Application note

Received: 22 March 2012 Revised: 9 July 2012 Accepted: 8 August 2012 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3084

Determination of polyamines in human plasmaby high-performance liquid chromatographycoupled with Q-TOF mass spectrometryRan Liu,a Kaishun Bi,a Ying Jia,b Qian Wang,a Ran Yina and Qing Lia*

A high-performance liquid chromatography coupled with Q-time of flight mass spectrometry (HPLC/Q-TOF MS) method was de-veloped and validated for the determination of 1, 3-diaminopropane, putrescine, cadaverine, spermidine and spermine in humanplasma. The plasma samples were first pretreated by 10% HClO4 and then derived by benzoyl chloride with 1, 6-diaminohexaneas internal standard. The derived polyamines were separated on a C18 column using a gradient program. The detection wasperformed on a Q-TOF MS by positive ionization mode. Calibration curve for each polyamine was obtained in the concentrationrange of 0.4 ~200.0ng • ml�1, with limit of detection of 0.02~0.1 ng • ml�1. The intra- and inter-day RSD for all polyamines were2.5–14.0% and 2.9~13.4%, respectively. The method was applied to determine the polyamines in human plasma from cancerpatients and healthy volunteers. Results showed that the mean levels of polyamines in the plasma of cancer patients were higherthan that of healthy volunteers, which suggested that the plasma polyamines could be employed as cancer diagnostic indicatorsin clinical testing. Copyright © 2012 John Wiley & Sons, Ltd.

Keywords: polyamines; human plasma; benzoyl chloride derivatization; cancer indicators; HPLC/Q-TOF MS

* Correspondence to: Qing Li, School of Pharmacy, Shenyang PharmaceuticalUniversity, 103 Wenhua Road Shenhe Dist, Shenyang 110016, China. E-mail:lqyxm@hotmail.com

a School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,Shenyang 110016, China

b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical Uni-versity, 103 Wenhua Road Shenhe Dist, Shenyang 110016, China

134

Introduction

Cancer is a life-threatening disease, and which has a chronic path-ogenic process. The early detection and diagnosis could greatlyimprove the odds of successful treatment and survival. Recentstudies indicated that cancer biomarkers could indicate the occur-rence of cancer at an early time, and which thus could play impor-tant roles in cancer progression and diagnosis. Cancer biomarkerscould be substances related to nucleic acid, acidic phospholipidand also protein. Among different cancer biomarkers, polyaminesare one group of such substances.[1–9] The polyamines of 1, 3-dia-minopropane, putrescine, cadaverine, spermidine and spermineare ubiquitous polycationic molecules that occur in living cells.They are the derivatives of ornithine and lysine. Polyamines aremetabolized and inactivated by two main enzymes, ornithinedecarboxylase and S-adenosylmethionine decarboxylase, whichmight lead to a series of metabolites from the protein prosoma -ornithine and lysine (see the summary in Fig. 1).

The pre-treatment methods of the plasma for polyamine detec-tion have been reported.[10–12] Because of the low concentrationsof polyamines, usually nano-gram per milliliter in biological sam-ples,[13–15] the time-consuming and complex preparation proce-dures for the samples have caused numerous difficulties. Accord-ingly, trace detection by mass spectrometry had been developedfor the polyamines determination. Q-time of flight (Q-TOF) massspectrometry possesses high resolution and accurate detection,and which is currently a useful tool analyzing compounds incomplex matrix.[16–19] The coupling of high-performance liquidchromatography (HPLC) and Q-TOF mass spectrometry has advan-tages over gas chromatograph and HPLC-coupled fluorescencedetector for high-resolution and narrow extracted-ion windows.By extracting the target ions from the total ion chromatogram,minor polyamines could be quantitative analyzed in complex

J. Mass Spectrom. 2012, 47, 1341–1346

matrices. Thus, the combination of sample pre-treatment andHPLC/Q-TOF MS method could greatly save the analytical time.

Among the numerous derivative reagents, benzoyl chloride hadbeen widely adopted for its easy obtaining and high activity inacylated reaction. Benzoyl polyamines in human urine samples hadalready been separated and detected by applying HPLC/Q-TOF MSin our former report.[20] By using benzoyl chloride for derivatization,the polyamines in human plasma were analyzed here.

Here, the polyamines in plasma could be separated and ana-lyzed by HPLC/Q-TOF MS using benzoyl chloride as derivatives.In particular, the method of determinate polyamines was appliedto distinguish the healthy volunteers and the cancer patients.

Material and methods

Chemicals, reagents and materials

1, 3-diaminopropane, putrescine, spermine, 1, 6-diaminohexane(used as an internal standard), cadaverine hydrochloride, spermi-dine hydrochloride and benzoyl chloride were obtained fromSigma-Aldrich (St. Louis, MO). Methanol and acetonitrile of HPLCgrade were purchased from Fisher Chemicals (Fair Lawn, NJ). All

Copyright © 2012 John Wiley & Sons, Ltd.

1

Figure 1. A schematic figure to show the possible metabolic roles ofpolyamines in cell. The notations here are ornithine decarboxylase(ODC) and S-adenosylmethionine decarboxylase (SAMOC).

R. Liu et al.

1342

the other reagents were of analytic grade. Redistilled and deio-nized water was used throughout the study.The human plasma samples were collected from 14 healthy

volunteers and 14 cancer patients, half male and half female.Fourteen healthy volunteers were from the authors’ university,and 14 cancer patients were collected from a cancer hospital inChina (from February 2010 to April 2010). Diagnoses of cancerwere made on the basis of usual clinical and laboratory resultsand confirmed by tissue biopsy.

Apparatus and operation conditions

Liquid chromatography

Liquid chromatography was performed on an Agilent 1200 HPLCsystem with auto-sampler and column oven. A Kromasil ODS C18column (5 mm, 250� 4.6mm id) was held at 35 �C. Chromato-graphic separation was achieved with gradient elution using amobile phase composed of water (A) and methanol (B). The gra-dient elution started at 55% B and changed linearly to 74% in14min, and maintained at 74% B for 10min. The total flow rateof the mobile phases was 1ml • min�1. 25% of the elution wassplit into the inlet of mass spectrometer. The injection volumewas 10ml.

Mass chromatography

Mass spectrometric detection was carried out on a Bruker Dal-tonics micrOTOF-Q mass spectrometer (Billerica, MA) with anelectrospray ionization (ESI) interface. The ESI source was set inpositive ionization mode (ESI+). The optimized ionization condi-tions were as follows: capillary voltage 4.5 kV. Nitrogen was usedas the desolvation and nebulizing gas at 230 �C by the gas flow of7.0 L • min�1. Full scan mode was employed in the mass range of50 – 900m/z. Formic sodium was used for mass correction. Thesoftware microTOF control was applied to system operation anddata collection.

Preparation of stock solutions and calibration standards

Standard stock solutions of polyamines and IS (1, 6-diaminohexane)were both prepared inmethanol at the concentration of 1mg •ml�1

wileyonlinelibrary.com/journal/jms Copyright © 2012 Joh

and 10mg • ml�1, respectively. All the stock solutions were stored at4 �C and further diluted with methanol to make working standards.

Calibration standards for polyamines were prepared by spikingblank plasma (250 ml) at 0.4, 2.0, 4.0, 20.0, 40.0 and 200.0 ng • ml�1

for each polyamine (250ml). Polyamines standards were evapo-rated to dryness at 30 �C under a stream of nitrogen beforeplasma preparation and derivatization. The quality control sam-ples were prepared with blank plasma (250 ml) at low, middleand high concentrations of 0.8, 2.0, 20.0 ng • ml�1.

Plasma samples preparation and derivatization

The IS solution (100 ng • ml�1) was prepared by dilution of IS stocksolution. Then, 250 ml IS (followed by evaporating to dryness at30 �C under a stream of nitrogen), 250ml plasma and 150ml 10%perchloric acid were added into a clean glass tube and vortexedfor 3min. After centrifugation at 15 000 rpm for 3min, the super-natant was transferred to another clean glass tube.

The perchloric supernatants were first alkalinized by adding400 ml of 2mol • ml�1 NaOH solution, followed by 125ml benzoylchloride (4% diluted with acetonitrile) and stood under ultrasoni-cation at 30 �C for 20min. After that, the equal volume of satu-rated sodium chloride solution was added to react with excessreagent, and the solvent was extracted into the equal volumeof diethyl ether, followed by evaporating to dryness at 30 �Cunder a stream of nitrogen. The residue was dissolved in 50mlmethanol and an aliquot of 10ml of the supernatant was injectedinto the HPLC/Q-TOF MS system for analysis.

Method validation

Linearity and limit of detection

To evaluate linearity, calibration standards in plasma at sixconcentration levels ranged from 0.4 to 200.0 ng • ml�1 for eachpolyamines were prepared and assayed in triplicate by derivativeprocedure as described above. The limit of detection (LOD) wasassessed by detecting the concentration of polyamine at variousconcentrations from 0.02 to 0.1 ng • ml�1.

Precision

The precision was expressed as relative standard deviation andassessed to determine QC samples at three concentration levels(0.8, 2.0, 20.0 ng • ml�1of each polyamines). Intra-day precisionwas determined by six replicate analyses of QC samples on oneday, while inter-day precision was determined by three replicateanalyses on three consecutive days.

Recovery and matrix effect

Recoveries of polyamines were determined by comparing therecovered polyamine concentrations with the nominal concentra-tions at QC levels. In order to evaluate the matrix effect on theionization of analysts, such as the potential ion suppression orenhancement due to the matrix components, three concentrationlevels (0.8, 2.0, 20.0 ng • ml�1of each polyamines) of polyamineswere tested. Thematrix effect was evaluated by comparing the peakarea ratio of post-spiked derivative polyamines standards in theplasma derivatives and IS to those of the neat standard solutions.

Stability

The stability tests were designed to cover the anticipatedconditions that real samples might experience. Stability was

n Wiley & Sons, Ltd. J. Mass Spectrom. 2012, 47, 1341–1346

The application of HPLC/Q-TOF MS

evaluated by analyzing the sample at QC levels. The sampleswere examined at 0, 2, 4, 8, 12 and 24 h after derivatization.During the analysis, the samples were stored at 4 �C. The stabilitywas also tested for three freeze–thaws and long term. In eachfreeze–thaw cycle, the plasma samples were frozen for at�20 �C 24 h and thawed at room temperature. The results oflong-term stability showed that the samples could be stable in30 days.

Results and discussion

Sample preparation and polyamines derivatization

Precipitation protein method was chosen here for the pre-treat-ment of the plasma sample. 10% HClO4 was chosen as extractionand deproteinization solvent; this acid solution could combinewith the basic group of polyamines to separate from other en-dogenous substances.

In our study, benzoyl chloride was used as the derivativereagent, which reacted with the amino group of polyamines toform acetated derivatives. Figure 2 described the compoundsformed after the benzoyl chloride addition. A standard solutionof six polyamines including IS (1, 6-diaminohexane, 10mg •

ml�1 each, 10ml) was used to investigate the optimized derivati-zation conditions. During the method development, the effectsof the parameters on the derivatization were evaluated on thederived polyamines peak area. The derivatization reaction shouldproceed in alkaline medium since hydrogen ion was continuouslycreated during the acetated reaction of polyamines and benzoylchloride. In order to remove excess benzoyl chloride, saturatedsodium chloride solution was then added. The derivatizationwas performed at four reaction temperatures (20, 30, 40, 50 �C)and monitored at four time points (10, 15, 20, 30min). As shownin Fig. 3, the derivatization could be completed at 30 �C in 20min.Longer time and higher temperature were not necessary in accel-erating the reaction. Various organic solvents, such as diethylether, ethyl acetate, n-butyl alcohol and n-hexane were evalu-ated. Finally, diethyl ether was found to be optimal for yieldingthe highest recovery of each polyamine.

The optimal HPLC/Q-TOF MS conditions

Benzoyl chloride derivatization procedure significantly increasedsensitivity since at least two benzoyl groups were added to themolecule. After derivatization, the benzoyl polyamines could beseparated from the endogenous substance. Buffer and acid didnot use in the mobile phase for they could not improve the MSresponse for polyamines. Figure 4(A) shows the base peak chro-matogram obtained from the derivatives of standard polyamines.As shown in mass spectra, the sodium adduct ions [M+Na]+ of allpolyamines derivatives were identified. The [M+Na]+ was mostlikely formed by combination of active nitrogen atom of benzoylpolyamines and sodium ion in reaction medium. The identified

Figure 2. Description the derived polyamines compounds formed after the

J. Mass Spectrom. 2012, 47, 1341–1346 Copyright © 2012 John

ions for quantify putrescine, 1, 3-diaminopropane, cadaverine, 1,6-diaminohexane, spermidine and spermine were selected atm/z 319.1, m/z 303.1, m/z 333.2, m/z 347.2, m/z 480.2 and m/z641.3, respectively.

In order to increase the selectivity, the condition of massspectrometric was optimized. Some parameters of the Q-TOFMS, including capillary voltage, dry gas flow, dry gas temperatureand nebulizer pressure, were investigated to improve thesensitivity of the method. Having the capillary voltage increasedfrom 3700 V to 4500 V, the peak area increased gradually andhad the maximum at 4500 V. Different dry gas flows werecompared and the optimum flows was 7.0 L • min�1. In the samemanner, 230 �C was chosen for the optimum dry gas tempera-ture. The nebulizer pressure was tested from 1.2 to 1.4 bar forpolyamines to obtain the highest ion intensity, and 1.3 bar wasselected as the incoming pressure of the nebulizer gas.

Method validation

Calibration curves for polyamines in plasma were generated byplotting the peak area ratio (y) of polyamines subtracted thoseof blank samples to IS versus nominal concentrations (x) ofpolyamines by 1/x2 weighted least square linear regressions.The calibration curves for linear from six different (i.e. 0.4, 2.0,4.0, 20.0, 40.0, 200.0 ng • ml�1) concentrations and the calibrationregression coefficients were ranged from 0.9937 to 0.9972, the REvalues were all less than 15%. According to the criteria of S/N= 3,the LOD was 0.02 ng • ml�1 for putrescine, 0.1 ng • ml�1 for 1, 3-diaminopropane, 0.1 ng • ml�1 for cadaverine, 0.1 ng • ml�1 forspermidine, 0.05 ng • ml�1 for spermine, respectively.

The intra- and inter- day precision for polyamines were listed inTable 1. The intra- and inter-day precision with RSD were all lessthan 14.0% and the RE values were all less than 15%, indicatingan acceptable precision of the method.

The recovery values from plasma at concentration of 0.8, 2.0and 20.0 ng • ml�1 were shown in Table 1. These results indicatedthat the recoveries of polyamines were consistent and notconcentration dependent.

In terms of matrix effect, all the ratios defined as in Section2.5.3 were between 85.1% and 113.5%, which meant no signifi-cant matrix effect in this method.

The polyamines were found to be stable at 4 �C after derivatiza-tion with RSD less than 9.9%within 24 h, which was the acceptancecriterion for stability measurements. Three freeze–thaw cycles andthe long-term stability of the plasma samples had no effect onquantification of polyamines.

Determination of polyamines in human plasma samples

The proposed method was applied to determine the concentra-tions of five polyamines in plasma from the cancer patients andthe healthy volunteers. The typical mass chromatograms of poly-amines in human plasma from cancer sufferers and healthy

benzoyl chloride addition.

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms

1343

Figure 3. The influence of reactive temperature and time on polyamines derivation. The derivatization reaction proceed in 2mol • l�1NaOH, theconcentration of polyamines and I.S. were 10mg • ml�1.

Figure 4. Base peak chromatogram (BPC) obtained from standard polyamines (A), healthy volunteers’ plasma (B) and cancer sufferers’ plasma (C) byHPLC-Q-TOF/MS. (1) Putrescine; (2) 1,3-diaminopropane; (3)Cadaverine; (4) 1,6-diaminohexane (I.S.); (5) Spermidine; (6) Spermine.

Table 1. Validation data for the analysis of polyamines using the proposed method

Polyamines Linearity(r) LOD(ng) Plasma recovery (%)b

0.8 ng • ml�1 2.0 ng • ml�1 20.0 ng • ml�1 Mean

PUTa 0.9941 0.02 76.90 82.5 82.5 80.6

DAPa 0.9952 0.1 79.79 81.3 85.4 82.2

CADa 0.9972 0.1 75.52 81.2 76.89 77.87

SPDa 0.9939 0.1 77.59 77.11 83.7 79.46

SPMa 0.9937 0.05 84.3 84.4 83.3 84.0

Intra-day precision (RSD c %) Inter-day precision (RSD c %)

0.8 ng • ml�1 2.0 ng • ml�1 20.0 ng • ml�1 0.8 ng • ml�1 2.0 ng • ml�1 20.0 ng • ml�1

PUTa 9.3 11.2 7.8 9.8 7.8 4.3

DAPa 10.1 2.5 2.5 11.3 12.5 13.4

CADa 11.7 4.2 5.1 5.5 10.9 8.7

SPDa 14.0 5.0 11.6 11.1 11.6 7.3

SPMa 3.9 5.0 7.9 2.9 3.2 3.5

aPUT (putrescine), DAP (1,3-diaminopropane), CAD (cadaverine), SPD (spermidine), SPM (spermine).bRecovery (%) = (A/B� 100)%. The data was presented as average of six independent determinations, where n=6.

A: the recovered polyamine concentrations. B: the nominal concentrations at QC levelscRelative Standard of Derivation, where n=6.

R. Liu et al.

wileyonlinelibrary.com/journal/jms Copyright © 2012 John Wiley & Sons, Ltd. J. Mass Spectrom. 2012, 47, 1341–1346

1344

Table

2.Amou

nts

ofpolyam

ine(ng

•ml�

1)in

plasm

afrom

14patients

and14

healthyvo

lunteer

Polyam

ines

Plasmaofpatients(n=14

)Plasmaofhealthyvo

lunteers(n=14

)

PUTa

*DAPa

*CADa

SPDa*

SPM

a*

Totalp

olyam

ines

PUTa

*DAPa

*CADa

SPDa*

SPM

a*

Totalp

olyam

ines

p15

108.0

8.92

28.11

22.37

9.10

176.5

h1

16.55

1.14

1.22

8.03

3.91

30.85

p16

140.0

8.03

1.18

18.01

10.96

178.2

h2

14.88

1.79

1.24

11.39

5.19

34.49

p17

109.1

9.15

1.26

6.30

10.25

136.1

h3

14.22

1.79

1.24

9.47

6.15

32.87

p18

178.9

18.03

1.36

11.31

5.93

215.5

h4

14.19

1.85

1.27

3.68

3.60

24.59

p19

196.4

30.97

1.44

9.54

19.16

257.5

h5

13.60

1.11

1.25

3.37

3.89

23.22

p20

179.2

37.01

1.60

19.91

12.33

250.1

h6

16.55

1.49

1.39

4.19

5.48

29.1

p21

192.9

37.86

1.52

15.84

14.16

262.3

h7

15.98

1.15

1.29

6.50

4.19

29.11

p22

178.8

7.54

1.45

4.81

5.98

198.6

h8

14.97

1.92

1.31

4.38

4.21

26.79

p23

194.0

37.54

1.58

6.41

14.15

253.7

h9

14.89

1.42

2.00

16.34

17.14

51.79

p24

104.5

15.22

2.59

9.97

8.83

141.1

h10

19.16

2.64

1.56

5.40

5.86

34.62

p25

182.1

37.27

4.03

29.51

22.23

275.1

h11

13.07

1.83

1.37

4.46

4.40

25.13

p26

187.8

32.35

2.08

16.77

13.28

252.3

h12

14.78

1.01

1.60

3.80

8.14

29.33

p27

152.0

17.86

1.96

10.74

10.97

193.5

h13

15.09

1.89

1.41

5.70

3.87

27.96

p28

194.8

29.84

1.23

11.95

13.09

250.9

h14

14.05

1.69

1.25

8.21

4.82

30.02

Mean�S.D.

164.2�34

.76

23.39�12

.38

3.67

�7.07

13.81�6.97

12.17�4.51

217.2�46

.62

Mean�S.D.15

.17�1.59

1.67

�0.42

1.37

�0.21

7.01

�3.71

5.59

�3.57

30.71�6.98

aPU

T(putrescine),D

AP(1,3-diaminopropan

e),C

AD(cad

averine),SPD

(spermidine),SPM

(spermine).

*Th

esignificantdifferen

cebetweencancersufferersan

dhealthyvo

lunteersas

usingindep

enden

t-samplest-test

andnonparam

etrictest.

The application of HPLC/Q-TOF MS

J. Mass Spectrom. 2012, 47, 1341–1346 Copyright © 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms

1345

R. Liu et al.

1346

volunteers were shown in Fig. 4 (B, C). The derivative polyamineswere identified from the comparison of MS characteristic ofstandard polyamines. As shown in Table 2, the mean levels ofeach polyamine (ng • ml�1) and the total polyamines of patientswere higher than those of healthy volunteers.Compared to the urine, which has been studied in our previous

paper,[20] plasma as extra-cellular fluid could indirectly reflect themetabolic activity of cell internal environment, and plasma had aclose relationship with the cancer located in the circulation. Thismethod had been applied successfully in plasma of patients andhealthy volunteers. It could be a promising assessing approach ofpolyamines levels in human plasma as well as urine.

Conclusion

This paper described the simultaneous determination of fivepolyamines in human plasma after derivatization with benzoylchloride by HPLC/Q-TOF MS. The present validated method wassimple, effective and sensitive. It could be successfully employedin assessing of anti-tumor drug monitoring. Moreover, thismethod might be a useful tool in clinical diagnosis and cancertreatment.

References[1] D. H. Russell, C. C. Levy, S. C. Schimpff. Urinary polyamines in cancer

patients. Cancer Res. 1971, 31, 1555.[2] D. H. Russell. Clinical relevance of polyamines as biochemical markers

of tumor kinetics. Clin. Chem. 1977, 23, 22.[3] G. Sulochana, R. Kalavathy, L. Padmanabhan, et al. Separation of

polyamines in gastric aspirate by TLC in the diagnosis of gastriccarcinoma. Med. Chem. Res. 1983, 77, 758.

[4] Y. Horn, S. L. Beal, N. Walach, et al. Further evidence for the use ofpolyamines as biochemical markers for malignant tumors. CancerRes. 1982, 42, 3248.

[5] D. H. Russell, P. M. Gullino, L. J. Marton, et al. Polyamine depletion ofthe MTW9 mammary tumor and subsequent elevation of spermi-dine in the sera of tumor-bearing rats as a biochemical marker oftumor regression. Cancer Res. 1974, 34, 2378.

[6] D. H. Russell, B. G. Durie, S. E. Salmon. J. Lancet. Polyamines aspredictors of success and failure in cancer chemotherapy. J. Lancet1975, 25, 797.

wileyonlinelibrary.com/journal/jms Copyright © 2012 Joh

[7] C. Moinard, L. Cynober, J. P. Bandt. Polyamines: metabolism andimplications in human diseases. J. Clin. Nutr. 2005, 24, 184.

[8] R. M. Reguera, B. L. Tekwani, R. Balaña-Fouce. Polyamine transport inparasites: a potential target for new antiparasitic drug development.Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol. 2005, 140, 151.

[9] N. Uehara, K. Kita, S. Shirakawa, et al. Elevated polyamine contentin erythrocytes of malignant lymphoma patients. J.Cancer. 1980,71, 393.

[10] F. Gaboriau, R. Havouis, J. P. Moulinoux. Atmospheric pressurechemical ionization-mass spectrometry method to improve thedetermination of dansylated polyamines. Anal. Biochem. 2003,318, 212.

[11] K. Dziarkowska, K. Dziarkowska. Single hollow fiber SLM extractionof polyamines followed by tosyl chloride derivatization and HPLCdetermination. Anal. Chim. Acta 2008, 606, 184.

[12] T. Ekegren, C. Gomes-Trolin. Determination of polyamines inhuman tissues by precolumn derivatization with 9-fluorenylmethylchloroformate and high-performance liquid chromatography. Anal.Biochem. 2005, 338, 179.

[13] L. Li, K. J. Hara, J. T. Liu, et al. Rapid and simultaneous determinationof hair polyamines as N-heptafluorobutyryl derivatives by gaschromatography–mass spectrometry. J. Chromatogr. B 2008, 876, 257.

[14] M. H. Choi, K. R. Kim, B. C. Chung. Determination of hair polyaminesas N-ethoxycarbonyl-N-pentafluoropropionyl derivatives by gaschromatography–mass spectrometry. J. Chromatogr. A 2000, 897,295.

[15] L. Hawel, C. V. Byus. A streamlined method for the isolation andquantitation of nanomole levels of exported polyamines in cellculture media. Anal. Biochem. 2002, 311, 127.

[16] H. R. Morris, T. Paxton, M. Panico, R. McDowell, A. Dell, M. Guihaus,V. Mlynski, D. Selby. A novel geometry mass spectrometer, theQ-TOF, for low-femtomole/attomole-range biopolymer sequencing.J. Protein Chem. 1997, 5, 469.

[17] K. Sugiura, J. Z. Min, T. Toyo’oka, et al. Rapid, sensitive and simulta-neous determination of fluorescence-labeled polyamines in human hairby high-pressure liquid chromatography coupled with electrospray-ionization time-of-flight mass spectrometry. J. Chromatogr. A 2008,1205, 94.

[18] M. S. Dopico-Garcíab, J. M. López-Vilariño, G. Fernández-Martínez.Liquid chromatography method to determine polyamines inthermosetting polymers. Anal. Chim. Acta 2010, 667, 123.

[19] A. P. Stevens, K. Dettmer, G. Kirovski. Quantification of intermedi-ates of the methionine and polyamine metabolism by liquidchromatography-tandem mass spectrometry in cultured tumorcells and liver biopsies. J. Chromatogr. A 2010, 1217, 3282.

[20] R. Liu, Y. Jia, W. Cheng, J. Ling, L. Liu, K. Bi, Q. Li. Determination ofpolyamines in human urine by precolumn derivatization with ben-zoyl chloride and high-performance liquid chromatography coupledwith Q-time-of-flight mass spectrometry. Talanta 2011, 83, 751.

n Wiley & Sons, Ltd. J. Mass Spectrom. 2012, 47, 1341–1346