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

Authors:
  • Sagar Institute of Research and Technology - Pharmacy, Bhopal

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%.
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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revision received July 7, 2007.
Journal of Chromatographic Science, Vol. 46, July 2008
504
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A stability-indicating UV spectroscopic and high-performance liquid chromatography (RP-HPLC) method is developed for the quantification ofPioglitazone, Glimepiride & Metformin Hydrochloride drug substances. UV spectroscopic method was developed and validated, the wavelength selected for simultaneous estimation were 226nm for pioglitazone, 229nm for glimepiride and 232nm for metformin hydrochloride. The isosbestic point found for the analysis was 229nm. Selected mobile phase was a combination of methanol and water with a ratio of 70% Methanol and 30 % HPLC water with the flow rate of 0.85ml/min. The analyte was analysed on the C18 HPLC column having the pore size of 5 microns at room temperature. The method is validated according to ICH guidelines, the retention time of about 4.0min for metformin, 5.5min for Pioglitazone and 6.8min for Glimepiride was observed. The linearity range with regression co-efficient for Pioglitazone, Glimepiride & Metformin Hydrochloride is 3-15 μg/mL,0.4-1.2 μg/mL and 100-500 μg/mL and 0.9998, 0.9991, 0.9991 respectively.
Article
A rapid and accurate HPLC method has been developed for simultaneous determination of pioglitazone and glimepiride. Chromatographic separation of the two pharmaceuticals was performed on a Cosmosil C18 column (150 mm 4.6 mm, 5 m) with a 45:35:20 (v/v) mixture of 0.01 m triammonium citrate (pH adjusted to 6.95 with orthophosphoric acid), acetonitrile, and methanol as mobile phase, at a flow rate of 1.0 mL min–1, and detection at 228 nm. Separation was complete in less than 10 min. The method was validated for linearity, accuracy, precision, limit of quantitation, and robustness [1, 2]. Linearity, accuracy, and precision were found to be acceptable over the ranges 2.50–30.00 g mL–1 for pioglitazone and 0.10–10.00 g mL–1 for glimepiride.
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A sensitive and selective method for quantitation of glimepiride in human plasma was established using liquid chromatography-electrospray ionization tandem mass spectrometry. Three different methods for the sample preparation of glimepiride and an internal standard were investigated (liquid-liquid extraction, solid-phase extraction and protein precipitation). Glipizide was used as an internal standard. Compounds were separated on a C18 column with 80% acetonitrile and 20% deionized water (adjusted to pH 3.5 with acetic acid), as mobile phase at a flow rate of 200 L min–1. By use of multiple reaction monitoring mode in MS-MS with liquid-liquid extraction and solid-phase extraction, glimepiride and glipizide were detected without severe interference from the human plasma matrix. Glimepiride produced a protonated precursor ion ([M+H]+) at m/z 491 and a corresponding product ion at m/z 352, and the internal standard produced a protonated precursor ion ([M+H]+) at m/z 446 and a corresponding product ion at m/z 321. The limit of quantitation was 0.1 ng mL–1, 0.5 ng mL–1 and 1.0 ng mL–1 when using liquid-liquid extraction, solid-phase extraction and protein precipitation, respectively. The validation, reproducibility, stability, and recovery of the different sample preparation methods were comparable and all the methods gave reliable results. The method has been successfully applied to pharmacokinetic study of glimepiride in human plasma.
Article
A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of pioglitazone (U-72107) and its potential metabolites (M-1 to M-6) in human serum was developed. The method involved a solid phase extraction (SPE) of pioglitazone, its metabolites, and the internal standard (U-92573) from serum using C18 SPE columns with an elution solvent of 0.5 ml of acetonitrile-water (35:65, v/v). Separation of the eight analytes was achieved within 20 min using a reversed-phase Zorbax RX-C8 analytical column (250 mm x 4.6 mm i.d., 5 microns particle size) with a mobile phase of acetonitrile-water (40:60, v/v) containing 3 ml acetic acid per liter mobile phase (apparent pH 5.5). An ultraviolet detector operated at 269 nm was used with a linear response observed from 0.02 to 2 micrograms ml-1 for these analytes except for M-4 which was best fitted with a polynomial regression. Limit of quantitation was found to be 0.02 microgram ml-1 for pioglitazone, M-3, M-5, and M-6; 0.04 microgram ml-1 for M-2 and M-4; and 0.5 microgram ml-1 for M-1 when using a 0.5 ml serum sample for extraction. Obtained from the method validation, intra- and inter-assay precision was < or = 9% and accuracy ranged from -8.2 to 13.4% for all analytes. The applicability of this method has been demonstrated by successfully analyzing clinical serum samples. The strategies in the HPLC characterization and in the SPE procedure development for this method are discussed as well.
Article
A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5-4000 ng/ml.
Article
Glimepiride (Amaryl), which is a new oral antidiabetic drug in the sulfonylurea class, was analysed by using second order derivative UV spectrophotometry. The quantification of glimepiride in dimethylformamide was performed in the wavelength range of 245-290 nm at N = 6, ?lambda = 21. The second order derivative spectra was calculated using peak to peak (lambdaDMF = 263.3-268.2 nm), peak to zero (lambdaDMF = 268.2 nm) and tangent (lambdaDMF = 263.3-271.8 nm) method for calibration curves, the linearity range of 1.00-500.00 microg ml(-1) by using the second order derivative UV spectrophotometric method. The developed method was applied to directly and easily to the analysis of the pharmaceutical tablet preparations. R.S.D. were found to be 4.18% (Amaryl tablet; 1 mg) and 2.21% (Amaryl tablet; 2 mg). The method was completely validated and proven to be rugged. The limit of quantitation and the limit of detection were found as 1.00 and 0.4 microg ml(-1), respectively. This validated derivative UV spectrophotometric method is potentially useful for a routine laboratory because of its simplicity, rapidity, sensitivity, precision and accuracy.
Article
A simple, precise and accurate high performance liquid chromatography (HPLC) method was developed for the simultaneous estimation of metformin with gliclazide and glipizide present in multicomponent dosage forms. The method was carried out on Inertsil C(18) column. A mobile phase composed of acetonitrile-water containing camphor sulphonic acid (adjusted to pH 7 using 0.1 N sodium hydroxide; 75 mM) at a flow rate of 1 ml min(-1) was used for the separation. Detection was carried out at 225 nm. Tolbutamide was used as internal standard. Validation of the developed HPLC method was carried out.
Article
A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of pioglitazone (PIO) and its two metabolites: M-III (keto-derivative) and M-IV (hydroxy-derivative) in human plasma. Human plasma samples of 0.2 ml were extracted by a single step liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography (HPLC) electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C-18 column, ionized using a positive ion atmospheric pressure electrospray ionization source and analyzed using multiple reaction monitoring mode. The ion transitions monitored were m/z 357-->134 for PIO, m/z 371-->148 for M-III, m/z 373-->150 for M-IV and m/z 413-->178 for the internal standard. The chromatographic run time was 2.5 min per injection, with retention times of 1.45, 1.02 and 0.95 min for PIO, M-III and M-IV, respectively. The calibration curves of pioglitazone, M-III and M-IV were well fit over the range of 0.5-2000 ng/ml (r(2)>0.998759) by using a weighted (1/x(2)) quadratic regression. The inter-day precisions of the quality control samples (QCs) were </=10.5% (N=15), coefficient of variation (CV) and the inter-day accuracy (%Nominal) ranged from 84.6 to 103.5% for PIO, 94.4 to 104.0% for M-III, and 96.8 to 101.0% for M-IV. All three analytes demonstrated acceptable short-term, long-term, and freeze/thaw stability. The method is simple, rapid and rugged, and has been applied successfully to sample analysis for clinical studies.
Article
A simple, rapid, and precise method is developed for the quantitative simultaneous determination of metformin and pioglitazone in a combined pharmaceutical-dosage form. Separation is achieved with a Zorbax XDB C(18), 15-cm analytical column using buffer-acetonitrile (66:34, v/v) of pH 7.1, adjusted with orthophosphoric acid as the mobile phase. The buffer used in the mobile phase contains 10mM disodium hydrogen phosphate and 5mM sodium dodecyl sulphate in double-distilled water. The instrumental settings are flow rate of 1 mL/min, column temperature at 40 degrees C, and detector wavelength of 226 nm. The internal standard method is used for the quantitation of the ingredients of this combination. Methylparaben is used as an internal standard. The method is validated and shown to be linear for metformin and pioglitazone. The correlation coefficients for metformin and pioglitazone are 0.9991 and 0.9999, respectively. The relative standard deviations for six replicate measurements in two sets of each drug in the tablets are always less than 2%.
Article
Five impurities in glimepiride drug substance were detected and quantified using a simple isocratic reverse phase HPLC method. For the identification and characterization purpose these impurities were isolated from a crude reaction mixture of glimepiride using a normal phase HPLC system. Based on the spectroscopic data like NMR, FTIR, UV and MS these impurities were characterized and used as impurity standards for determining the relative response factor during the validation of the proposed isocratic reverse phase HPLC method. The chromatographic separation was achieved on a Phenomenex Luna C8 (2) 100 A, 5 microm, 250 mm x 4.6 mm using a mobile phase consisting of phosphate buffer (pH 7.0)-acetonitrile-tetrahydrofuran (73:18:09, v/v/v) with UV detection at 228 nm and a flow rate of 1 ml/min. The column temperature was maintained at 35 degrees C through out the analysis. The method has been validated as per international guidelines on method validation and can be used for the routine quality control analysis of glimepiride as active pharmaceutical ingredient (API).