2.2.58. Inductively Coupled Plasma-Mass Spectrometry

2.2.58. Inductively coupled plasma-mass spectrometry EUROPEAN PHARMACOPOEIA 10.
LINEARITY to the ion optics. The ion optics consist of an electrostatic

Prepare and analyse not fewer than 4 reference solutions over lens, which takes ions from an area at atmospheric pressure
the calibration range plus a blank. Perform not fewer than to the mass filter at a vacuum of 10-8 Pa or less, maintained
5 replicates. with a turbomolecular pump. After their filtration, ions of the
The calibration curve is calculated by least-square regression selected mass/charge ratio are directed to a detector (channel
from all measured data of the calibration test. The regression electromultiplier, Faraday cup, dynodes), where ion currents
curve, the means, the measured data and the confidence are converted into electrical signals. The element is quantified
interval of the calibration curve are plotted. The operating according to the number of ions arriving and generating
method is valid when : electrical pulses per unit time.
The sample-introduction system and data-handling techniques
– the correlation coefficient is at least 0.99 ;
of an ICP-AES system are also used in ICP-MS.
– the residuals of each calibration level are randomly
distributed around the calibration curve. APPARATUS
Calculate the mean and relative standard deviation for the The apparatus consists essentially of the following elements :
lowest and for the highest calibration level. – sample-introduction system, consisting of a peristaltic
When the ratio of the estimated standard deviations of the pump delivering the solution at constant flow rate into a
lowest and the highest calibration level is less than 0.5 or nebuliser ;
greater than 2.0, a more precise estimation of the calibration – radio-frequency (RF) generator ;
curve may be obtained using weighted linear regression. Both
linear and quadratic weighting functions are applied to the – plasma torch ;
data to find the most appropriate weighting function to be – interface region including cones to transport ions to the
employed. ion optics ;
If the means compared to the calibration curve show a – mass spectrometer ;
deviation from linearity, two-dimensional linear regression – detector ;
is used. – data-acquisition unit.
ACCURACY
Verify the accuracy preferably by using a certified reference INTERFERENCE
material (CRM). Where this is not possible, perform a test Mass interference is the major problem, for example by
for recovery. isobaric species that significantly overlap the mass signal of the
Recovery. For assay determinations a recovery of 90 per ions of interest, especially in the central part of the mass range
cent to 110 per cent is to be obtained. The test is not valid (for example 40-80 a.m.u.). The combination of atomic ions
if recovery, for example for trace-element determination, leads to polyatomic or molecular interferences (i.e. 40Ar16O
is outside of the range 80 per cent to 120 per cent of the with 56Fe or 40Ar40Ar with 80Se). Matrix interference may also
theoretical value. Recovery may be determined on a suitable occur with some analytes. Some samples have an impact on
reference solution (matrix solution) spiked with a known droplet formation or on the ionisation temperature in the
quantity of analyte (concentration range that is relevant to plasma. These phenomena may lead to the suppression of
the samples to be determined). analyte signals. Physical interference is to be circumvented by
using the method of internal standardisation or by standard
REPEATABILITY addition. The element used as internal standard depends on
The repeatability is not greater than 3 per cent for an assay the element to be measured : 59Co and 115In, for example, can
and not greater than 5 per cent for an impurity test. be used as internal standards.
LIMIT OF QUANTIFICATION The prime characteristic of an ICP-MS instrument is its
Verify that the limit of quantification (for example, determined resolution, i.e. the efficiency of separation of 2 close masses.
using the 10 σ approach) is below the value to be measured. Quadrupole instruments are, from this point of view, inferior
to magnetic-sector spectrometers.
PROCEDURE
01/2008:20258 SAMPLE PREPARATIONS AND SAMPLE INTRODUCTION
The sample preparation usually involves a step of digestion of
the matrix by a suitable method, for example in a microwave
oven. Furthermore, it is important to ensure that the
analyte concentration falls within the working range of the
2.2.58. INDUCTIVELY COUPLED instrument through dilution or preconcentration, and that the
sample-containing solution can be nebulised in a reproducible
PLASMA-MASS SPECTROMETRY manner.
Several sample-introduction systems tolerate high acid
concentrations, but the use of sulfuric and phosphoric acids
can contribute to background emission. Therefore, nitric
Inductively coupled plasma-mass spectrometry (ICP-MS) is a and hydrochloric acids are preferable. The availability of
mass spectrometry method that uses an inductively coupled hydrofluoric acid-resistant (for example perfluoroalkoxy
plasma (ICP) as the ionisation source. The basic principles of polymer) sample-introduction systems and torches also allows
ICP formation are described in chapter 2.2.57 on inductively the use of hydrofluoric acid. In selecting a sample-introduction
coupled plasma-atomic emission spectrometry (ICP-AES). method, the requirements for sensitivity, stability, speed,
ICP-MS utilises the ability of the ICP to generate charged sample size, corrosion resistance and resistance to clogging
ions from the element species within a sample. These ions have to be considered. The use of a cross-flow nebuliser
are then directed into a mass spectrometer, which separates combined with a spray chamber and torch is suitable for
them according to their mass-to-charge ratio (m/z). Most most requirements. The peristaltic pumps usually deliver the
mass spectrometers have a quadrupole system or a magnetic standard and sample solutions at a rate of 20-1000 μL/min.
sector. Ions are transported from the plasma through 2 cones In the case of organic solvents being used, the introduction of
(sampler and skimmer cones, forming the interface region) oxygen must be considered to avoid organic layers.
108 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 10.0 2.2.59. Glycan analysis of glycoproteins
CHOICE OF OPERATING CONDITIONS When the ratio of the estimated standard deviations of the
The standard operating conditions prescribed by the lowest and the highest calibration level is less than 0.5 or
manufacturer are to be followed. Usually, different sets of greater than 2.0, a more precise estimation of the calibration
operating conditions are used for aqueous solutions and for curve may be obtained using weighted linear regression. Both
organic solvents. Suitable operating parameters are to be linear and quadratic weighting functions are applied to the
properly chosen : data to find the most appropriate weighting function to be
– selection of cones (material of sampler and skimmer) ; employed.
– support-gas flow rates (outer, intermediate and inner tubes If the means compared to the calibration curve show a
of the torch); deviation from linearity, two-dimensional linear regression
is used.
– RF power ;
ACCURACY
– pump speed ;
Verify the accuracy preferably by using a certified reference
– selection of one or more isotopes of the element to be material (CRM). Where this is not possible, perform a test
measured (mass). for recovery.
ISOTOPE SELECTION Recovery. For assay determinations a recovery of 90 per
Isotope selection is made using several criteria. The most cent to 110 per cent is to be obtained. The test is not valid
abundant isotope for a given element is selected to obtain if recovery, for example for trace-element determination, is
maximum sensitivity. Furthermore, an isotope with the least outside the range 80 per cent to 120 per cent of the theoretical
interference from other species in the sample matrix and value. Recovery may be determined on a suitable reference
from the support gas should be selected. Information about solution (matrix solution) spiked with a known quantity of
isobaric interferences and interferences from polyatomic ions analyte (concentration range that is relevant to the samples
of various types, for example hydrides, oxides, chlorides, etc., to be determined).
is usually available in the software of ICP-MS instrument REPEATABILITY
manufacturers. The repeatability is not greater than 3 per cent for an assay
and not greater than 5 per cent for an impurity test.
CONTROL OF INSTRUMENT PERFORMANCE
LIMIT OF QUANTIFICATION
System suitability Verify that the limit of quantification (for example, determined
– Tuning of the instrument allows to monitor and adjust using the 10 σ approach) is below the value to be measured.
the measurement before running samples. ICP-MS mass
accuracy is checked with a tuning solution containing
several isotopes covering the whole range of masses, for
example 9Be, 59Co, 89Y, 115In, 140Ce and 209Bi.
– Sensitivity and short- and long-term stability are recorded.
The instrument parameters (plasma condition, ion lenses 01/2011:20259
and quadrupole parameter) are to be optimised to obtain
the highest possible number of counts.
– Tuning for resolution and mass axis is to be done with a
solution of Li, Y and Tl to ensure an acceptable response
over a wide range of masses. 2.2.59. GLYCAN ANALYSIS OF
– Evaluation of the efficiency of the plasma to decompose GLYCOPROTEINS
oxides has to be performed in order to minimise these
interferences. The ratio Ce/CeO and/or Ba/BaO is a good 1. INTRODUCTION
indicator, and a level less than about 3 per cent is required. Glycan analysis is a test to analyse glycan moieties of
– Reduction of the formation of double-charged ions is made glycoproteins. It may involve :
with Ba and Ce. The ratio of the signal for double-charged – whole glycoprotein analysis ;
ions to the assigned element should be less than 2 per cent. – separation and detection of protein glycoforms ;
– Long-term stability is checked by running a standard – analysis of glycopeptides obtained after enzymatic
first and at the end of the sample sequence, controlling treatment of the glycoprotein ;
whether salt deposits on the cones have reduced the signal
throughout the run. – analysis of released glycans obtained after chemical or
enzymatic treatment of the glycoprotein.
VALIDATION OF THE METHOD Monosaccharide analysis may complement information
Satisfactory performance of methods prescribed in obtained by glycan analysis.
monographs is verified at suitable time intervals. Glycosylation can play a predominant role in determining the
LINEARITY function, pharmacokinetics, pharmacodynamics, stability,
Prepare and analyse not fewer than 4 reference solutions over and immunogenicity of biotherapeutics. Glycosylation,
the calibration range plus a blank. Perform not fewer than unlike transcription, is a non-template-driven enzymatic
5 replicates. modification process that results in glycan heterogeneity. The
manufacturing procedure also has an influence on glycan
The calibration curve is calculated by least-square regression heterogeneity. Glycoprotein glycan analysis may therefore be
from all measured data of the calibration test. The regression an important test to identify variations in the glycosylation
curve, the means, the measured data and the confidence pattern of the glycoprotein and/or monitor the consistency of
interval of the calibration curve are plotted. The operating the glycosylation pattern during production.
method is valid when :
Glycan analysis can be a comparative procedure, because
– the correlation coefficient is at least 0.99 ; the information obtained, compared to a similarly treated
– the residuals of each calibration level are randomly reference substance, confirms product consistency.
distributed around the calibration curve. This chapter provides approaches used for glycoprotein glycan
Calculate the mean and relative standard deviation for the analysis and requirements for the application of methods and
lowest and for the highest calibration level. validation of methods.
General Notices (1) apply to all monographs and other texts 109

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2.2.58. Inductively coupled plasma-mass spectrometry EUROPEAN PHARMACOPOEIA 10.

LINEARITY to the ion optics. The ion optics consist of an electrostatic

108 See the information section on general monographs (cover pages)

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