WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Chunduri et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041

Volume 5, Issue 5, 838-850 Research Article ISSN 2278 – 4357

DEVELOPMENT AND VALIDATION OF LC-MS/MS METHOD FOR

QUANTIFICATION OF TENELIGLIPTIN IN HUMAN PLASMA AND
ITS APPLICATION TO A PHARMACOKINETIC STUDY

Raja Haranadha Babu Chunduri1* and Gowri Sankar Dannana2

1
School of Pharmaceutical Sciences and Technologies, Jawaharlal Nehru Technological
University, Kakinada-533 003, Andhra Pradesh, India.
2
A. U College of Pharmaceutical Sciences, Andhra University, Vishakapatnam-530 003,
Andhra Pradesh, India.

ABSTRACT
Article Received on
01 March 2016, A selective, sensitive and rapid liquid chromatographic method with
Revised on 21 March 2016,
electro spray ionization tandem mass spectrometric detection has been
Accepted on 11 April 2016
DOI: 10.20959/wjpps20165-6637 developed and validated for quantification of teneligliptin in human
plasma using repaglinide as internal standard (IS). The analytes and IS
were extracted from 50 µL of plasma by liquid-liquid extraction
*Corresponding Author
Raja Haranadha Babu technique using methyl tert - butyl ether which offers high sensitivity,
Chunduri wide linearity without interferences form endogenous matrix
School of Pharmaceutical components. In a short chromatographic run of 1.50 min run time,
Sciences and Technologies,
separation was achieved on a Hypersil Gold C18 column using a
Jawaharlal Nehru
mobile phase composed of 0.1% formic acid in Milli-Q water/0.1%
Technological University,
Kakinada-533 003, Andhra formic acid in acetonitrile in gradient elution mode. The quantification
Pradesh, India. of teneligliptin was performed in a positive electro spray ionization
mode and multiple reaction monitoring (MRM). Response was a linear
function of concentration in the ranges of 0.1 to 4000 ng/mL for teneligliptin with r2> 0.99.
The intra- and inter-day precision and accuracy results were below 15% and acceptable as per
Food and Drug Administration (FDA) guidelines. Stability of teneligliptin was established in
a battery of stability studies, i.e. bench top, auto sampler and long term storage stability as
well as freeze thaw cycles. The validated method can be used as a routine method to support
pharmacokinetic studies.

KEYWORDS: Teneligliptin; LC-MS/MS.

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INTRODUCTION
Type 2 diabetes mellitus is the 7th leading cause of death in world wide. Approximately 1.5
million deaths occurred in 2014 are directly due to type 2 diabetes mellitus. More than 80%
of diabetes deaths occurring in developing countries. Unhealthy diet, lack of physical
exercise, stress and more usage of tobacco increasing the risk of developing type 2 diabetes
mellitus.[1]

Type 2 diabetes mellitus is a chronic metabolic disease characterized by impaired insulin

secretion and insulin resistance in insulin-targeting tissues (liver, skeletal muscle and
adipocytes).[2] Insulin resistance in these tissues is accompanied by decreased glucose
utilization by muscle and fat cells, and increased hepatic glucose output, leads to
hyperglycemia. Insulin resistance is compensated by excessive insulin secretion. The over
secretion of insulin gradually decreases the islet beta-cell reserve and its function.[3] The
complexity in treatment of type 2 diabetes mellitus is welcoming new drugs for specific cure.

Teneligliptin is chemically {(2S,4S)-4-[4-(3-methyl-1-phenyl-1H- pyrazol-5-yl) piperazin-1-

yl] pyrrolidin-2-yl} (1,3-thiazolidin-3-yl) methanone, exhibits a unique structure that is
characterized by five consecutive rings. Teneligliptin, a novel DPP-4 inhibitor, a new class of
oral hypoglycemic drug used in the treatment of type 2 diabetes mellitus.[4] Dipeptidyl
peptidase-4 (DPP-4) inhibitors improve glycemic control with a minimal risk of
hypoglycemia and weight gain in patients with type 2 diabetes mellitus.[5]

Incretin hormones, namely glucagon-like peptide-1 (GLP-1) and glucose-dependent

insulinotropic polypeptide (GIP), are released from enteroendocrine cells and enhance insulin
secretion.[6] Incretins are rapidly inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4),
and have a very short half-life (t1/2) as a result.[7] DPP-4 inhibitors increase the levels of
active GLP-1 and GIP by inhibiting DPP-4 enzymatic activity; thus, in patients with diabetes,
these inhibitors improve hyperglycemia in a glucose-dependent manner by increasing serum
insulin levels and decreasing serum glucagon levels.[8-11] Therefore, incretin-related agents
such as DPP-4 inhibitors are promising drugs that can decrease glucose fluctuations in
diabetic patients and have emerged as a new class of ant diabetic drugs. So there is a need for
the method to estimate the concentration of teneligliptin from plasma.

Literature survey reveals that, till date there is no LC-MS/MS method has been reported in
literature for the determination of teneligliptin in human plasma. Hence, authors attempted to

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develop a simple and reproducible LC-MS/MS method for quantification of teneligliptin

which helps the researchers for therapeutic drug monitoring and pharmacokinetics.

The aim of this work is, to develop a simple, selective and sensitive method, which employs
liquid-liquid extraction technique for sample preparation and liquid chromatography with
electrospray ionisation-tandem mass spectrometry for quantification of teneligliptin in human
plasma. The proposed method has significant advantages like 1) Shorter run time, 2) Wider
linearity range with a more sensitivity, 3) Simple reproducible extraction. The present method
has been validated as per the current USFDA guidelines.[12] The application of this assay in a
clinical pharmacokinetic study following oral administration of teneligliptin is described.

EXPERIMENTAL
Materials and reagents
Reference standard of teneligliptin (Figure. 1) was purchased from Rxn chemicals (Mumbai,
India) and internal standard (Repaglinide) was purchased from Sigma-Aldrich (Hyderabad,
India). HPLC grade acetonitrile was procured from J.T Baker (Phillipsburg, USA). HPLC
grade dimethyl sulfoxide (DMSO) and formic acid was procured from Sigma-Aldrich
(Hyderabad, India). Milli-Q water (18.2 mΩ and TOC≤50 ppb) from Milli-Q purification
system, (Millipore, Bangalore, India) was used throughout the study. Methyl tert - butyl ether
was purchased from Rankem (Hyderabad, India). Drug free human plasma was procured
from Biochemed (Winchester, USA).

Chromatographic and mass spectrometric conditions

Waters Acquity UPLC system (Waters corporation, Milford, USA) consisting of binary
solvent manager, sample manager and column manger was used for solvent and sample
delivery. Mobile phase A consisted of 0.1% formic acid in Milli-Q water and mobile phase B
was 0.1% formic acid in acetonitrile. The analyte and IS were separated by the following
gradient (minutes, % mobile phase B) (0.01, 10) (0.20, 10) (0.90, 90) (1.10, 90) (1.20, 10)
(1.50, 10) delivered with a flow rate of 0.5 mL/min on a Hypersil Gold C18 column (50
mm×3.0 mm, 1.9 µm; Thermo scientific, India) maintained at 40°C.The sample manager was
maintained at 5°C and injection volume was 2 µL. The total chromatographic run time was
1.50 min. The analytes and IS were detected using a Waters XEVO TQ mass spectrometer
(Waters corporation, Milford, USA) equipped with Z spray source. The quantification of
analyte and IS were achieved by operating the mass spectrometer in positive ion electrospray
ionization (ESI) with MRM mode. Nitrogen gas was used as both cone gas and desolvation

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gas with a flow rate of 50 L/Hr and 800 L/Hr respectively. The source dependent parameters
capillary voltage, extractor voltage, source temperature and desolvation temperature were set
at 3.50 KV, 3 V, 150°C and 500°C respectively. The precursor to product ion transitions
along with the cone voltage and collision energy for both analytes and IS were as follows:
Teneligliptin m/z 427.2→243.2, 29 V, 19 eV; Repaglinide m/z 453.2→ 230.2, 26 V, 22 eV
with dwell time 100 ms. Data acquisition and calculations were performed using Masslynx
software, version 4.0.

Preparation of calibration standards and quality control samples

Standard stock solution of teneligliptin was prepared by dissolving requisite amount in
dimethyl sulfoxide (DMSO) to obtain final drug concentration of 1mg/ml. Working stock
solution of teneligliptin with concentrations in the range of 0.005 to 200 µg/mL was prepared
by serial dilutions with acetonitrile: water (50:50). A nine point calibration curve standards of
teneligliptin with concentrations in the range of 0.1 to 4000 ng/mL was prepared by spiking
(2% of total plasma volume) in blank human plasma with respective working stock solutions.
The quality control (QC) samples were similarly prepared for teneligliptin at concentration of
3000 ng/mL (high quality control, HQC), 1500 ng/mL (middle quality control), 0.3 ng/mL
(low quality control) and 0.1 ng/mL (lower limit of quantification quality control, LLOQ QC)
respectively with blank human plasma by a separate weighing of standards. Stock solution (1
mg/mL) of the internal standard (repaglinide) was prepared by dissolving appropriate amount
in DMSO and its working stock solution (250 ng/mL) was prepared by diluting the stock
solution in acetonitrile: water (50:50).

Sample preparation
A simple liquid-liquid extraction method was developed for extraction of analyte and IS from
human plasma. Prior to analysis all frozen subject samples, calibration standards and quality
control samples were thawed at ambient temperature. In the following order 10 µL of IS
working stock solution (250 ng/mL repaglinide) was added into each 1.5 mL eppendorf tube
except for blank plasma. 50 µL of standards, QCs, study samples and blank plasma were
transferred into eppendorf tubes. After vortexing for 30 seconds, 100 µL of Milli-Q water
was added to each tube and vortexed to mix. The analyte and IS were extracted with 1mL of
methyl tert - butyl etherby vortexing for 10 min and followed by centrifugation at 5000 g for
10 min. The organic layer was transferred into a clean test tube and evaporated to dryness at
40°C under a gentle stream of nitrogen in the Turbo vap evaporator (Caliper life sciences,

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USA).The dried extract was reconstituted with 0.2 mL of mobile phase and 2 µL of aliquot
was injected onto the LC-MS/MS system for analysis.

Method validation
The bioanalytical method was thoroughly validated to meet the acceptance criteria of
industrial guidance for the bioanalytical method validation (US Food and Drug
Administration, 2001).The method was validated for selectivity, linearity, precision and
accuracy, recovery, matrix effect, dilution integrity and stability. Selectivity is the ability of
analytical method to differentiate and quantify the analytes in the presence of other expected
components in the sample. This was evaluated by comparing the chromatograms of eight
different blank human plasma (which includes one hemolyzed, one lipemic and six normal
lots) with corresponding spiked plasma at LLOQ QC level. Peak areas of endogenous
compounds co eluting with the analytes should be less than 20% of the peak area of LLOQ
response. Peak areas of endogenous compounds co eluting with IS should be less than 5% of
the mean response of internal standard in LLOQ samples. Linearity was tested for
teneligliptin in the concentration range of 0.1 to 4000 ng/mL. For the determination of
linearity five standard calibration curves containing at least nine non-zero standards were
constructed by a weighed (1/x2) least squares linear regression method through the
measurement of the peak area ratio of analyte to IS. In addition blank and blank+IS were
analyzed to conform the absence of direct interferences, these data were not included to
construct calibration curves. The acceptance limit of accuracy for each of the back calculated
concentrations were ±15% except for LLOQ, where it was ±20%. For a calibration run to be
accepted at least 75% of the standards, including the LLOQ and ULOQ were required to meet
the acceptance criterion, otherwise the calibration curve was rejected. Intra-day precision and
accuracy were determined by analyzing six replicate analysis of each quality control (LLOQ,
LQC, MQC and HQC) samples of two different batches on same day. Inter-day precision and
accuracy were determined by analyzing six replicate analysis of each quality control (LLOQ,
LQC, MQC and HQC) samples of five different batches.The acceptance criteria included
accuracy with in ±15% deviation from the nominal values, except the LLOQ where it should
be ±20% and a precision of ±15% coefficient of variance (%CV), except for LLOQ, where it
should be ±20%. The extraction recovery of each analyte was estimated at three different QC
levels (six replicates of each LQC, MQC and HQC) by comparing the peak area response of
extracted analytes with unextracted analytes (extracted blank sample spiked with the
analytes) that represents 100% recovery. Similarly recovery of IS was estimated by

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comparing the mean peak area of extracted QC samples (n=18) with mean peak area of
unextracted QC samples. Recovery of the analytes and IS need not be 100% but it should be
precise and reproducible at all QC levels. The matrix effect was evaluated by comparing the
peak area ratios (analyte/IS) obtained from post extraction spiking with mean peak area ratio
(analyte/IS) obtained at the same concentration in neat solution. The matrix effect was
determined at all three QC levels using six replicates at each level for each analyte, where IS
was determined at a single concentration of 250 ng/mL. Dilution integrity was performed to
extend the upper concentration limits with acceptable precision and accuracy. The dilution
integrity experiment was carried out for each analyte by analysing six replicate samples at
concentration of two times the ULOQ concentration was prepared and diluted to 2- and 4-
fold with blank plasma.Stability tests were conducted to evaluate the analyte stability in stock
solutions and in plasma samples under different conditions. The stock solution stability at
room temperature and refrigerated conditions (2–8°C) was determined by comparing the area
response of the analytes (stability samples) with the response of the sample prepared from
fresh stock solution. Bench-top stability (8 h), processed sample stability (autosampler
stability for 24 h), dry extract stability (8 h), freeze–thaw stability (four cycles) and long-term
stability (60 days) were tested at LQC and HQC levels using six replicates at each level.
Samples were considered to be stable if assay values were within the acceptable limits of
accuracy (±15%) and precision (±15% CV).

Pharmacokinetic study
The pharmacokinetic study was carried out in healthy male volunteers (n = 6). Six volunteers
were administered a single oral dose of teneligliptin (20 mg) tablet. Blood samples were
collected at pre-dose and 0.18, 0.33, 0.50, 0.67, 0.83, 1, 1.25, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10 and
12 hours in K2-EDTA vacutainer collection tubes (BD, Franklin, NJ, USA).The tubes were
centrifuged (Thermo Scientific, Germany) at 3200 rpm for 10 min and the plasma was
collected. The collected plasma samples were stored at –80C till their use. This study was
carried out as per the approval and guidelines of the local ethical committee. Plasma samples
were spiked with the IS and processed as per the extraction procedure described earlier.
Along with the clinical samples, the QC samples at low, middle and high concentration levels
were also assayed in triplicate. Plasma concentration-time profile of teneligliptin was
analyzed by non-compartmental method using WinNonlin Version 5.3 (Pharsight
Corporation, CA, USA).

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RESULTS AND DISCUSSION

Table 1. Precision and accuracy data for back - calculated concentrations of calibration
standards.
Concentration added Teneligliptin
(ng/ml) Mean (n=5) CV (%) Accuracy (%)
0.100 0.103 6.97 103.20
0.200 0.203 4.33 101.40
1.000 1.024 5.73 102.36
5.000 5.036 1.09 100.73
50.000 50.740 2.64 101.48
400.000 393.351 5.40 98.34
1600.000 1579.733 2.81 98.73
3200.000 3260.480 2.74 101.89
4000.000 4060.913 2.22 101.52

Figure 1: Chemical structures of Teneligliptin.

Figure 2: Product ion mass spectra of [M+H] + of teneligliptin.

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Figure 3: Typical multiple reaction monitoring mode chromatograms [A] Blank

chromatograms of teneligliptin and IS, and [B] Human plasma spiked with teneligliptin
at LLOQ level and IS.

Figure 4: Mean plasma concentration-time profile of teneligliptin.

Table 2. Intra -day and inter -day accuracy and precision of teneligliptin.
Teneligliptin
Concentration
LLOQ LQC MQC HQC
added (ng/mL)
0.100 0.300 1500.000 3000.000
Intra-day (n=6)
Mean 0.104 0.297 1464.232 3024.667
CV (%) 8.32 4.27 4.84 4.36
Accuracy (%) 103.83 99.03 97.62 100.82
Inter-day (n=30)
Mean 0.103 0.300 1485.301 3037.203
CV (%) 8.59 4.37 5.36 3.92
Accuracy (%) 103.20 100.09 99.02 101.24

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Table 3. Mean overall recoveries of analyte and IS.

Sample Response Response
Analyte Overall recovery
concentration unextracted extracted Recovery
name (Mean ± CV (%))
(ng/mL) (Mean ± CV (%)) (Mean ± CV (%))
0.3 927 ± 1.44 836 ± 2.59 90.20
Teneligliptin 1500 4574317 ± 0.98 4138383 ± 1.90 90.47 91.25 ± 1.74
3000 9232620 ± 1.57 8593684 ± 3.25 93.08
IS 250 5670294 ± 4.09 5009824 ± 2.52 88.35

Table 4. Summary of stability data of teneligliptin in human plasma.

Teneligliptin
Stability
LQC HQC
Bench top (27°C, 8 h)
Mean (n=6) 0.300 2966.315
CV (%) 3.09 3.61
Change (%) 0.22 -2.56
Auto sampler (4°C, 24 h)
Mean (n=6) 0.303 3038.783
CV (%) 3.63 3.75
Change (%) 1.28 -0.18
Dry extract (4°C, 24 h)
Mean (n=6) 0.303 3069.759
CV (%) 3.81 4.09
Change (%) 1.39 0.84
Freeze-thaw (-80°C, After 4th cycle)
Mean (n=6) 0.308 3093.467
CV (%) 4.93 3.77
Change (%) 3.01 1.62
Long term (-80°C, 60 days)
Mean (n=6) 0.302 3050.696
CV (%) 3.04 2.91
Change (%) 1.29 0.43

Table 5. Pharmacokinetic data of teneligliptin (n-6, Mean ± SD).

Estimated value
Parameter
Teneligliptin
Cmax (ng/ml) 189.353±5.16
Tmax (h) 1.00±0.00
T1/2 (h) 6.54±1.45
AUC0-t (ng.h/mL) 1061.764±156.65
AUC0-α (ng.h/mL) 1189.234±179.71

Method development and optimization

Optimization of the mass spectrometric conditions
Mass spectrometric conditions were optimized so as to achieve the maximum stable response
of the precursor ions and the major product ions of the analytes. Both analytes in this study

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were basic in nature so mass spectrometer was operated in positive ion mode to get good
response. The inherent selectivity of MRM mode for quantification of analytes was expected
to be beneficial in developing a selective and sensitive method. All analytes showed the
singly charged protonated ions [M+H]+as the prominent ion in the full scan of Q1 spectrum
and was used as the precursor ion to obtain Q3 product ion spectra. The cone voltage and
collision energy was optimized to get highest intensity for precursor ion and product ion
respectively. The mass transition ion pair was selected as m/z 427.2→243.2 for teneligliptin.
The product ion mass spectrum of teneligliptin was presented in Figure. 2.

Optimization of the chromatographic conditions

Chromatographic conditions, especially the composition of mobile phase and column were
optimized inorder to achieve good chromatographic resolution; symmetric analyte peak
shapes within a short run time. The feasibility of various mixtures of solvents such as
methanol and acetonitrile with different buffers such as ammonium acetate, ammonia
solution with altered flow rates on different types of columns such as C18 and C8 were tested
for complete chromatographic resolution of analytes and IS from interfering biological
matrix. Finally the gradient mobile phase system consisting of 0.1% formic acid in Milli-Q
water- 0.1% formic acid in acetonitrile mixture (minutes, % mobile phase B) (0.01, 10) (0.20,
10) (0.90, 90) (1.10, 90) (1.20, 10) (1.50, 10) delivered with a flow rate of 0.5 mL/min on a
Hypersil Gold C18 column achieved the good chromatographic separation of analyte and IS
with desired response. The retention times of teneligliptin and IS were 0.78 and 0.83 min
respectively. Several compounds were investigated to find a suitable IS and finally
repaglinide was found to be compatible with targeted analytes in terms of extraction
efficiency, chromatographic behavior and ionization yield.

Optimization of the sample extraction procedure

Due to the complex nature of plasma, a sample pre treatment was often needed to remove
protein and potential interferences prior to LC-MS/MS analysis. Currently, the most widely
employed biological sample preparation methodologies were protein precipitation (PPT),
solid phase extraction (SPE) and liquid-liquid extraction (LLE). LLE procedure gives more
clean and matrix free sample compared to other techniques. Hence liquid-liquid extraction
was used for sample preparation in this study. It produces a clean chromatogram of a blank
sample and yield good, reproducible recovery for analytes from the plasma.

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METHOD VALIDATION
Selectivity: Representative chromatograms obtained from blank plasma sample and blank
plasma spiked with LLOQ standard of teneligliptin and IS were presented in Figure. 3. There
is no significant endogenous interferences observed in the respective MRM channel at the
retention time of teneligliptin and IS in blank plasma sample.

Linearity: Linearity of each calibration curve was determined by plotting the peak area ratio
of analyte to IS (y) versus the nominal concentration(x) of the calibration points, and fitted to
the y = mx+c using a regression factor (1/x2). The nine point calibration curve was found to
be linear over the concentration range of 0.1 to 4000 ng/mL for teneligliptin. Correlation
coefficients were in the range 0.980<r2<0.995 for teneligliptin. The percentage accuracy
values ranged from 98.34% to 103.20%, while the precision (%CV) values ranged from 1.09
to 6.97 for teneligliptin. Table 1 summarizes the calibration curve results for teneligliptin.

Precision and Accuracy: The intra- and inter-day precision and accuracy values were within
the acceptance limit for teneligliptin and summarized in Table 2. The intra-day accuracy
ranged between 97.62% and 103.83% with a precision of 4.27% to 8.32%, the inter-day
accuracy between 99.02% and 103.20% with a precision of 3.92% to 8.59%.

Recovery: The extraction recoveries of teneligliptin and IS were good and reproducible. The
mean overall recovery (with the precision) of teneligliptin was summarized in Table 3.

Matrix effect: There was no effect of endogenous contribution from blank plasma in the
measurement of both the teneligliptin and IS. The average matrix factor values ranged from
0.99 to 1.01, while the precision (%CV) values ranged from 2.03 to 2.88 for teneligliptin.

Dilution integrity: The upper concentration limit was extended to 8000 ng/mL for
teneligliptin by a half and quarter dilution with screened human blank plasma. The mean
back calculated concentrations for half and quarter dilution samples within 85-115% of
nominal value, while precision values ranged from 3.67% to 4.01% for teneligliptin.

Stability studies: Analysis of stock solution stability was performed at 4000 ng/mL for
teneligliptin. After storage for 24 days at 2-8°C and at room temperature for 6 h, more than
97% of teneligliptin remained unchanged. The results of bench-top stability (8 h), processed
sample stability (Auto sampler stability for 24 h), dry extract stability (8 h), freeze–thaw

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stability (four cycles) and long-term stability (60 days) were summarized in Table 4 and
found to be within the acceptance limit.

APPLICATION OF THE METHOD IN PHARMACOKINETIC STUDY

The established UPLC-MS/MS method was successfully applied to the determination of
teneligliptin concentrations in human plasma samples collected from healthy volunteers
(n=6). The mean plasma concentrations vs time profiles of teneligliptin was shown in Figure.
4. In addition, the pharmacokinetic parameters were presented in Table 5.

CONCLUSION
In summary, a selective, sensitive and rapid LC-MS/MS method for quantification of
teneligliptin in human plasma was developed and fully validated as per FDA guidelines. Till
date there was no reported LC-MS/MS method for quantification teneligliptin in any
biological matrix. This method offers significant advantages, in terms of wide range of
linearity, recovery, rapid extraction and shorter run time. Finally the shorter chromatographic
runtime gives the method capability for high sample throughput. From the results of all the
validation parameters we can conclude that the present method can be useful for
pharmacokinetic/bioequivalence studies with desired precision and accuracy.

ACKNOWLEDGEMENT
The authors gratefully acknowledge Chandra Laboratories, India for providing necessary
facilities to carry out this work.

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