Simultaneous Estimation of Metformin Hydrochloride, Pioglitazone Hydrochloride, and Glimepiride by RP-HPLC in Tablet Formulation

Abstract

A simple, precise, rapid, and reproducible reversed-phase high-performance liquid chromatography method is developed for the simultaneous estimation of metformin hydrochloride (MET), pioglitazone hydrochloride (PIO), and glimepiride (GLP) present in multicomponent dosage forms. Chromatography is carried out isocratically at 25 degrees C +/- 0.5 degrees C on an Inertsil-ODS-3 (C-18) Column (250 x 4.60 mm, 5 microm) with a mobile phase composed of methanol-phosphate buffer (pH 4.3) in the ratio of 75:25 v/v at a flow rate of 1 mL/min. Detection is carried out using a UV-PDA detector at 258 nm. Parameters such as linearity, precision, accuracy, recovery, specificity, and ruggedness are studied as reported in the International Conference on Harmonization guidelines. The retention times for MET, PIO, and GLP are 2.66 + 0.5 min, 7.12 + 0.5 min, and 10.17 + 0.5 min, respectively. The linearity range and percentage recoveries for MET, PIO, and GLP are 10-5000, 10-150, and 1-10 microg/mL and 100.4%, 100.06%, and 100.2%, respectively. The correlation coefficients for all components are close to 1. The relative standard deviations for three replicate measurements in three concentrations of samples in tablets are always less than 2%.

Full text

A simple, precise, rapid, and reproducible reversed-phase
high-performance liquid chromatography method is developed for
the simultaneous estimation of metformin hydrochloride (MET),
pioglitazone hydrochloride (PIO), and glimepiride (GLP) present in
multicomponent dosage forms. Chromatography is carried out
isocratically at 25°C ± 0.5°C on an Inertsil-ODS-3 (C-18) Column
(250 ×
×4.60 mm, 5 µm) with a mobile phase composed of
methanol–phosphate buffer (pH 4.3) in the ratio of 75:25 v/v at a
flow rate of 1 mL/min. Detection is carried out using a UV-PDA
detector at 258 nm. Parameters such as linearity, precision,
accuracy, recovery, specificity, and ruggedness are studied as
reported in the International Conference on Harmonization
guidelines. The retention times for MET, PIO, and GLP are 2.66 +
0.5 min, 7.12 + 0.5 min, and 10.17 + 0.5 min, respectively. The
linearity range and percentage recoveries for MET, PIO, and GLP
are 10–5000, 10–150, and 1–10 µg/mL and 100.4%, 100.06%, and
100.2%, respectively. The correlation coefficients for all
components are close to 1. The relative standard deviations for
three replicate measurements in three concentrations of samples in
tablets are always less than 2%.
Introduction
Metformin hydrochloride (MET) (N,N-dimethylimidodicar-
bonimidic diamide hydrochloride) (Figure 1A) is an orally
administered biguanide widely used in the treatment of type 2
(non-insulin dependent) diabetes mellitus. It improves hepatic
and peripheral tissue sensitivity to insulin without the problem
of serious lactic acidosis commonly found with its analogue,
phenformin. MET is a hydrophilic drug with an oral bioavail-
ability of 50–60% and a relatively short half-life of 1.5–4.5 h (1).
Pioglitazone hydrochloride (PIO) [(±)-5-[[4-[2-(5-ethyl-2-
pyridinyl) ethoxy] phenyl] methyl]-2,4-] thiazolidine-dione
monohydrochloride (Figure 1B) is an oral anti-hyperglycemic
agent which acts primarily by decreasing insulin resistance. It is
used in the treatment of type-II diabetes mellitus (2).
Glimepiride (GLP) 1-[[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrro-
line-1-carboxamido)ethyl]-phenyl]-sulfonyl]-3-(trans-4-me-
thylcyclohexyl) urea (Figure 1C) is a new oral anti-diabetic drug
in the sulfonylurea class, with the advantage of being completely
bioavailable, being effective at low doses in patients with non-
501
Abstract
Simultaneous Estimation of Metformin Hydrochloride,
Pioglitazone Hydrochloride, and Glimepiride by
RP-HPLC in Tablet Formulation
Deepti Jain1,*, Surendra Jain2, Deepak Jain1, and Maulik Amin1
1School of Pharmaceutical Sciences, Rajiv Gandhi Technical University, Pharmacy; 2Shri Ravishankar College of Pharmacy, Pharmacy
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
Journal of Chromatographic Science, Vol. 46, July 2008
* Author to whom correspondence should be addressed: email deepti2515@yahoo.com.
Table I. System Suitability
Serial No. Parameters MET PIO GLP
1 No. of theoretical plates 991 4599 4907
2 HETP 0.251 0.055 0.051
3 Tailing factor 1.13 1.04 0.97
Figure 1. Chemical structures of MET HCl (A), PIO HCl (B), GLP (C).
Figure 2. Representative chromatogram of MET, PIO, and GLP.

Journal of Chromatographic Science, Vol. 46, July 2008
502
insulin-dependent diabetes mellitus, showing linear pharma-
cokinetics, and having a prolonged effect. As with the other
sulphonylureas, glimepiride appears to lower blood glucose
levels by stimulating insulin release from the pancreas (3).
Tablet dosage forms containing MET, PIO, and GLP in ratio of:
500 mg, 15 mg, and 1 mg; and 500 mg, 15 mg, and 2 mg, respec-
tively, of various brands are available in market. MET has been
reported to be determined by HPLC (4,5) from formulations and
in biological fluids. PIO determination has been done by high-
performance liquid chromatography (HPLC) (6,7) and liquid
chromatography (LC)–mass spectrometry (MS)–MS (8) in a
variety of samples, while GLP determinations have been reported
by UV derivative spectrophotometry (9), HPLC (10), and
LC–MS–MS (11). Simultaneous determination of GLP, PIO (12),
and MET, PIO (13) in pharmaceutical dosage forms was reported
by HPLC. However, there is no method available for the simulta-
neous determination of these three drugs. Therefore, an attempt
was made to develop a new, rapid, and sensitive method for the
simultaneous determination of MET, PIO, and GLP. To access the
reproducibility and wide applicability of the developed method, it
was validated as per ICH norm, which is also mandatory (14–15)
Experimental
Instrumentation
The LC system was from Shimadzu (Kyoto, Japan) and was
comprised of a manual injector, double reciprocating plunger
pump LC10 ATvp for constant flow and constant pressure
delivery, and a photodiode array (PDA) detector SPD-M10 Avp
connected to software Class M10A for controlling the instru-
mentation as well as processing the data generated was used.
Reagents and chemicals
PIO and GLP were obtained as pure sam-
ples from Cadila Health Care, Ahemdabad
and MET Hydrochloride was obtained from
Ranbaxy Labs, Dewas as a gift sample.
Acetonitrile, methanol, and glacial acetic
acid were of HPLC grade and supplied by
Merck Ltd., India. Triple distilled water was
generated in house. Tablets, Tribet-1, and
Tribet-2 of Nicholas Piramal India Limited
containing MET, PIO, and GLP in ratio of:
500 mg, 15 mg, and 1 mg; and 500 mg, 15
mg, 2 mg, respectively, were purchased
from a local market.
Chromatographic condition
The isocratic mobile phase consisted of
methanol–phosphate buffer (pH 4.3) in the
ratio of 75:25, v/v, flowing through the
column at a constant flow rate of 1.0
mL/min. An Inertsil-ODS-3 (C-18)
Column (250 ×4.60 mm, 5 µm) was used
as the stationary phase. MET, PIO, and GLP
have different λmax (viz 235, 265, and 227
nm, respectively), but considering the
chromatographic parameter, sensitivity,
and selectivity of the method for all three
drugs, 258 nm was selected as the detec-
tion wavelength for UV–PDA detector.
Standard preparation
Standard stock solution
Standard stock solutions of 10000, 1500,
Table II. Results of Recovery Experiments
Conc. of drug Recovered
Serial. in preanalyzed Std. drug sol. amount*
No. samples (µg/mL) Added (µg/mL) (µg/mL) %Recovered
MET PIO GLP MET PIO GLP MET PIO GLP MET PIO GLP
1 1000 30 2 1000 30 2 1003.4 30.02 2.2 100.3 100.0 100.2
2 2000 60 4 2000 60 4 2001.1 59.56 4.1 100.0 97.6 100.3
3 3000 90 6 3000 90 6 3002.5 89.92 6.3 100.0 99.9 100.2
Mean 100.1 99.16 100.23
S.D. 0.17 1.35 0.057
%RSD 0.16 1.36 0.056
* Mean of three readings.
Table III. Results of Precision
% Mean* S.D. % R.S.D.
MET PIO GLP MET PIO GLP MET PIO GLP
1 Repeatability 100.5 99.1 97.69 0.39 0.20 0.68 0.38 0.2 0.69
2 Intermediate 100.4 99.19 98.60 0.31 0.26 1.10 0.31 0.27 1.11
precision
(day to day)
3 Intermediate 100.6 99.33 100.6 0.30 0.52 0.99 0.29 0.52 0.98
precision
(analyst to analyst)
* Mean of fifteen determinations (3 replicates at 5 concentration levels).
Serial.
no.
Validation
parameter
Figure 3. Representative chromatogram of Metformin, Pioglitazone, and
Glimepiride (by minimizing the scale).

1000 µg/mL of MET, PIO, and GLP were prepared in methanol
respectively.
Working standard solution
Working standard solutions were prepared by taking dilutions
ranging from 10–5000, 10–150, 1–10 µg/mL for MET, PIO, and
GLP, respectively.
Sample preparation
Twenty tablets of Tribet-1 and Tribet-2 of Nicholas Piramal
India Limited containing MET, PIO, and GLP: 500 mg, 15 mg,
and 1 mg; 500 mg, 15 mg, and 2 mg, respectively, were weighed
and crushed to fine powder. Powder equivalent to 500 mg MET
was weighed and dissolved in 100 mL methanol, sonicated for 10
min, and filtered through Whatmann filter paper No. 42; finally,
different concentrations of tablet sample were prepared by the
serial dilution technique.
Results and Discussion
Chromatography
Initially, reversed-phase LC separation was tried using
methanol and water (75:25) as the mobile phase, in which GLP
gave tailing of 2.6, although the other two drugs responded prop-
erly, and the resolution was also poor. The organic content of
mobile phase was also investigated to optimize the separation of
PIO and GLP. To improve the tailing factor, the pH of mobile
phase becomes an important factor. At pH 6.4, the signal-to-noise
ratio for GLP was less, and the retention time was also 14 min.
Thereafter, methanol–phosphate buffer of pH 4.3 in the ratio of
75:25 v/v was selected to improve the resolution, and the tailing
for the three peaks were reduced considerably and brought close
to 1, and the retention time of GLP was also reduced to 10 min.
To analyze these three drugs, detection were tried at various
wavelengths: from 233 nm to 260nm. Initially, 233 nm was
selected, considering the λmax of three drugs (λmax of MET 235
nm, λmax of PIO 227 nm and 265 nm, and λmax of GLP 227).
At 233 nm, MET was found to merge with a component, which
is structurally similar to MET, as it showed a similar spectra but
did not absorb at 258 nm. Because of this component, base-to-
base separation between MET and PIO was also not observed
below 258 nm.
The spectra of MET extended from below 200 nm to 267 nm.
Although the absorbance is less at 258 nm, it is considerable,
and secondly, the concentration of MET in combination is also
very high.
Therefore, 258 nm was found to be suit-
able where all the three drugs could be
detected simultaneously. The sensitivity of
the detector is 0.5.
The concentration of GLP is low, hence
the AUC is not noticeable in comparison
to MET and PIO; therefore, the peak is not
clearly visible on the same scale in chro-
matogram (Figure 2). By minimizing the
scale, the peak corresponding to GLP is
clearly visible (Figure 3).
System suitability
System suitability parameters, such as number of theoret-
ical plates, HETP, and peak tailing, are determined. The results
obtained are shown in Table I. The number of theoretical
plates for MET, PIO, and GLP were 991, 4599, and 4907,
respectively.
Linearity
MET, PIO, and GLP showed a linearity of
response between 10–5000, 10–150, and
1–10 µg/mL, respectively. The linearity
was represented by a linear regression
equation as follows.
Y(MET) = 3706.27 conc. + 98586.40
(r2= 0.9998)
Y(PIO)= 16231.16conc. + 5021.31
(r2= 0.9981)
Y(GP) = 7647.59conc. + 41.12 (r2= 0.9995)
Table IV. Results of Robustness
% Mean* S.D. % R.S.D.
MET PIO GLP MET PIO GLP MET PIO GLP
1 Robustness 98.87 100.59 100.22 0.64 0.99 1.30 0.64 0.98 1.29
* Mean of six determinations.
Journal of Chromatographic Science, Vol. 46, July 2008
503
Serial.
no.
Validation
parameter
Table V. Stability Data of MET, PIO, and GLP
AUC ± %RSD
Hours
MET PIO GLP
(1000 µg/mL) (30 µg/mL) (2 µg/mL)
0 3858142 ± 0.31 483857 ± 0.53 15474 ± 1.25
6 3858231 ± 0.42 483745 ± 0.68 15467 ± 2.23
12 3858443 ± 0.35 483687 ± 0.93 15479 ± 2.25
Table VI. Results of the HPLC Analysis for Tablets
Parameters
TRIBET-1 TRIBET-2
MET PIO GLP MET PIO GLP
1 % Mean* 100.7 99.95 98.77 100.06 98.68 98.88
2 S.D. 0.44 1.21 0.77 0.40 0.98 1.6
3 % R.S.D 0.43 1.20 0.78 0.39 0.99 1.63
4 SEσ0.17 0.49 0.31 0.16 0.39 0.65
* Mean of fifteen determinations (3 replicates at 5 concentration levels).
Serial.
no.

Accuracy
Recovery studies were performed to validate the accuracy of
the developed method. To a pre-analyzed sample solution, a def-
inite concentration of standard drug was added, and the recovery
was studied. These results are summarized in Table II.
Precision
Repeatability
Five dilutions in three replicates were analyzed in same day for
repeatability, and the results were found within acceptable limits
(RSD < 2), as shown in Table III.
Intermediate precision
Five dilutions in three replicates were analyzed on two dif-
ferent days and by two analysts for day-to-day and analyst-to-ana-
lyst variation. Although the relative standard deviation (RSD)
value for GLP is higher than that of MET and PIO, this is because
of its low concentration; however, all results fall within accept-
able limits (RSD < 2), as shown in Table III.
Robustness
As per ICH norms, small but deliberate variations, by altering
the pH or concentration of the mobile phase, were made to check
the method’s capacity to remain unaffected (method stability).
The change was made in the ratio of mobile phase: instead of
methanol–phosphate buffer (pH 4.3) (75:25 v/v), methanol–
phosphate buffer (pH 4.3) (70:30 v/v) was used as the mobile
phase. Results of analysis are summarized in Table IV.
Stability of sample solution
The sample solution injected after 12 h did not show any
appreciable change. Results are shown in Table V.
Tablet analysis
Contents of MET, PIO, and GLP found in the tablets by the
proposed method are shown in Table VI. The low RSD values
indicate that the method is precise and accurate.
Conclusions
An RP-HPLC method was developed and validated for simulta-
neous estimation of MET, PIO, and GLP in tablet dosage form.
The proposed method is fast, accurate, precise, and sensitive, as
it could estimate GLP concentration, which is far less when com-
pared to the other two components; hence, it can be employed
for routine quality control of tablets containing these three
drugs in industries.
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Manuscript received February 7, 2007;
revision received July 7, 2007.
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