Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 99103

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Determination of a novel dipeptidyl peptidase IV inhibitor in monkey

plasma by HPLCMS/MS and its application in a pharmacokinetics
study
Jifeng Deng a , Jiayin Guo b , Renke Dai a , Guicheng Zhang c , Hui Xie d,
a

School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510640, PR China
Zhongshan PharmaSS Corporation, Zhongshan 528437, PR China
c
Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, PR China
d
The First Afliated Hospital of Guangzhou Medical University, 151 Yanjiang Road, Guangzhou 510120, PR China
b

a r t i c l e

i n f o

Article history:
Received 24 June 2015
Received in revised form 20 August 2015
Accepted 21 August 2015
Available online 29 August 2015
Keywords:
Type 2 diabetes
LCMS/MS
DPP-IV inhibitor
Pharmacokinetics
HWH-10d

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 determined to have comparable efcacy to the marketed drug (alogliptin) in ICR male mice. Therefore, a
sensitive and rapid liquid chromatography-tandem mass spectrometric method was developed and
validated for the further evaluation of HWH-10d in monkey. The analytes were extracted through a
liquidliquid extraction with ethyl acetate. The linear detection range for HWH-10d in monkey plasma
was from 0.5 to 2000 ng/mL with the lower limit of quantication of 0.5 ng/mL. The relative standard
deviation was measured to be less than 10.4% for determination of inter- and intra-day precisions, and
the relative error was determined to be within 7.2% for all accuracy measurements. The simple and rapid
LCMS/MS method for HWH-10d in monkey plasma could be used for the pharmacokinetics studies of
HWH-10d in monkeys. The oral bioavailability of HWH-10d in monkeys is 57.8%.
2015 Elsevier B.V. All rights reserved.

1. Introduction
The diabetes has become a serious life threat to human society, causing near to 4 million deaths per year, similar to the death
by HIV/AIDS [1]. Population living with diabetes is rising and diabetes becomes the epidemic of the 21st century [1]. It has been
known that the type 2 diabetes accounts for 9095% of diabetes
[2]. Treatment of type 2 diabetes (non-insulin-dependent) is now
possible with orally administered hypoglycemic agents that help
to decrease the blood glucose levels. The synthetic hypoglycemic
agents have some side effects in clinical practices [3]. It is an imminent need for new drug discovery and development for diabetes.
It is well known that glucagon-like peptide 1 (GLP-1) plays an
important role in reducing glucose concentrations for treatment of
type 2 diabetes [4]. Discontinuation of GLP-1 infusion in type 2 diabetics leads to a rapid reversion to hyperglycemia because GLP-1
activity is quickly quenched by the action of dipeptidyl peptidase
IV (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).
http://dx.doi.org/10.1016/j.jpba.2015.08.033
0731-7085/ 2015 Elsevier B.V. All rights reserved.

inactive GLP-1 amide through cleavage of the N-terminal dipeptide

of GLP-1 [5,6]. Therefore, inhibition of DPP-IV becomes a new therapeutic approach for type 2 diabetes [713]. The selectivity between
DDP-IV and other dipeptidyl peptidases, such as DDP-VIII and DDPIX, is also a desirable factor for screening DPP-IV inhibitors in new
drug discovery and development since inhibition of DPP-VIII and
DPP-IX may cause toxicity [14,15].
Sitagliptin from Merck, the DPP-IV inhibitor for treatment of
type 2 diabetes was rst proved by FDA to market in 2006 [14].
Since then, actually, several drug candidates functioned as the
DPP-IV inhibitors have been progressing in the clinical trials, such
as Vildagliptin (Novartis) [16], Saxagliptin (Bristol-Myers Squibb)
[17], and Alogliptin (Takeda) [18]. The mechanism of inhibition
of DPP-IV activity has been focused for new drug discovery and
development.
(R)-1-(3-(2-cyanobenzyl)-4-oxo-3,4-dihydrothieno[3,2d]pyrimidin-2-yl)
piperidin-3-aminium
benzoate,
named
HWH-10d, was found to be a potent DPP-IV inhibitor. HWH10d displayed much higher in vitro inhibition potential toward
DPP-IV than alogliptin [19]. In the past few years, the drug
metabolism and drugdrug interaction of HWH-10d were studied
in liver microsome system (data not shown). In this study, in

100

J. Deng et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 99103

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

order to further characterize its pharmacokinetic property, a

LC-MS/MS method for HWH-10d was developed and validated.
The pharmacokinetic study of HWH-10d in monkeys is conducted
and the results are also discussed.

2. Experimental
2.1. Materials and reagents
HWH-10d (purity, >99.0%) were synthesized and puried
at Guangzhou Institutes of Biomedicine and Health, Chinese
Academy of Sciences. The structure of HWH-10d is shown in the
Fig. 1. Alogliptin used as internal standard was purchased from
SigmaAldrich (St. Louis, MO, USA). HPLC grade ethyl acetate,
methanol and formic acid were purchased from Dikma Technology
Inc., Lake Forest, CA, USA. Capcell Pak C18 column was purchased
from Shiseido China Co., Ltd., Beijing, China. Water was glassdouble distilled and ltered with 0.2 m membranes.
2.2. Instrumentation and analytical conditions
The LCESIMS/MS system used was composed of a Nanospace
HPLC system (Shiseido, Tokyo, Japan) coupled to a Q TrapTM 4000
hybrid triple quadruple linear ion trap mass spectrometer (Applied
Biosystems/MDS Sciex, Concord, Ontario, Canada). Data processing was performed with AnalystTM 1.5 software package (Applied
Biosystems, MA, USA).
Chromatographic separation was performed on a Capcell Pak
C18 column (5 m, 2.0 mm ID 50 mm, Tokyo, Japan) at room temperature with a ow rate of 0.2 mL/min. The mobile phase A was
composed of methanol and water at ratio of 5/95 (v/v) and B,
methanol and water, 95/5 (v/v). Both contained 0.1% formic acid. In
the LC gradient prole, the mobile phase B was 5% (v/v) for 0.3 min
and linearly increased to 100% from 0.3 to 1.0 min, held 100% from
1.0 to 3.0 min and returned to 5% from 3.0 to 3.2 min, then held 5%
from 3.2 to 5 min. The total run time was 5 min.
HWH-10d and IS were all detected by monitoring the precursor product ion transition at unit resolution using multiple
reaction monitoring (MRM) scan mode. Both analyte and its own
IS responded best to the positive ionization mode, with the protonated molecular ions [M + H]+ as the major species. The monitoring
ion was set as m/z 366.2/349.2 for HWH-10d and m/z 340.1/232.2
for IS. HWH-10d and its product ions mass spectra of [M + H]+ were
shown in Fig. 2. Mass spectrometric conditions were optimized to
obtain maximum sensitivity using the following ESI conditions:
declustering potential, 85 eV; ionspray voltage, 5500 V; entrance
voltage, 7 V; source temperature, 500 C; curtain gas, 25 psi; nebulizing gas, 80 psi; turbo ion spray gas, 70 psi; collision energy, 27 eV;
dwell 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 solution
were prepared separately by dissolving the accurately weighed
reference compound in methanol to give a nal concentration of
1 mg/mL. These two stock solutions were used for calibration standards and QC standards, respectively. The solutions were then
serially diluted with methanol/water (50:50, v/v) to obtain the
desired concentrations. A 1 mg/ml stock solution of internal standard alogliptin was also prepared in methanol. This was diluted
with methanol/water (50:50, v/v) to obtain a 1.5 g/mL working
solution. The analytical standard and QC samples were prepared
by spiking blank heparinized monkey plasma (100 L) with standard working solutions (20 L) during validation and during each
experiment for the pharmacokinetic study. The lower limit of quantication of the samples was prepared at the concentration of
0.5 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 prepared
at the concentrations of 1.0, 50 and 1600 ng/mL. The analytical
standards 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 by
shaking for 15 min. After centrifugation at 14000 g for 5 min,
the upper organic layer was separated and evaporated to dryness
at 45 C under a stream of nitrogen in the TurboVap evaporator
(Zymark, Hopkinton, MA, USA). The residue was reconstituted in
100 L of the mobile phase, and then vortex-mixed. A 20 L aliquot
of the resulting solution was injected into the LCMS/MS system for
analysis.
2.5. Application to pharmacokinetics study
Cynomologus monkeys (7.07.9 kg) were provided by Guangdong Landau Biotechnology Co., Ltd. They were fasted overnight
and allowed free access to water before administration. All procedures involving animals were in accordance with the Regulations of
Experiment Animal Administration issued by the State Committee
of Science and Technology of China. HWH-10d was dissolved with
distilled water: ethanol (95:5, v/v) as stock solution (1 mg/mL). The
stock solution was orally and intravenous administrated to six male
cynomologus monkeys and six female cynomologus monkeys at a
single dose of 2 mg/kg. Blood was collected from the suborbital vein
before 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) 99103

101

Fig. 3. Typical MRM chromatograms of HWH-10d and IS in monkey plasma. The retention times were approximately 3.32 min and 2.95 min, respectively. (a-1) Blank plasma
sample of HWH-10d; (a-2) Blank plasma sample of HWH-10d; (b-1) plasma sample spiked with 0.5 ng/mL of HWH-10d; (b-2) plasma spiked with 300 ng/mL of IS.

0.5, 1, 2, 4, 6, 8, 12, 16 and 24 h after dosing. About 500 L blood

samples were collected into heparinized tubes and then immediately centrifuged at 4000 g for 10 min. The plasma obtained was
stored at 20 C until analysis.

2.6. Method validation

To ensure the selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effect and stability, the method was validated
according to the Food and Drug Administration Guidance on Bioanalytical Medical Method Validation in May 2001 [20] as follows:
Selectivity was performed by analyzing the blank plasma from six
different sources to detect interference at the retention times of
the analyte and internal standard. The linearity of the assay method
was determined by plotting the peak area ratios of HWH-10d and IS
against the concentrations of HWH-10d in plasma in duplicate on
three consecutive days. Inter- and intra-day accuracy and precision
for the assay were determined by the performance of three levels
of QCs run on three validation days, and on each day six replicates
were analyzed together with an independently prepared calibration curve. Recovery of HWH-10d was evaluated by comparing the
mean peak areas of the regularly prepared QC samples (n = 6) at
1.0, 50 and 1600 ng/mL with the mean peak areas of spiked-after
extraction samples which represented the 100% recovery value. The
stabilities of HWH-10d in monkey plasma were evaluated by analyzing replicates (n = 3) of plasma samples at the concentrations
of 1.0, 50 and 1600 ng/mL, which were exposed to different conditions (time and temperature). The spiked plasma samples were
analyzed after storage at room temperature for 6 h, at 20 C for
one month and after three freezethaw cycles from 20 C to room
temperature. The autosampler stability of HWH-10d in monkey
plasma was evaluated by re-injecting the previously injected quality control samples after a period of storage in the autosampler.
The matrix effect was evaluated at three concentrations (1.0, 50

and 1600 ng/mL in plasma). Matrix effects were evaluated by comparing the peak area of known concentration of working standards
(A) with that of the same analyte concentration spiked with blank
plasma extract after extraction (B). The ratio (B/A 100%) is dened
as absolute matrix effects.
3. Results and discussion
3.1. Method development
HWH-10d and Alogliptin (IS) all displayed higher sensitivity in
the positive ion mode than in the negative ion mode with the protonated molecular ion [M + H]+ under the ESI conditions. The mobile
phase played a critical role in achieving good chromatographic
behavior and appropriate ionization. HWH-10d and Alogliptin were
found to have their highest response in the mobile phase with 0.1%
formic acid.
In this study, LiquidLiquid extraction (LLE) was used for the
isolation of HWH-10d and IS from monkey plasma samples. Ethyl
acetate gave the highest recovery among the solvents such as nbutyl alcohol, ether, ethyl acetate and tert-butyl methyl ether.
Therefore, ethyl acetate proved to be a simple, efcient solvent for
extracting HWH-10d and IS.
3.2. Method validation
3.2.1. Selectivity
Alogliptin was used as the IS, which is similar to HWH-10d
in structure. It has no interference in plasma samples. The typical chromatograms of blank monkey plasma and a spiked plasma
sample with 0.5 ng/mL of HWH-10d and 300 ng/mL of IS are shown
in Fig. 3. The retention times for HWH-10d and IS were 3.32 and
2.95 min, respectively. No signicant interference from endogenous was observed.

102

J. Deng et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 99103

Table 1
Precision and accuracy of HWH-10d in monkey plasma (n = 6).
Concentration added (ng/mL) Within-batch
Analyte

HWH-10d

0.5
1.0
50
1600

Between-batch

Concentration
found (ng/mL)
(mean SD)

Precision
RSD
(%)

Accuracy
(%)

Concentration
found (ng/mL)
(mean SD)

Precision
RSD
(%)

Accuracy (%)

0.0492 0.0019
0.928 0.049
51.9 2.6
1708 82.8

3.9
5.3
5.0
4.8

98.4
92.8
104
107

1.06 0.11
52.5 3.3
1654 121

10.4
6.2
7.3

106
105
103

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

Concentration added (ng/mL)

Recoveries (mean SD)(%)

PrecisionRSD(%)

HWH-10d

1.0
50
1600

85.7 8.4
92.0 6.1
94.7 3.4

9.8
6.6
3.6

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

HWH-10d

Freeze and thaw

stability
Autosampler stability

One month stability

Concentration added (ng/mL)

mean SD

RSD (%)

Accuracy(%)

1.0
50
1600
1.0
50
1600
1.0
50
1600
1.0
50
1600

0.954 0.021
49.7 3.7
1620 125
1.02 0.03
52.4 1.2
1470 80.9
0.949 0.043
48.6 3.9
1529 50.3
0.931 0.032
47.9 2.8
1543 97.1

2.2
7.4
7.7
2.9
2.3
5.5
4.5
8.0
3.3
3.4
5.8
6.3

95.4
99.4
101
102
105
91.9
94.9
97.2
95.6
93.1
95.8
96.4

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

AUC(0t)

Units
p.o
i.v.

g/L h
1164 157.1
2014 289.4

MRT(0t)
h
4.68 0.47
3.99 0.48

VRT(0t)
2

h
23.9 4.11
25.5 4.96

t1/2z

Tmax

CLz

Vz

Cmax

h
5.42 1.57
5.12 0.95

h
1.00 0.0
0.033 0.0

L/h/kg
1.69 0.26
0.99 0.14

L/kg
12.9 2.61
7.24 1.39

g/L
333.1 55.5
1164 216.7

3.2.2. Linearity of calibration curves & lower limits of

quantication
Calibration standards containing HWH-10d over the concentration range of 0.52000 ng/mL were prepared using working
solutions of HWH-10d in duplicate. A typical regression equation
was obtained as y = 7.98 103 x + 2.63 103 using a weighting
factor of 1/x2 , where y represents the peak area ratio of HWH-10d to
that of IS and x represents the plasma concentration of HWH-10d.
The correlation coefcient (r2 ) was obtained to be 0.9959 for the
equation. LLOQ was determined to be 0.5 ng/mL. All plasma samples from the pharmacokinetic studies can be determined within
this linear concentration range.

3.2.3. Precision & accuracy, extraction recovery

The intra-day precision was determined to be 7.0% or less, and
the inter-day precision was 10.4% or less for each QC level of HWH10d. The accuracy was measured to be between 92.8% and 107%
for all assays. The results are shown in Table 1. Referring to FDA
guideline [20], the present method has good accuracy, precision,
and reproducibility. The recovery of HWH-10d obtained from the
spiked plasma samples were also shown in Table 2. The RSD was
less than 9.8% for all recoveries throughout the entire standard
concentration ranges, showing good consistency.

3.2.4. Stability
As shown in Table 3, the deviation was measured to be within
8.1% of nominal concentrations at each QC concentrations of HWH10d for stability determination. These results demonstrated that
HWH-10d was stable in monkey plasma at room temperature for
at least 6 h and at 20 C for one month, respectively. It is also stable after three freeze-thaw cycles. Stability was also assessed using
the processed plasma samples in the autosampler at approximately
4 C for 24 h.
3.2.5. Matrix effect
The 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 from
the plasma matrix was negligible under the current conditions.
3.2.6. Pharmacokinetic study in monkeys
The pharmacokinetic property of HWH-10d was studied in
monkeys following i.v. (2 mg/kg) and oral (2 mg/kg) administrations. Non-compartmental mode was used to calculate the
pharmacokinetics parameters with DAS 2.0 software (Pharmacokinetics Institute of China). The mean plasma concentration-time
proles of HWH-10d by the two routes of administration are

J. Deng et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 99103

103

References

Fig. 4. The mean plasma concentration-time proles of HWH-10d after an intravenous dose of 2 mg/kg HWH-10d (n = 6) and an oral dose of 2 mg/kg HWH-10d for
monkeys (n = 6).

shown in Fig. 4. The pharmacokinetic parameters of HWH10d are shown in Table 4. Its estimated main pharmacokinetic
parameters are presented as follows: the elimination t1/2 was estimated as 5.12 0.95 h for the intravenous dose of 2 mg/kg and
5.42 1.57 h for the oral dose of 2 mg/kg. The AUC0t of intravenous administration was found to be 2014 289.4 g/(L h).
In contrast, The AUC0t of oral administration was found to be
1164 157.1 g/(L h).
4. Conclusion
A specic, sensitive, accurate and rapid LCMS/MS method for
determination of HWH-10d in monkey plasma was rst developed
and validated, which had been successfully applied in determination of pharmacokinetic property for HWH-10d. The oral
bioavailability of HWH-10d is determined to be 57.8% in monkeys. The pharmacokinetic parameters indicated that HWH-10d
displayed good absorption and slow elimination and probably low
tissue distributions.
Acknowledgments
This work was supported in part by the National High-tech
R&D Program [Grant 2006AA02Z339] and [Grant 2008AA02Z314];
the Guangzhou Science and Technology Bureau [Grant 2006Z1E4031, 2006P067]; and the Guangzhou Development District
[Grant 2006Ss-P067].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jpba.2015.08.033.

[1] International diabetes federation. http://www.idf.org/

[2] D.E. Moller, New drug targets for type 2 diabetes and the metabolic
syndrome, Nature 414 (6865) (2001) 821827.
[3] J. Rachman, B.A. Barrow, J.C. Levy, R.C. Turner, Near-normalisation of diurnal
glucose concentrations by continuous administration of glucagon-like
peptide-1 (GLP-1) in subjects with NIDDM, Diabetologia 40 (2) (1997)
205211.
[4] K.L. Balkan, R. Miserendino, J.J. Holst, X. Li, Inhibition of dipeptidyl peptidase
IV with NVP-DPP728 increases plasma GLP-1 (736 amide) concentrations
and improves oral glucose tolerance in obese Zucker rats, Diabetologia 42
(1999) 13241331.
[5] T.J. Kieffer, C.H. Mclntosh, R.A. Pedersion, Degradation of glucose-dependent
insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and
in vivo by dipeptidyl peptidase IV, Endocrinology 136 (8) (1995) 35853596.
[6] R. Mentlein, B. Gallwitz, W.E. Schmidt, Dipeptidyl-peptidase IV hydrolyses
gastric inhibitory polypeptide glucagon-like peptide-1(7-36) amide peptide
histidine methionine and is responsible for their degradation in human
serum, Eur. J. Biochem. 214 (3) (1993) 829835.
[7] B. Ahren, R. Gomis, E. Standl, D. Mills, A. Schweizer, Twelve- and 52-week
efcacy of the dipeptidyl peptidase IV inhibitor LAF237 in metformin-treated
patients with type 2 diabetes, Diabetes Care 27 (12) (2004) 28742880.
[8] B. Ahren, M. Landin-Olsson, P.A. Jansson, M. Svensson, D. Holmes, A.
Schweizer, Inhibition of dipeptidyl peptidase-4 reduces glycemia sustains
insulin levels and reduces glucagon levels in type 2 diabetes, J. Clin.
Endocrinol. Metab. 89 (5) (2004) 20782084.
[9] B. Ahren, G. Pacini, J.E. Foley, A. Schweizer, Improved meal-related beta-cell
function and insulin sensitivity by the dipeptidyl peptidase-IV inhibitor
vildagliptin in metformin-treated patients with type 2 diabetes over 1 year,
Diabetes Care 28 (8) (2005) 19361940.
[10] B. Ahren, E. Simonsson, H. Larsson, M. Landin-Olsson, H. Torgeirsson, P.A.
Jansson, M. Sandqvist, P. Bavenholm, S. Efendic, J.W. Eriksson, Inhibition of
dipeptidyl peptidase IV improves metabolic control over a 4-week study
period in type 2 diabetes, Diabetes Care 25 (5) (2002) 869875.
[11] C.F. Deacon, T.E. Hughes, J.J. Holst, Dipeptidyl peptidase IV inhibition
potentiates the insulinotropic effect of glucagon-like peptide 1 in the
anesthetized pig, Diabetes 47 (5) (1998) 764769.
[12] J.J. Holst, C.F. Deacon, Inhibition of the activity of dipeptidyl-peptidase IV as a
treatment for type 2 diabetes, Diabetes 47 (11) (1998) 16631670.
[13] C.F. Deacon, J.J. Holst, Dipeptidyl peptidase IV inhibitors: a promising new
therapeutic approach for the management of type 2 diabetes, Int. J. Biochem.
Cell. Biol. 38 (56) (2006) 831844.
[14] N.A. Thornberry, A.E. Weber, Discovery of JANUVIA (Sitagliptin): a selective
dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes, Curr.
Top Med. Chem. 7 (6) (2007) 557568.
[15] J.J. Wu, H.K. Tang, T.K. Yeh, C.M. Chen, H.S. Shy, Y.R. Chu, C.H. Chien, T.Y. Tsai,
Y.C. Huang, Y.L. Huang, Biochemistry pharmacokinetics and toxicology of a
potent and selective DPP8/9 inhibitor, Biochem. Pharmacol. 78 (2) (2009)
203210.
[16] E. Bosi, R.P. Camisasca, C. Collober, E. Rochotte, A.J. Garber, Effects of
vildagliptin on glucose control over 24 weeks in patients with type 2 diabetes
inadequately controlled with metformin, Diabetes Care 30 (4) (2007)
890895.
[17] A. Mari, W.A. Scherbaum, P.M. Nilsson, G. Lalanne, A. Schweizer, B.E. Dunning,
S. Jauffret, J.E. Foley, Characterization of the inuence of vildagliptin on
model-assessed -cell function in patients with type 2 diabetes and mild
hyperglycemia, J. Clin. Endocrinol. Metab. 93 (1) (2008) 103109.
[18] R.E. Pratley, Alogliptin:a new highly selective dipeptidyl peptidase-4 inhibitor
for the treatment of type 2 diabetes, Expert Opin. Pharmacother. 10 (3) (2009)
503512.
[19] J. Deng, L. Peng, G. Zhang, X. Lan, C. Li, F. Chen, Y. Zhou, Z. Lin, L. Chen, R. Dai,
H. Xu, L. Yang, X. Zhang, W. Hu, The highly potent and selective dipeptidyl
peptidase IV inhibitors bearing a thienopyrimidine scaffold effectively treat
type 2 diabetes, Eur J Med Chem :. 46 (2011) 7176.
[20] FDA, Guidance for industry, Bioanal. Method Validation (2001), http://www.
fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM070107.pdf.