Simultaneous Determination of Rosiglitazone and Metformin in ...

Simultaneous Determination of Rosiglitazone and Metformin in Pharmaceutical Preparations by LC 2007, 66, 589–593

¨ zaltın Ceren Yardımcı&, Nuran O Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhıye, Ankara, Turkey; E-Mail: [email protected]

Received: 4 May 2007 / Revised: 8 June 2007 / Accepted: 27 June 2007 Online publication: 31 July 2007

Abstract In the present study, a novel, fast and simple liquid chromatographic method was developed and validated for the simultaneous determination of rosiglitazone and metformin in pharmaceutical preparations. The separation was achieved on a phenyl column (250 · 4.6 mm i.d., 5 lm) using a mobile phase composed of acetonitrile:10.0 mM phosphate buffer pH 5.5 (70:30, v/v). The flow rate was 1 mL min)1. UV detection was performed at 245 nm and verapamil was used as internal standard. The developed method was validated in terms of stability, specificity, sensitivity, linearity, accuracy, precision and robustness. The limit of quantification was 0.02 lg mL)1 for both drugs. The method developed was successfully applied to the simultaneous determination of rosiglitazone and metformin in pharmaceutical preparations. The results were compared to two methods reported in the literature and no significant difference was found statistically.

Keywords Column liquid chromatography Rosiglitazone and metformin Validation Drug analysis

Introduction Many patients with type 2 diabetes require treatment with more than one antihyperglycaemic drug to achieve optimal glycaemic control. The thiazolidinediones are novel oral antihyperglycaemic drugs that improve glycaemic control primarily by decreasing insulin resistance by sensitizing the skeletal muscle, liver and adipose tissue to the actions of insulin. They also improve beta-cell function. The

Full Short Communication DOI: 10.1365/s10337-007-0347-y 0009-5893/07/10

combination of metformin hydrochloride (M), a biguanide that enhances glucose uptake in peripheral tissues and reduces hepatic gluconeogenesis, with rosiglitazone maleate (R), one of the newly available members of the thiazolidinedione family, offers a rational therapeutic approach for the treatment of type 2 diabetes [1]. The two agents can be used in combination to achieve additive glucose-lowering efficacy in the treatment of type 2 diabetes, without stimulating insulin secretion and

without causing hypoglycaemia [2]. A fixed-dose formulation of R/M was recently approved in the EU and the US for the treatment of type 2 diabetes mellitus in patients inadequately controlled on M monotherapy. Several methods have been reported for the determination of R in pharmaceutical preparations including liquid chromatography (LC) [3, 4], micellar electrokinetic chromatography (MEKC) [3], high performance thin layer chromatography (HPTLC) [5–7] and spectrophotometry [8]. There are also numerous methods for the determination of M either alone or in combination with various drugs, such as LC [9], spectrophotometry [10–13] and gas chromatography (GC) [14]. In a recent publication we developed a novel method for the simultaneous determination of R and M in human plasma by LC [15]. However, only two studies have been published for the simultaneous determination of these two drugs in pharmaceutical preparations. One of them is an ion-pair LC (IPC) method reported by Kolte et al. [16]. The other method is a capillary zone electrophoresis (CZE) method which reported by us [17] for the simultaneous determination of R and M as an alternative to the published IPC method. The CZE is suitable for routine use and has the advantage of simplicity of operation, flexibility and low cost. However, LC is more common in quality control laboratories because of its high sensitivity and precision. IPC methods have some drawbacks, including slow column

Chromatographia 2007, 66, October (No. 7/8) 2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

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formed and the validated method was successfully applied to the pharmaceutical preparations. Although there is a large difference in the label claims of M and R per tablet, simultaneous analysis of these two drugs has been achieved in the same sample preparation. The method described here comprises the advantage of easy and simple determination of R and M with an isocratic LC method. The developed and validated method is superior to previously described methods with rapid and simple mobile phase and sample preparation step, improved sensitivity and a short chromatographic run time.

Experimental Instrumentation and Chromatographic Conditions

Fig. 1. Representative chromatograms of a tablet excipients (placebo), b tablet excipients after being spiked with standards, c standard solutions, d commercial pharmaceutical preparations (Avandamet containing 2 mg R/500 mg M). Chromatographic conditions: Ace 5 phenyl column (250 · 4.6 mm i.d., 5 lm); mobile phase: acetonitrile:10 mM pH 5.5 KH2PO4 (70:30 v/v); flow rate 1 mL min)1; detection wavelength: 245 nm (R 0.2 lg mL)1, M 50.0 lg mL)1 and V (IS) 5.0 lg mL)1)

equilibrium, irreversible adsorption of ion pair reagents to the stationary phase resulting short column lifetime and poor reproducibility. The described IPC method [16] also requires difficult sample and mobile phase preparation steps. There is no official pharmacopeia method for the simultaneous determination of R and M in pharmaceutical preparations to apply for routine quality control analyses. Therefore, our objective in the

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present study was to develop a novel and simple LC method for the simultaneous determination of R and M in pharmaceutical preparations. For this purpose, the influence of column type, mobile phase composition, buffer type, buffer pH and flow rate was systematically investigated and the simultaneous determination of R and M were achieved without using an ion-pair reagent. The method validation studies were per-

The LC system consisted of a SpectraSYSTEM P2000 gradient pump, a SpectraSYSTEM SCM 1000 degasser, a Rheodyne manual injector with a 20 lL injection loop and a SpectraSYSTEM UV2000 detector (Thermo Separation Products, USA). The analytical column was a Ace 5 phenyl column (250 · 4.6 mm i.d., 5 lm, Advanced Chromatography Technologies, UK) protected by a phenyl guard column (4.0 · 3.0 mm i.d., Phenomenex, USA). A mixture of acetonitrile:10 mM pH 5.5 KH2PO4 (adjusted with 10 mM Na2HPO4) (70:30 v/v) was used as a mobile phase. The flow rate was 1 mL min)1. The detection wavelength was set to 245 nm. Operation, data acquisition and analysis were performed using ChromQuest software. For pH measurements, a pH meter (Mettler Toledo MA 235, Switzerland) was employed. Mobile phase was filtered through a 0.45 lm nylon membrane filter (Advantec MFS, CA, USA) under vacuum and degassed by ultrasonication (Sonorex, Bandelin, Germany).

Reagents Rosiglitazone maleate reference standard and the tablets containing rosiglitazone maleate and metformin hydrochloride (Avandamet containing 2 mg R/500 mg M) were kindly supplied by Glaxo SmithKline (Istanbul, Turkey). Metfor-

Chromatographia 2007, 66, October (No. 7/8)

Full Short Communication

min hydrochloride and verapamil which was used as internal standard (IS) were purchased from Sigma (St Louis, MO, USA) and Acros (New Jersey, USA), respectively. Acetonitrile and methanol were LC grade (Merck, Germany). All other chemicals were analytical reagent grade. Deionized water was prepared using a Barnstead Nanopure Diamond Analytical (IA, USA) ultrapure water system.

Standard and Sample Solutions Standard Solutions

Standard stock solutions (1,000 lg mL)1) of M and verapamil (IS) were prepared in water, R (1,000 lg mL)1) in methanol. These solutions were kept at 4 C. Various aliquots of standard solutions were taken, the IS added and then diluted to 1 mL with mobile phase to give a final desired analyte concentration. Sample Preparation

Ten tablets were weighed and finely powdered in a mortar. A quantity of the powder equivalent to one tablet was accurately weighed and transferred to a 100 mL volumetric flask. The flask was sonicated for 15 min and diluted to the mark with methanol. Then an aliquot was centrifuged at 5,000 rpm for 10 min. Clear supernatant of about 10 lL was transferred to a vial, 25 lL 200 lg mL)1 IS added and diluted with mobile phase to 1 mL. This solution was injected into the LC system.

Results and Discussion

ary phase. Higher polarity of phenyl columns with respect to alkyl-bonded columns can provide alternative retention mechanism in addition to hydrophobic interaction. This retention mechanism is based upon p–p interactions occurring between the analyte molecules and the phenyl-bonded phase [18]. As expected, the use of a phenyl column improved the retention and the peak shape of M. Various mobile phases with different compositions were tried on a phenyl column and the best chromatographic conditions for the simultaneous separation of R and M were achieved with a mobile phase composed of acetonitrile:10.0 mM pH 5.5 KH2PO4 (70:30 v/v). It was found that further increase in acetonitrile composition caused a decrease in the retention time of R, but an increase in the retention time of M. Seventy-percent acetonitrile content was chosen for the best resolution. The effect of pH was studied within the pH range of 5.0–6.0 at a fixed buffer concentration of 10 mM KH2PO4. Since R have a acidic and M have a basic character, their retention times were inversely affected, the former’s decreased and the latter’s increased with increasing pH. The resolution was below 1.0 at pH 6.0, hence pH 5.5 was chosen as the optimized pH. The effect of the concentration of phosphate buffer on the separation of R and M was also investigated. Capacity factor of M decreased distinctly with the increased buffer concentration while the capacity factor of R was not changed. Therefore, 10.0 mM KH2PO4 was selected for further experiments. Under the optimized conditions, the retention times of R, M and V (IS) were 4.26, 3.33 and 5.54 min, respectively, at a flow rate of 1.0 mL min)1.

Specificity, described as the ability of a method to discriminate the analyte from all potential interfering substances, was evaluated by preparing the analytical placebo and it was confirmed that the signals measured were caused only by the analytes. A solution of an analytical placebo (containing all the ingredients of the formulation except the analyte) was prepared according to the sample preparation procedure and injected. To identify the interference by these excipients, a mixture of the inactive ingredients (placebo), before (Fig. 1a) and after being spiked with standards (Fig. 1b), standard solutions (Fig. 1c) and the commercial pharmaceutical preparations including R and M (Fig. 1d) were analyzed by the proposed method. The representative chromatograms show no other peaks which confirm the specificity of the method.

Linearity

The proposed method was validated with respect to stability, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness according to the ICH Guidelines [19].

Stability

LOD and LOQ

Stability of the standard solutions of R and M was evaluated under different

The limits of detection defined as signal-to-noise ratio of 3:1 were about

Method Validation


Specificity

Considering drug content (2 mg R/500 mg M), linearity was studied simultaneously in the concentration range of 0.05–1.2 lg mL)1 for R and 5.0–80.0 lg mL)1 for M. In all cases 5.0 lg mL)1 of verapamil were added as IS. Peak area ratios of the analytes to the internal standard were used to construct calibration curves. The linearity curves were defined by the following equations: y = (0.9294 ± 0.018)x ) (0.0096 ± 0.0015), r = 0.9999 for R and y = (1.327 ± 0.012)x + (0.6495 ± 0.1335), r = 1 for M, where y is the ratio of peak areas and x is the concentration expressed in lg mL)1 (n = 10).

Optimization of Chromatographic Conditions Since M is a highly polar and strongly basic compound (pKa = 12.4), it is poorly retained in reversed-phase LC mode. Preliminary experiments indicated that using different concentrations of acetonitrile or methanol with different pHs of the buffers, did not produce a suitable retention and peak shape of M on a C18 column. Polar molecules elute early on alkylbonded reversed-phase columns (C18 and C8), because they cannot form hydrophobic interactions with apolar station-

storage conditions. For short-term stability, working standard solutions were kept at room temperature for 24 h. The long-term stability was assessed after storage of stock solutions at 4 C for 4 months. The stability results were evaluated by comparing peak area ratios of R and M to IS with those of freshly prepared standard solutions. The results found within 98.14–101.91% of initial values indicate that R and M can be considered stable under the conditions investigated.


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Table 1. System precision data for R and M

Retention time (min) Peak area Capacity factora Peak asymmetry Resolution

R

M

x = 4.26 ± 0.001 SD = 0.004 RSD = 0.10% x = 136274.67 ± 316.69 SD = 950.07 RSD = 0.70% x = 1.03 ± 0.001 SD = 0.004 RSD = 0.43% x = 1.21 ± 0.003 SD = 0.008 RSD = 0.69 % x = 2.74 ± 0.01 (R and V) SD = 0.020 RSD = 0.62%

x = 3.33 ± 0.002 SD = 0.005 RSD = 0.15% x = 164559.56 ± 441.82 SD = 1325.46 RSD = 0.81% x = 0.59 ± 0.001 SD = 0.003 RSD = 0.57% x = 1.35 ± 0.004 SD = 0.011 RSD = 0.83% x = 2.26 ± 0.01 (R and M) SD = 0.019 RSD = 0.77%

x Mean ± standard error, SD standard deviation, RSD relative standard deviation a Methanol was used for the determination of dead volume

Accuracy and Recovery Studies

Table 2. Intra-day and inter-day accuracy and precision data of R and M (n = 6) Added Intra-day (lg mL)1) Founda (lg mL)1) R

0.1 0.4 1.0 M 10.0 30.0 60.0

0.11 0.40 1.00 9.87 29.86 60.08

± ± ± ± ± ±

0.004 0.004 0.004 0.02 0.03 0.06

Inter-day Precision Accuracyb Founda RSD (%) (Bias %) (lg mL)1)

Precision Accuracyb RSD (%) (Bias %)

4.84 1.28 0.75 0.65 0.25 0.22

8.94 1.31 2.89 1.49 0.35 0.47

10.00 0 0 )1.30 )0.47 0.13

0.10 0.39 1.05 9.96 30.14 60.11

± ± ± ± ± ±

0.004 0.004 0.01 0.06 0.04 0.11

0 )2.50 5.0 )0.40 0.47 0.18

RSD Relative standard deviation a Mean ± standard error b Bias %: [(found ) added)/added] · 100

Table 3. Data from the analysis of commercial tablets by the developed LC method and the comparison to the reference methods (n = 6) Label claim

Found (mg) Developed LC method

X = 2.01 ± 0.01 RSD = 1.39% Bias % = 0.50 CL = 1.98–2.04 M (500 mg/tablet) X = 497.74 ± 3.75 RSD = 1.85% Bias % = )0.45 CL = 488.10–507.38 No difference (one-way ANOVA, p > 0.05) R (2 mg/tablet)

Reference IPC method [16]

Reference CZE method [17]

X = 1.96 ± 0.02 RSD = 2.55% Bias % = )2.00 CL = 1.91–2.01 X = 500.28 ± 3.42 RSD = 1.68% Bias % = 0.06 CL = 491.49–509.07

X = 1.98 ± 0.02 RSD = 2.02% Bias % = )1.00 CL = 1.93–2.03 X = 495.36 ± 2.24 RSD = 1.11% Bias % = )0.93 CL = 489.60–501.12

Bias %: [(found ) added)/added] · 100, X Mean ± standard error, RSD relative standard deviation, CL confidence limit

0.01 lg mL)1 for R and 0.005 lg mL)1 for M. The limits of quantification were determined as 0.02 lg mL)1 for R and M with acceptable precision (RSD £ 15.0%, n = 10) and accuracy (%Bias £ 10, n = 10) under the stated conditions.

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standard solution containing 2.0 lg mL)1 R and M. The results were evaluated by considering retention time, peak area, capacity factor, peak asymmetry and resolution values of R and M. The data show good precision of the system with a RSD £ 1% (Table 1). Three different concentrations of R and M (in the linear range) were analyzed in six independent series in the same day (intra-day precision) and six consecutive days (inter-day precision) within each series every sample was injected three times. The RSD values of intra- and inter-day studies varied from 0.75 to 8.94% showed that the precision of the method was satisfactory (Table 2).

Precision The assay was investigated with respect to repeatability and intermediate precision. In order to measure repeatability of the system (injection repeatability), ten consecutive injections were made with a

The accuracy of the proposed method is determined by calculating the percent difference (bias %) between the measured mean concentrations and the corresponding nominal concentrations. Table 2 shows the results obtained for intra and inter-day accuracy. The accuracy of the proposed method was also tested by recovery experiments. Recovery experiments were performed by adding known amounts of R, M and IS to the analytical placebo solution. R and M were spiked at three different concentrations (1 mg R/500 mg M, 2 mg R/ 500 mg M, 4 mg R/500 mg M) according to label claim in the pharmaceutical preparations. Six samples were prepared for each recovery level. Samples were treated as described in the procedure for sample preparation. The obtained recoveries were between 101.67–98.14% with RSD between 0.55–2.68% (n = 3).

Robustness Robustness relates to the capacity of the method to remain unaffected by small but deliberate variations introduced into the method parameters. Several experimental parameters, like mobile phase acetonitrile ratio (68–72%), buffer pH (5.3–5.7), buffer concentration (9–11 mM) and flow rate (0.9–1.1 mL min)1), were varied around the value set in the method to reflect changes likely to arise in different test environments. Analyses were carried out in triplicate and only one parameter was changed in the experiments at a time. The determination of 0.4 lg mL)1 R and



30.0 lg mL)1 M under the various conditions was performed. Each mean value was compared with the mean value obtained by optimum conditions. The statistically comparison was done with t test [20] and no difference was found between results (p = 0.05). Therefore, the method is robust to the small changes in experimental conditions.

Analysis of Pharmaceutical Preparations Developed and validated method was applied to the simultaneous determination of R and M in pharmaceutical preparations. Satisfactory results were obtained for each compound and were found to be in agreement with label claims (Table 3). The IPC method [16] and the CZE method [17] mentioned in the literature were used as comparison methods to evaluate the validity of the method developed. A comparison of the results obtained by three methods was carried out using the one-way ANOVA test. It was indicated that there were no significant differences between the results obtained by three methods (p > 0.05).

Conclusion In this work, a simple, fast and reliable LC method was developed for the simultaneous determination of R and M in pharmaceutical preparations. The simultaneous determination of R and M


could be achieved successfully, although there was a great difference in polarity between the compounds. The developed method exhibits excellent chromatographic performance (e.g. good peak shapes and short analysis time) without requiring ion-pair reagents, thereby avoiding the inherent disadvantages of diminished column lifetime and long equilibration times. The method also shows a good performance with respect to specificity, sensitivity, linearity, accuracy, precision and robustness. It was concluded that the developed method offers several advantages such as rapid and simple mobile phase and sample preparation step, improved sensitivity and a short chromatographic run time as compared to previous methods. This makes the method suitable for routine analysis in quality control laboratories.

Acknowledgments This work is a part of the project (04D05301001) supported by Hacettepe University, Scientific Research Unit.

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