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Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 99–103

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j o ur na l ho mepage: www.elsev ier .com/ locate / jpba

etermination of a novel dipeptidyl peptidase IV inhibitor in monkeylasma by HPLC–MS/MS and its application in a pharmacokineticstudy

ifeng Deng a, Jiayin Guo b, Renke Dai a, Guicheng Zhang c, Hui Xie d,∗

School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510640, PR ChinaZhongshan PharmaSS Corporation, Zhongshan 528437, PR ChinaGuangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, PR ChinaThe First Affiliated Hospital of Guangzhou Medical University, 151 Yanjiang Road, Guangzhou 510120, PR China

r t i c l e i n f o

rticle history:eceived 24 June 2015eceived in revised form 20 August 2015ccepted 21 August 2015vailable online 29 August 2015

a b s t r a c t

A lot of attention has been given to novel diabetes treatment, which is used to replace injectable insulin.A novel dipeptidyl peptidase IV inhibitor (HWH-10d) for treating type 2 diabetes was previously deter-mined to have comparable efficacy to the marketed drug (alogliptin) in ICR male mice. Therefore, asensitive and rapid liquid chromatography-tandem mass spectrometric method was developed andvalidated for the further evaluation of HWH-10d in monkey. The analytes were extracted through a

eywords:ype 2 diabetesC–MS/MSPP-IV inhibitorharmacokineticsWH-10d

liquid–liquid extraction with ethyl acetate. The linear detection range for HWH-10d in monkey plasmawas from 0.5 to 2000 ng/mL with the lower limit of quantification of 0.5 ng/mL. The relative standarddeviation was measured to be less than 10.4% for determination of inter- and intra-day precisions, andthe relative error was determined to be within ±7.2% for all accuracy measurements. The simple and rapidLC–MS/MS method for HWH-10d in monkey plasma could be used for the pharmacokinetics studies ofHWH-10d in monkeys. The oral bioavailability of HWH-10d in monkeys is 57.8%.

. Introduction

The diabetes has become a serious life threat to human soci-ty, causing near to 4 million deaths per year, similar to the deathy HIV/AIDS [1]. Population living with diabetes is rising and dia-etes becomes the epidemic of the 21st century [1]. It has beennown that the type 2 diabetes accounts for 90–95% of diabetes2]. Treatment of type 2 diabetes (non-insulin-dependent) is nowossible with orally administered hypoglycemic agents that helpo decrease the blood glucose levels. The synthetic hypoglycemicgents have some side effects in clinical practices [3]. It is an immi-ent need for new drug discovery and development for diabetes.

It is well known that glucagon-like peptide 1 (GLP-1) plays anmportant role in reducing glucose concentrations for treatment of

ype 2 diabetes [4]. Discontinuation of GLP-1 infusion in type 2 dia-etics leads to a rapid reversion to hyperglycemia because GLP-1ctivity is quickly quenched by the action of dipeptidyl peptidaseV (DPP-IV) [5,6]. It may due to DPP-IV-mediated formation of an

∗ Corresponding author.E-mail address: xie hui126@163.com (H. Xie).

ttp://dx.doi.org/10.1016/j.jpba.2015.08.033731-7085/© 2015 Elsevier B.V. All rights reserved.

© 2015 Elsevier B.V. All rights reserved.

inactive GLP-1 amide through cleavage of the N-terminal dipeptideof GLP-1 [5,6]. Therefore, inhibition of DPP-IV becomes a new thera-peutic approach for type 2 diabetes [7–13]. The selectivity betweenDDP-IV and other dipeptidyl peptidases, such as DDP-VIII and DDP-IX, is also a desirable factor for screening DPP-IV inhibitors in newdrug discovery and development since inhibition of DPP-VIII andDPP-IX may cause toxicity [14,15].

Sitagliptin from Merck, the DPP-IV inhibitor for treatment oftype 2 diabetes was first proved by FDA to market in 2006 [14].Since then, actually, several drug candidates functioned as theDPP-IV inhibitors have been progressing in the clinical trials, suchas Vildagliptin (Novartis) [16], Saxagliptin (Bristol-Myers Squibb)[17], and Alogliptin (Takeda) [18]. The mechanism of inhibitionof DPP-IV activity has been focused for new drug discovery anddevelopment.

(R)-1-(3-(2-cyanobenzyl)-4-oxo-3,4-dihydrothieno[3,2-d]pyrimidin-2-yl) piperidin-3-aminium benzoate, namedHWH-10d, was found to be a potent DPP-IV inhibitor. HWH-

10d displayed much higher in vitro inhibition potential towardDPP-IV than alogliptin [19]. In the past few years, the drugmetabolism and drug–drug interaction of HWH-10d were studiedin liver microsome system (data not shown). In this study, in

100 J. Deng et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 99–103

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distilled water: ethanol (95:5, v/v) as stock solution (1 mg/mL). The

Fig. 1. The structure of HWH-10d.

rder to further characterize its pharmacokinetic property, aC-MS/MS method for HWH-10d was developed and validated.he pharmacokinetic study of HWH-10d in monkeys is conductednd the results are also discussed.

. Experimental

.1. Materials and reagents

HWH-10d (purity, >99.0%) were synthesized and purifiedt Guangzhou Institutes of Biomedicine and Health, Chinesecademy of Sciences. The structure of HWH-10d is shown in theig. 1. Alogliptin used as internal standard was purchased fromigma–Aldrich (St. Louis, MO, USA). HPLC grade ethyl acetate,ethanol and formic acid were purchased from Dikma Technology

nc., Lake Forest, CA, USA. Capcell Pak C18 column was purchasedrom Shiseido China Co., Ltd., Beijing, China. Water was glass-ouble distilled and filtered with 0.2 �m membranes.

.2. Instrumentation and analytical conditions

The LC–ESI–MS/MS system used was composed of a NanospacePLC system (Shiseido, Tokyo, Japan) coupled to a Q TrapTM 4000ybrid triple quadruple linear ion trap mass spectrometer (Appliediosystems/MDS Sciex, Concord, Ontario, Canada). Data process-

ng was performed with AnalystTM 1.5 software package (Appliediosystems, MA, USA).

Chromatographic separation was performed on a Capcell Pak18 column (5 �m, 2.0 mm ID × 50 mm, Tokyo, Japan) at room tem-erature with a flow rate of 0.2 mL/min. The mobile phase A wasomposed of methanol and water at ratio of 5/95 (v/v) and B,ethanol and water, 95/5 (v/v). Both contained 0.1% formic acid. In

he LC gradient profile, the mobile phase B was 5% (v/v) for 0.3 minnd linearly increased to 100% from 0.3 to 1.0 min, held 100% from.0 to 3.0 min and returned to 5% from 3.0 to 3.2 min, then held 5%rom 3.2 to 5 min. The total run time was 5 min.

HWH-10d and IS were all detected by monitoring the pre-ursor → product ion transition at unit resolution using multipleeaction monitoring (MRM) scan mode. Both analyte and its ownS responded best to the positive ionization mode, with the proto-ated molecular ions [M + H]+ as the major species. The monitoring

on was set as m/z 366.2/349.2 for HWH-10d and m/z 340.1/232.2or IS. HWH-10d and its product ions mass spectra of [M + H]+ werehown in Fig. 2. Mass spectrometric conditions were optimized tobtain maximum sensitivity using the following ESI conditions:

eclustering potential, 85 eV; ionspray voltage, 5500 V; entranceoltage, 7 V; source temperature, 500 ◦C; curtain gas, 25 psi; nebu-izing gas, 80 psi; turbo ion spray gas, 70 psi; collision energy, 27 eV;well time, 200 ms.

Fig. 2. HWH-10d and its product ions mass spectra of [M + H]+.

2.3. Stock standards, standard(s) and QC samples

The analytical standard stock solution and QC stock solutionwere prepared separately by dissolving the accurately weighedreference compound in methanol to give a final concentration of1 mg/mL. These two stock solutions were used for calibration stan-dards and QC standards, respectively. The solutions were thenserially diluted with methanol/water (50:50, v/v) to obtain thedesired concentrations. A 1 mg/ml stock solution of internal stan-dard alogliptin was also prepared in methanol. This was dilutedwith methanol/water (50:50, v/v) to obtain a 1.5 �g/mL workingsolution. The analytical standard and QC samples were preparedby spiking blank heparinized monkey plasma (100 �L) with stan-dard working solutions (20 �L) during validation and during eachexperiment for the pharmacokinetic study. The lower limit of quan-tification of the samples was prepared at the concentration of0.5 ng/mL. Calibration samples were made at concentrations of 0.5,2.0, 10.0, 50, 200, 1000 and 2000 ng/mL. QC samples were preparedat the concentrations of 1.0, 50 and 1600 ng/mL. The analyticalstandards and QC samples were stored at −20 ◦C.

2.4. Sample preparation

100 �L of plasma sample, 20 �L aliquot of the IS solution(2 �g/mL) and 20 �L of methanol/water (50:50, v/v) were mixed.The mixture was then extracted with 800 �L of ethyl acetate byshaking for 15 min. After centrifugation at 14000× g for 5 min,the upper organic layer was separated and evaporated to drynessat 45 ◦C under a stream of nitrogen in the TurboVap evaporator(Zymark, Hopkinton, MA, USA). The residue was reconstituted in100 �L of the mobile phase, and then vortex-mixed. A 20 �L aliquotof the resulting solution was injected into the LC–MS/MS system foranalysis.

2.5. Application to pharmacokinetics study

Cynomologus monkeys (7.0–7.9 kg) were provided by Guang-dong Landau Biotechnology Co., Ltd. They were fasted overnightand allowed free access to water before administration. All proce-dures involving animals were in accordance with the Regulations ofExperiment Animal Administration issued by the State Committeeof Science and Technology of China. HWH-10d was dissolved with

stock solution was orally and intravenous administrated to six malecynomologus monkeys and six female cynomologus monkeys at asingle dose of 2 mg/kg. Blood was collected from the suborbital veinbefore administration and at 0.083 (0.033 for i.v.), 0.25 (0.17 for i.v.),

J. Deng et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 99–103 101

F entions spike

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ig. 3. Typical MRM chromatograms of HWH-10d and IS in monkey plasma. The retample of HWH-10d; (a-2) Blank plasma sample of HWH-10d; (b-1) plasma sample

.5, 1, 2, 4, 6, 8, 12, 16 and 24 h after dosing. About 500 �L bloodamples were collected into heparinized tubes and then immedi-tely centrifuged at 4000× g for 10 min. The plasma obtained wastored at −20 ◦C until analysis.

.6. Method validation

To ensure the selectivity, sensitivity, linearity, accuracy, preci-ion, recovery, matrix effect and stability, the method was validatedccording to the Food and Drug Administration Guidance on Bio-nalytical Medical Method Validation in May 2001 [20] as follows:electivity was performed by analyzing the blank plasma from sixifferent sources to detect interference at the retention times ofhe analyte and internal standard. The linearity of the assay methodas determined by plotting the peak area ratios of HWH-10d and IS

gainst the concentrations of HWH-10d in plasma in duplicate onhree consecutive days. Inter- and intra-day accuracy and precisionor the assay were determined by the performance of three levelsf QCs run on three validation days, and on each day six replicatesere analyzed together with an independently prepared calibra-

ion curve. Recovery of HWH-10d was evaluated by comparing theean peak areas of the regularly prepared QC samples (n = 6) at

.0, 50 and 1600 ng/mL with the mean peak areas of spiked-afterxtraction samples which represented the 100% recovery value. Thetabilities of HWH-10d in monkey plasma were evaluated by ana-yzing replicates (n = 3) of plasma samples at the concentrationsf 1.0, 50 and 1600 ng/mL, which were exposed to different con-itions (time and temperature). The spiked plasma samples werenalyzed after storage at room temperature for 6 h, at −20 ◦C for

ne month and after three freeze–thaw cycles from −20 ◦C to roomemperature. The autosampler stability of HWH-10d in monkeylasma was evaluated by re-injecting the previously injected qual-

ty control samples after a period of storage in the autosampler.he matrix effect was evaluated at three concentrations (1.0, 50

times were approximately 3.32 min and 2.95 min, respectively. (a-1) Blank plasmad with 0.5 ng/mL of HWH-10d; (b-2) plasma spiked with 300 ng/mL of IS.

and 1600 ng/mL in plasma). Matrix effects were evaluated by com-paring the peak area of known concentration of working standards(A) with that of the same analyte concentration spiked with blankplasma extract after extraction (B). The ratio (B/A × 100%) is definedas absolute matrix effects.

3. Results and discussion

3.1. Method development

HWH-10d and Alogliptin (IS) all displayed higher sensitivity inthe positive ion mode than in the negative ion mode with the proto-nated molecular ion [M + H]+ under the ESI conditions. The mobilephase played a critical role in achieving good chromatographicbehavior and appropriate ionization. HWH-10d and Alogliptin werefound to have their highest response in the mobile phase with 0.1%formic acid.

In this study, Liquid–Liquid extraction (LLE) was used for theisolation of HWH-10d and IS from monkey plasma samples. Ethylacetate gave the highest recovery among the solvents such as n-butyl alcohol, ether, ethyl acetate and tert-butyl methyl ether.Therefore, ethyl acetate proved to be a simple, efficient solvent forextracting HWH-10d and IS.

3.2. Method validation

3.2.1. SelectivityAlogliptin was used as the IS, which is similar to HWH-10d

in structure. It has no interference in plasma samples. The typi-

cal chromatograms of blank monkey plasma and a spiked plasmasample with 0.5 ng/mL of HWH-10d and 300 ng/mL of IS are shownin Fig. 3. The retention times for HWH-10d and IS were 3.32 and2.95 min, respectively. No significant interference from endoge-nous was observed.

102 J. Deng et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 99–103

Table 1Precision and accuracy of HWH-10d in monkey plasma (n = 6).

Concentration added (ng/mL) Within-batch Between-batch

Analyte Concentrationfound (ng/mL)(mean ± SD)

PrecisionRSD(%)

Accuracy(%)

Concentrationfound (ng/mL)(mean ± SD)

PrecisionRSD(%)

Accuracy (%)

HWH-10d 0.5 0.0492 ± 0.0019 3.9 98.4 – – –1.0 0.928 ± 0.049 5.3 92.8 1.06 ± 0.11 10.4 10650 51.9 ± 2.6 5.0 104 52.5 ± 3.3 6.2 1051600 1708 ± 82.8 4.8 107 1654 ± 121 7.3 103

Table 2Extraction recoveries of HWH-10d in monkey plasma (n = 3).

Analyte Concentration added (ng/mL) Recoveries (mean ± SD)(%) PrecisionRSD(%)

HWH-10d 1.0 85.7 ± 8.4 9.850 92.0 ± 6.1 6.61600 94.7 ± 3.4 3.6

Table 3The stability of HWH-10d under storage conditions.

Analyte Concentration added (ng/mL) mean ± SD RSD (%) Accuracy(%)

HWH-10d Room temperaturestability

1.0 0.954 ± 0.021 2.2 95.450 49.7 ± 3.7 7.4 99.41600 1620 ± 125 7.7 101

Freeze and thawstability

1.0 1.02 ± 0.03 2.9 10250 52.4 ± 1.2 2.3 1051600 1470 ± 80.9 5.5 91.9

Autosampler stability 1.0 0.949 ± 0.043 4.5 94.950 48.6 ± 3.9 8.0 97.21600 1529 ± 50.3 3.3 95.6

One month stability 1.0 0.931 ± 0.032 3.4 93.150 47.9 ± 2.8 5.8 95.81600 1543 ± 97.1 6.3 96.4

Table 4The pharmacokinetic parameters of HWH-10d by oral and intravenous administration in monkeys.

Paramters AUC(0−t) MRT(0−t) VRT(0−t) t1/2z Tmax CLz Vz Cmax

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p.o 1164 ± 157.1 4.68 ± 0.47 23.9 ± 4.11 5.4i.v. 2014 ± 289.4 3.99 ± 0.48 25.5 ± 4.96 5.1

.2.2. Linearity of calibration curves & lower limits ofuantification

Calibration standards containing HWH-10d over the concen-ration range of 0.5–2000 ng/mL were prepared using workingolutions of HWH-10d in duplicate. A typical regression equationas obtained as y = 7.98 × 10−3x + 2.63 × 10−3 using a weighting

actor of 1/x2, where y represents the peak area ratio of HWH-10d tohat of IS and x represents the plasma concentration of HWH-10d.he correlation coefficient (r2) was obtained to be 0.9959 for thequation. LLOQ was determined to be 0.5 ng/mL. All plasma sam-les from the pharmacokinetic studies can be determined withinhis linear concentration range.

.2.3. Precision & accuracy, extraction recoveryThe intra-day precision was determined to be 7.0% or less, and

he inter-day precision was 10.4% or less for each QC level of HWH-0d. The accuracy was measured to be between 92.8% and 107%or all assays. The results are shown in Table 1. Referring to FDA

uideline [20], the present method has good accuracy, precision,nd reproducibility. The recovery of HWH-10d obtained from thepiked plasma samples were also shown in Table 2. The RSD wasess than 9.8% for all recoveries throughout the entire standardoncentration ranges, showing good consistency.

h L/h/kg L/kg �g/L7 1.00 ± 0.0 1.69 ± 0.26 12.9 ± 2.61 333.1 ± 55.55 0.033 ± 0.0 0.99 ± 0.14 7.24 ± 1.39 1164 ± 216.7

3.2.4. StabilityAs shown in Table 3, the deviation was measured to be within

8.1% of nominal concentrations at each QC concentrations of HWH-10d for stability determination. These results demonstrated thatHWH-10d was stable in monkey plasma at room temperature forat least 6 h and at −20 ◦C for one month, respectively. It is also sta-ble after three freeze-thaw cycles. Stability was also assessed usingthe processed plasma samples in the autosampler at approximately4 ◦C for 24 h.

3.2.5. Matrix effectThe absolute matrix effects for HWH-10d at concentrations of

0.5, 50 and 1600 ng/mL were 91.7%, 98.3 and 96.5%, respectively.The absolute matrix effects for IS (300 ng/mL in plasma) were 96.2%.These results showed that ion suppression or enhancement fromthe plasma matrix was negligible under the current conditions.

3.2.6. Pharmacokinetic study in monkeysThe pharmacokinetic property of HWH-10d was studied in

monkeys following i.v. (2 mg/kg) and oral (2 mg/kg) adminis-trations. Non-compartmental mode was used to calculate thepharmacokinetics parameters with DAS 2.0 software (Pharmacoki-netics Institute of China). The mean plasma concentration-timeprofiles of HWH-10d by the two routes of administration are

J. Deng et al. / Journal of Pharmaceutical and B

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ig. 4. The mean plasma concentration-time profiles of HWH-10d after an intra-enous dose of 2 mg/kg HWH-10d (n = 6) and an oral dose of 2 mg/kg HWH-10d foronkeys (n = 6).

hown in Fig. 4. The pharmacokinetic parameters of HWH-0d are shown in Table 4. Its estimated main pharmacokineticarameters are presented as follows: the elimination t1/2 was esti-ated as 5.12 ± 0.95 h for the intravenous dose of 2 mg/kg and

.42 ± 1.57 h for the oral dose of 2 mg/kg. The AUC0−t of intra-enous administration was found to be 2014 ± 289.4 �g/(L × h).n contrast, The AUC0−t of oral administration was found to be164 ± 157.1 �g/(L × h).

. Conclusion

A specific, sensitive, accurate and rapid LC–MS/MS method foretermination of HWH-10d in monkey plasma was first developednd validated, which had been successfully applied in deter-ination of pharmacokinetic property for HWH-10d. The oral

ioavailability of HWH-10d is determined to be 57.8% in mon-eys. The pharmacokinetic parameters indicated that HWH-10displayed good absorption and slow elimination and probably lowissue distributions.

cknowledgments

This work was supported in part by the National High-tech&D Program [Grant 2006AA02Z339] and [Grant 2008AA02Z314];he Guangzhou Science and Technology Bureau [Grant 2006Z1-4031, 2006P067]; and the Guangzhou Development DistrictGrant 2006Ss-P067].

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.jpba.2015.08.033.

[

iomedical Analysis 117 (2016) 99–103 103

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