter- and the presence of metabolite or protein. In addition,
it is unlikely that a single genetic marker will

characterize all of the mutations that produce a
particular disease. Furthermore, randomly occurring
mutations may not be detected. The false-negative
rate of current molecular methods that analyze
genetic mutations can be high. Many mutations or
known alterations in a gene fragment may have no
clinical consequence if not expressed (benign poly-
morphisms).37 This fact suggests a high false-positive
rate. Nonetheless, some traditional biochemical tests
may not be available or adequate for the analysis and
detection of some inherited disorders. Detection of
these genetic diseases will most likely be performed
using primary DNA analysis.
The interplay among metabolome, proteome, and
genome analyses will be the challenge of this and
future decades. Most likely, clinical chemists will
embrace all three forms of analysis. In some situa-
tions, the clinical chemist may actually use all these
analyses sequentially to first identify the metabolome
associated with the disorder, then to confirm this
identification using protein and/or DNA analysis.
With other clinical information, laboratory data will
be used to characterize disease severity and treat- Figure 2. Organic acids commonly measured in urine and
ment strategies. Currently, MS plays an important plasma by GC/MS for the diagnosis of inherited disorders
role in disorders of metabolism, where diseases are of organic acid metabolism.
diagnosed by abnormal concentrations of biomarkers.
These methods are described below. for interpretation of results provided by different
laboratories. A key metabolite measured in labora-
B. Organic Acids tory A might not be measured in laboratory B. If this
key metabolite is diagnostic for a particular disease
Chalmers19 defines organic acids as carboxylic acids and a physician orders the analysis from laboratory
that may contain hydroxyl, oxo, and non-amino B, then a strong likelihood exists for a false diagnosis.
functional groups. This definition includes short and This lack of standardization may certainly increase
medium-chain fatty acids and excludes R-amino with the rapid expansion of MS into clinical labora-
acids. Unconjugated organic acids are characterized tories. Further discussion of issues in the standard-
by their acidity, high water solubility, and relatively ization of MS methods is provided in Section V.
low molecular mass (<250 Da). Nearly 250 organic Fortunately, for organic acid analysis, there already
acids have been identified in urine.33 Some important is a core set of diagnostic metabolites that is mea-
organic acids frequently analyzed in clinical labora- sured in most GC/MS assays.
tories are shown in Figure 2. a. Metabolic Profiling. Historically, GC/MS has
Metabolic disorders that produce high concentra- proven to be the most valuable clinical chemistry tool
tions of organic acids in blood and urine are described to diagnose disorders of organic acid metabolism.18,43
as organic acidurias.5,19,33 Organic acids are involved GC/MS provides two means for assisting in the
in nearly all pathways of intermediary metabolism. identification of key metabolites in derivatized urine
Their analysis provides important diagnostic infor- extracts: high-resolution capillary gas chromatogra-
mation for many inherited diseases.38 GC/MS has phy and MS. The combination of chromatography and
served a vital role in the diagnosis and recognition mass spectrometric detection is a major reason for
of many metabolic disorders39 and remains today a reduced numbers of false-positives and for the high
cornerstone of routine clinical laboratory analyses. accuracy for these assays. Another advantage of
combined GC/MS techniques is a diagnostic approach
1. GC/MS Applications known as metabolic profiling.16 Metabolic profiling
Applications of GC/MS to the analysis of urinary is a method of diagnosing a disease by identifying
organic acids are numerous. Several excellent books, and quantifying important disease markers or me-
chapters, and reviews have been written.16,18,19,21,33,39-42 tabolites in a single analysis. In this manner, me-
Many of these reviews include clinical correlations tabolite relationships, i.e., concentration of metabolite
to disease diagnosis.18,38 A notable fact in the review A relative to metabolites B and C, indicate a specific
of organic acid assays using GC/MS is the substantial disorder. In addition, metabolic profiling provides a
number of publications that differ slightly in the visual pattern of metabolite relationships, and this
method of analysis (i.e., sample preparation, chro- pattern has not been fully integrated in computer-
matographic separation) or pertain to a highly spe- assisted data interpretation. For some metabolic
cific analysis for an individual disorder. This fact is disorders, interpretation is very simple because a key
important if one considers that most clinical analyses metabolite is present in urine at an extremely high
rely upon standardization or harmonization of meth- concentration. Conversely, other disorders are char-
odology. Harmonization of methods provides the basis acterized by subtle or mild increases of several
different metabolites. Some physicians and clinical

laboratory experts that are experienced in complex
pattern recognition may observe subtle changes in
metabolite concentrations;44 however, many other
clinical chemists will not detect these differences. The
need for computer-assisted interpretation will be
important in detecting subtle metabolic patterns in
several ways. These may include metabolite ratios
and integration of patient physical data such as age,
clinical data such as liver disease, cardiomyopathy,
or other laboratory tests that detect vitamin deficien-
cies, hypoglycemia, or hyperammonemia. Some com-
puter-assisted interpretation systems have been
developed.45-47 Recently, neural networks have been
used to assist in identification of new patterns of
metabolic profiles that indicate disease.48
b. Urine Organic Acids. Organic acids are iso- Figure 3. Illustration of common fragments produced by
lated from urine by solvent extraction or anion- electron ionization of TMS derivatives of organic acids.
exchange chromatography.42,49-51 In addition, organic
acids have been directly extracted from urine that alternative to forming silyl derivatives is methyla-
has been applied to filter paper.52 The most common tion, using diazomethane and other reagents.56 These
methods for extraction of organic acids use ethyl derivatives are very stable and produce informative
acetate or diethyl ether and salt-saturated, acidified mass spectra. Other derivatization schemes have
urine specimens. Recently, ion-exchange chromatog- been used frequently.41,57,58 Certain classes of organic
raphy51 has been used to extract urine organic acids. acids are difficult to derivatize with standard tech-
This technique is based on the retention of organic niques. For example, ketoacids are unstable and
acids, while neutral and basic compounds are washed require methods that convert these compounds to
with aqueous buffers or distilled water from the methoxime and ethoxime TMS derivatives.59 These
column. Organic acids are eluted from the ion- derivatives provide characteristic fragment ions, M
exchange resins with organic solvent. Some research- - 15, M - 31, or M - 45 that help to identify the
ers have used robotic workstations for sample prepa- molecular ions and keto groups.
ration but have had limited success.53 Due to increased Chromatography40 of organic acids is performed
volatility following many derivatization techniques, using a variety of capillary columns and stationary
short-chain and medium-chain free fatty acids (acetic phases, including DB-1, SE-30, OV-1, and OV-17.
through decanoic) and other very volatile organic Mass spectrometric analyses are most commonly
compounds are not detected because they coelute performed on quadrupole MS instruments with elec-
with solvent peaks or are lost during sample prepa- tron ionization. This use of quadrupole MS analysis
ration. is significant because most searchable MS reference
Quantitation of urinary organic acids is nearly libraries are composed of EI spectra generated using
always expressed relative to the concentration of quadrupole mass spectrometers. Practitioners of GC/
creatinine. This method of quantifying creatinine is MS routinely use these libraries as a reference in the
important because the concentration of organic acids identification of organic acids. GC quadrupole ion
will vary substantially, depending upon the volume trap MS (GC-QIT) and GC/MS/MS methods are both
of urine produced in a defined time period (typically used in several laboratories.60 Other techniques rely
24 h). Creatinine is an excellent reference because it on different methods of ionization such as chemical
is produced in plasma at a constant rate and within ionization (CI),57,61,62 which offers the advantage of
a narrow concentration range. Creatinine has been producing observable molecular ions. Selected ion
used to estimate the glomerular filtration rate and, monitoring (SIM)63 is the standard method for com-
as such, is an excellent reference standard for urine pound specific analysis with improved sensitivity.
output. Creatinine is most often measured by non- Isotope dilution mass spectrometry (IDMS) tech-
MS-based assays,54 although one procedure used GC/ niques provide more accurate quantitative results for
MS.55 many analytes.64 Other improvements in the quan-
Following extraction, organic acids are derivatized titation of urine organic acids have also been de-
most commonly by trimethylsilylation. TMS deriva- scribed.65
tives are characterized by their ease of preparation, c. Plasma Organic Acids and Free Fatty Acids
good chromatographic separation, and characteristic by GC/MS. Organic acids are isolated from biological
M - 15 fragment ion (loss of CH3) as shown in Figure fluids which contain protein, e.g., plasma. These
3. The most frequent alternative to TMS derivatiza- sample preparation methods require deproteinization
tion is tert-butyldimethylsilylation. This derivative either by solvent precipitation, membrane ultra
offers improvements in hydrolytic stability and better filtration,66 or other techniques.67 The analysis of
GC separation. This method of derivatization is short-chain and very long-chain fatty acids in plasma,
helpful in the analysis of more volatile organic acids using GC/MS, has been described,68-70 with recent
and short-chain fatty acids by increasing their reten- improvement in plasma free fatty acid analysis
tion time and making them slightly less volatile. An described by Jones et al.71
d. Clinical Applications. Ozand and Gascon

define an organic acidemia as the accumulation of
organic acids in cells or body fluids and is indicative
of a disorder of intermediary metabolism.72,73 More
than 50 phenotypically different organic acidemias37
(organic acidurias) are known primarily because of
metabolic profiling with GC/MS. Organic acidemias
arise from (i) defects in branched-chain amino acid
metabolism, e.g., isovaleric acidemia, propionic aci-
demia, β-ketothiloase deficiency, and 3-methylcroto-
nyl-CoA carboxylase deficiency; (ii) vitamin metabo-
lism, e.g., methylmalonic acidemia, vitamin B12
deficiency, and other cobalamin defects; (iii) flavopro-
tein metabolism disorders, e.g., short-, medium-, and
very long-chain acyl-CoA dehydrogenase deficiencies,
glutaric acidurias type I and II; (iv) lipid metabolism
disorders; (v) disorders in glycolysis, e.g., pyruvate
dehydrogenase deficiency; (vi) gluconeogenesis; (vii)
citric acid cycle disorders, e.g., fumaric aciduria,
2-ketoglutaric aciduria; and (viii) disorders of glu-
tathione metabolism.
Propionic acidemia is an inherited metabolic dis-
order of propionyl CoA metabolism caused by a
deficiency of propionyl CoA carboxylase.37 Symptoms
of the acute disorder that presents early in infants
are lethargy, vomiting, acidosis, hypogylcemia, hy-
perammonemia and possibly death.37 Patients with
chronic propionic acidemia present symptoms of
hyperglycinemia, ketoacidosis, and vomiting after
protein ingestion, developmental delay, and seizures.
Treatment involves protein restriction and adminis-
tration of biotin and L-carnitine. Propionyl-CoA is an
intermediate in the catabolic pathway of leucine,
isoleucine, valine, methionine, and threonine. Pro- Figure 4. Total ion chromatograms (TIC) from derivatized
pionyl-CoA is converted to methylmalonyl-CoA by urine extracts of a control patient and a patient with
propionyl-CoA carboxylase (an enzyme that requires propionic acidemia. TMS derivatives of 3-hydroxypropionic
biotin as a coenzyme). Deficiency of this enzyme acid (a), propionylglycine (b), and methylcitric acid (c) are
results in a marked accumulation of propionyl-CoA diagnostic metabolites identified by retention time and
and propionic acid. The accumulation of propionic mass spectra. C-24 (n-tetracosane) is the reference stan-
dard. Laboratory data from E. Prence, E. Naylor, and S.
acid causes acidosis in blood. Propionic acid is Singleton.
removed by metabolism through other pathways. The
disorder is diagnosed by using an organic acid profile
obtained by GC/MS, as shown in Figure 4, when
compared to control urine specimens. The most
diagnostic compounds for this disease are propionic
acid, methylcitric acid, and 3-hydroxypropionic acid
derivatized as their TMS derivatives (Figure 5); their
respective mass spectra are provided in Figure 6. The
molecular ions of these compounds are generally
small or absent, with (M - 15) ions produced by the
loss of CH3• indicating the molecular weight (Figure
3). In addition, timethylsilyl derivatives are charac-
terized by an intense ion (Figure 3) at m/z 73, (CH3)3-
Si+. Compounds with two or more silyl groups
produce an ion at m/z 147 that is formed by rear-
rangement (Figure 3)19 to give TMS-O+dSi(CH3)2. Figure 5. Structures of TMS derivatives of 3-hydroxypro-
Propionic acid is not normally detected as a TMS pionic acid, propionylglycine, and methylcitric acid.
derivative because of its high volatility. However,
propionic acid can be indirectly detected as a glycine addition to identifying organic acidemias, GC/MS
conjugate using GC/MS. analysis of organic acids has been helpful in the
Early detection of organic acidemias can prevent diagnosis of vitamin B12 deficiency75 and in the
severe complications. Some investigators have used management of diabetes.76 The analysis of fatty acids,
GC/MS in newborn screening by developing a rapid using GC/MS, has been helpful in prostate cancer
simplified GC/MS analysis of urine samples.74 In diagnosis.77
MS methods even though their analysis is often

performed on a stationary viscous liquid (LSI) or
crystalline matrix (MALDI) and placed in the end of
a stainless steel probe or target and manually
inserted into a mass spectrometer. To reduce some
confusion, where possible, HPLC will be used to
broadly categorize those methods in which chroma-
tography is performed. Methods that use flow injec-
tion analysis or liquid secondary ionization will be
part of the general categories of either LC-MS or MS/
MS. Where possible, the ionization mode will be
included, e.g., electrospray (ESI), ionspray, atmo-
spheric pressure chemical ionization (APCI), and
liquid secondary ionization methods (FAB or FIB).
One final note concerning LC-MS methods. The
commonality between LSI, FAB, FIB, ESI, MALDI,
etc. is that they are soft ionization techniques rather
than out-and-out LC-MS methods. These techniques
gave mass spectrometrists the first possibility to
analyze biological molecules directly without deriva-
tization to aid volatility.
a. Organic Acids. Methods that use HPLC-MS
techniques to analyze organic acids are few because
HPLC offers little improvement over clinical analysis
of organic acids by GC/MS. Liquid chromatographic
resolution is relatively poor as compared to capillary
GC. Mass spectra formed by LC-MS analysis are
primarily protonated molecules and do not contain
any important fragment ion information. The use of
LC-MS/MS to generate these fragment ions is limited
because of the unavailability of product ion libraries
of organic acids. Because organic acids generate a
negative charge at neutral pH, thermospray or elec-
trospray ionization produces negative ions that are
not detected with the same degree of sensitivity as
positive ions. To overcome the limitations of negative
ions, very acidic or ammonium acetate-based mobile
phases78 have been used to assist in protonation.
Figure 6. Mass spectra of the TMS derivatives of 3-hy- Negative ionization methods that produce intense (M
droxypropionic acid (a), propionylglycine (b), and methyl- - H)- molecular ions are not sensitive and yield little
citric acid (c). Molecular ions are characterized by loss of fragmentation data.79 Recently, Johnson80 developed
methyl groups (CH3). Laboratory data from E. Prence, E. a new derivatization scheme for use in positive ion
Naylor, and S. Singleton. electrospray MS. This scheme produced dimethy-
laminoethyl esters of carboxylic acids. This applica-
2. LC-MS Applications tion primarily focused on fatty acids. However, this
technique may potentially lead to new methods
The term and abbreviation, LC-MS, is used to developed with LC-MS in the analysis of organic
broadly categorize methods that use a mass spec- acids. Recently, Johnson described a method to
trometer and a liquid interface or delivery system. elucidate peroxisomal disorders,81 using dimethy-
Liquid chromatography is clearly implied in the laminoethyl esters of long-chain fatty acids. Organic
abbreviation LC and creates confusion because many acid disorders have also been detected with LC-MS
“LC-MS” methods use direct flow injection techniques techniques in other approaches that measured gly-
without chromatography. In addition, liquid mobile cine conjugates in urine or acylcarnitines in blood.
phases may be delivered via a syringe pump rather These methods are described in Section II.D.
than a high-pressure liquid chromatographic “pump”.
Flow injection techniques use a high-pressure, pulse- C. Amino Acids
less delivery of mobile phases composed of aqueous
and organic solvents to the mass spectrometer through Amino acids are compounds characterized by amino
an uncoated, deactivated, fused-silica capillary, PEEK, and carboxylic acid functional groups.82 Physiologi-
or other small inner diameter tubing. No chroma- cally important amino acids are known as R-amino
tography is intended in these analyses. Furthermore, acids. Structurally, R-amino acids are composed of
older ionization techniques such as fast atom or ion an amine, a carboxylic acid, and a side chain denoted
bombardment (FAB or FIB), also known as liquid as the “R” group, which is attached to a common
secondary ionization (LSI), and matrix-assisted laser “central” carbon atom in the R-position relative to the
desorption (MALDI) are often characterized as LC- carboxylic acid. These R-amino acids are essential
components of all peptides and proteins. In solution amounts of urea adversely affect chromatographic
at physiological pH, amino acids are dipolar (zwitter) and mass spectrometric analysis of several amino
ions. Amino acids are categorized as neutral, basic, acids. A solution to this problem has been the
or acidic by either their pKa or pKb values, which pretreatment of urine with urease, which reduces the
directly correlate with the composition of the R group. concentration of urea in a specimen.47,74 Due to their
Amino acids are the essential components of proteins. similarity and common derivatization schemes, or-
Twenty-two different amino acids have been found ganic acid and amino acid analysis have been com-
in varying amounts in human proteins. These and bined in a single analytic run.84
other important amino acids participate in interme- In addition to forming TMS derivatives, amino
diary metabolism and are precursors to active bio- acids can be converted to their tert-butyldimethylsilyl
molecules such as vitamins, nucleic acids, and neu- (TBDMS) derivatives.85 Also, a single-step esterifi-
rotransmitters. cation procedure, using ethyl chloroformate, has been
The primary source of amino acids for protein developed.86 Many GC/MS methods employ isotope
synthesis is dietary protein. Proteins are enzymati- dilution techniques for quantitative amino acid
cally converted to amino acids in the gastrointestinal analyses52,87-90 with excellent reproducibility and
tract (proteolysis), and are absorbed in blood, where precision. Ionization methods include electron impact
they are available to participate in catabolic and (EI) and chemical ionization (CI) for positive and
anabolic pathways. Approximately 10 of the 20 most negative ions.91 Modified amino acids have also been
common amino acids are essential amino acids and found in disease states, and their measurement was
must be obtained in the diet. Many amino acids are obtained with GC/MS; for example, identification of
readily interconverted to other amino acids by tran- N-acetyl amino acids in urine.92
samination. Metabolism includes conversion of amino
acids to ammonia and organic acids by deamination. 2. LC-MS Applications
Many inherited disorders of amino acid metabolism
a. MS/MS Applications. MS/MS was first used
are characterized by a significant elevation in the
in the clinical analysis of amino acids in plasma and
concentration of certain amino acids in blood (amino
blood, using liquid secondary ionization (fast ion
acidemias) and urine (amino acidurias), ammonia,
bombardment, FIB).24,93-95 The early studies required
and organic acids (organic acidemias).12,19,20 Abnor-
the use of manual sample introduction techniques.
mal concentrations of amino acids may also be found
Semi-automated flow injection,96 using FIB ioniza-
in blood or urine as a result of organ failure and
tion, was developed to facilitate automated sample
impaired function from disease or immature develop-
introduction and higher throughput. However, this
ment.21,82
method was somewhat tedious because of problems
HPLC and ion-exchange chromatography are the regarding sample retention on the probe tip and
predominant methods for quantitative amino acid frequent blockage in the capillary at the probe
analysis in clinical laboratories.11,21,38 Improved speci- surface. With the introduction of electrospray ioniza-
ficity can be achieved by combining chromatographic tion, significant improvement in sample throughput
and mass analysis with GC/MS. This approach is and automated sample analysis was realized.97-99
similar to that for organic acid analysis and requires ESI-MS/MS has been subsequently shown to be a
extensive sample preparation and derivatization. robust, rapid, and accurate method for rapid through-
More recently, MS/MS has been applied to the very put, high sample volume, and amino acid analyses
rapid screening for amino acids.54 This method of as demonstrated by validated methods used success-
analysis requires no chromatography and is accurate, fully in newborn screening or clinical amino acid
sensitive, and selective. IDMS techniques are used analyses.2,98-105
in quantitation. The method is limited to screening
Historically, GC/MS has played the primary role
or analyte-specific quantitation because it does not
in clinical chemistry for the diagnosis of inherited
detect all important amino acids. Special sample
metabolic disorders. However, applications of MS/MS
preparation techniques are required prior to the
in the analysis of dried filter paper blood samples for
analysis of disulfide-forming amino acids, e.g., ho-
purposes of screening inborn errors of metabolism is
mocysteine and cysteine. Chromatography is neces-
now sharing in this primary role. Clearly, MS/MS
sary to separately measure isomeric amino acids, e.g.,
applications are making an extraordinary impact in
leucine, isoleucine, alloisoleucine, and isobaric amino
increasing the number of metabolic disorders screened
acids (e.g., hydroxyproline). Recently, some develop-
in newborns.2,102,103 The MS/MS analysis of amino
ment toward rapid capillary HPLC methods with MS/
acids requires no chromatographic separation and is
MS detection has occurred as reported at the 4th
extremely rapid (∼2 min per sample). It is highly
International Society of Neonatal Screening Confer-
accurate, selective, and precise. For example, the MS/
ence.83
MS analyses of newborn blood samples for PKU has
1. GC/MS Applications been shown to significantly lower the false-positive
rate by at least 10-fold as compared to other tech-
Amino acids are often analyzed by GC/MS by using niques that use HPLC, fluorometry, and bacterial
methods similar to that for organic acids47,74 Most inhibition.83,106 MS/MS also demonstrated the ability
amino, carboxylic acid, and hydroxyl functional groups to accurately detect PKU in samples collected within
of amino acids easily form TMS derivatives.47 Analy- the first 24 h. Blood specimens collected from infants
sis of amino acids in urine has been problematic less than 24 h after birth increase the risk of a false-
because of the high concentration of urea. Large negative result because the diagnostic metabolite,
phenylalanine, may not have reach sufficient con-

centration to be diagnostic for PKU. Time of collection
is also important for other disorders characterized
by the accumulation of toxic metabolites, e.g., MSUD,
homocystinuria, etc. For MS/MS techniques, the
threshold concentration, i.e., the concentration that
is used to decide whether a sample result is pre-
sumptive for a disease, can be altered in such a
manner to reduce false negatives without raising the
false-positive rates substantially.
The sample preparation required for the MS/MS
analysis of amino acids in blood specimens is rela-
tively simple. The use of methanol to extract amino
acids and other organic compounds from the dried
blood imbedded in a filter paper matrix results in
high extraction efficiencies. This extract is also free
of proteins and high salt concentrations that could
otherwise form adducts with important analytes,
thereby reducing sensitivity.24 Furthermore, stable
isotope-labeled amino acid standards are added to the
methanol extract and are subsequently used to
quantify amino acids.24,83 Note, however, that meth-
ods that use extraction solvent that contains internal
standards will not account for the losses of analyte
during the extraction process. Therefore, this method
of quantitation of amino acids is not entirely the same
as traditional IDMS methods, in which the internal
standard is mixed with liquid samples. The use of
the term, pseudo-isotope dilution mass spectrometry
(pseudo-IDMS) should be considered as a way to
indicate that the manner in which stable isotopes are
used in filter paper blood specimen analyses is a
variation of traditional IDMS methods. Figure 7. Product ion ESI-MS/MS of butyl esters of
phenylalanine (M + H)+ ) 222 Da and citrulline (M + H+)
b. Filter Paper Blood Specimens. Although the ) 232 Da. Laboratory data from D. Chace and E. Naylor.
analysis of dried filter paper blood samples with
pseudo-IDMS is less accurate than liquid specimens dicarboxylic acid groups greatly improves ionization
with IDMS, there are some advantages in their use. efficiencies and hence analytical sensitivity, espe-
These advantages include ease of collection, reduced cially for LSI-MS methods. Furthermore, esterifica-
biohazards for some bacteria and viruses that are tion improves the mass differentiation of dicarboxylic
killed on exposure to air, and improved storage and acids such as glutamate and aspartate from other
pre-sample cleanup for methods that use organic amino acids. Reconstitution of analytes in acetoni-
solvent extraction. Nevertheless, there are some trile:water (50:50) with 0-1% formic acid has been
limitations that require mention. The volume of dried used as the mobile phase in most procedures.83
blood samples on filter paper is imprecise.107,108 The selective MS/MS analysis of several diagnosti-
Therefore, much of the high accuracy of MS methods cally important amino acids is obtained via a neutral
that use isotope dilution techniques, i.e., addition of loss of 102 Da (NL 102) scan function. Butyl formate
known concentrations of internal standard to known (102 Da) and a product ion that is 102 Da less than
volumes of liquid, is lost. The estimated volume of its precursor ion are produced by collision-induced
blood obtained from a dried filter blood sample can dissociation (CID) of protonated butylated R-amino
be calculated from the area of the excised sample and acids. An example of the product ion spectra of the
the mean blood volume per square inch or square butyl esters of amino acids, e.g., phenylalanine, is
centimeter. This estimated volume is based on a 50% shown in Figure 7. A schematic of the CID for
hematocrit and a quantity of liquid blood applied to phenylalanine is shown Figure 8. The neutral loss
the paper.109 Blood spot diameters and blood hema- of 102 Da MS/MS profile of a normal newborn and a
tocrit that are different from the NCCLS reference newborn with the inherited metabolic disorder PKU
standard109 will produce errors in the calculated blood (phenylketonuria) is presented in Figure 9. PKU is
volume of the excised filter paper dot.108 Even with an inherited disorder produced by the absence of
these limitations, the use of filter paper blood speci- phenylalanine hyrdoxylase, resulting in a reduction
mens is increasing, especially in the age of the in phenylalanine (Phe) metabolism and a subsequent
genome, where DNA can be extracted and analyzed. hyperphenylalaninemia. Tyrosine (Tyr) is the end
Following amino acid extraction, analytes are de- product of phenylalanine hydroxylase, and the ty-
rivatized to butyl esters with acidified butanol (3 N rosine concentration in blood is potentially reduced
HCl in butanol or butanol with acetyl chloride). in conditions where phenylalanine hydroxylase is
Butylation of the amino acids that contain mono- and deficient. However, this expected tyrosine deficiency
Figure 8. Schematic of collision-induced dissociation (CID)

of the protonated butyl esters of phenylalanine and citrul-
line.
may not be substantial if significant amounts of

tyrosine are derived from dietary sources. Because
these two amino acids are the substrate and product Figure 9. Neutral loss of 102 ESI-MS/MS of butylated,
dried-blood spot extracted amino acids from a normal
of phenylalanine hydroxylase, their simultaneous newborn (top) and newborns diagnosed with phenylketo-
measurement and concentration ratio (Phe/Tyr) is a nuria (middle) and acute neonatal citrullinemia (bottom).
very sensitive indicator of PKU.106 In the analysis of Stable isotope internal standards are underlined italic.
a sample obtained from a patient with PKU, a Laboratory data from D. Chace, E. Naylor, and J. DiPerna.
significant elevation in phenylalanine (m/z 222) rela- of ammonia or other basic side chains in addition to
tive to its internal standard, phenylalanine-d5 (m/z butyl formate. A NL 119 Da scan function is used to
227) is observed in Figure 9. Other amino acids are detect ornithine, citrulline, and homocitrulline. Cit-
also measured, including tyrosine (m/z 238), alanine rulline can also be detected in a NL 102 scan from
(m/z 146), leucine + isoleucine (m/z 188), glutamic the (M + H - 17)+ produced by nozzle-skimmer or
acid (m/z 260), and their respective internal stan- orifice-induced dissociation in the electrospray source.
dards. The product ion scan for citrulline butyl ester (m/z
Some basic amino acids are analyzed with alterna- 232) is presented in Figure 7. A representation of CID
tive scan functions. These scans account for the loss of citrulline is shown in Figure 8. An ion at m/z 113
(Figure 7) represents the loss of 119 Da from the

precursor ion of citrulline at m/z 232, and its CID is
represented in Figure 8. Other important fragments
of citrulline include m/z 215, representing the neutral
loss of ammonia (M - 17), and m/z 70, representing
the neutral loss of 162 Da produced by losses of butyl
formate104 and H2N-CO-NH2. Interestingly, citrul-
line is also detected in a NL 102 scan analyses at
m/z 215. Citrulline loses ammonia in the electrospray
source prior to analysis in the first quadrupole.
Therefore, the source-induced fragment ion with a
mass/charge ratio of 215 is detected in MS-1. This
ion is subject to a neutral loss of 102 Da in the Figure 10. Chemical structures of homocysteine and
collision cell producing a product ion at m/z 113. homocystine.
Acute neonatal citrullinemia is characterized by a
substantial increase in the concentration of citrulline period but do become more apparent with age.
in blood and plasma. A MS/MS of a NL 102 profile of Premature death can arise from venous and arterial
citrulline, using the electrospray source-induced frag- thrombosis. Disorders of the folate-dependent me-
mentation pathway described above, is shown in thylation of homocysteine, part of the methionine
Figure 9. A significant elevation of citrulline butyl recycling pathway, produce mental retardation. Neo-
ester (M - 17, m/z 215) is shown relative to its natal screening is the only practical means to screen
internal standard, d2-citrulline butyl ester (M - 17, for these disorders early.
m/z 217). In addition to a NL of 102 profile, a NL Recently, the importance of plasma levels of ho-
119 profile or MRM analysis of the ions 232/113 mocysteine has emerged as an indicator of risk for
(precursor/product or Pre/Pro) can be used to mea- cardiovascular disease, although the relationship
sure citrulline. Other amino acids, including orni- between elevated homocysteine and cardiovascular
thine and arginine, are detected via similar ap- disease is not completely understood.82 In addition,
proaches, e.g., ornithine using NL 119 and arginine elevated plasma homocysteine may indicate folate
using NL 161 scan functions.2,102 and cobalamin cofactor deficiencies, which can con-
tribute to neural tube defects. Increasing folic acid
c. HPLC-MS and HPLC-MS/MS. In addition to
in the diet is believed to decrease levels of homocys-
MS/MS approaches that use flow injection analysis,
teine in plasma and thereby reduce the risk for
other methods use chromatographic separation prior
cardiovascular disease and neural tube defects. With
to MS or MS/MS analyses. Tuchmann110 described a
either newborn screening for inherited metabolic
pseudo-IDMS method for the analysis of phenylala-
defects or adult population screening for risk factors
nine and tyrosine in filter paper blood samples by
in cardiovascular disease, MS is emerging as an
selected ion monitoring. A benzoylation derivatiza-
accurate and precise method for measuring homocys-
tion procedure has been used prior to HPLC-MS
teine in plasma and (potentially) newborn blood
analysis111,112 to simplify the analysis of liquid speci-
samples.
mens. This procedure avoids many tedious steps such
a. GC/MS and LC-MS Applications. More than
as protein precipitation, drying, and the use of
70% of homocysteine is bound to plasma proteins via
pyridine. The combination of MS/MS and HPLC is
disulfide bonds with other thiol-containing amino
under development in several laboratories and may
acids. It is also present as free circulating disulfide
be an even more powerful technique for the quantita-
(homocystine) and as a mixed disulfide with cysteine.
tive clinical analysis of amino acids.
Measurement of homocysteine requires reduction of
3. Homocysteine the disulfide bonds that are formed by homocysteine
and other thiols prior to analysis. Preventing refor-
Homocysteine is a branch-point metabolic inter- mation of the disulfide bond is necessary with GC/
mediate in a pathway that produces cysteine from MS and LC-MS methods.113
methionine or reconversion back to methionine. Ho- Several GC/MS methods used to quantify homocys-
mocysteine is unstable in solution. In excess, ho- teine in plasma and urine have been published.114-120
mocysteine undergoes conversion to its disulfide, Many of these assays use β-mercaptoethanol118,119 to
homocystine. Structures of homocysteine and ho- reduce disulfides to the free thiol compounds. Sample
mocystine are presented in Figure 10. Homocysteine purification includes the use of cation- or anion-
is the main biochemical marker for inborn errors of exchange chromatography. Isotope dilution tech-
transulfuration. Homocystinurias are disorders char- niques and selected ion monitoring are used to
acterized by increased concentrations of homocys- provide accurate, precise, and sensitive quantitation.
teine in urine and plasma. The most common ho- Ubbink121 compared several homocysteine assays and
mocystinuria is produced by cysteine β-synthase found poor agreement of a comparison between GC/
deficiency. This enzyme deficiency produces a meta- MS and other techniques such as HPLC, fluores-
bolic block of the conversion of homocysteine to cence, and enzyme immunoassays. These methods
cystathionine,82 resulting in the accumulation of expressed different biases in homocysteine quanti-
homocystine and methionine in plasma. Clinical tation for fasted patients or those subjected to me-
symptoms that include ocular, skeletal, and vascular thionine loading. The study concluded that the
abnormalities are not detectable in the newborn results of each method could not be interchanged. The
Figure 12. Chemical structures of thyroid hormones T3

and T4.
4. Thyroid Hormones
The thyroid gland produces two major hormones,
thyroxine (T4, tetraiodothyronine) and triiodothyro-
nine (T3). The chemical structures of these com-
pounds are shown in Figure 12. T3 is the most
Figure 11. MRM flow injection profiles of homocysteine biologically active of these hormones and is 5-fold
(HCY) and its internal standard (IS), D4HCY, for normal more potent than T4. The thyroid gland primarily
plasma (a), abnormal plasma (b), normal urine (c), and (d) secretes T4; peripheral deiodination of T4 accounts for
abnormal urine. Laboratory data from P. Rinaldo and M.
Magera.
nearly 80% of T3 production.128 These thyroid hor-
mones have many important actions, including regu-
lation of metabolic rate, growth, maturation, and
Centers for Disease Control (CDC)122,123 and others124
development.
produced a similar report and conclusions.
Disorders of thyroid hormone metabolism include
Newer developments have been made to improve hyper- and hypothyroidism.128 Each metabolic disor-
the analysis of homocysteine125 and related metabo- der is expressed by different clinical symptoms and
lites.126 Recent publications by Magera et al.125 and produces characteristic physiological effects. Hy-
Gempel127 used continuous flow ESI-MS/MS to ana- pothyroidism is defined as a deficiency of thyroid
lyze total homocysteine from plasma and urine activity. It is relatively common in mild or severe
samples with isotope dilution techniques. Disulfides forms and occurs predominantly in women with
were reduced with dithiothreitol. The product ion advancing age. Primary hypothyroidism occurs as a
tandem mass spectrum of underivatized homocys- result of decreased production in T3 and T4. Deficien-
teine (m/z 136) is characterized by a prominent cies of T3 and T4 cause hypersecretion of thyroid-
fragment ion at m/z 90 (neutral loss of formic acid). stimulating hormone (TSH). Congenital hypothyroid-
Other ions represent the additional loss of either ism is produced from inherited defects in the synthesis
ammonia or hydrogen sulfide and loss of formic acid, of thyroid hormones or the absence of a thyroid
resulting in product ions at m/z 73 and 56, respec- gland.37 Irreversible neurological damage will occur
tively. This CID is similar to the fragmentation of if this disorder is not detected in the newborn period.
R-amino acids shown in Figures 7 and 8, with the Detection of hypothyroidism relies upon measure-
exception that homocysteine is underivatized in this ment of high TSH levels or low thyroid hormone (T3
application.125 Data were acquired in the SRM mode and T4) concentrations in blood. Hyperthyroidism is
with the transitions (Pre/Pro) of 136/90 for homocys- characterized by elevated levels of thyroid hormones
teine (HCY) and 140/94 for d4-homocysteine (D4HCY). together with suppression of TSH concentrations in
Figure 11 shows an overlay of SRM-extracted ion blood. It is a relatively rare disorder with hyper
chromatograms of HCY and the internal standard D4- “stress” symptoms such as weight loss, fatigue,
HCY for control (A) and abnormal (B) plasma samples nervousness, and restlessness.
and control (C) and abnormal urine samples (D).125 The clinical analysis of thyroid hormones in plasma
This MS method125 demonstrated good correlation is used to diagnose thyroid diseases.38 Total T4 is the
with the other methods that are used routinely in sum of free T4 plus T4 bound to plasma proteins. The
clinical laboratories. determination of total T4 reflects thyroid hormone
production.128 However, measurement of free T4

provides improved diagnostic information because it
is unaffected by the elevated concentrations of plasma
proteins that occur in various clinical states such as
pregnancy. T3 is a poor measurement of thyroid
activity because its concentration in blood is highly
variable; it fluctuates rapidly in situations of physi-
ological stress or other diseases. TSH analysis may
be a better indicator for detection of thyroid disorders
because TSH reflects the integrative action of all
thyroid hormones. Many laboratories measure plasma
thyroid hormone and TSH concentrations to provide
the most complete diagnostic information.
Mass spectrometric methods have been developed
to analyze the thyroid hormones T3 and T4 primarily
by GC/MS129-139 and most recently by LC-MS.131,140
The GC/MS methods used in T3 and T4 quantification
are similar to procedures use to analyze amino acids.
Derivatization methods include trimethylsilylation,136
trifluoroacetylation,134,135 methylation,132,133 and the
formation of N,O-diheptafluorobutyryl methyl es-
ters.129,130 Mass spectrometric analysis is primarily
characterized by isotope dilution GC/MS with SIM
of the thryoid hormones and their associated isoto-
pically labeled internal standards. The results from
these studies demonstrate excellent precision and
compare well to other methods such as radioimmu-
nossay.133 MS has been suggested as a reference
method for thyroid hormone analysis.132
Recently, the use of LC-MS/MS has been applied
to the analyses of thyroid hormones. Thienpont131,140
Figure 13. Schematic of β-oxidation of fatty acids.435 CU,
describes a flow injection ESI-MS/MS analysis fol- carnitine uptake transporter; AS, acyl-CoA synthase; CPT
lowing protein precipitation and HPLC column chro- I and II, carnitine palmitoyl transferase I and II; T,
matography for sample purification. In both methods, carnitine, acylcarnitine translocase; VLCAD, MCAD, and
an SRM analysis is used to quantify T3 by obtaining SCAD, very long-chain, medium-chain, and short-chain
mass spectra of T3 (m/z 652/661) and its internal acyl-CoA dehyrdogenases, respectively.
standards 13C9-T3 (m/z 606/614). T4 quantification is
obtained similarly by measuring T4 (m/z 777/731) and strates (ketone bodies) that are utilized by other
its internal standard, 13C9-T4, (m/z 783/737). A com- tissues for energy metabolism. Very long-chain fatty
parison of GC/MS and LC-MS/MS demonstrated acids, primarily 20-26 carbons, are metabolized by
limits of detection of 100 and 18 pg, respectively, with peroxisomal β-oxidation to a chain length that is
excellent recoveries, precision, and accuracy.131 within the range for mitochondrial β-oxidation.38,69
Intermediates of mitochondrial β-oxidation or per-
D. Acylcarnitines and Acylglycines oxisomal β-oxidation are important clinical diagnostic
markers for many disorders of fatty acid and organic
The oxidation of fat plays a major role in energy acid metabolism. Knowledge of these pathways helps
metabolism especially during fasting periods.37,141 in understanding disease process as well as assisting
Fatty acids with carbon chain lengths of primarily in the interpretation of complex metabolic profiles.
18 carbons or less are metabolized in the mitochon- For example, in the inherited metabolic disorder,
dria by a process known as β-oxidation (Figure 13). MCAD (medium-chain acyl-CoA dehydrogenase) de-
They are transported through the cellular membrane ficiency, the metabolism of medium-chain length
into the cell cytosol and are translocated across the fatty acids is impaired. As shown in Figure 13, this
outer mitrochondrial membrane to form fatty acyl- disorder primarily produces an increase in mitochon-
CoA thioesters.37 The fatty acyl group is transferred drial 8-carbon fatty acids as well as significant
to carnitine and transported into the inner mitochon- concentration of 6- and 10-carbon saturated and
drial matrix, where it is transferred back to CoA to unsaturated fatty acylcarnitines. These mitochon-
reform a fatty acyl-CoA plus free unesterified car- drial fatty acids can be found in blood and plasma
nitine. The fatty acyl-CoA metabolites undergo oxi- and detected using methods such as MS/MS. Knowl-
dation by a complex of membrane-bound and matrix- edge of β-oxidation assists in the recognition that an
soluble enzymes (that are “size”-specific), producing impairment of medium-chain length fatty acids would
acetyl-CoA and fatty acyl-CoA thioesters. Each round lead to an accumulation of these fatty acylcarnitines.
of metabolism produces a single molecule of acetyl- Likewise, very long- or short-chain acylcarnitines and
CoA and a fatty acyl-CoA that is two carbons shorter. numerous other disorders would likely produce dis-
Acetyl-CoA is converted in the liver to other sub- ease-specific metabolic patterns.37,141
1. Inherited Disorders of Fatty Acid and Organic Acid Another approach to analyze acylcarnitines by GC/
Metabolism MS is based on the preparation of volatile lactones
Inherited disorders of fatty acid oxidation are an via cyclization.146-149 Isolated acylcarnitines are trans-
important class of metabolic diseases; between 20 and formed into acyloxylactones and are analyzed by
30 disorders have been characterized.38,141 These positive CI-GC/MS, using isobutane as reactant gas.
inherited metabolic diseases can produce hypoglyce- The selected ion monitoring of a common ion at m/z
mia, vomiting, liver disease, cardiomyopathy, devel- 85 and its molecular ions enabled a selective and
opmental delay, hypotonia, seizures, coma, and pre- sensitive detection of all C2-C18 acylcarnitines.
mature sudden death.37 Symptoms can occur early Alternatively, Huang developed a novel approach
in the newborn period through adult life in varying that uses N-demethylated derivatives.150,151 This ap-
degrees of severity. Alternatively, these diseases may proach involved esterification followed by ion-pair
be asymptomatic until a life-threatening episode. extraction with potassium iodide into chloroform and
Exacerbation of the fatty acid oxidation disorders subsequent on-column N-demethylation of the result-
occurs especially during fast or inadequate caloric ing acylcarnitine propyl ester iodides. The products,
intake whereas organic acidemias are exacerbated by acyl demethylcarnitine propyl esters, are volatile and
high protein intake. Several disorders in mitochon- are amenable to CI-GC/MS analysis.
drial β-oxidation (Figure 13) have been characterized b. LC-MS and HPLC-MS. Although GC/MS offers
and include very long-chain acyl-CoA dehydrogenase the advantage of characterizing acylcarnitines by
(VLCAD) deficiency, medium-chain acyl-CoA dehy- chromatography and MS, these methods are tedious
drogenase (MCAD) deficiency, short-chain acyl-CoA and time-consuming. Because carnitine is a pre-
dehydrogenase (SCAD) deficiency, multiple acyl-CoA formed cation, it is readily detected with LC-MS as
dehydrogenase deficiency (MADD, GA-II), carnitine a positive ion with high sensitivity. Therefore, meth-
transporter defects, long- and short-chain hydroxy- ods utilizing LC-MS and LC-MS/MS have grown
acyl-CoA dehydrogenase (LCHAD and SCHAD, re- rapidly during the past 10 yr for several reasons that
spectively) deficiencies, carnitine-palmitoyl trans- include the following: a relatively simple sample
ferase type I and II deficiencies (CPT-I and CPT-II), preparation and analysis; multiple compound and
HMG CoA-lyase, and 2,4-dienoyl-CoA reductase de- chemical class analysis; and an accurate, selective,
ficiencies.37 sensitive, and rapid analysis.
Disorders of fat metabolism produce significant One of the earliest clinical applications for acyl-
elevations of free fatty acids in plasma.37 These fatty carnitine analysis was reported by Roe et al.,152,153
acids are eliminated in urine predominantly as a who used a high-resolution mass spectrometer to
conjugate with glycine (an acylglycine). Fatty acids detect propionylcarnitine in urine. This work was
in the mitochondria form acylcarnitines and are followed by research that identified several other
exported into the cell cytosol and plasma. Acylcar- acylcarnitines with similar methods.154-156 Acylcar-
nitines are eliminated in bile and urine. In metabolic nitines were analyzed underivatized or as methyl
disorders, carnitine deficiency results in many cases esters. HPLC thermospray MS was used to separate
from the continued formation of fatty acylcarnitine on-column mixtures of acylcarnitines157 and subse-
and its subsequent loss in urine and bile. Ameliora- quently used to analyze acylcarnitines in biological
tion of these deficiencies may include enhancing fluids using isotope dilution techniques.158 In another
elimination of toxic fatty acids as acylglycines or application, desorption chemical ionization was used
acylcarntines by administration of glycine and car- for the analysis of acylcarnitines.159 FAB-MS was
nitine or reducing metabolism of fatty acids by subsequently applied to the analysis of acylcar-
restoring and maintaining blood glucose at normal nitines,145,160,161 using a quadrupole MS rather than
levels. In the past decade, MS has played a critical high-resolution sector mass spectrometers. With the
role in the analysis of these important biomarkers introduction of continuous-flow FAB technology, car-
and is rapidly becoming the method of choice in nitines were soon analyzed with these techniques in
newborn and clinical screening.2,22,23 combination with HPLC.162,163 Recently, separation
2. Acylcarnitines of acylcarnitines by capillary electrophoresis com-
a. GC/MS. Carnitine is a highly water-soluble and bined with MS detection has been demonstrated.164
polar quaternary ammonium compound that com- c. LC-MS/MS. Substantial improvements in the
bines with fatty acids to form acylcarnitines of analysis of acylcarnitines and clinical diagnosis of
different carbon chain length. The high polarity inherited disorders of fatty acid metabolism occurred
makes these compounds particularly suitable for LC- with the use of a tandem quadrupole mass spectro-
MS analysis. However, early studies relied upon meter.165-169 Several organic acidemias and fatty acid
hydrolysis of the fatty acyl group followed by GC/MS oxidation defects were found in the plasma or urine
or, alternatively, upon specialized derivatization of patients with these disorders. It was recognized
techniques. Bieber first characterized acylcarnitines that detection of metabolic disorders in the newborn
in 1977 with GC/MS analysis of acyl residues follow- period, prior to clinical symptoms, could prevent
ing hydrolysis142 and later characterized short-chain hospitalization and premature death. Therefore, the
acylcarnitines.143 The therapeutic value of measuring newborn screening MS/MS applications of acylcar-
these fatty acids released from acylcarnitines was nitines extracted from dried filter paper blood samples
recognized by Roe et al.144 Kerner characterized was developed, using liquid secondary ionization
acylcarnitines in urine with GC/MS following saponi- tandem mass spectrometry (LSI-MS/MS) in manual
fication.145 introduction and dynamic modes.9,93,96 The LSI MS/
MS methods (including FAB and FIB ionization) were

further refined including variations and improve-
ments in derivatization93 (i.e., methyl and butyl
esters, validation of clinical methodology for MCAD
deficiency,170 clinical studies,171-175 and the combina-
tion of amino acid and acylcarnitines in a single
assay9,93).
The number of samples analyzed per day was
limited with manual sample introduction LSI-MS/
MS (static FAB or FIB). The analysis of hundreds of
samples per day required multiple instruments and
adequate staff. Automated sample introduction was
achieved with continuous flow LSI-MS/MS (dynamic
FAB or FIB) although the method was problematic
because of sample retention on the probe tip or
clogging at the end of the capillary.96 The use of
electrospray ionization MS/MS methods98-100,176,177
enabled high throughput analyses of clinical samples,
without problems encountered in continuous flow
FAB- or FIB-MS/MS, and improved sensitivity for
several acylcarnitines. Numerous applications of MS/
MS to the analysis of acylcarnitines have been
published recently that demonstrate the scope of ESI- Figure 14. Schematic of collision-induced dissociation
(CID) of the protonated butyl esters of carnitine and
MS/MS for clinical and newborn screening in plasma, acylcarnitines. R indicates a fatty acid of 2-20 carbons.
urine, or filter paper blood samples.2,101-104,178-183
Recently, acylcarnitine analysis has been applied to
derivatization technique share a common mass for
cultured cells184-187 obtained from patients with
that product ion, e.g., m/z 99 or 85 for methyl or butyl
suspected or known metabolic disorders. Further
esters of acylcarnitines, respectively. Precursor ion
improvement of acylcarnitine analysis by MS/MS is
scans are therefore used to generate a selective MS/
still occurring. One problem identified by Johnson188
MS analysis of acylcarnitines.
is the hydrolytic instability of acylcarnitines during
One of the most important developments that
storage and sample preparation. Short-chain acyl-
occurred during the 1990s was multiple metabolite
carnitines are highly susceptible to hydrolysis to fatty
analysis in a single sample injection. For example,
acids and carnitine.
both butyl esters of amino acids and acylcarnitines
d. MS/MS Analyses of Acylcarnitines in a are analyzed concurrently. Furthermore, additional
Dried Blood Sample. The principals and funda- scan functions have been developed for free carnitine
mentals of MS/MS analyses of acylcarnitines have and basic amino acids.2,102 These multiple scan func-
been relatively unchanged since the first studies were tions have enabled a comprehensive multiple meta-
published.93 Acylcarnitines were extracted with etha- bolic profile in newborn screening.103 Disorders of
nol or methanol directly from plasma or dried filter amino acid metabolism, organic acid metabolism, and
paper blood samples. Isotopically labeled acylcar- fatty acid metabolism are measured in a single test.
nitine internal standards are used for the quantifica- As described previously, the introduction of electro-
tion of individual acylcarnitines. Acylcarnitines con- spray ionization led to rapid throughput systems and
tain a quaternary nitrogen that carriers a positive sample introduction robotics. Automated data reduc-
charge. However, carnitine also has a carboxyl group tion and computer-assisted interpretation are com-
that could also carry a negative charge, resulting in monplace.98,99,102
a zwitterion. Esterification of acylcarnitines to methyl An example of an acylcarnitine analyses, using flow
or butyl esters will prevent the formation of zwitte- injection ESI-MS/MS from dried filter paper blood
rions, leaving acylcarnitines with a net positive samples of a normal newborn and newborn subse-
charge. The prevention of formation of a net negative quently diagnosed with MCAD deficiency, is shown
charge is especially true for dicarboxylic acid acyl- in Figure 15. The method includes several MS/MS
carnitines. scan modes.102 There are two full scans (precursors
The MS/MS analysis of acylcarnitines is character- of 85 for acylcarnitines and NL 102 for amino acids)
ized by a stable product ion at m/z 99 or 85 for methyl and three specialized MRM analyses (Pre 103 for free
and butyl esters, respectively. An example of the CID carnitine, NL 119 for basic amino acids, and NL 161
for butyl esters of carnitine and acylcarnitines is for arginine). An example of a the full-scan NL 102
shown in Figure 14. Underivatized acylcarnitines profile for amino acids is shown in Figure 9.
also produce an intense product ion at m/z 85. It is Disorders are indicated when the ratio of diagnos-
important to ensure complete derivatization if butyl tically important metabolites to internal standards
esters are chosen as the derivative because of possible is high. In Figure 15, MCAD deficiency was indicated
loss of selectivity from interference between butyl because of the substantially increased concentration
esters and underivatized acylcarnitines, which both (8 µM) of octanoylcarnitine (C8, m/z 344) and other
share a common fragment ion. On the basis of the metabolites (hexanoylcarnitine, C6, m/z 316, and
product ion spectra, all acylcarnitines of the same decenoylcarnitine, C10:1, m/z 370) as compared to a
Figure 16. Examples of the bile acid, choline, and its

taurine and glycine conjugates.
measuring acylglycines, investigators have developed

methods to detect acyl-CoA thioester intermediates
of fatty acid β-oxidation as the N-acylglycines by
negative-ion chemical ionization GC/MS199 and acyl-
glucuronides as TMS derivatives by GC/MS.200
E. Bile Acids
Bile acids serve important roles in cholesterol
metabolism and digestion of lipids.201 Produced by the
liver and secreted in the intestine, bile acids solubi-
lize lipids to facilitate their absorption. Bile acids also
serve as the major mechanism for cholesterol homeo-
Figure 15. ESI-MS/MS of the butyl esters of carnitine and stasis. The major route of elimination of cholesterol
acylcarnitines from a filter paper blood spot extract of a is by excretion of bile acids into the intestine.
normal newborn and newborn with MCAD deficiency. Cholesterol elimination/conservation is regulated in
MRM transitions are shown (Pre/Pro) for free carnitine part by the extent of reabsorption of bile acids in
(CN) and its internal standard, acetyl (C2), and propionyl- enterohepatic circulation. Therefore, bile acids serve
carnitine (C3) and internal standards, and a full-scan
acylcarnitine profile (Pre 85 Da) of mass range 255-500 as a major mechanism to regulate cholesterol levels
Da. Stable isotope internal standards are underlined italic. in blood. Most disorders of bile acid metabolism are
Laboratory data from D. Chace, J. DiPerna, and E. Naylor. reflected in an abnormal liver function. There are
several metabolic disorders characterized by defective
control samples, where the mean octanoylcarnitine bile acid synthesis or diseases that produce elevated
concentration is less than 0.15 µM. Molecular meth- concentrations of bile acids, i.e., certain peroxisomal
ods were also used to support this presumptive defects.
diagnosis of MCAD by mutation analysis for the most There are two primary bile acids, cholic and cheno-
common genetic mutation, A985G.189 deoxycholic acid. These acids are conjugated with the
amino acids taurine and glycine in the liver to form
3. Acylglycines and Other Fatty Acyl Conjugates taurocholic and glycocholic acids, respectively. Figure
Another method to diagnose patients with meta- 16 provides the chemical structures of cholic acid and
bolic disorders is to measure by GC/MS glycine its taurine and glycine conjugates. Bile acids are
conjugates of diagnostic fatty acids and organic acids synthesized from cholesterol and are produced in the
in urine.169,190-197 An LC-MS method for acylglycines liver primarily as four different bile acid conjugates.
was described by Rinaldo et al.198 In addition to These acids contain polar and nonpolar groups that
together serve to solubilize biliary lipids via their

detergent action. Conjugation with amino acids in-
creases the water solubility and further augments the
detergent properties of bile acids. Several assays have
been developed to quantify bile acids in blood.202 MS
has been important in the identification and quan-
tification of individual bile acids in body fluids such
as plasma and serum.203 Methods include GC/MS,
LC-MS, and MS/MS. Arguments have been made to
include bile acid analyses in newborn screening
panels.204
1. GC/MS
Bile salts are complex biomolecules of which there
are three major classes: (i) unconjugated bile acids,
(ii) glycine- or taurine-amidated conjugates, and (iii)
sulfate, oxo, and glucuronide conjugates. GC/MS
analysis of biles acids provides unequivocal identifi-
cation through retention time and mass spectra and Figure 17. ESI-MS/MS analysis of bile acid conjugates
forms the basis for the claim that GC/MS is a extracted from a filter paper blood spot obtained from a
reference method, especially for the determination of normal neonate (top), a newborn with idiopathic neonatal
hepatitis (middle), and a newborn with extrahepatic biliary
stereochemistry.203 Setchell developed the first ap- atresia (EHBA) (bottom). MRM transitions are shown for
plications of MS to the analysis of bile acids.205-207 GC and GCC (Pro 74) and TC and TCC (Pro 80). See text
The technique uses solid-liquid/liquid gel extrac- for abbreviations. Stable isotope internal standards are
tions, ion-exchange chromatography, and bile acid underlined italic. Laboratory data from M. Morris.
analysis by capillary GC/MS. The techniques dem-
onstrate increased sensitivity as compared with other taurine conjugates produce a product ion at m/z 80.
conventional methods. Several papers demonstrate MRM analyses of bile acid conjugates and their
the applicability of clinical bile acid analysis and internal standards extracted from dried blood spots
disease detection with GC/MS.208-220 of a normal neonate, a neonate with idiopathic
neonatal hepatitis, and extrahepatic biliary atresia
2. LC-MS (EHBA) are shown in Figure 17. The method is
simple, sensitive, and accurate. However, the use of
The number and scope of LC-MS techniques for the this method in newborn screening is limited by the
analysis of bile acids has increased substantially due probability of high false-positive rates estimated at
to improvements in sensitivity, simplicity, and speed. 10%.230 Introducing a confirmatory test in addition
The earliest LC-MS methods for bile acid and bile to bile acid screening would enable the consideration
acid conjugate analyses were published by Setchell of newborn screening for bile acid disorders.
et al. using either FAB208 or thermospray221 MS.
Other applications of thermospray MS have been F. Cholesterol and Steroids
described.222,223 In addition, tandem mass spectro-
metric methods have been used to further character- Steroid hormones are synthesized from cholesterol
ize bile acids.224,225 Continuous-flow FAB-MS with in the adrenal glands and gonads. Structures of
flow injection sample introduction was developed for cholesterol and other clinically relevant steroids are
shown in Figure 18. The type and quantity of
high-throughput applications.226 More efficient ion-
individual steroids produced in plasma depends on
ization and sample analysis is achieved with spray
which gonads (testes or ovaries) are present and on
ionization techniques, i.e., ESI-MS/MS227,228 and ion
the activity of the adrenal gland.234 Steroids are
spray MS.229 These methods have been applied to the
biosynthesized by a complex group of enzymes that
diagnosis of cholestatic hepatobiliary disease230 as includes hydroxylases, lyases, isomerases, and de-
well as Smith-Lemli-Opitz (SLO) syndrome231 and hydrogenases. Catabolism of steroids occurs prima-
other disorders of bile acid metabolism.232,233 rily in the liver where the potency of steroid hor-
a. Clinical Application of Bile Acid Analysis mones is reduced by addition of hydroxyl groups,
Using ESI-MS/MS. The conjugated bile acids gly- dehydrogenation, reduction, and conjugation with
cocholic (GC), taurocholic (TC), glycochenodeoxycholic sulfuric or glucuronic acid. Most steroids are excreted
(GCC), and taurochenodeoxycholic (TCC) were ana- as water-soluble sulfate and glucuronide conjugates.
lyzed in dried filter paper blood specimens in infants Disorders of steroid biosynthesis and metabolism
with cholestatic hepatobiliary disorders with ESI-MS/ that occur in the adrenal cortex are recognized by
MS.228,230 Bile acids were eluted with methanol that depressed or excess hormone production. Adrenal
contained deuterium-labeled standards of the bile hypofunction is a result of adrenal insufficiency of
acid conjugates named above. Negative ion ESI-MS/ key steroids (Addison’s disease), whereas adrenal
MS was performed, using 50:50 acetonitrile:water as hyperfunction is characterized by the excess of pro-
the mobile phase. Glycine conjugates of bile acids duction of glucocorticoids, mineralcorticoids, and
produce an intense product ion at m/z 74, whereas androgens (Cushing’s syndrome). The genetically
Figure 19. ToF spectrum of cholesterol (m/z analyzed

directly from a filter paper blood sample used as the target
matrix). The [M - H]+ and [M - OH]+ ions of cholesterol
are observed at m/z 385.35 and 369.35, respectively.
Laboratory data from D. Hercules and P. Zimmerman.
Many other studies have focused on the analysis of

androgenic steroids243-246 as well as testosterone.247
In addition to the use of quadrupole mass analyzers,
ion trap GC/MS methods have now been used in the
detection of urinary anabolic agents.248
2. LC-MS
The LC-MS analysis of steroids was introduced by
Shackleton in the early 1980s using FAB ioniza-
tion.249,250 HPLC and thermospray ionization was
used in the MS analysis of corticosteroids.251,252
Comparison of GC/MS analysis with thermospray
MS253 demonstrated that LC-MS was a satisfactory
Figure 18. Steroids commonly measured in the clinical and simple analysis but was not nearly as precise as
laboratory. GC/MS for cortisol measurements. Plasmaspray (dis-
charge-assisted thermospray) has been used to quan-
inherited disorder, congenital adrenal hyperplasia tify 17OH-progesterone present in samples from the
(CAH), is routinely assayed in many newborn screen- saliva of patients diagnosed with CAH.254 Substantial
ing laboratories. This disorder produces an excess of quantitative improvements were observed when elec-
17OH-progesterone as a result of a deficiency of 21- trospray ionization240 was coupled to microbore HPLC.
hydroxylase enzyme. The SLO syndrome is a disorder The analysis of anabolic steroids using atmospheric
of cholesterol metabolism that is characterized by low pressure chemical ionization (APCI) LC-MS, has been
blood cholesterol and a marked increase in 7-dehy- described by Joos et al.255 Several methods that use
drocholesterol. The analysis of steroid hormones has HPLC-MS256-259 and HPLC APCI-MS/MS260 have
been traditionally performed with GC/MS. Diagnosis been described.
of these disorders has often involved GC/MS analyses
of steroids.235-237 3. Time-of-Flight MS
1. GC/MS The use of time-of-flight MS analysis (ToF-MS) in
the analysis of proteins and oligonucleotides is un-
Isotope dilution GC/MS has been shown to be a dergoing a rapid expansion. The number of applica-
reliable, important method for the quantitation of tions for ToF-MS of metabolomes are relatively few.
steroids in serum.14 There have been several publica- One important application that has been described
tions that describe steroid-specific applications that is the ToF-MS analysis of cholesterol and metabolites
have been available for decades. For example, GC/ for the diagnosis of the SLO syndrome.261,262 Choles-
MS techniques have been used to quantify progest- terol and 7-dehyrocholesterol standard are both
erone since the early 1980s.236,238,239 Several recent measured in this assay. An example of a ToF spec-
clinical GC/MS papers have been published that focus trum for cholesterol directly analyzed from a filter
on mineralcorticoid analysis and on its relationship paper blood spot (no sample preparation was used)
to hypertension,240 whereas other studies have de- is shown in Figure 19. (Note also that the filter paper
scribed methods for disease detection such as the blood spot was used as the target.) The [M - H]+ and
SLO syndrome235,241 and others.242 Steroid analysis [M - OH]+ ions of cholesterol are detected a m/z
has been used to screen infants with suspected 385.35 and 369.35, respectively. In the SLO syn-
disorders of the metabolism of these compounds.237 drome, the relative ratio of 7-dehydrocholesterol to
cholesterol is significantly elevated relative to control H. Other Classes of Small Biomarkers

and is diagnostic for that disorder.261
1. Lipids
G. Biogenic Amines Lipids are characterized by their high solubility in
organic solvent and near insolubility in water. They
Catecholamines serve an important role as neu- yield fatty acids on hydrolysis or are esters of fatty
rotransmitters for a wide range of physiological acids with complex alcohols.279 Clinically important
processes.263 The three naturally occurring catechola- lipids include sterols (Section II.F), fatty acids (Sec-
mines are epinephrine, norepinephrine, and dopam- tion II.B.), glycerol esters, ceramides,280 phospholip-
ine. The major sites of synthesis of catecholamines ids, sphingosine derivatives, and terpenes. MS is
are the adrenal medulla and the sympathetic nervous commonly used for the analysis of triglycerides and
system. Epinephrine is the most important amine other lipids,281 especially by isotope dilution GC/
produced by the adrenal medulla and, like other MS.282,283 LC-MS applications for triglyceride analysis
catecholamines, is rapidly metabolized (T1/2 ) 2 min) include ESI-MS284 and MALDI-ToF-MS.285 Many
by catechol O-methylation and oxidative deamina- other applications of GC/MS, LC-MS, and MS/MS
tion. Most biogenic amines and metabolites are have been developed for prostaglandins and throm-
excreted in urine as conjugates of sulfuric and glu- boxanes. An extensive series has been published by
curonic acid. Murphy et al.286 and other investigators.287-293
Disorders of catecholamine overproduction produce
the following clinical sequellae: increased stress, 2. Carbohydrates
decreased blood pressure, decreased blood volume, Carbohydrates are aldehyde or ketone derivatives
thyroid hormone deficiency, congestive heart failure, of polyhydroxy alcohols or are compounds that when
and arrhythmias. Low levels of these neurotransmit- subjected to hydrolysis produce these derivatives.
ters can result in postural hypotension. Pheochro- Monosaccharides are simple sugars, e.g., glucose, and
mocytomas and neuroblastomas are tumors that consist of a single polyhyddroxy aldehyde or ketone
produce excess catecholamines. Elevated concentra- unit that cannot be hydrolyzed. Disaccharides are
tions of catecholamines following quantitative plasma two monosaccharides joined covalently by an O-
or urine analysis provide clinical data that may be glycosidic bond.294 Polysaccharides are multiple
used to diagnose these disorders. In addition, mea- monosaccharide units linked together via glycosidic
surements of catecholamine concentrations in blood bonds. The metabolism of carbohydrates produces
or urine may provide information that is useful in glucose, the primary energy source in humans. In
the diagnosis of orthostatic hypotension and some addition, carbohydrates serve important functions in
psychiatric disorders. glycoproteins such as antibody recognition. Carbo-
hydrates are also incorporated in cell membranes and
Pheochromocytomas arise from neurochromaffin
participate in cell surface recognition. GC/MS and
cells of the autonomic nervous system or the adrenal
ESI-MS have both been used to measure carbohy-
medulla and produce excess catecholamines and
drates in blood.295-300 Carbohydrate analyses by MS
metabolites. The classical symptoms of this disorder has recently been reviewed.301
are sustained hypertension, weight loss, headache, Glucose is routinely measured with a wide variety
palpitations, and anxiety. Ninety percent of these of methods. Rapid glucose measurements are impor-
rare tumors occur in the adrenal medulla. Neuro- tant in disease monitoring, especially for diabetic
blastomas are one of the most common malignant patients. Although MS will not likely be used to
tumors in pediatric patients. Approximately 80% are routinely measure a single carbohydrate such as
found in children under 5 years of age. More than glucose, it is more likely that glucose will be part of
90% of these tumors produce excess norepinephrine, a multi-analyte test of several markers for diabetes.
dopamine, and vanillylmandelic acid (VMA). The The identification of unusual carbohydrates produced
important analytes that are measured with MS in by bacteria has been performed by MS in several
the clinical laboratory for these diseases are de- applications of infectious disease screening. GC/MS
scribed.264,265 has been used to analyze carbohydrates unique to
GC/MS is the most commonly used mass spectro- bacteria for the identification of the microbes respon-
metric method for catecholamine and catecholamine sible for infection.302-306 Other techniques for carbo-
metabolite analysis.266-269 Pheochromocytomas have hydrate analysis that use a mass spectrometer have
been diagnosed with these methods.270 Furthermore, been developed.307,308
applications of GC/MS has also been used for the
diagnosis of neuroblastomas.271,272 Most methods 3. Trace Elements
incorporate isotope dilution techniques in their mass Trace elements are essential elements that are
spectrometric analysis, with special attention to present in milligram per kilogram amounts or less,
quality assurance.273 Analyte-specific methods have and if deficiencies occur, then physiological impair-
been developed for specific disorders. For example, ment results.309 Important trace elements in bio-
GC/MS has been used to analyze dopamine274,275 in chemistry include iron, zinc, copper, iodine, cobalt,
research and clinical investigations of Parkinson’s molybdenum, and selenium. The quantification of
disease. Recently, LC-MS methods have been used trace elements is important for diagnosing disorders
to analyze neurogenic amines and include thermo- produced by their deficiencies. One of the key aspects
spray276 and electrospray MS277,278 applications. of the clinical analyses of trace elements is sample
handling. Important considerations are given to may not have adverse consequences. With the power
preventing contamination of environmental sources of µs analysis for analyzing intact hemoglobin in
of these trace elements. Two methods are most often hemoglobinapathies, the question remains: why is
used in the clinical laboratory, atomic absorption it not used routinely in the clinical laboratory? One
spectrophotometry (AA) and inductively coupled answer may be the lack of experience by clinical
plasma emission spectrometry (ICP-ES). However, chemists in performing these assays or the difficulty
inductively coupled plasma mass spectrometry (ICP- of integrating them in routine clinical screening.
MS) is replacing these other methods because of Second, other assays perform these analysis equally
its high specificity and accuracy. The analysis of well and at lower cost. Third, no laboratory has taken
several trace elements in biological samples has been the lead in developing an MS-based hemoglobin assay
developed.310-316 that is robust, is validated, and can be applied to
hundreds of sample analyses per day. The final
III. Diagnostic Proteins and Glycoproteins answer may be the most important. Currently, intact
hemoglobin MS methods cannot detect several im-
A. Proteome Analyses portant variant hemoglobins. Altered amino acids of
many variant hemoglobin associated with disease
The important role of MS in the identification of (HbC, HbE, and HbO) alter the mass by only 1
proteins and their correlation to the genes that Da.5,328 The resolution of mass spectrometers are not
encode them is discussed in the paper “Mass Spec- sufficiently adequate to separate the globins by mass
trometry in the Age of the Proteome” by Yates.317 and, as a result, cannot detect some hemoglobinapa-
With near completion of Human Genome Project, thies.
emphasis will shift from genomics to proteomics. MS Further advances in MS will permit more rapid
will have an important role in the characterization amino acid sequencing and more accurate, higher
of expressed proteins.6,318-320 Gene expression in- resolution analyses of intact large biomolecules.34
volves the production of mRNA that translates DNA- Ionization techniques are playing key roles in this
based information into protein synthesis.35,321 Abnor- area and include ESI and MALDI.329 New develop-
mal proteins are produced from errors in gene ments in mass analyzers (including quadrupole MS
sequences provided that these sequences are used in and MS/MS systems, ion trap MS, Fourier transform
the translation of information from DNA to RNA. In MS, and ToF-MS1) and analyzer configurations, e.g.,
addition, the “predicted” abnormal sequences may be Q-ToF, will demonstrate improved sensitivity and
altered or removed by post-translational modifica- resolution at higher masses. This section of this
tions.322 Proteins with altered amino acid composition review will emphasize MS analyses of hemoglobin
may or may not have clinical consequence, depending and glycohemoglobin as an example of protein analy-
on which amino acids are altered, whether these sis via MS in the clinical laboratory.
altered amino acids change substrate specificity, or
other changes that affect protein activity, specificity, B. Hemoglobin
or function.
MS is important in protein identification and HbA comprises 96% of normal adult hemoglobin
characterization.34,318,319,323-326 In clinical research, whereas hemoglobin A2 (HbA2) accounts for 3%. HbA2
mass information and structure elucidation is often is composed of two R and two β chains. During fetal
used to understand the function of a protein and can development, the dominant hemoglobin is fetal he-
be used to correlate the protein composition with a moglobin (hemoglobin F, HbF). Fetal hemoglobin is
gene sequence. It is likely that, on completion of the composed of two R and two γ globin chains. After
human genome project, research dollars will be birth, the amount of HbF diminishes to levels of less
redirected to the investigation and characterization than 1% in adults.330
of proteins coded by human genes. MS will play a Thalassemias are inherited disorders characterized
dominant role in this area, especially with regard to by decreased synthesis of either R or β globin chains.
post-translational modifications.327 As proteins are R-Thalassemias are the most common genetic abnor-
identified as important biomarkers of disease, the use malities of hemoglobin in humans. Symptoms of R-
of MS to screen proteins that are diagnostic for these and β-thalasemias are categorized as minor to severe
disorders will increase. The MS analysis of hemoglo- with the latter including lethal anemia, growth
bin328 is an example of the potential role of protein retardation, and bone malformation. In contrast,
analyses in a clinical screen. hemoglobinopathies are disorders characterized by
Normal adult hemoglobin (hemoglobin A, HbA) is structural alternations in either one or more globin
a tetrameric protein that is composed of two sets of chains. Over 700 structural variants of hemoglobin
polypeptide chains, two R and two β chains. Hemo- have been found, with the majority having no clinical
globin exists in various forms that are characterized or hematological manifestations.328 Some hemoglo-
by the composition of the globin chains. Some hemo- binopathies, however, have significant clinical se-
globin variants exists normally and, in fact, are quellae. Hemoglobin S (HbS) is a form of hemoglobin
predominant at defined stages of development, i.e., that forms long rope-like polymers with other HbS
hemoglobin F is the dominant hemoglobin in the molecules. These hemoglobins aggregate and distort
fetus. Other variants of the globin chain produce the shape of red blood cells to form a characteristic
serious disease, i.e., hemoglobin S is the dominant “sickle” shape. Clinical symptoms of sickle cell ane-
hemoglobin in sickle cell disease. MS has played a mia include joint pain and organ damage in patients
role in identifying many more variants that may or that are homozygous for this disease.330 Heterozy-
gotes (carriers of one copy of the altered gene) are

generally unaffected unless another hemoglobin vari-
ant is present, i.e., HbC, HbO-Arab. These affected
patients are classified as compound heterozygotes for
a sickle cell hemoglobinapathy and often exhibit
symptoms of sickle cell disease. A combination of HbS
with a thalassemia may produce a sickle cell disorder.
This is because HbS becomes the dominant hemo-
globin in the absence of HbA. Due to the extremely
large number of hemoglobin variants discovered, a
classification system was developed.328 Variants are
classified by the globin chain affected and the sites
of substitution. In addition, a characterization of the
mutation is often included in the notation. HbS, for
example, is denoted as β6GlufVal.
Traditionally, hemoglobins have been analyzed
with electrophoresis, isoelectric focusing, and ion- Figure 20. ESI-MS spectra of extracted hemoglobin from
exchange HPLC. With the advent of LC-MS tech- a dried filter paper blood sample for a normal newborn and
niques such as electrospray and more recently MAL- a newborn categorized as a carrier (heterozygote) or
DI-ToF, MS has been used to identify specific homozygous for sickle cell disease. Laboratory data from
M. Morris.
hemoglobin variants. It has also been used as a
confirmatory method. With further improvements, may provide further information for protein identi-
MS may become a primary screening tool to analyze fication. Fourth, these peptic digests are analyzed
blood for hemoglobinopathies and thalassemias. How- with HPLC-MS, HPLC-MS/MS, or MS/MS without
ever, it should be noted, that simple and accurate chromatography.
non-MS-based methods exist for hemoglobinopathies,
that the comprehensiveness of MS results are not 2. Intact Globin Analysis
always needed, and that the expertise and resources
needed to perform these analyses are high. Therefore, Electrospray MS has been used to characterize
implementation of MS analysis of proteins such as intact hemoglobin.331,348-350 The mass spectrum gen-
hemoglobins may require the consideration of new erated by electrospray ionization is characterized by
models for laboratory services before MS testing can a series of molecular ions of different charge states,
be widely implemented. ranging from approximately 11 to 21 under the most
common operating conditions.5 Adjacent peaks differ
Several excellent reviews on the subject of hemo-
by 1 charge in the “molecular envelope” of hemoglobin
globin analysis are available.5,331,332 The analysis of
multiply protonated molecules. Software transforms
hemoglobin can be achieved in two ways: (i) pro-
these data to a unique mass that characterizes the
teolytic fragment analysis and (ii) intact hemoglobin
average molecular mass of hemoglobin. An example
analysis. It is worth noting that a comprehensive
of transformed mass spectra for the R and β chains
analysis of hemoglobin includes proteolytic fragment
of hemoglobin, using a quadrupole mass spectrom-
and intact hemoglobin analysis. Because certain
eter, is shown in Figure 20. Wada describes the use
limitations exist in both methods, a comprehensive
of a high-resolution sector instrument for this analy-
hemoglobin analysis will use both methods to provide
sis.351 TNBA (trinitrobenzenesulfonic acid) and TNB
the most accurate clinical information.
(5,5′-dithiobis(2-nitrobenzoic acid)) conjugates of he-
1. Proteolytic Fragment Analysis moglobin have also been analyzed.352
In an alternative approach to proteolysis, MS/MS
The study of proteolytic fragments of hemoglobin with electrospray ionization has been used to analyze
were describe by Wada et al.333 and others,334-336 hemoglobin fragments produced by CID of the
using desorption ionization techniques. In addition, hemoglobin chains.353-355 Recent progress has led
FAB-MS337-343 and more recently, electrospray344-346 to the development of nano-ESI-MS/MS analysis.346
and MALDI-ToF MS347 have been used to rapidly The potential use of ToF analysis of hemoglobin
analyze protein digests. In general, the proteolytic has been recently investigated.356 The use of MS
MS analysis of hemoglobin is comprised of a few analysis for screening of hemoglobinopathies has
important steps. First, preparative LC techniques are been described.357-359
used to separate globin chains. Second, derivatization a. Clinical Screening of Hemoglobinopathies.
of some amino acids are required to prevent proteoly- Although MS shows great promise in newborn screen-
sis, i.e., alkylation of cysteine residues. Third, various ing, its use has been limited to confirmation of known
proteolytic enzymes, e.g. trypsin are used to hydro- variants or the identification of new variants. An ESI-
lyze proteins at specified locations to form defined MS spectra of filter paper-extracted hemoglobin from
peptide fragments. Trypsin is the most commonly a control patient and patients that are heterozygote
used enzyme because it produces peptide fragments and homozygote for sickle cell disease are shown in
in a mass range that is most suitable for MS Figure 20. HbS is easily identified in both the
analyses. This enzyme hydrolyzes the peptide bonds homozygote and heterozygote in this rapid analysis.
of basic amino acids (i.e., lysine and arginine). Other Heterozygotes are differentiated from homozygotes
enzymes hydrolyze proteins at different sites and by the presence of a sickle β chain and a normal β
adduct, glutathionyl hemoglobin, is also useful as an

index of oxidative stress often found in diabetes and
other hyperlipidemias. However, others have ob-
served increases in glutathionylated β-chain on pro-
longed storage of liquid blood (M. Morris, personal
communication).
C. Specific Disease Diagnostics Using MS

MS has been used in several other clinical applica-
tions for the diagnosis of disease. In the area of cancer
diagnostics, MALDI-ToF has been used to character-
ize glycoproteins and tumor markers.367-369 Other
areas include peptide analysis of insulin370 or neu-
ropeptides.371,372 The use of MS in the characteriza-
tion of proteins in cerebrospinal fluid has also been
Figure 21. ESI-MS spectra of glycated hemoglobin from described.373-375 There are numerous other applica-
a patient with diabetes. Laboratory data from M. Morris. tions in clinical research and protein analysis that
are more appropriate for other reviews.5,34,329
chain. The method is simple because hemoglobins are
extracted from a dried blood sample and followed by 1. Protein Profiling
flow injection ESI-MS analyses. The methods have MALDI-ToF is a technique that is amenable to
limitations in newborn screening. MS cannot detect direct analysis of proteins or peptides from cells, cell
mutations that differ by only a few mass units due lysates, protein digests, etc.323 This ease and versatil-
to inadequate mass resolution. For example, HbC, ity has led to the development of protein profiling,
HbE, HbO-Arab, and D-Punjab may be not dif- which is essentially a qualitative technique that uses
ferentiated because of a mass difference of 1 Da.5 MS to identify the mass of hundreds of proteins
However, with extreme care, some recent work sug- and peptides from a specimens in a single
gests that it is possible to differentiate heterozygotes analysis.324,325,329,376-378 This profile is analogous to a
from homozygotes with a 1-Da mass change even fingerprint for the identification of cells or cellular
though the individual components are not resolved preparations. Examples of emerging applications
(M. Morris, personal communication). include identification of bacteria379-383 and tumor
markers.384,385
3. Glycohemoglobins
a. Identification of Baterial Isolates. An ex-
Glycation of hemoglobin primarily occurs in dia- ample of protein profiling that is emerging in pro-
betic patients.330 The degree of glycation may serve teomics and MS is shown in Figure 22. Bacteria are
as an index of disease control during treatment, i.e., isolated from patients with infections, grown over-
increased glycation indicates poorer control when night on agar plates, and washed. A 500-nL sample
compared to previous measurements. It has been of a single colony is washed and diluted. Then 500
shown that low concentrations of glycated Hb are ml of the dilute solution was mixed with an equal
associated with fewer complications from the dis- volume of sinapinic acid matrix and placed on a
ease.330 The ability of MS to measure glycated and stainless steel target and analyzed by MALDI-ToF.
nonglycated normal hemoglobin offers an improve- The top panel shows a spectrum of Escherichia coli
ment over other methods that require an external while the bottom panel shows a spectrum of Staphy-
reference standard to quantify glycohemoglobin. In lococcus aureus. S. aureus was digested with lysozyme
addition, problems with sample quality (i.e., specimen (identified in the spectrum as the large peak at 14000
degradation or the effect of therapeutic interventions Da).
that interfere with other glycohemoglobin assays) are Although, most individual proteins/peptides are not
reduced by using MS-based analyses.360-362 identified in these spectra, the profiles for each
Glycohemoglobin (GHb) is the term used to denote bacterial isolate are unique and hence suggest the
the binding of glucose to hemoglobin as a ketoamine. ability of MS to identify bacteria on the basis of a
Primarily, glucose condenses predominantly with the unique peptide pattern or fingerprint. Data has been
N-terminal valine of the β-chains and to a lesser shown to be reproducible, and much research and
extent with R-chains and other residues such as development is being devoted to the development of
lysine. MS can detect the number of glycations in a industrial high-throughput methods and fingerprint
given hemoglobin by examining its mass shift but (interpretation) design.386,387 Efforts to standardize
cannot identify the actual site of binding. Mass protocols are being coordinated by analytical chem-
spectrometric analysis with electrospray ionization ists at the FDA.
methods has been performed.360,361 Recently, MALDI- b. Identification of Tumor Markers. Another
ToF has been used to identify glycated hemo- example of protein profiling that has potential in the
globin.363-365 An example of the application of LC- clinical laboratory is characterization and identifica-
MS to the analysis of glycated hemoglobins is shown tion of tumor markers. An example of a procedure
in Figure 21. This analytical approach is similar to for identifying normal tissue from malignant tissue
that used to detect hemoglobinopathies. Recently, is shown in Figure 23. These MALDI-ToF mass
Niwa et al.366 demonstrated that another hemoglobin spectra were directly acquired from cells that are
useful in the identification of the site of origin for

cancers that have metastasized.
IV. Diagnostic Biopolymers

The basis for an inherited metabolic disease can
be found in an abnormal human gene.388 The abnor-
mal gene produces an abnormal protein (either an
enzyme, structural, or functional protein). If the
protein is an enzyme and its activity is impaired,
then abnormal concentrations of metabolites will
occur (Figure 1). The clinical laboratory provides data
for the diagnosis of disease by measuring the con-
centration of metabolites, detecting abnormal func-
tional and structural proteins associated with dis-
ease, or identifying abnormal gene products known
to be associated with specific diseases. Which method
is used for disease diagnosis will primarily depend
on whether a specific biomarker strongly correlates
with a disorder and whether technology is available
to accurately measure that biomarker. Laboratory
data will be combined with clinical observations for
the final assessment by a physician. For diseases that
are not solely genetically based such as infection, the
use of molecular, protein, and metabolite analysis of
foreign DNA, proteins, and metabolites will be used.
Molecular, protein, and metabolite analysis will also
be important analytical tools for risk assessment of
many disorders such as cancer, heart disease, and
Figure 22. Representative spectra from obtained from diabetes.
freshly grown bacteria analyzed by MALDI-ToF MS.
Bacteria were obtained from overnight growth on agar A. Genome Analyses
plates and washed to remove interfering components such
as salts. Gram-negative organisms such as E. coli (A) were The fundamental components of DNA and RNA are
spotted immediately while Gram-negative organisms such nucleic acids. Nucleic acids form nucleosides with the
as S. aureus (B) were incubated with lysozyme prior to
spotting and subsequent MALDI analysis. Each spectrum
addition of ribose and deoxyribose sugars. Nucle-
has 50-100 individual peaks in a unique pattern. Labora- otides are subsequently formed by addition of a
tory data from S. C. Smole, L. A. King, and P. E. Leopold. phosphate residue.54 Nucleic acids are either purine
analogues (adenine (A) and guanine (G)) or pyrimi-
laser capture microdissected384 from a single patient’s dine analogues (cytosine (C), thymine (T), and uracil
frozen tissue: normal breast epithelium, invasive (U)). DNA is composed of deoxyribose sugars and the
ductal carcinoma of breast, and metastatic ductal four bases A, G, C, and T, whereas RNA is composed
carcinoma. The spectra obtained from tumor cells is of a ribose sugar and G, C, T, and U. Nucleotides are
clearly different from normal. This “fingerprint” joined together by phosphodiester bonds and become
serves as a mechanism of identifying various types either a DNA or an RNA polymer. DNA exists as a
of tissue in a rapid manner. It may be especially double-stranded polymer in its native form while
Figure 23. MALDI-ToF mass spectra directly acquired from cells that are laser capture microdissected from a single
patient’s frozen tissue: normal breast epithelium (top), invasive ductal carcinoma of breast (middle), and metastatic ductal
carcinoma (bottom). The low mass region (5000-10 000 m/z) is shown at a lower magnification than the high mass region
(20 000-60 000 m/z). Note the features at 6100-6900, 7700-7900, and 45 000-58 000 that distinguish the normal cell
profiles from those of the carcinomas. Laboratory data from D. E. Palmer-Toy.
RNA is a much shorter single-stranded polymer. practical mass range for analysis of oligonucleotides
Short segments of DNA are called oligonucleotides; is based on the ability of the mass spectrometer to
with the average size of these polymers (oligomer) resolve an A to T transversion (change of A to T).
ranging between 15 and 45 bases. A common term The mass difference of A and T is 9 Da. A resolving
that is often used to denote the number of bases in power of 1 in 1000 is necessary to distinguish these
an oligonucleotide is the suffix “mer”. The number two bases. The analyses therefore has a practical,
of bases followed by mer in addition to the abbrevia- working mass range of 9000 Da or less.
tions ss and ds for single- and double-stranded DNA Advances in the next few years will include the use
is used. Therefore a 15-base double-stranded DNA of chip technology (micro-arrays),396 especially in the
is denoted 15-mer ds-DNA and is composed of deoxy- area of clinical screening. A DNA chip is a small
A, G, C, and T. device that holds a regular array of DNA molecules
The role of MS in DNA-based analysis and screen- that are chemically attached to the surface. These
ing is currently being defined.389-394 Potential routine DNA molecules generally contain known probe se-
applications of MS include detection of DNA modi- quences that will hybridize with their target comple-
fications or mutations and their sequencing. Re- mentary sequences. These arrays are analyzed by a
cently, the use of MS to analyze short DNA fragments variety of techniques, including MS.395,397 Chip tech-
for single nucleotide polymorphisms (SNPs)395 has led nology is amenable to automated sample analyses
to major advances in the automated analysis of DNA and high-throughput screening. MS may be advanta-
via MS. The mass range of mass spectrometric geous in analyzing multiple DNA probes on a single
analysis of oligonucleotides increases each year in chip because of its high selectivity and specificity.
part due to advances in MS technology and also due Multi-probe analyses is as important for screening
to methods for analysis and sample preparation. known genetic mutations in a single analysis in a
Mass resolution and the instability of long DNA similar manner that MS/MS has been used to screen
polymers that are easily fragmented limit the size of for multiple metabolites.
the oligonucleotide that can be analyzed. Most MS articles related to DNA analysis have
New methods in DNA array technology396 are been in the areas of biomedical and clinical research,
rapidly expanding mutation analysis capabilities for whereas the use of DNA analysis with MS detection
genetic diseases by inclusion of multiple genetic in the clinical lab is quite rare. During this decade,
probes for a disease and as a consequence reducing it is likely that screening for single nucleotide poly-
the false-negative rate.397 However, these methods morphisms for risk assessment of cancer and heart
will identify many carriers of disease (unaffected disease will be more commonplace.400 As molecular
heterozygotes). The implications of this technology methods enter routine screening, so will high-
require discussion before implementation. Questions throughput MS applications.
such as “Will carriers (heterozygotes) be reported?”
must be answered. The major contribution that MS B. Current MS Applications
will have in the DNA diagnostics area may be its Clearly, MS is an ideal tool for the analysis of
capacity to analyze multiple genetic probes or gene modified or unusual nucleic acids, nucleosides, or
fragments in a single test. Further into the future, nucleotides. Electrospray ionization is especially
improvements in MS may enable direct analysis of suited for the analysis of these DNA components.
gene fragments without the need for amplification Several methods for the measurement of nucleic
or excessive sample preparation. The question that acids401-404 have been published, especially in the
must be answered is whether a genetic mutation in area of urinalysis of tumor markers.5,405-407 With
question correlates with a disease and if so then how. regard to oligonucleotide analyses, the analysis of
Most likely, MS applications will be primarily focused these small DNA fragments are highly indicative of
on the gene expression. In the near term, the use of mutations that cause disease. New assays have been
DNA as the primary tool for diagnosing disease states developed for clinical disorders,408 including Tay-
will largely depend on the availability and accuracy Sachs disease,409 cardiovascular disorders,410,411 cystic
of other methods that analyze proteins or metabo- fibrosis,391,412 cancer,413 and pathogens.400 There are
lites. For diseases that are characterized by few several other genomic applications of MS in clinical
mutations and that cannot be screened by conven- research that are more appropriately and compre-
tional methods, DNA-based assays will provide a hensively presented in other reviews.414
solution. DNA-based analysis will clearly be used as
a confirmation method as well as prenatal diagnosis
and genetic counseling. More importantly, DNA V. Quantitative Analysis and Quality Assurance
analysis will play a critical role in risk assessment
of disorders such as cancer, neurological disorders, A. Quantification in Clinical Chemistry
and heart disease. The importance of quantification in clinical chem-
Advances in MALDI -ToF analysis has been the istry was described by Yergey at the 11th Sanibel
major contributor to the growth of MS applications Conference on Mass Spectrometry.4 At this confer-
in genomics.398,399 Most MALDI-ToF applications ence, clinical analyses were described as either
have been used to analyze DNA, including fragments qualitative or quantitative. Results from qualitative
as small as a 15-mer for a short oligonucleotide assays are characterized either as positive and nega-
sequence (∼1500 Da) to greater than a 500-mer tive or as black and white. Quantitative analyses are
(>100 000 Da) in most recent studies. Currently, the characterized by results that measure the degree of
a response such as the intensity of light emission, In clinical chemistry, the quantitative limits for
height of a peak plotted on a graph, or the number analytical accuracy must be also be defined.425 Con-
of events (i.e., counts in a defined period).54 A fidence in an analytical result is highest if the result
”semiquantitative” analysis is a category used com- is within the upper and lower range of standards.426
monly to define an assay that measures a response Methods that use ratios of ratios (a ratio of an analyte
but is not a highly precise term.415 to its internal standard versus ratio of reference
Assays often characterized as “qualitative” are material to its internal standard) provide the best
some drugs of abuse or metabolite screening tests statistics. The standard error of the estimates should
that are based on immunoassays or other assay that be used in linearity assessments rather than correla-
are based on biological responses such as bacterial tion coefficients. Data that have nonlinear responses
inhibition assays.23 In these assays, a positive or should not be used; rather, alternative assays should
negative result is most often reported. However, be developed.
these assays still have some basis in accurate quan-
tification, i.e., the concentration of the analyte above B. Quality Assurance
or below the cutoff but sufficiently accurate in order The other major requirement of clinical analyses
for the test to be useful. What may be less stringent is quality assurance.427 Quality assurance distin-
in qualitative assays is the linearity of the concentra- guishes methods used in clinical diagnostics from
tions over a large range of concentrations. It is clear clinical research.54,428 Quality assurance ensures that
that there is some difficulty in distinguishing a the result obtained in an analysis is reliable and
qualitative, semiquantitative, and quantitative assay. accurate. Furthermore, it must detect methodological
A more clear example for qualitative analysis is the errors and other problems that may occur at any
application of MS in hemoglobin analyses. The pres- point between the handling of a specimen and
ence or absence of one or two sickle globin chains reporting of a result. For example, errors can occur
(HbS) is indicative of a carrier or sickle cell disease. during specimen collection, delivery, storage, sample
Quantitative analyses are used to determine the preparation, sample analysis, data reduction, result
concentration of substrates in biological fluids (i.e., interpretation, communication of results to physi-
glucose, phenylalanine, homocysteine) in blood, urine, cians, and follow up. Quality assurance is necessary
CSF, or tissues.25 Quantitative data are generated because undetected errors can result in a false result
by a measurement of a response.416 The reliability of that may harm a patient. The medicolegal implica-
this response is based on statistical confidence pa- tions or a laboratory error can be serious.
rameters such as precision. Semiquantitative analy-
Examples of quality assurance systems developed
ses are actually quantitative assays that do not have
for mass spectrometric assays have been described
sufficient statistical power to generate high confi-
for applications that involve newborn screen-
dence in the precision of the result because of
ing.107,108,273,429 It is noteworthy that, to ensure imple-
analytical limitations. This technique is most used
mentation of a quality assurance program in a
in rapid metabolite screening or therapeutic drug
laboratory that performs clinical analysis, the labora-
monitoring, where the desired results do not need to
tory must meet Federal laboratory accreditation
be highly precise but rather to answer the question
requirements as defined by CLIA (Clinical and
of whether a metabolite falls within a certain range.
Laboratory Improvement Act) of 1988. Research
Standardization is required for all quantitative assays must be clearly delineated from routine clini-
assays. Concentration measurements are based on cal assays and cannot be used to report patient
the relationship of a measured response of a com- results. Several investigators have addressed quality
pound relative to the response of a standard or assurance and the use of mass spectrometric based
reference. This relationship of unknown to standard, assays by developing standards, reference materials,
often expressed as an intensity ratio, is compared to isotope dilution techniques, laboratory comparisons,
the relationship of a known amount of analyte to the and external assessments.107,108,273,427,429-434
standard. The plot of concentration of known analytes Two important applications of MS are (i) its use
versus standard is defined as the standard curve. as a confirmatory test for other assays or (ii) its use
Results from an unknown are obtained by interpola- as part of a quality assurance methods examining
tion from this standard curve. the production and quality of reagents or comparison
Standards are either external or internal. External of results from non-MS-based assays such as immu-
standards417 are measured separately and are used noassays and molecular assays. MS methods devel-
to calibrate the signal of an instrument relative to oped for these applications are critically important
known concentrations. Internal standards417 are because its serves as a means for identification,
present in the same matrix as the unknown and are characterization, and confirmation of analytes or
co-analyzed. Internal standards are typically materials that are used or found in less specific
homologues or analogues of the analyte being assays.
assayed. Standards that differ only by their
isotopic content are the most ideal standards for VI. Conclusions
use in MS applications because they differ only
by their isotopic content rather than chemical prop- As new technology is developed by MS manufac-
erties in most instances. MS applications that use turers working in conjunction with private industry
stable isotopes in quantitation are defined as IDMS and academic institutions, opportunities for innova-
techniques.135,295,415,416,418-424 tive research become available. The development of
novel methods enables the discovery of new of a disease. Other important issues in harmonization
biochemical and physiological processes. With a bet- and quality assurance include clinical interpretation,
ter understanding of these processes, knowledge and result reporting, physician education, and clinical
insight into the basis and mechanism of disease arise. and genetic follow up; those areas where mass
New biomarkers for disease are soon discovered, and spectrometrists are generally not experts. These are
methods are subsequently developed to measure issues that will require attention as MS expands the
these compounds in blood, plasma, or urine. The clinical lab.
methods used in the discovery of disease processes
are the methods that are adapted to their laboratory A. Outlook of Clinical MS
diagnosis. It is clear therefore that advancements in
technology directly impact clinical analysis, albeit It is clear that MS has served an important role in
delayed. Delays in method implementation are a the clinical laboratory, especially in areas of metabo-
result of stringent method validation that includes lome analyses. However, most of these analyses are
still being performed in specialized reference labo-
statistical assessments, method comparisons, pilot
ratories rather than hospital-based clinical labora-
screening studies, and QA/QC implementation. Fur-
tories. This trend is likely to continue in part because
thermore, new methods must be integrated into
MS and the associated specimen preparation and
laboratory protocols and information systems. Ad-
result interpretation requires a high degree expertise
vances made during the past decade are just now
not found routinely among clinical chemists. Never-
being applied to laboratory diagnostic medicine.
theless, there are efforts to automate and enable
These new applications are evidenced by the adapta-
turn-key mass spectrometric bases systems that do
tion and integration of MS/MS methodology used in
not require the extensive experience in MS. The
newborn screening.2 Developed nearly 10 years ago
success of turn-key systems in MS as total solutions
and originally applied to a few dozen plasma samples to a clinical analysis will be more apparent in this
per day, metabolic profiling using MS/MS for the decade.
analysis of dried blood samples is just now being
The heart of the mass spectrometer is the mass
implemented. Over 1 000 000 infants/year will be
analyzer, whether that analyzer is a quadrupole, ion
screened worldwide with MS/MS in 2000, and this
trap, or ToF. This past decade has shown that
growth is expected to continue.2
electrospray and MALDI are rugged, reproducible,
In addition to the benefits of MS in the clinical lab, efficient, and versatile ionization systems. The dra-
there are some limitations. First, mass spectrometric matic changes in mass spectrometer analyzer and
methods are generally not turn-key. Nevertheless, source configurations that occurred in the previous
progress will be made in this area as the demands two decades will shift to equally important develop-
for MS-based solutions increase. Second, there is ments in specimen handling, sample preparation,
limited availability of scientists that have expertise analyte isolation and purification, high-throughput
in laboratory medicine and in analytical chemistry/ automation, data processing, library searching, and
MS. Third, the number of users and methods will computer-assisted interpretation. Methods will be
increase as MS technology becomes more accessible. available to analyze a whole host of metabolites. The
With the development of slightly different variations question remains as to whether these developments
of a particular test used by different laboratories, will make mass spectrometric methods cost-effective
there is a greater likelihood of different results. The and as inexpensive as immunoassays, which are most
chance increases for a miscommunication or misdi- often applied in the “one disease/one analyte” clinical
agnosis between health care provider and laboratory. assay. Clearly, mass spectrometric methods, that
Required harmonization of MS methods will ensure include multiple analytes as observed in the area of
that if a physician orders test A, then the results will newborn screening of amino acids and acylcarnitines,
always include the concentration of compounds X, Y, will continue to grow and become a more common
and Z. Harmonization begins with the development clinical tool.
of an independent quality assessment program.107,108 We are now embarking on the age of proteomics.
Does harmonization mean that a single MS instru- With the Human Genome initiative essentially com-
ment or system be used to perform a particular plete, the next major endeavor will be research in
assay? The answer is no. Mass spectrometric methods gene expression. Clearly, specialty analyses will be
allow many variations of sample preparation and developed to identify specific proteins that are mark-
analyses and yet produce comparable end points, i.e., ers of disease. An example is hemoglobin and sickle
the concentration of phenylalanine in blood. In fact, cell disorders. Will MS replace robust, well-estab-
recent work in newborn screening has indicated that lished techniques such as isoelectric focusing or
acylcarnitines may be analyzed either as a butyl ester HPLC? Improvements in resolution, mass accuracy,
or directly without esterification The advantages of and sensitivity will be required if MS is to become
the removal of the esterification step in the procedure competitive on a routine basis for such assays.
is simplification and sample preparation time. The However, new approaches to identification of cellular
disadvantages may be loss of poorly ionizable me- profiles through protein fingerprinting may rapidly
tabolites, unexpected interferences, and inability to enter the clinical diagnostic field due to simplicity
co-analyze some amino acids. Therefore, MS allows and potential cost-effectiveness. Methods that can
creativity in method applications while still achieving qualitatively identify microorganisms or characterize
the same end points, e.g., obtaining the accurate tumorogenic tissue or other diseased tissues may
quantification of a metabolite that is used to diagnose become routine if methodology for specimen prepara-
tion is standardized. The key to the success of these (7) Sparkman, O. D. Mass Spectrometry Desk Reference, 1st ed.;
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Chem. Rev.

2001, 101, 445−477 445

Mass Spectrometry in the Clinical Laboratory

Received April 4, 2000

Contents VI. Conclusions 470

10.1021/cr990077+ CCC: $36.00 © 2001 American Chemical Society

versatile, and robust analytical systems make mass

accepted abbreviations developed by groups such as

II. Diagnostic Metabolites

it is unlikely that a single genetic marker will

different metabolites. Some physicians and clinical

d. Clinical Applications. Ozand and Gascon

MS methods even though their analysis is often

phenylalanine, may not have reach sufficient con-

Figure 8. Schematic of collision-induced dissociation (CID)

may not be substantial if significant amounts of

(Figure 7) represents the loss of 119 Da from the

Figure 12. Chemical structures of thyroid hormones T3

production.128 However, measurement of free T4

MS methods (including FAB and FIB ionization) were

Figure 16. Examples of the bile acid, choline, and its

measuring acylglycines, investigators have developed

together serve to solubilize biliary lipids via their

Figure 19. ToF spectrum of cholesterol (m/z analyzed

Many other studies have focused on the analysis of

cholesterol is significantly elevated relative to control H. Other Classes of Small Biomarkers

gotes (carriers of one copy of the altered gene) are

adduct, glutathionyl hemoglobin, is also useful as an

C. Specific Disease Diagnostics Using MS

useful in the identification of the site of origin for

IV. Diagnostic Biopolymers

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