Hindawi

Journal of Analytical Methods in Chemistry

Volume 2018, Article ID 1902510, 7 pages
https://doi.org/10.1155/2018/1902510

Research Article
Development and Validation of an HPLC Method for
Determination of Antidiabetic Drug Alogliptin Benzoate in
Bulk and Tablets

Hani Naseef ,1,2 Ramzi Moqadi ,2 and Moammal Qurt 2

1
Samih Darwazah Institute for Pharmaceutical Industries, Faculty of Pharmacy, Nursing and Health Professions,
Birzeit University, Birzeit, State of Palestine
2
Pharmacy Department, Faculty of Pharmacy, Nursing and Health Professions, Birzeit University, Birzeit,
State of Palestine

Correspondence should be addressed to Moammal Qurt; mqurt@birzeit.edu

Received 25 July 2018; Accepted 3 September 2018; Published 24 September 2018

Academic Editor: Josep Esteve-Romero

Copyright © 2018 Hani Naseef et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Alogliptin benzoate, a member of dipeptidyl peptidase-4 inhibitors, is a recent drug developed by Takeda Pharmaceutical
Company for the treatment of Type 2 diabetes; it potentiates the effect of incretin hormones through the inhibition of their
degradation. Alogliptin can be used alone or in combination therapy. A new sensitive and rapid HPLC method was developed for
the determination of alogliptin benzoate in bulk and pharmaceutical dosage forms; it was validated according to ICH and FDA
guidelines. The HPLC analysis was performed on the Agilent 1200 system equipped with a Hypersil Gold Thermo Scientific C18
(250 cm × 4.6 mm) 5 µm column, with a mixture of acetonitrile and ammonium carbonate buffer in the ratio of 55 : 45 v/v as the
mobile phase, at the flow rate of 1.0 mL/min. The detection was performed at the wavelength (λ) of 277, and the retention time of
alogliptin benzoate was around 4 min. The total run time was 6.0 min. The calibration plot gave linear relationship over the
concentration range of 85–306 µg/ml. The LOD and LOQ were 0.03 and 0.09 μg, respectively. The accuracy of the proposed
method was determined by recovery studies and was found to be 100.3%. The repeatability testing for both standard and sample
solutions showed that the method is precise within the acceptable limits. RSD% of the determination of precision was <2%. The
results of robustness and solutions stability studies were within the acceptable limits as well. The proposed method showed
excellent linearity, accuracy, precision, specificity, robustness, LOD, LOQ, and system suitability results within the acceptance
criteria. In addition, the main features of the developed method are low run time and retention time around 4 min.

1. Introduction the pancreas (β-cells) [2, 3]. The first line of therapy for the
treatment of Type 2 diabetes is metformin, but as the disease
Since the first evidence about a known case of diabetes progresses, a drug combination may be a must [4].
mellitus nearly 3000 years ago and despite the great deal of Incretin hormones are secreted in response to eating
research that has been done recently, diabetes mellitus is still food from the gastrointestinal tract to the blood stream and
a wide spread serious disease that affect the life quality of can stimulate insulin secretion and help control glucose
millions of people worldwide. It is estimated that the number levels; that is, they prepare the body against increase in blood
of patients with diabetes mellitus will rise to about 592 glucose. These hormones include glucagon-like peptide-1
millions by the year 2035 [1, 2]. and glucose-dependent insulin tropic polypeptide [5, 6].
It was until the year 1936 that diabetes mellitus was Dipeptidyl peptidase-4 is an enzyme found in the human
distinguished to Type 1 and Type 2 [1]. Two main features of body that helps inactivate the incretin hormones, thus
Type 2 diabetes mellitus is the increased cell resistance to terminating their hypoglycemic effect [2]. Alogliptin
insulin and the dysfunction of the insulin-producing cell in a member of dipeptidyl peptidase-4 inhibitors is a recent
2 Journal of Analytical Methods in Chemistry

drug developed in 2010 by Takeda Pharmaceutical O

Company [2, 7], which is used for the treatment of Type 2 N
diabetes, and it potentiates the effect of incretin hor- H2N
mones through inhibition of their degradation by the N N O
dipeptidyl peptidase-4 enzyme [2, 4]. Alogliptin can be
used alone or in combination therapy, and it is now
approved in the USA and Europe also [5]. Alogliptin is 2- N
({6-[(3R)-3-aminopiperidin-1-yl]-3-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1yl}methyl)benzonitrile (C18H21N5O2), Figure 1: Chemical structure of alogliptin.
and its structure is shown in Figure 1 [8].
Analytical method validation ensures that various HPLC 250 × 4.6 mm with a detection wavelength of 277 nm. The
analytical techniques shall give reliable and repeatable re- operating temperature of the column was set at 30°C. The
sults; it is a crucial step in developing new dosage forms as it injection volume was 10 μL, and the flow rate was main-
provides information about accuracy, linearity, precision, tained at 1.0 mL/min. The run time was 6 minutes.
detection, and quantitation limits. According to the ICH
guideline, “the objective of validation of an analytical pro-
cedure is to demonstrate that it is suitable for its intended 2.4. Preparation of Standard Solution. A standard solution of
purpose.” It is now obligatory in the process of drug de- alogliptin benzoate was prepared by dissolving an accurately
velopment to supply the validation data for the responsible weighed amount of alogliptin benzoate (42.5 mg, which is
authorities. Guidelines for analysis method validation in- equivalent to 31.25 mg alogliptin) in 50 ml of the mobile
clude ICH and USP guidelines [9–12]. phase, and then 5 mL of the resulting solution was diluted to
Literature survey revealed a few methods reported for 25 mL by the same solvent to obtain a standard solution of
determination of alogliptin benzoate in bulk drug as well as alogliptin benzoate (170 µg/ml).
pharmaceutical preparation [2, 5, 13–16].
In this research, a new sensitive and rapid HPLC method
was developed for the determination of alogliptin benzoate 2.5. Preparation of Sample Solution. Twenty alogliptin tablets
in pharmaceutical dosage forms, and this method was were weighed, triturated in porcelain mortar, and mixed,
validated according to ICH and FDA guidelines. and the average weight of tablet was calculated. Accurately
weighed amount of powder equivalent to 25 mg of alogliptin
2. Materials and Methods (34 mg alogliptin benzoate) was transferred completely to
a 200 mL volumetric flask, and 150 mL of the mobile phase
2.1. Instrumentation. Agilent 1200 HPLC system was used was added and sonicated for 30 minutes. The volume was
for liquid chromatography method development and vali- completed to mark by the same solvent to obtain a solution
dation (Santa Clara, USA), equipped with a pump (model of alogliptin benzoate with a concentration of 170 µg/ml. The
G1312A), an auto sampler (ALS) (model G1329A), and prepared solution was filtered through 0.45 μm membrane
a Hypersil Gold Thermo Scientific C18 (250 cm × 4.6 mm) filters.
5 µm column (Paisley, UK), and the detector consisted of
UV/VIS operated at 277 nm. Chemstation Software (Version
Rev B.04.03 (16)) was used for data processing and 2.6. Method Validation. The method was validated as per
evaluation. ICH and FDA guidelines, and the validation parameters
included specificity, linearity, range, accuracy, precision,
sensitivity (LOQ and LOD), and robustness [9, 17].
2.2. Chemicals and Reagents. A pharmaceutical grade sample
of alogliptin benzoate (assigned purity 99.4%) was obtained
as gift from Jordan Hikma Pharmaceuticals (Amman, Jor- 2.6.1. Specificity. Specificity is one of the significant features
dan). NESINA tablets containing 8.5 mg alogliptin benzoate of HPLC, and it refers to the ability of the analytical method
were purchased from the local market. Acetonitrile HPLC to discriminate between the analyte and the other compo-
grade and ammonium carbonate were purchased from nents in the complex mixture [18]. Specificity of the method
Merck (Merck Serono Amman, Jordan). The double distilled was evaluated by injecting 10 μl solutions of standard,
water was obtained from a local pharmaceutical company. sample, blank, and placebo separately.

2.3. Chromatographic Conditions. The mobile phase was 2.6.2. Linearity. To evaluate the linearity and range of the
prepared by dissolving 1.0 gm ammonium carbonate in method, different standard solutions were prepared by di-
1000 ml water. From the previous solution, 450 ml was luting the standard stock solution with the mobile phase in
mixed with 550 ml of acetonitrile. Prior to use the mobile deferent concentrations of alogliptin benzoate: 85, 136, 170,
phase was filtered through 0.45 μm membrane filters and 204, 255, and 306 µg/ml, which cover 50%, 80%, 100%, 120%,
degassed by sonication for 10 min. The analysis was carried 150%, and 180% of the target concentration, respectively.
out on an Agilent 1200 series HPLC system. The analytes Three injections from each concentration were analysed
were conducted on an analytical column C18, 5 µm, under the same conditions. Linear regression analysis was
Journal of Analytical Methods in Chemistry 3

used to evaluate the linearity of the calibration curve by chromatographic conditions, including detection wave-
using the least square linear regression method. length, mobile phase, stationary phase, and sample prepa-
ration procedure. For this purpose, a series of trials were
performed by varying the ratio of acetonitrile and ammo-
2.6.3. Sensitivity. Limit of detection (LOD)/limit of quan-
nium carbonate buffer and optimizing the chromatographic
titation (LOQ) of alogliptin benzoate were determined by
conditions on the Hypersil Gold Thermo Scientific C18
analysing different solutions of alogliptin benzoate and
(250 cm × 4.6 mm) 5 µm column. The results of method
measuring the signal-to-noise ratio. The limit of detection
optimization are summarized in Table 1.
(LOD) is the concentration that gives a signal-to-noise ratio
The mobile phase consisting of acetonitrile and am-
of approximately 3 : 1, while the limit of quantification
monium carbonate buffer in the ratio 55 : 45 v/v with a flow
(LOQ) is the concentration that gives a signal-to-noise ratio
rate of 1 mL/min, injection volume 10 µl, run time 6 min,
of approximately 10 : 1 with %RSD (n � 3) of less than 10%.
and column temperature 30°C at wavelength (λ) 277 was
optimized as the best chromatographic conditions for the
2.6.4. Accuracy. The accuracy of the assay method was entire study where alogliptin benzoate was eluted forming
determined by recovery studies at three concentration levels symmetrical peak shape, resolution and suitable analysis
(50%, 100%, and 150%), i.e., 85, 170, and 255 μg/ml, and time with retention time about 4 min (Figure 2).
three samples from each concentration were injected. The
percentage recovery of added alogliptin benzoate and RSD
were calculated for each of the replicate samples. 3.2. Method Validation
3.2.1. Specificity. Specificity was evaluated by comparing the
2.6.5. Precision. The system precision and method precision chromatograms of mobile phase blank, placebo solution,
(repeatability) of the proposed methods were determined by standard solution, and sample solution (alogliptin
several measurements of standard solution and sample 170 μg/ml). For this purpose, 10 μl from solutions mobile
solution, respectively [19–22]. System precision was estab- phase blank, standard solution, and sample solution were
lished by ten measurements of the standard solution at the injected into the HPLC system separately, and the chro-
100% concentration levels on the same day. Method pre- matogram results are shown in Figures 2–5. It can be ob-
cision was established by six assay determinations of the served that there no coeluting peaks at the retention time of
sample solution at the 100% concentration levels on the alogliptin benzoate interference. This result indicates that
same day [23]. The RSD of obtained results was calculated to the peak of the analyte was pure and this confirmed the
evaluate repeatability results. specificity of the method.

2.6.6. Robustness. Robustness of the method was verified by 3.2.2. Linearity and Range. Analytical method linearity is
applying minor and deliberate changes in the experimental defined as the ability of the method to obtain test results that
parameters, for example: are directly proportional to the analyte concentration, within
a specific range. The mean peak area obtained from the
(i) Column temperature: ±5°C HPLC was plotted against corresponding concentrations to
(ii) Flow rate: ±0.2 mL/min obtain the calibration graph. The results of linearity study
(iii) Wavelength: ±3 nm (Figure 6) gave linear relationship over the concentration
(iv) Mobile phase composition, organic composition range of 85–306 µg/ml for alogliptin benzoate. From the
±5% regression analysis, a linear equation was obtained:
y � 17412x + 1.1377, and the goodness-of-fit (r2) was found
Change was made to evaluate its effect on the method. to be 1.00, indicating a linear relationship between the
Obtained data for each case was evaluated by calculating % concentration of analyte and area under the peak.
RSD and percent of recovery.
3.2.3. Limit of Detection and Limit of Quantification (LOD
2.6.7. Stability of Analytical Solutions. The stability of an- and LOQ). The limit of detection (LOD) is the lowest
alytical solutions was determined by analysing the standard amount of analyte in a sample that can be detected, but not
and sample preparations at 0 h and after one day in re- necessarily quantitated, while the limit of quantification
frigerator and at ambient room temperature 30°C. Three (LOQ) is the lowest amount of analyte in a sample that can
injections from each solution were analysed, and the average be quantitatively determined with suitable precision [24].
of the peak and the RSD were calculated. The results showed an LOD and LOQ for alogliptin of 0.03
and 0.09 μg, respectively.
3. Results and Discussion
3.1. Method Development and Optimization. Several physical 3.2.4. Accuracy. The accuracy of an analytical procedure
and chemical properties of alogliptin benzoate were ob- expresses the closeness of results obtained by that method to
tained from the literature. The analytical method was de- the true value. The results of accuracy showed percentage
veloped to select preliminary reversed phase HPLC-UV recovery at all three levels in the range of 99.4–101.9%, and %
4 Journal of Analytical Methods in Chemistry

Table 1: Results of method optimization.

Column used Mobile phase Flow rate Wavelength Observation Result
(Buffer : methanol)
Restek C18, 125 × 4.0 mm i.d., 5 µm 1.0 ml/min 216 nm Poor resolution 1.4 Method rejected
(45 : 55) v/v
Thermo Scientific C18, (Buffer : acetonitrile)
1.0 ml/min 277 nm Poor resolution 1.6 Method rejected
250 × 4.6 mm i.d., 5 µm (25 : 75) v/v
Thermo Scientific C18, (Buffer : acetonitrile)
1.0 ml/min 277 nm Good resolution 2.4 Method accepted
250 × 4.6 mm i.d., 5 µm (45 : 55) v/v

500
1.5
Absorbance (mAU)

400 3.954 - alogliptin

Absorbance (mAU)
1
300

200 0.5

100
0
0
0 1 2 3 4 5 –0.5
Retention time (min)
0 1 2 3 4 5
Figure 2: Chromatogram of alogliptin standard solution. Retention time (min)
Figure 5: Chromatogram of placebo solution.

6000.0
500 y = 17412x + 1.1377
5000.0
Avg. peak area

3.994 - alogliptin 4000.0 R2 = 1

Absorbance (mAU)

400
3000.0
300 2000.0
1000.0
200 0.0
0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00
100 Concentration (μg/ml)

0 Figure 6: Standard calibration curve of alogliptin benzoate.

0 1 2 3 4 5
Retention time (min) RDS values were in the range of 0.06–0.43% as shown in
Table 2. The results of percentage recovery and %RSD were
Figure 3: Chromatogram of alogliptin sample solution.
within the accepted limits from 98.0% to 102.0% and not
more than 2.0%, respectively, which indicates the applica-
bility of the method for routine drug analysis.
0.4
0.3 3.2.5. Precision. The precision of the method is defined as
Absorbance (mAU)

0.2 “the closeness of agreement between a series of measure-

ments obtained from multiple sampling of the same ho-
0.1
mogeneous sample under the prescribed conditions,” and it
0 is normally expressed as the relative standard deviation [25].
–0.1 The results of both system and method precision showed
–0.2
that the method is precise within the acceptable limits. The
RSD, tailing factor, and number of theoretical plats were
–0.3 calculated for both solutions; all the results are within limits.
0 1 2 3 4 5
Acceptable precision was not more than 2.0% for the RSD
Retention time (min)
and the tailing factor and not less than 1000 for number of
Figure 4: Chromatogram of blank solution. plates, as shown in Tables 3 and 4.
Journal of Analytical Methods in Chemistry 5

Table 2: Recovery data of the proposed HPLC method.

% spiked level Replicate number Peak area % recovery Mean %RSD
1 1508.4 101.9 101.5
50 2 1495.5 101.0
0.43
3 1503.5 101.6
1 2950.7 99.4 99.4
100 2 2950.8 99.4
0.06
3 2953.8 99.5
1 4443.5 100.2 100.1
150 2 4435.4 100.0
0.13
3 4431.9 99.9
Mean (% of recovery) 98.0–102.0 100.318
%RSD Max 2.00 0.964149

Table 3: System precision data from the standard solution of the proposed HPLC method.
Replicate number RT Peak area Number of theoretical plates Tailing factor
1 3.954 2952 1.32 6274
2 3.956 2951 1.36 6388
3 3.961 2951 1.35 6363
4 3.959 2960 1.33 6364
5 3.961 2953 1.36 6386
6 3.965 2946 1.36 6441
7 3.962 2949 1.38 6479
8 3.965 2950 1.35 6486
9 3.965 2954 1.35 6464
10 3.969 2958 1.33 6471
Average 3.962 2952 1.3 6412
%RSD — 0.10 — —

Table 4: Method precision data from the sample solution of the proposed HPLC method.
Alogliptin 6.25 mg tablet
Replicate number RT Peak area Tailing Plates % assay
1 4.025 3009 1.54 8086 99.2
2 4.024 3012 1.52 8049 99.2
3 4.027 3009 1.48 8101 99.2
4 4.027 3009 1.49 8105 98.6
5 4.028 3015 1.50 8039 99.3
6 4.027 3012 1.50 8107 99.5
Average 4.026 3011.0 1.5 8081 99.2
%RSD — 0.1 0.31

3.2.6. Robustness. The analytical method robustness was number of theoretical plates were found within acceptable
tested by evaluating the influence of minor modifications in limits as well.
HPLC conditions on system suitability parameters of the
proposed method, as mentioned in Section 2.6.6. The re-
sults of robustness testing showed that a minor change of 3.2.7. Solution Stability. The percent of recovery was within
method conditions, such as the composition of the mobile the range of 98.0% to 102.0% and RSD was not more than
phase, temperature, flow rate, and wavelength, is robust 2.0%, indicating a good stability of the sample and standard
within the acceptable limits. The results are summarized in solutions for 24 hr at both conditions. The percent of re-
Table 5. In all modifications, good separation of alogliptin covery was within acceptable limits, and the %RSD is within
benzoate was achieved, and it was observed that the percent the limit of not more than 2.0%. The tailing factors and
of recovery was within acceptable limits and the %RSD is number of theoretical plates were found within acceptable
within limit of not more than 2.0%. The tailing factors and limits as well. The results are shown in Table 6.
6 Journal of Analytical Methods in Chemistry

Table 5: Robustness data of the proposed HPLC method.

Parameter %RSD of standard peak area %RSD of assay
25°C 0.07 0.15
Column temperature 30°C (normal) 0.03 0.19
35°C 0.04 0.2
274 nm 0.06 0.07
Wavelength 277 nm (normal) 0.03 0.19
280 nm 0.06 0.17
−5% acetonitrile 0.05 0.20
Mobile phase composition Normal 0.03 0.19
+5% acetonitrile 0.02 0.14
0.8 ml/min 0.04 0.11
Flow rate 1 ml/min (normal) 0.03 0.19
1.2 ml/min 0.08 0.23

Table 6: Solutions stability data of the proposed HPLC method.

Avg. peak RSD peak Number of
Parameter RT Tailing factor Recovered (%)
area area (%) theoretical plates
0h 4.034 3022.7 0.07 1.5 — 8058
Standard solution After 24 h at 30°C 4.035 3021.7 0.2 1.6 100.0 8143
After 24 h at refrigerator 4.049 2983.7 0.08 1.5 98.7 8137
0h 4.034 2995.7 0.07 1.5 — 8142
Sample solution After 24 h at 30°C 4.035 3001.3 0.3 1.5 100.2 8179
After 24 h at refrigerator 4.036 3000.0 0.2 1.6 100.1 8188

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