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Journal of Chromatography B, 945– 946 (2014) 193– 198

Contents lists available at ScienceDirect

Journal of Chromatography B

j ourna l h om epage: www.elsev ier .com/ locate /chromb

hort Communication

imultaneous determination of SYL-1119 and SYL-1119-P in ratlasma using HPLC coupled with tandem mass spectrometry

hu Yang, Jinping Hu, Yan Li ∗

eijing City Key Laboratory of Active Substances Discovery and Drugability Evaluation, State Key Laboratory of Bioactive Substance and Function of Naturaledicines, Department of Drug Metabolism of Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing

00050, China

r t i c l e i n f o

rticle history:eceived 30 August 2013eceived in revised form 31 October 2013ccepted 2 November 2013vailable online 26 November 2013

eywords:YL-1119YL-1119-PC–MS/MSharmacokinetic

a b s t r a c t

SYL-1119 is a sphingosine-1-phosphate receptor 1 modulator for the treatment of autoimmune dis-ease with better selectivity, while SYL-1119-P is its active phosphate. A sensitive and specific liquidchromatography–tandem mass spectrometry method was developed and validated for the simultaneousdetermination of SYL-1119 and SYL-1119-P in rat plasma. SYL-1110, an analogue of SYL-1119, was usedas the internal standard. Plasma samples were prepared by protein precipitation using acetonitrile. Theanalytes and internal standard were separated on a Zorbax SB-C18 column (3.5 �m, 100mm × 2.1 mm)with a gradient mobile phase consisting of methanol and water containing 0.1% formic acid at a flow rateof 0.2 ml/min with an operating temperature of 20 ◦C. The detection was performed on a triple quadrupoletandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring modeof the transitions at m/z 364 → 259 for SYL-1119, m/z 444 → 259 for SYL-1119-P, and m/z 378 → 273

for the IS. Calibration curves were linear in the range of 0.2–50 ng/ml for SYL-1119 and 10–1000 ng/mlfor SYL-1119-P. The lower limit of quantification (LLOQ) was 0.2 ng/ml for SYL-1119 and 10 ng/ml forSYL-1119-P. The intra- and inter-day precisions were 5.4–12.8% for two analytes with accuracies within±10%. The recoveries for two compounds were 91.3–104.5%. The analytes were proved to be stable dur-ing all sample storage, preparation, and analytic procedures. The method was successfully applied to thepharmacokinetic study of SYL-1119 and SYL-1119-P in rats after oral administration of SYL-1119.

. Introduction

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediatorhat could regulate a variety of cellular functions, including dif-erentiation, survival, proliferation, and chemotaxis by interactingith five receptor subtypes (sphingosine-1-phosphate receptor

ubtype 1–5, S1P1–5) [1,2]. Recently, sphingosine-1-phosphateeceptor 1 (S1P1) has been pursued as an important therapeuticarget because of its essential role played in immune regulation3]. One of the S1P1 modulators, FTY720 was approved in thenited States for the therapy of relapsing multiple sclerosis [4,5].

t was found to undergo the reversible biotransformation to a

ctive phosphate (FTY720-P) [6,7], which acts as a potent agonistf S1P1 on lymphocytes, resulting in the suppression of lympho-ytes egress from lymph nodes and prevention of their recirculation

∗ Corresponding author at: Department of Metabolism, Institute of Materia Med-ca, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xianong Tan Street, Xicheng District, Beijing 100050, China. Tel.: +86 10 63165172;

ax: +86 10 63165172.E-mail address: yanli@imm.ac.cn (Y. Li).

570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jchromb.2013.11.005

© 2013 Elsevier B.V. All rights reserved.

to other organs. However, interacting with three other S1P recep-tors (S1P3–5) may lead to the side effects such as bradycardia andhypertension [8].

SYL-1119, a novel chemical entity, was found to be phosphory-lated to SYL-1119-P in vivo, which showed equivalent lymphopeniaactivity and less effect on heart rate compared with FTY720 in vivo[9]. The objective of the present study was to explore a simple andsensitive LC–MS/MS method for the simultaneous determinationof both SYL-1119 and SYL-1119-P with good accuracy to meet therequirements of pharmacokinetic study in rats after oral adminis-tration of SYL-1119.

2. Experimental

2.1. Chemical and reagents

SYL-1119, SYL-1119-P, and internal standard SYL-1110 wereprovided by Laboratory of Chemical Synthesis (Institute of Mate-

ria Medica, Chinese Academy of Medical Science). Methanol andacetonitrile were of HPLC grade (Fisher, USA). All other chemicalswere of analytical reagent grade. HPLC grade water was obtainedusing a Milli Q system (Millipore, MA, USA).

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.2. HPLC condition

The separation was carried out on an Agilent 1260 series HPLCystem (Agilent, USA). A reversed-phase Zorbax SB-C18 analyticalolumn (3.5 �m, 100mm × 2.1 mm, Agilent, Santa Clara, USA) with

0.5 online filter (Upchurch Scientific Ltd.) was used. Mobile phase was 0.1% formic acid, mobile phase B was methanol with 0.1%

ormic acid, and the flow rate was 0.2 ml/min with an operatingemperature of 20 ◦C. The initial condition for the HPLC gradientas 80:20 (A:B). From 0.5 to 0.6 min, the mobile phase composition

hanged linearly to 5:95 (A:B). This condition was held until 4 min.he gradient was returned in a linear fashion to 80:20 (A:B) from 4o 4.1 min and re-equilibrated until 11 min.

.3. Mass conditions

An API 4000 triple quadruple mass spectrometer (AB SCIEX,SA) equipped with a TurboIonSpray interface (MDS Sciex, Sanrancisco, USA) consists of a quaternary pump, automatic sol-ent degasser, autosampler, and an automatic thermostatic columnompartment. Mass spectrometric analysis was performed inositive electrospray ionization (ESI) mode. The following werehe optimum ESI conditions: ion spray voltage, 5500 V; tem-erature, 500 ◦C; CAD gas, 6 psi; CUR gas, 25 psi; both GS1 andS2, 50 psi. The MS recordings were carried out in MRM modeith specific ion transitions of protonated precursor ion toroduct ion at m/z 364 → 259 for SYL-1119, m/z 444 → 259 forYL-1119-P, and m/z 378 → 273 for SYL-1110. Automated datacquisition and data analysis were performed using Analyst 1.5.2oftware.

.4. Preparation of standard and quality control samples

SYL-1119, SYL-1119-P, and IS were dissolved in methanol at mg/ml as the stock solution. Working standards were preparedy the dilution of stock solution in methanol to obtain the desiredoncentrations of 1, 2.5, 10, 25, 50, 100, 200, 250 ng/ml for SYL-1119nd 50, 125, 250, 500, 1000, 2500, 4000, 5000 ng/ml for SYL-1119-. The IS spiking solution was prepared at 500 ng/ml by the dilutionf stock solution with methanol. All working solutions were storedt −20 ◦C until use.

Calibration standard samples were prepared by adding 20 �Lf IS working solution and working standards at different con-entrations into 100 �L of blank rat plasma, giving the finaloncentrations of 0.2, 0.5, 2, 5, 10, 20, 40, 50 ng/ml for SYL-1119nd 10, 25, 50, 100, 200, 500, 800, 1000 ng/ml for SYL-1119-P inlasma. The quality control (QC) samples (0.4, 4, 40 ng/ml for SYL-119 and 20, 150, 800 ng/ml for SYL-1119-P) were prepared in

manner similar to that used for preparation of the calibratoramples.

.5. Preparation of samples

A 20 �L of IS working solution (500 ng/ml) and 180 �L ace-onitrile were added to 100 �L of plasma sample. The mixtureas vortexed for 30 s followed by centrifugation at 14,000 rpm for

min. A 10 �L aliquot of each supernatant was injected into theC–MS/MS system for the analysis.

.6. Method validation

.6.1. Selectivity

Selectivity was evaluated using six independent blank rat

lasma. Chromatograms of these blank samples were comparedith the corresponding spiked plasma for the test of endogenous

nterferences.

– 946 (2014) 193– 198

2.6.2. Linearity, precision and accuracyThe linearity of each calibration curve was determined by plot-

ting the peak area ratio (analyte/internal standard) versus thenominal concentration of analytes with weighed (1/X2) least squarelinear regression. The range of calibration curve was 0.2–50 ng/mlfor SYL-1119 and 10–1000 ng/ml for SYL-1119-P. The calibrationcurves require the correlation coefficients of 0.99 or better.

The precision and accuracy of the assay were determined byanalysis of the QC samples. The intra-day precision and accuracywere evaluated by analysis of five replicates QC samples at low,medium, and high concentration levels within one day. The inter-day precision and accuracy were assessed by repeating the analysisof three concentration levels of QC samples on five consecutivedays. Precision was expressed as a percentage of relative standarddeviation (RSD%), while the accuracy was expressed as the rela-tive error (RE%). The accuracy was required to be within ±15% andthe intra- and inter-day precision values were not to exceed 15%,except for LLOQ, where it should not exceed ±20% of the accuracyas well as precision.

2.6.3. RecoveryThe recoveries of SYL-1119 and SYL-1119-P from plasma were

calculated by comparing the peak area of extracted three levels QCsamples to that of the analytes spiked to the blank sample extractsat the same concentration. The recoveries of the two compoundsin rat plasma were examined at least five times.

2.6.4. Matrix effectsThe matrix effect was defined as the ion suppres-

sion/enhancement on the ionization of analytes, which wasevaluated by comparing the responses of deproteinized samplesof blank plasma from six rats spiked QC samples (n = 5) with thoseof the standard samples at equivalent concentrations.

2.6.5. Stability studiesPost-extraction, three freeze-thaws, long-term and short-term

stabilities of analytes in plasma were tested using QC samples atthree concentration levels of 0.4, 4, 40 ng/ml for SYL-1119 and20, 150, 800 ng/ml for SYL-1119-P. The post-extraction stabilitywas done by comparing the concentration of deproteinized QCsamples after being kept at autosampler for 24 h with the nom-inal concentration. For short-term stability, QC plasma sampleswere kept at 4 ◦C for 24 h before sample preparation. Long-termstorage stability was acquired by assaying QC plasma samplesafter storage at −20 ◦C for 30 days. Freeze-thaw stability wasinvestigated by comparing the value of QC samples after threefreeze-thaw cycles with the nominal value (from −20 ◦C to roomtemperature).

2.7. Pharmacokinetic study

All animal protocols were approved by Institute Animal Care andWelfare Committee. Five male Sprague-Dawley rats (200 ± 20 g)were purchased from Beijing Vital River Experimental Animal Co.,Ltd. The rats were fasted overnight but had free access to water.SYL-1119 was dissolved in ultrapure water for the pharmacokineticstudies. The rats were orally administered with SYL-1119 at a doseof 1 mg/kg. Blood samples were taken from each rat at 0.5, 1, 2, 4, 6,8, 12, 24, 48, 96, 144, 192, 240, 288, and 336 h after dosing. The bloodsamples were immediately centrifuged at 5000 rpm for 10 min and

the plasma was frozen at −20 ◦C until analysis. The plasma sampleswere treated as described in Section 2.5. The plasma concentrationsof SYL-1119 and SYL-1119-P were expressed as mean ±SD. Datafitting and pharmacokinetic parameter estimates were carried out

S. Yang et al. / J. Chromatogr. B 945– 946 (2014) 193– 198 195

H]+ of

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Fig. 1. Product ion mass spectra of [M +

sing DAS 2.0 pharmacokinetic program (Chinese Pharmacologicalociety).

. 3 Results and discussion

.1. LC–MS/MS conditions

By monitoring the mass spectra of analytes in Q1 scan, muchigher signal intensity was found using the ESI source in the pos-

tive ionization mode with protonated molecular ion [M + H]+ at

(A) SYL-1119, (B) SYL-1119, and (C) IS.

m/z 364, 444, and 378 for SYL-1119, SYL-1119-P and IS. As shownin Fig. 1, m/z 311 and 259 were the two most abundant ions in theproduct ion spectrum of SYL-1119 and SYL-1119-P. For IS, the twoobvious product ions were at m/z 325 and 273. Finally, ion transi-tions at m/z 364 → 259 for SYL-1119, m/z 444 → 259 for SYL-1119-P,and m/z 378 → 273 for SYL-1110 were selected for much higher

signal response of chromatographic peak. The mass spectromet-ric parameters were optimized, including ion spray voltage, sourcetemperature, nebulizer gas, collision gas, and collision energy formore strong and stable signal.

196 S. Yang et al. / J. Chromatogr. B 945– 946 (2014) 193– 198

F lank r( 1119;

a(gf

ig. 2. Representative MRM chromatograms of SYL-1119, SYL-1119-P, and IS: (A) bC) rat plasma sample at 4 h postdose of SYL-1119 (1 mg/kg) spiked with IS. a: SYL-

Different mobile phase and category of columns were tested

nd compared. Methanol and Zorbax SB-C18 analytical column3.5 �m, 100mm × 2.1 mm) were selected for the better chromato-raphic separation without any interference peak. Addition of 0.1%ormic acid in methanol/water provided higher signal response

at plasma; (B) LLOQ sample (0.2 ng/ml for SYL-1119 and 10 ng/ml for SYL-1119-P); b: SYL-1110 (IS); c: SYL-1119-P.

and more stable retention time for two analytes and IS. The buffer

solution such as ammonium acetate was also investigated with noimprovement for S/N ratios. Compared with isocratic elution, bet-ter results in terms of peak shape, sensitivity, and retention timewere obtained using the present gradient elution.

S. Yang et al. / J. Chromatogr. B 945– 946 (2014) 193– 198 197

Table 1Intra- and Inter-day accuracies and precisions of SYL-1119 and SYL-1119-P in ratplasma (n = 5).

Sample Spiked Measured Accuracy Precision(ng/ml) (Mean ±SD) (RE%) (RSD%)

SYL-11190.4 0.38 ± 0.03 −5.0 7.9

Intra-day 4 3.96 ± 0.38 −1.0 9.640 42.4 ± 2.3 6.0 5.40.4 0.39 ± 0.05 −2.5 12.8

Inter-day 4 3.85 ± 0.35 −3.8 9.140 39.6 ± 4.6 −1.0 11.6

SYL-1119-P20 20.5 ± 2.4 2.5 11.7

Intra-day 150 168.8 ± 9.1 12.5 5.4800 814.8 ± 51.9 1.9 6.420 20.9 ± 1.8 4.5 8.6

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infinity (AUC ) was 874.6 and 20428.7 �g/L h for SYL-1119 and

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Inter-day 150 157.9 ± 11.7 5.3 7.4800 831 ± 74.8 3.9 9.0

.2. Method validation

.2.1. SelectivityAs shown in Fig. 2, there are no significant interference from rat

lasma observed at the retention times of two analytes and IS. Theentention time of SYL-1119, SYL-1119-P, and SYL-1110 were 8.0,.3, and 8.8 min, respectively.

.2.2. Linearity and rangeThe calibration curve was linear over the concentration range

f 0.2–50 ng/ml for SYL-1119 and 10–1000 ng/ml for SYL-1119-P.he regression equations obtained by least squared regres-ion were Y = 0.063 × X + 0.00531 (r2 = 0.9946) for SYL-1119 and

= 0.00133 × X + 0.00722 (r2 = 0.9930) for SYL-1119-P using weigh-ng factor (1/X2). The observed deviations were within ±15% for allalibration concentrations. The lower limit of quantification (LLOQ)as set to be 0.2 ng/ml for SYL-1119 and 10 ng/ml for SYL-1119-P,hich are sufficient for pharmacokinetic studies of two compounds

n rats.

.2.3. Precision and accuracyThe accuracies and intra- and inter-day precisions were

xpressed in Table 1. The accuracies (RE%) of low, medium, andigh QC levels of two analytes were within ±10%. The intra- and

nter-day precisions (RSD%) of analytes ranged from 5.4 to 12.8%or SYL-1119 and 5.4 to 11.7% for SYL-1119-P, which were within

he acceptable limits. The results indicated that the present methodas reliable and reproducible for the simultaneous quantitative

nalysis of SYL-1119 and SYL-1119-P in rat plasma samples.

able 2tability of SYL-1119 and SYL-1119-P in rat plasma (n = 5).

SYL-1119

Conditions Spiked Measured

(ng/ml) (Mean ±SD

Post-extraction (ambient temperature for 24 h) 0.4 0.43 ± 0.02

4 3.89 ± 0.18

40 39.6 ± 1.8

At 4 ◦C (24 h) 0.4 0.43 ± 0.03

4 4.31 ± 0.04

40 41.1 ± 1.3

Three freeze-thaw cycles 0.4 0.38 ± 0.04

4 3.52 ± 0.06

40 34.9 ± 0.5

Stored at −20 ◦C (1 month) 0.4 0.42 ± 0.05

4 3.71 ± 0.02

40 35.2 ± 0.7

Fig. 3. Mean plasma concentration-time profiles of SYL-1119 and SYL-1119-P in ratsafter a single oral dose of 1 mg/kg of SYL-1119. Data are expressed as mean ±SD.

3.2.4. Recovery and stabilityThe mean absolute recoveries were 91.3–102.2% for SYL-1119

and 98.4–104.5% for SYL-1119-P at three QC levels.SYL-1119 and SYL-1119-P in rat plasma were found to be stable

for 30 days at −20 ◦C, for 24 h at 4 ◦C, and for three freeze-thawcycles. Furthermore, the response of sample extracts was stableafter storage in an autosampler at ambient temperature for a 24 hperiod, which indicated that a large number of samples could bedetermined in each analytical run (Table 2).

3.2.5. Matrix effectThe mean matrix effect values obtained were 109.3, 107.2,

108.4% for SYL-1119 and 115.9, 91, 97.7% for SYL-1119-P at low,medium, and high QC levels, respectively. The results suggestedthat the matrix effect on the ionization of two analytes was negli-gible in this method.

3.2.6. Pharmacokinetic studyThe present method was successfully applied to the phar-

macokinetic studies of SYL-1119 and SYL-1119-P in rats afteroral administration of SYL-1119 at 1 mg/kg. The mean plasmaconcentration-time curve (n = 5) was illustrated in Fig. 3. The maxi-mum plasma concentration (Cmax) was 6.7 ng/ml for SYL-1119 and135 ng/ml for SYL-1119-P. The time of maximum plasma concen-tration (Tmax) was 8 h for SYL-1119 and 12 h for SYL-1119-P. Thearea under the plasma concentration-time curve from 0 h to the lastmeasurable concentration (AUC0–t) was 826.7 and 18976.6 �g/L h,the area under the plasma concentration-time curve from 0 h to

0–∞SYL-1119-P, respectively. The half-life of drug elimination at theterminal phase (t1/2) was 73.3 h for SYL-1119 and 78.1 h for SYL-1119-P.

SYL-1119-P

Accuracy Spiked Measured Accuracy) (RE%) (ng/ml) (Mean ±SD) (RE%)

7.5 20 22.2 ± 0.9 11−2.8 150 167.6 ± 4.8 11.7−1.0 800 864.8 ± 9.8 8.17.5 20 21.5 ± 1.7 7.57.8 150 154.6 ± 17.2 3.12.8 800 764.8 ± 49.8 −4.4−5.0 20 18.4 ± 0.8 −8.0−12.0 150 163.2 ± 5.2 8.8−12.8 800 880 ± 12.8 10.05.0 20 20.3 ± 2.4 1.5−7.3 150 170.2 ± 7.4 13.5−12 800 828.2 ± 26.1 3.5

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. Conclusions

A sensitive and simple LC–MS/MS method for simultaneouseasurement of SYL-1119 and SYL-1119-P in rat plasma has

een developed and validated. The method was found to beccurate, precise and specific, and was successfully applied tohe pharmacokinetic investigations of SYL-1119 in rats after oraldministration.

cknowledgments

This work was supported by the National Science andechnology Major Project of China (2012ZX09301002-001-007,

011ZX09102-001-01, 2012ZX09301002-006, 2012ZX09103-101-01) and National Natural Science Foundation of China (NSFC no.1102322). The authors would like to acknowledge Ms. Sheng, Ms.hen and Mr. Wang for their technical assistance.

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– 946 (2014) 193– 198

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