Quantification of 1-13C-L-methionine in ratserum with hydrophilic interaction liquidchromatography coupled with tandem massspectrometry and its application inpharmacokinetic studyYing Xu,a Xiao Huang,a Xiuli Nie,b,c Li Yang,a* Weili Yan,b,c* Zhengtao Wang,a

Changhong Wanga and Zhibi Hua

ABSTRACT: A rapid, selective and sensitive hydrophilic interaction liquid chromatography (HILIC) coupled with tandem massspectrometry (MS/MS) method was developed to determine 1-13C-L-methionine in rat serum. Proteins in serum were precipi-tated using acetonitrile and the supernatant was separated after centrifugation. 1-13C-L-phenylalnine was used as the internalstandard. HILIC–tandem mass spectrometry analysis was performed on a hydrophilic interaction silica column (TSK-GELAMIDE-80) using a linear gradient elution system, acetonitrile-5 mM ammonium acetate containing 0.1% formic acid andmultiple reaction monitoring mode for 1-13C-L-methionine and 1-13C-L-phenylalnine. The assay was validated with a linearrange between 10 and 150 ng mL-1 (r � 0.99) and a lower limit of quantification of 10 ng mL-1, calculated with weighted (1/x2)least squares linear regression. The RSD of intra-day precision was smaller than 3.6% and the inter-day RSD less than 6.5%,while the average recovery was 100.48% with an RSD of accuracy within 2.9%, determined from quality control samples. TheHILIC-MS/MS method was fully validated and successfully applied to the in vivo pharmacokinetic study of stable-isotope1-13C-L-methionine in rats. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: 1-13C-L-methionine; hydrophilic interaction liquid chromatography; tandem mass spectrometry; pharmacokinetics

IntroductionMethionine is an essential amino acid and serves as a universalmethyl donor in bio-metabolism (Milazzo et al., 2005). Forexample, the sulfur-containing amino acid, homocysteine, is syn-thesized from methionine through transmethylation catalyzedby methionine adenosyltranferase (Candito et al., 1997; Cattaneoet al., 1999; Mansoor et al., 1992; Van Der Griend et al., 1998).Because of the importance of methionine in metabolism andclinical diagnosis, pharmacokinetics has been used to evaluatethe serum concentration–time relationship of methionine(Banasch et al., 2008a, b; Shinohara et al., 2001).

There existed analytical methods for determination ofmethionine, including amino acid-analyzer, thiol-specificanalysis, ion exchange liquid chromatography (IEX-LC), high-performance liquid chromatography–mass spectrometry (HPLC-MS) and gas chromatography–mass spectrometry (GC-MS) (Toueet al., 2006; Fukagawa et al., 2000; Waterval et al., 2009). Thosemethods usually require an expensive derivatization process andwere not appropriate for routine clinical use. HPLC-MS has beensuccessfully employed in recent pharmaceutical analysis.However, methionine is absorbed with difficulty in a commonreversed-phase C18 column, and in order to obtain ideal separa-tion, the mobile phase requires ion-pairing reagents. The addi-tion of ion-pairing to mobile phase would affect the performanceof mass spectrometry. The newly developed analytical method of

hydrophilic-interaction chromatography (HILIC) is suitable forretaining highly polar analytes without the need for mobilephase modifiers such as ion-pairing reagents. Furthermore, HILICcoupled with mass spectrometry (MS) has the advantage ofimproving MS sensitivity due to the high content of organic

* Correspondence to: Li Yang, The MOE Key Laboratory for Standardization ofChinese Medicines, Institute of Chinese Materia Medica, Shanghai Universityof Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, China. E-mail:yangli7951@hotmail.com

Weili Yan, Department of Nuclear Medicine, Renji Hospital, Shanghai Jiao-tong University of Medicine, 1630 Dongfang Road, Shanghai, China. E-mail:wlyan@sjtu.edu.cn

a The Ministry of Education Key Laboratory for Standardization of ChineseMedicines and the State Administration of TCM Key Laboratory for NewResources and Quality Evaluation of Chinese Medicines, Institute of ChineseMateria Medica, Shanghai University of Traditional Chinese Medicine, Shang-hai 201210, China

b Department of Nuclear Medicine, Renji Hospital, Shanghai Jiaotong Univer-sity School of Medicine, Shanghai 200127, China

c Institute of Clinical Nuclear Medicine, Renji Hospital, Shanghai Jiaotong Uni-versity School of Medicine, Shanghai 200127, China

Abbreviations used: ESI, electrospray ionization; HILIC, hydrophilic interac-tion liquid chromatography; MRM, multiple reaction monitoring

Research article

Received 4 August 2010, Revised 26 September 2010, Accepted 28 September 2010 Published online in Wiley Online Library: 02 February 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1563

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solvent in the mobile phase which will evaporate easily (Wanget al., 2008). Compared with previous reported methods, aminoacids analysis based on HILIC-MS has proved to be much simpler,rapid, sensitive and selective, without the need for a complexprocess of derivatization.

The quantification of 1-13C-L-methionine using HILIC-MS/MScan be effectively used to study methionine metabolism. In thepresent study, hydrophilic interaction liquid chromatographycoupled with triple quadrupole mass spectrometry was appliedto the determination of serum concentration of 1-13C-L-methionine to assess its pharmacokinetic behavior.

Experimental

Chemicals and reagents

1-13C-L-methionine and 1-13C-L-phenylalnine were purchased from Isotec(Miamisburg, OH, USA). Acetonitrile of HPLC-grade and formic acid ofHPLC-grade were obtained from Tedia Co. (Fairfield, OH, USA). Ammo-nium acetate was purchased from Fisher Scientific Co. (Nepean, Ont.,Canada). Water was purified with a Milli-Q system (Millipore, MA, USA).

Animals

Male Sprague–Dawley (SD) rats (210–310 g) were obtained from theLaboratory Animal SHUTCM. All animals were fed with a commercialpellet diet and ad libitum water. Animal welfare and the animal experi-mental protocols were strictly consistent with the Guide for the Care andUse of Laboratory Animals (US National Research Coucil, 1996) and therelated ethics regulations of SHUTCM.

Instrumentation and conditions

The HPLC separation was performed on a Waters Acquity system (WatersCorp., Milford, MA, USA) with a cooling autosampler and a binary pump.A hydrophilic interaction silica column (TSK-GEL AMIDE-80, 50 ¥ 2.0 mm,3 mm) was used for HPLC separation at 45°C. The mobile phase consistedof acetonitrile (A) and 5 mM ammonium acetate containing 0.1% formicacid (B) at a flow rate of 0.2 mL min-1, using a gradient elution system:0–2 min (70% A), 2–4 min (70–60% A). The autosampler temperature waskept at 4°C and the injected volume was 2 mL.

A triple quadrupole tandem mass spectrometer (Quattro Premier,Waters Corp., Milford, MA, USA) equipped with an electrospray ionization(ESI) interface was used. The ESI source was set in a positive ionizationmode. Quantification was performed using the multiple reaction moni-toring (MRM) mode of the transitions of m/z 151.04 → 134.00 for 1-13C-L-methionine and m/z 167.23 → 120.07 for internal standard (IS),respectively. A scan time of 0.4 s per transition was set. The tuning param-eters were optimized and set as follows: capillary voltage, 3.2 kV; conevoltage, 20 V; source temperature, 120°C; and desolvatation temperature,350°C. Nitrogen was used as the desolvatation and cone gas with flowrates of 600 and 50 L h-1, respectively. The optimized collision energy forthe analyte and IS was 15 eV. All data were acquired in centroid mode andprocessed using MassLynx™ 4.1 software (Waters Corp., Milford, MA, USA).

Stock solutions, standards and quality control samples

The stock solution of the chemical reference standard (CRS), 1-13C-L-methionine, was prepared by dissolving approximately 5 mg of accu-rately weighed 1-13C-L-methionine in 5 mL double-distilled (dd) water.The stock solution was diluted with the initial mobile phase to provideeight working solution of different concentrations. The stock solution ofIS CRS, 1-13C-L-phenylalnine, was prepared by dissolving approximately5 mg IS, accurately weighed, in 5 mL dd water, which was diluted with ddwater to yield the working solution with final concentration of25 ng mL-1. The test samples for standard curves were prepared with theaddition of IS working solution and eight working solution of 1-13C-L-

methionine to blank serum. The test samples for quality controls (QC)were prepared similarly using IS working solution and 1-13C-L-methionineworking solution of low, medium and high concentrations. After depro-teinization process, the supernatants were diluted with initial mobilephase to provide the final test samples of appropriate concentrations.

Calibration standards were prepared daily by spiking appropriatequantities of working solutions (10 mL 1-13C-L-methionine and 10 mL IS) to20 mL of blank serum to obtain final concentrations of 1-13C-L-methionineat 10, 15, 20, 25, 50, 80, 100 and 150 ng mL-1 and IS at 25 ng mL-1. The QCsamples were prepared with blank serum at low (QC-L), medium (QC-M)and high (QC-H) concentrations of 1-13C-L-methionine at 10, 50 and100 ng mL-1 and stored at -80°C.

Sample preparation

Internal standard (10 mL) and dd water (10 mL) were added to serum(20 mL) and mixed well before deproteinization with 60 mL acetonitrile.The mixture was vortex-mixed and centrifuged at 20,000g for 10 min. Thesupernatant (60 mL) was centrifuged at 20,000g for 10 min again. Fivemicroliters of the supernatant was diluted with 995 mL of initial mobilephase, an aliquot of which (2 mL) was injected into the HILIC-MS/MSsystem for analysis.

Selectivity, linearity and sensitivity

The selectivity was evaluated by comparing the MRM chromatograms ofblank serum with IS-spiked serum samples after intravenous administra-tion of 1-13C-L-methionine in rats.

The calibration curves of 1-13C-L-methionine were established usingeight concentrations containing serum samples in the range of10–150 ng mL-1, calculated with weighted (1/x2) least squares linearregression for three different days. The limit of detection (LOD) was deter-mined using the signal-to-noise ratio (S/N) of 3:1. The low limit of quan-tification (LLOQ) is defined as the lowest concentration on the calibrationcurve with an acceptable RSD of accuracy (within 20%), based on an S/Nof higher than 10:1.

Precision and accuracy

Precision and accuracy were determined from six replicates of eachquality control sample (QC-L, QC-M and QC-H). The intra-day precisionwas worked out on the basis of the variation coefficient obtained throughthe analysis of six replicate QC-L, QC-M and QC-H samples. For inter-dayprecision, analyses of the same six replicate samples at low, medium andhigh concentrations were performed on three different days over aperiod of 2 weeks. The accuracy of the assay was evaluated by recovery ofQC samples. The samples for recovery determination were prepared atthree concentrations in six replicates by adding the analytes to blankserum before deproteinization. Recovery was determined as the ratio ofthe measured concentration vs the initial concentration. Accuracy wasdeemed acceptable when the measured concentration was within �15%of the initial concentration, except under the low limit of quantification(LLOQ), where it should not deviate by more than �20%. Similarly, preci-sion was acceptable when the coefficient of variation of replicates wassmaller than �15%, except under the LLOQ, where it should not exceed�20%.

Matrix effect

The matrix effect was examined by comparing the MRM peak response ofeach concentration of the analyte (at three concentrations of QC samples)or supernatants spiked with IS after deproteinization of serum (A) withthat of the analyte dissolved in the mobile phase (B). The value A/B ¥100% was represented as the matrix effect.

Stability studies

Long-term sample stability. Six quality control samples at threeconcentration levels (QC-L, QC-M and QC-H) were kept in a deep freezer

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at -80°C and thawed after 7 days of storage, then centrifuged if necessarybefore subjecting to HILIC-MS/MS analysis.

Short-term sample stability. Similar to the long-term sample sta-bility study, six quality control samples at low, medium and high concen-trations were analyzed after leaving at room temperature (25 � 3°C)for 4 h.

Autosampler stability. Six replicates of quality control samples atthree concentrations were set in the thermostated controlled autosam-pler for 8 h at 4°C and then analyzed.

Freeze–thaw stability. Six replicates of quality control samples atlow, medium and high concentrations were stored at -80°C. Thawing wasperformed at room temperature, followed by freezing at -80°C for 24 h.The samples were subjected to two more freeze–thaw cycles for analysis.

Application to pharmacokinetic study

Male Sprague–Dawley rats (n = 6, 210–310 g) were given no food but freeaccess to water for 12 h prior to intravenous administration of 1-13C-L-methionine (50 mg kg-1). Blood samples (each ca 0.15 mL) were collectedbefore and at 1.5, 3, 7, 10, 15, 20, 30, 45, 60 and 90 min after administra-tion of 1-13C-L-methionine. Serum samples were obtained by centrifuga-tion of blood at 3000g for 10 min and stored at -80°C before use.

The pharmacokinetic (PK) parameters such as apparent volume of dis-tribution (Vd), clearance rate (Cl), mean residence time (MRT), the areaunder the serum concentration–time curve (AUC0-t), the area under theserum concentration–time curve from zero to infinity (AUC0-•) and half-life of drug elimination (t1/2) were analyzed using the non-compartmentalPK data analysis software of PK Solution 2 ™ (Summit Research Services,CO, USA). The initial peak concentration (Cinitial) was obtained directly fromthe analytical results.

Results and discussion

Method development

To achieve the quantification of 1-13C-L-methionine with highanalytical efficiency and speed, different columns and mobile

phases were tested to optimize the separation conditions.Because of the strong polar properties of amino acids, methion-ine is absorbed with difficulty in common reverse-phase C18

columns. The recently developed analytical method, HILIC-MS/MS, was chosen to overcome these difficulties with improved MSsensitivity. Compared with previous methods, amino acid analy-sis based on HILIC-MS/MS proved to be much simpler, morerapid, with higher sensitivity and selectivity, and with no need forcomplex process of derivatization. A TSK-Gel Amide-80 column(50 ¥ 2.0 mm, i.d., 3 mm) and a linear gradient elution system ofacetonitrile–0.1% formic acid containing 5 mM ammoniumacetate were employed as the optimized method to obtain suf-ficient separation within 4.0 min.

Selectivity, linearity and sensitivity

The representative MRM chromatograms of blank serum andIS-spiked serum after intravenous administration of 1-13C-L-methionine are shown in Fig. 1. No interference from endog-enous substance was observed at each analyte MRM function.

The peak area ratios (analyte/IS, y) of analytes to IS varied lin-early over the concentration range (10–150 ng mL-1), calculatedwith weighted (1/x2) least squares linear regression method. Thecorrelation coefficient (r) for all test samples was above 0.99. Theresults of calibration curve are summarized in Table 1. The LOD ofthe method was determined to be 4 ng mL-1 based on an S/Nratio of 3:1. The LLOQ was found to be 10 ng mL-1, with a recoveryof 96.07 � 3.4%.

Precision and accuracy

The intra- and inter-day precision and accuracy of the method aresummarized in Table 2. The intra-day precision (RSD) was smallerthan 3.6% and inter-day precision smaller than 6.7% for eachconcentration. The average recovery at three concentrations was100.48 � 2.9%.

Figure 1. Representive MRM chromatograms of: (A) blank serum; (B) blank serum spiked with 1-13C-L-methionine at a concentration of 50 ng mL-1 andIS (25 ng mL-1); (C) serum sample from rats 3.0 min after intravenous administration of 1-13C-L-methionine and IS (25 ng mL-1).

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Matrix effect and stability

In terms of matrix effect, all the ratios defined in the Matrix Effectsection ranged from 105 to 112%. No significant matrix effect on1-13C-L-methionine determination was observed, indicating thatno co-eluting substance could influence the ionization of theanalyte.

The results from all stability tests are shown in Table 3, anddemonstrated a good stability of 1-13C-L-methionine through theanalysis. The method therefore proved to be applicable to routineanalysis.

Pharmacokinetics study

This validated HIILC-MS/MS method was successfully applied to aPK study of 1-13C-L-methionine in rat serum. The concentration–

time curve is shown in Fig. 2. The initial serum concentration(Cinitial) was 149.91 � 26.98 mg mL-1, the area under the serumconcentration–time curve from 0 min to the time of last measur-able concentration (AUC0–t) was 3198.09 � 615.64 mg min mL-1,the area under the serum concentration–time curve from 0 minto infinity (AUC0–•) was 4731.88 � 1232.23 mg min mL-1, and thehalf-life of drug elimination at the terminal phase (t1/2) was 79.04� 36.91 min. Clearance rate and MRT were 11.21 �3.09 mL min kg-1 and 86.25 � 42.93 min, respectively. Apparentvolume of distribution was 1205.75 � 473.47 mL kg-1.

ConclusionsA sensitive, selective and rapid HILIC-MS/MS method was devel-oped based on validated methodology and successfully applied

Table 1. Calibration curve data for standard 1-13C-L-methionine

Concentration (ng/mL) Slope (�SD) Intercept (�SD) Regression coefficient (r) LOD (ng/mL) LLOQ (ng/mL)

10–150 0.0211 � 0.02 0.0357 � 0.32 0.9959 � 0.11 4 10

Table 2. Summary of precision and accuracy for the HILIC-MS/MS method

Sample Nominalconcentration

(ng/mL)

Accuracy(%)

Mean accuracy� SD (%)

Intra-dayprecision(RSD%)

Inter-dayprecision(RSD%)

QC-L 10 96.07 � 3.44 100.48 � 2.94 3.58 5.75QC-M 50 102.94 � 2.99 2.84 6.78QC-H 100 102.41 � 2.40 2.64 5.26

Table 3. Stability of 1-13C-L-methionine in quality control samples (n = 6)

Stability Recovery determination (mean � SD)QC-L QC-M QC-H

Post-preparative (4°C for 8 h) 103.11 � 4.43 97.42 � 4.39 90.54 � 4.86Short-term (room temperature for 4 h) 98.57 � 10.62 101.61 � 4.02 101.59 � 4.97Three freeze–thaw cycles 92.02 � 2.59 96.96 � 7.60 99.74 � 3.07Long-term (-80°C for 7 days) 100.51 � 3.99 92.09 � 6.03 96.63 � 6.01

Figure 2. Mean serum concentration–time curve of 1-13C-L-methionine after intravenous administra-tion (n = 6).

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to the determination of 1-13C-L-methionine in rat serum for PKstudy. It is the first time that a quantitative analysis method hasbeen reported using HILIC-MS/MS to determine 1-13C-L-methionine. This method has overcome the disadvantage of thedifficulty of analyzing highly polar analytes with commonHPLC-MS methods. Furthermore, sufficient selectivity and sensi-tivity for accurate quantitative analysis can be achieved bydetecting the stable isotope methionine in MRM mode.

AcknowledgementsThe financial support of the Natural Science Foundations of China(30701104 and 30770599), the Shanghai Rising-Star Program(09QA1405500) and the Scientific Innovation Project from Shang-hai Municipal Education Commission (09YZ125 and 09YZ82) isgratefully acknowledged. The authors owe a deep debt to DrShouming Zhong for his great help with the English.

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