Amino Acids

DOI 10.1007/s00726-014-1717-z

AMINO ACIDS PROTOCOLS

Analysis of polyamines in biological samples

by HPLC involving pre-column derivatization
with o-phthalaldehyde and N-acetyl-L-cysteine
Zhaolai Dai • Zhenlong Wu • Junjun Wang •
Xiaoqiu Wang • Sichao Jia • Fuller W. Bazer •

Guoyao Wu

Received: 7 February 2014 / Accepted: 25 February 2014

Ó Springer-Verlag Wien 2014

Abstract Polyamines (putrescine, spermine and spermi- replicate measurement) of the HPLC method are 2.5–4.2 %
dine) play a crucial role in the regulation of cell growth, and 0.5–1.4 %, respectively, for biological samples,
differentiation, death and function. Accurate measurement depending on polyamine concentrations and sample type.
of these substances is essential for studying their metabo- Our HPLC method is highly sensitive, specific, accurate,
lism in cells. This protocol describes detailed procedures easily automated, and capable for the analysis of samples
for sample preparation and HPLC analysis of polyamines with different characteristics and small volume/amount,
and related molecules (e.g., agmatine and cadaverine) in and provides a useful research tool for studying the bio-
biological samples. The method is optimized for the chemistry, physiology, and pharmacology of polyamines
deproteinization of samples, including biological fluids and related substances.
(e.g., 10 ll), plant and animal tissues (e.g., 50 mg), and
isolated/cultured cells (e.g., 1 9 106 cells). The in-line Keywords Polyamines
Derivatization

reaction of polyamines with o-phthalaldehyde and N- o-Phthalaldehyde
N-Acetyl-L-cysteine
HPLC
acetyl-L-cysteine yields fluorescent derivatives which are
separated on a reversed-phase C18 column and detected by Abbreviations
a fluorometer at an excitation wavelength of 340 nm and an HPLC High-performance liquid chromatography
emission wavelength of 450 nm. The total running time for NAC N-Acetyl-L-cysteine
each sample (including column regeneration on the auto- OPA o-Phthaldialdehyde
mated system) is 30 min. The detection limit is 0.5 nmol/ PBS Phosphate-buffered saline
ml or 0.1 nmol/mg tissue in biological samples. The assays SDS Sodium dodecyl sulphate
are linear between 1 and 50 lM for each of the poly-
amines. The accuracy (the nearness of an experimental
value to the true value) and precision (agreement between Introduction

Electronic supplementary material The online version of this Major polyamines in cells are putrescine, spermidine, and
article (doi:10.1007/s00726-014-1717-z) contains supplementary spermine which are found in almost all living organisms
material, which is available to authorized users. (Hamana and Matsuzaki 1992; Ignarro et al. 2001; Wallace
et al. 2003). Polyamines contain polycations that can
Z. Dai
Z. Wu
J. Wang
G. Wu (&)
State Key Laboratory of Animal Nutrition, China Agricultural interact with polyanionic macromolecules such as DNA,
University, Beijing 100193, China RNA, ion channels, and protein kinases, thus regulating
e-mail: [email protected] gene expression, post-transcriptional modifications, cell
Z. Wu cycle, membrane structure, and function (Pegg and Casero
e-mail: [email protected] 2011; Koponen et al. 2012; Wallace et al. 2003; Wu 2009).
As a result, polyamines play a crucial role in the regulation
X. Wang
S. Jia
F. W. Bazer
G. Wu
Department of Animal Science, Texas A&M University, of growth, development, and function of animals, plants,
College Station, TX 77843-2471, USA and microorganisms (Agostinelli 2012; Wu et al. 2009).

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 Z. Dai et al.

The composition and concentrations of polyamines in the combined use of OPA and NAC offers advantages for
biological samples vary greatly with cell type and physi- rapid, sensitive, and reproducible analysis of polyamines in
ological conditions of the organism (Seiler and Raul 2005; biological samples.
Wei et al. 2001; Wu and Morris 1998). Therefore, there is Sample-processing methods may also affect the accuracy
growing interest in the biology and pathobiology of poly- of polyamine analysis. Relevant factors are (1) chemical
amines (Correa-Fiz et al. 2012; Do et al. 2013; Levillain composition and temperatures of buffers used to dilute the
et al. 2012; Tavladoraki et al. 2012), including amino acid samples and dilution factor, (2) temperatures and containers
biochemistry and nutrition (Wu 2013). Sensitive and (e.g. glass and plastic) used for grounding samples, (3)
accurate determination of polyamines is crucial for con- chemical characteristics and concentrations of acids used to
ducting biological science research. deproteinize samples, (4) temperatures and speed of centri-
The analysis of polyamines has been a challenge for sci- fugation of the acidified samples, (5) chemical characteris-
entists over the past decades because these molecules are not tics and concentrations of alkalines used to adjust the pH of
chromophores or fluorophores themselves and cannot be the acid extracts for derivatization, and (6) derivatization
readily analyzed by spectrophotometric or fluorescent detec- methods used (manually or automated) for HPLC analysis. It
tion methods (Liu et al. 2012; Seiler 1971). Many techniques was reported that polyamines could bind to glass and,
have been developed to determine polyamines, such as thin- therefore, use of plastic tubes and vials for treating and
layer chromatography, enzymatic assay, high-performance storage of polyamine-containing samples is critically
liquid chromatography (HPLC) coupled with pre-column or important for their analysis (Flores and Galston 1982). Also,
post-column derivatization methods [forming fluorophores deproteinization method, such as trichloroacetic acid–ether
with dansyl chloride, fluorescamine, and o-phthaldialdehyde extraction, has been shown to affect the accuracy of poly-
(OPA); forming chromophores with benzoyl chloride, dabsyl amine analysis (Marton et al. 1974). Of note, perchloric acid
chloride, and tosyl chloride] and liquid chromatography– is widely used for the deproteinization of various biological
electrospray ionization tandem mass spectrometry (LC–ESI– samples (including fluids, plant and animal tissues, isolated/
MS/MS) or high-performance liquid chromatography with cultured cells, as well as bacteria and solid food/feed stuff),
quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF and is known to stabilize polyamines for at lest 6 months
MS) methods (Acheampong et al. 2011; Cerrada-Gimenez (Acheampong et al. 2011; Codoñer-Franch et al. 2011;
et al. 2012; Isobe et al. 1987; Jung et al. 2012; Liu et al. 2012; Flores and Galston 1982). Moreover, potassium carbonate
Marton and Lee 1975; Sánchez-López et al. 2009; Schenkel (K2CO3) has been used with perchloric acid to remove extra
et al. 1995; Seiler 1971; Wu et al. 2000a, b). The HPLC amounts of acid and Cl- in acidified samples in our and other
methods have been used widely due to their high sensitivity laboratories (Chen et al. 1987; Masukawa et al. 2006; Sase
and reproducibility, as well as ease of automation. However, et al. 2013; Wu and Knabe 1995). The combined use of
the setbacks are also obvious, such as long derivatization liquid nitrogen, cold perchloric acid, and cold potassium
times and instability of derivatives (Acheampong et al. 2011). carbonate to grind and deproteinize samples offers an
The addition of sodium dodecyl sulfate (SDS) or phosphate advantage of preventing the degradation/transformation of
buffer (pH 4) to the HPLC solvents or extraction procedures to polyamines in biological samples while removing proteins.
stabilize the o-phthalaldehyde-derivatives has been reported Therefore, this protocol optimizes and integrates sample
to be ineffective (Campı́ns-Falcó et al. 2001). Also, the processing methods that are suitable for the deproteiniza-
inclusion of SDS in the HPLC solvents is corrosive to the tion and derivatization of a variety of biological samples
plunger of the HPLC pump. with different characteristics and a small volume/amount,
Polyamines can react with OPA and N-acetyl-L-cysteine and also achieves simple, fast, sensitive, reproducible, and
(NAC) to form relatively stable derivative products, accurate analysis of polyamines in agricultural and bio-
namely OPA–NAC-polyamines (Campı́ns-Falcó et al. medical research. The work flow diagram of the protocol is
2001). This reaction is very rapid to allow in-line pre- shown in Fig. 1.
column derivatization which eliminates errors in manual
operations and shortens the time interval between the for-
mation of the reaction products and detection of fluores- Materials
cence. Based on our experience, the amount of OPA (the
final concentration of 1.95 mg/ml or 14.5 mM) used in the Reagents
reaction with polyamines has been found to be optimal for
rapid derivatization with substances containing amino or HPLC-grade water (Fisher Scientific; Cat. # W7-4).
amine groups (e.g., Choi et al. 2014; Dai et al. 2013; Lei HPLC-grade methanol (Fisher Scientific; Cat. # A452-4).
et al. 2013; Rezaei et al. 2013; Satterfield et al. 2013; Wu Tetrahydrofuran, CHROMASOLVÒ Plus, for HPLC,
and Knabe 1994; Wu and Thompson 1987, 1988). Thus, C99.9 % (Sigma-Aldrich; Cat. # 34865-1L.

123
Determination of polyamine

Agmatine sulfate salt (Sigma-Aldrich; Cat. # A7127-

1G).
Cadaverine dihydrochloride (Sigma-Aldrich; Cat. #
33220-10G-F).
1,6-Hexanediamine (Sigma-Aldrich; Cat. # H11696-25G).
N-Acetylputrescine hydrochloride (Sigma-Aldrich; Cat.
# A8784-25MG).
N1-Acetylspermine trihydrochloride (Sigma-Aldrich;
Cat. # 01467-100MG).

Equipment

AriumÒ pro VF Ultrapure Water System (Sartorius AG;

http://www.sartorius.com).
SorvallÒ LegendÒ Micro 21R Microcentrifuge (Thermo
Scientific; http://www.thermoscientific.com).
Waters Alliance e2695 Separation Module (Waters;
Fig. 1 Work flow diagram on the procedures described in this http://www.waters.com).
protocol for HPLC analysis of polyamines Waters 2475 Multi k Fluorescence Detector (Waters;
http://www.waters.com).
Benzoic acid (Sigma-Aldrich; Cat. # 242381-500G). Empower 3TM software (Waters; http://www.waters.com).
Potassium tetraborate tetrahydrate (K2B4O7
4H2O) Vortex-Genie 2 (Scientific Industries; http://www.scien
(Sigma-Aldrich; Cat. # P5754-500G). tificindustries.com).
Sodium tetraborate decahydrate (Na2B4O7
10H2O) ThermomixerÒ comfort (Eppendorf; http://www.eppen
(Sigma-Aldrich; Cat. # B9876-500G). dorf.com).
N-Acetyl-L-cysteine (Sigma-Aldrich; Cat. # A7250-5G). Analytical balance (METTLER TOLEDO; http://www.
o-Phthaldialdehyde (OPA) (Sigma-Aldrich; Cat. # mt.com; Cat. # ML204).
P0657-5G). OPA should be stored in a brown bottle to FiveEasyTM pH meter (METTLER TOLEDO; http://
protect it from light. www.mt.com; Cat. # FE20).
BrijÒ L23 solution (BrijÒ 35 solution) (Sigma-Aldrich;
Cat. # B4184-100ML). Preparation of reagents
Sodium acetate trihydrate (Sigma-Aldrich; Cat. #
S7670-1KG). HPLC-grade water is used to prepare the following
Perchloric acid (HClO4; 70 %) (Sigma-Aldrich; Cat. # solutions:
244252-500ML; or Fisher Scientific, Cat # A229-8LB). 6 N HCl: add slowly 49.1 ml of concentrated HCl
Potassium carbonate (K2CO3) (Sigma-Aldrich; Cat. # (37–38 %) to 50.9 ml H2O.
P5833-500G). 1.5 M HClO4: add 32.2 ml of 70 % HClO4 to 150 ml
Hydrochloric acid (HCl) (Sinopharm Chemical Reagent; H2O. Make up to a final volume of 250 ml with H2O.
Cat. # 10011018). 2 M K2CO3 dissolve 69.11 g K2CO3 in 150 ml H2O.
Sodium chloride (NaCl) (Sigma-Aldrich; Cat. # S5886- Make up to a final volume of 250 ml with H2O.
1KG). Phosphate-buffered saline (PBS): dissolve 8.01 g NaCl,
Potassium chloride (KCl) (Sigma-Aldrich; Cat. # 0.2 g KCl, 1.42 g Na2HPO4, and 0.27 g KH2PO4 in 800 ml
P5405-500G). H2O. Adjust the pH of the solution to 7.4 using 6 N HCl. Make
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich; up the final volume of 1 l with H2O. Autoclave the solution at
Cat. # S5136-500G). 121 °C for 20 min and cool the solution to 25 °C before use.
Potassium phosphate monobasic (KH2PO4) (Sigma- 1.2 % (w/v) benzoic acid: dissolve 8.4 g benzoic acid in
Aldrich; Cat. # 60218-100G). 525 ml H2O. Add 175 ml of saturated K2B4O7 (saturated
Putrescine dihydrochloride (Sigma-Aldrich; Cat. # K2B4O7 is prepared in H2O).
P7505-25G). 40 mM sodium borate buffer (pH 9.5): dissolve 30.51 g
Spermidine trihydrochloride (Sigma-Aldrich; Cat. # Na2B4O7
10H2O in 2 l H2O.
85578-1G). o-Phthalaldehyde–N-acetyl cysteine (OPA–NAC)
Spermine tetrahydrochloride (Sigma-Aldrich; Cat. # reagents: dissolve 50 mg OPA and 50 mg NAC in 1.25 ml
85605-1G). methanol. Add 11.2 ml of 40 mM sodium borate buffer

123
 Z. Dai et al.

(pH 9.5) and 0.4 ml of BrijÒ-35 to the solution. Mix the Table 1 HPLC gradients for the analysis of polyamines
solution gently. (Use a brown bottle to prepare and store Solvent (%) Time (min)
the OPA–NAC solution. Prepare the solution on the day of
HPLC analysis and store it at 4 °C for use within 24 h). 0 12 16 18 23 25 30
Solvent A (0.1 M sodium acetate; pH 7.2): add 27.3 g A 70 35 30 0 0 70 70
sodium acetate (trihydrate) and 96 ll of 6 N HCl to 1.6 l B 30 65 70 100 100 30 30
H2O. Add 180 ml methanol and 10 ml tetrahydrofuran. Make
Flow rate: 1.0 ml/min (or 0.8 ml/min under high pressure)
up the final volume of 2 l with H2O. Mix the solution well.
Solvent B: 100 % HPLC-grade methanol.
1 mM Hexanediamine: mix 50 ll of 20 mM hexane-
diamine with 950 ll H2O.
Polyamine standard solutions
Mixed polyamine standards (100 nmol/ml for each
polyamine).
HPLC-grade water is used to prepare the following
Add the following to a microcentrifuge tube:
solutions.
100 ll of 1 mM Putrescine.
Polyamine standards are dissolved in H2O in autoclaved
100 ll of 1 mM Spermidine.
tubes. The polyamine standard solution can be stored at -
100 ll of 1 mM Spermine.
80 °C for 6 months.
100 ll of 1 mM Agmatine.
1. 20 mM Putrescine: dissolve 16.12 mg putrescine- 100 ll of 1 mM N1-Acetylputrescine.
2HCl (MW = 161.1) in 5 ml H2O. 100 ll of 1 mM N1-Acetylspermine.
100 ll of 1 mM Cadaverine.
1 mM Putrescine: mix 50 ll of 20 mM putrescine with
100 ll of 1 mM Hexanediamine.
950 ll H2O.
200 ll H2O.
2. 20 mM Spermidine: dissolve 25.5 mg spermidine- Mixed polyamine standards (10 nmol/ml for each
3HCl (MW = 254.6) in 5 ml H2O. polyamine).
Add 100 ll of the above mixed polyamine standards
1 mM Spermidine: mix 50 ll of 20 mM spermidine
(100 nmol/ml) with 900 ll H2O.
with 950 ll H2O.
3. 20 mM Spermine: dissolve 34.9 mg spermine-4HCl Chromatographic equipment
(MW = 348.2) in 5 ml H2O.
The HPLC system is a Waters Alliance e2695 HPLC system
1 mM Spermine: mix 50 ll of 20 mM spermine with
equipped with a guard column (5 cm 9 4.6 mm ID) filled
950 ll H2O.
with Supelco Pellicular packing (40 lm, Cat. # 58232) and an
4. 20 mM Agmatine: dissolve 22.9 mg agmatine (sulfate analytical column (Supelco SUPELCOSILTM LC-18 HPLC
salt; MW = 228.3) in 5 ml H2O. column; 15 cm 9 4.6 mm ID, 3 lm, Supelco Cat. # 58985).
The HPLC is run at a gradient mode (Table 1). The total
1 mM Agmatine: mix 50 ll of 20 mM agmatine with
running time for each sample (including column regeneration
950 ll H2O.
on the automated system) is 30 min. A Waters 2475 multi k
5. 20 mM N1-Acetylputrescine: dissolve 6.7 mg N-acet- fluorescence detector is used to detect the fluorescence of each
ylputrescine-HCl in 2 ml H2O. polyamine derivative, with excitation k being set at 340 nm
and emission k at 450 nm. The Empower 3TM software is used
1 mM N-Acetylputrescine: mix 50 ll of 20 mM N-
to record and analyze the HPLC chromatography data.
acetylputrescine with 950 ll H2O.
6. 20 mM N1-Acetylspermine: dissolve 7.1 mg N1-acet-
ylspermine-3HCl in 1 ml H2O. Procedures
1 mM N1-Acetylspermine: mix 50 ll of 20 mM N1-
Sample preparations
acetylspermine with 950 ll H2O.
7. 20 mM Cadaverine: dissolve 17.5 mg cadaverine- Methods for preparation of samples depend on their char-
2HCl in 5 ml H2O. 1 mM Cadaverine: mix 50 ll of acteristics (Li et al. 2001; Wu et al. 2000a, b). Here,
20 mM cadaverine with 950 ll H2O. effective procedures are given for biological fluids, tissues,
8. 20 mM Hexanediamine: dissolve 11.6 mg 1,6-hexane- and isolated/cultured cells. All the procedures described
diamine in 5 ml H2O. below are carried out on ice.

123
Determination of polyamine

Preparation of biological fluids (c) Add 100 ll of ice-cold 2 M K2CO3 to the mixture.
Leave the cap open for 1 min to allow the evapo-
(a) Transfer 200 ll of a biological fluid to a 1.5-ml ration of CO2.
Eppendorf tube. Centrifuge the tubes at 15,0009g at (d) Proceed with steps d–f described for the preparation
4 °C for 10 min, and transfer 100 ll of the super- for biological fluids.
natant fluid to a new 1.5-ml eppendorf tube.
(b) Add 100 ll of ice-cold 1.5 M HClO4 to a tube
Preparation of HPLC vials for auto-sampling
containing 100 ll of the supernatant fluid; vortex the
and analysis
tube for 1 min at 25 °C.
(c) Add 50 ll of ice-cold 2 M K2CO3 to the mixture and
(a) To a 2-ml vial, add the following:
leave the cap open for 1 min to allow the evapora-
tion of CO2. 50 ll of a sample, polyamine standard solution
(d) Seal the cap of the tube and vortex the tube for 1 min or water (blank).
at 25 °C. Open the cap of the tube to release extra 50 ll of 1.2 % (w/v) benzoic acid.
gas and then re-seal the cap. 700 ll of H2O.
(e) Centrifuge the tube at 15,0009g at 4 °C for 10 min.
(b) Vortex each vial for 10 s at 25 °C.
Transfer 100 ll of the supernatant fluid to a new 1.5-
(c) Place the vials onto the autosampler: the OPA–
ml Eppendorf tube. The dilution factor for the
NAC reagent vial at position #1, the blank vial at
sample is 2.5.
position #2, the polyamine standard vial at
(f) Add adequate amounts of H2O to the resulting
position #3, sample vials at positions #4 to #14,
supernatant fluid from step (e) above to make the
and the polyamine standard vial at position #15.
appropriate dilution for HPLC analysis of polyamines.
The ratio of 1:1 (g/g) for OPA:NAC is optimal
for the response factors of polyamines in terms of
Preparation of tissues their peak areas.
(d) The in-line pre-column derivatization of poly-
(a) Snap-freeze small pieces of fresh tissue samples in amines is accomplished by setting the ‘‘auto
liquid nitrogen and store the frozen tissues at -80 °C. addition’’ option in the sample set method of the
(b) Grind a small piece of the frozen sample (*200 mg) software Empower 3, an injection volume of
in liquid-nitrogen cooled mortar. 10 ll was set for both the derivatization reagent
(c) Weight *50 mg of the grounded sample powder in (vial #1) and standard/sample (e.g., vial #2 to
a 1.5-ml Eppendorf tube cooled by liquid nitrogen. #15). After mixing in the derivatization loop, an
(d) Add 200 ll of ice-cold 1.5 M HClO4 to a tube injection (a total volume of 20 ll) was automat-
containing 50 mg sample and then seal the cap of the ically performed with no delay time.
tube. Vortex the tube for 1 min at 25 °C. (e) After each sample set is completed, use the
(e) Add 100 ll of ice-cold 2 M K2CO3 to the mixture. Empower 3 software to quantify polyamines in
Leave the cap open for 1 min to allow the evapo- the samples.
ration of CO2.
(f) Proceed with steps d–f described for the preparation
HPLC chromatograms
for biological fluids.
All the polyamines in the assay mixture are well separated, as
Preparation of isolated/cultured cells indicated in the HPLC chromatography of polyamine stan-
dards and related substances, including agmatine and
(a) Transfer medium containing *5 9 106 cells to a cadaverine (Fig. 2). This method is specific for polyamines
new 1.5-ml Eppendorf tube, centrifuge the tube at and is free of interference by amino acids. The detection limit
10,0009g at 4 °C for 1 min and discard the super- for this method is 0.5 nmol/ml or 0.1 nmol/mg tissue in
natant fluid. Wash the cell pellet twice with 0.5 ml of biological samples. OPA used in the derivatization solution
ice-cold PBS (pH 7.4) by centrifugation at has no adverse effect on the HPLC system, and one HPLC
10,0009g at 4 °C for 1 min after each wash. column can be used to analyze at least 500 samples. The
(b) Vortex the tube containing cells for 30 s at 25 °C, procedures described in this protocol have been used suc-
then add 200 ll of ice-cold 1.5 M HClO4 to the tube, cessfully in our laboratory for the determination of poly-
and seal the cap of the tube. Vortex the tube for amines in sow’s milk, as well as tissue samples from the liver
1 min at 25 °C. and small intestine of growing pigs (Table 2).

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 Z. Dai et al.

Fig. 2 HPLC chromatography

for analysis of polyamine
standards (10 nmol/ml)

Table 2 Concentrations of Sow’s milka Piglet liverb Piglet jejunumb Piglet ileumb
polyamines in various (nmol/ml) (nmol/g tissue) (nmol/g tissue) (nmol/g tissue)
biological samples
N1-Acetylputrescine ND ND ND ND
1
N -Acetylspermine ND ND ND ND
Values are as mean ± SD, Agmatine 6.30 ± 1.11 ND ND ND
n=6 Cadaverine 0.26 ± 0.07 ND ND ND
ND not detectable Hexanediamine ND ND ND ND
a
Obtained from sows on Day Putrescine 2.22 ± 0.41 164 ± 10 171 ± 15 211 ± 12
14 of lactation Spermidine 28.4 ± 4.9 1244 ± 133 966 ± 72 1177 ± 93
b
Obtained from 28-day-old Spermine 11.8 ± 1.4 6125 ± 355 2565 ± 106 2672 ± 137
piglets

The deproteinization procedures described in this pro- known amounts of polyamine standards and expressed as
tocol have been optimized for the processing and analysis of the relative errors [(measurement value - true value)/true
small volume samples. Examples for the volume or amount value 9 100 %)], is 2.5–4.2 % for the biological samples,
of a biological sample required for the analysis of poly- depending on polyamine concentrations and sample type
amines are 10 ll of a physiological fluid (e.g., plasma and (Supplemental Table 1). The precision (agreement between
fetal fluids), 50 mg of a plant or animal tissue, and 1 9 106 replicate measurement) of the analysis, as evaluated by the
cells. An insert within an HPLC vial may be used for a small relative deviation (mean of absolute deviation/mean of
volume or amount of a sample. Additionally, the volumes of replicate measurements 9 100 %), is 0.5–1.4 %, depend-
HClO4 and K2CO3 used for sample preparation can be ing on polyamine concentrations and sample type (Sup-
reduced to minimize the dilution of a sample. For example, plemental Table 2). Our HPLC method is highly sensitive,
the sample to 1.5 M HClO4 ratio can be 1:1 (v/v) for specific, accurate, easily automated, and provides a useful
physiological fluids and 1:4 (w/v) for a tissue. The 1.5 M research tool for studying the biochemistry, physiology,
HClO4 to 2 M K2CO3 ratio is 2:1 (v/v) for all types of and pharmacology of polyamines and related substances.
samples. The OPA–NAC reagent to sample ratio is 1:1 (v/v)
for the in-line pre-column derivatization. The rapid and Acknowledgments Work in our laboratories was supported by the
sensitive analysis of polyamines is satisfactorily achieved China Postdoctoral Science Foundation (2012T50163), National
through the automated in-line pre-column derivatization of Natural Science Foundation of China (u0731001, 30810103902,
31172217, 31272449, and 31272450), Chinese Universities Scientific
polyamines using the OPA–NAC reagents. The assays are Funds (2012RC024), the Thousand-People Talent program at China
linear between 1 and 50 lM for each polyamine. Agricultural University, National Research Initiative Competitive
Grants from the Animal Reproduction Program (2008-35203-19120
Accuracy and precision of the HPLC method and 2011-67015-20028) and Animal Growth & Nutrient Utilization
Program (2008-35206-18764) of the USDA National Institute of Food
and Agriculture, and Texas AgriLife Research Hatch project (H-
The accuracy (the nearness of an experimental value to the 8200). We are grateful to Gang Lin, Bin Wang, and Ji Yun for
true value) of the HPLC method, as determined with technical assistance.

123
Determination of polyamine

Conflict of interest The authors declare that they have no conflict enzymes in the different zones of male and female mouse
of interests. kidneys. Amino Acids 43:2153–2163
Li H, Meininger CJ, Hawker JR Jr et al (2001) Regulatory role of
arginase I and II in nitric oxide, polyamine, and proline
syntheses in endothelial cells. Am J Physiol Endocrinol Metab
280:E75–E82
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