TY - JOUR
AU - Abbulu,, Konde
AB - Abstract A sensitive, rapid and cost-effective method based on HPTLC with UV detection was developed for the quantitation of Glibenclamide (GLIBEN), Rosiglitazone maleate (ROSI) and Metformin hydrochloride (MET) from a combined dosage form. Pre-coated RP-18 F254s aluminum sheets were used as the stationary phase. Methanol–tetrahydrofuran–water–glacial acetic acid (16: 3.6: 4: 0.4, v/v) used as the mobile phase, along with chamber saturation of 10 min offered an optimum migration (Rf = 0.54, 0.62 and 0.80 for GLIBEN, ROSI and MET, respectively). TLC Scanner 3 was used for densitometric evaluation of the chromatograms. DigiStore 2 Documentation System with winCATS software version 1.4.10 was used for the quantitation and photodocumentation. The LOD for GLIBEN, ROSI and MET was found to be 80 ng, 80 ng and 48 ng, respectively. Moreover, the LOQ was 200 ng, 200 ng and 120 ng for GLIBEN, ROSI and MET, respectively. The method was linear for GLIBEN (r = 0.9991), ROSI (r = 0.9993) and MET (r = 0.9988) within the tested range (200–1000, 200–1000 and 120–600 ng/band, respectively). The method was found to be precise and accurate for all the three drugs. The method was applied for the analysis of Triglucored tablets, and it proved to be a reliable quality control tool for the routine analysis of GLIBEN, ROSI and MET in a combined dosage form. Introduction Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, glycosuria, hyperlipidemia, negative nitrogen balance and ketonemia (1). Combination therapy helps in patient compliance and better relief from the ailment. Combination of GLIBEN, ROSI and MET is formulated to control high blood glucose levels in patients with type 2 diabetes (2). Glibenclamide (Figure 1A) {1-[[4-[2-[(5-chloro-2-methoxybenzoyl)amino]ethyl]phenyl]sulfonyl]-3- cyclohexylurea} (GLIBEN) is a second-generation sulfonylurea antidiabetic which exerts both pancreatic (short-term) and extrapancreatic (long-term) actions. By binding to the sulfonylurea receptors on the pancreatic β cell membrane, it results in rapid increase in insulin release. It also reduces hepatic gluconeogenesis and glycogenolysis. GLIBEN increases the glucose uptake in the liver and its utilization in the skeletal muscles. Figure 1 Open in new tabDownload slide Chemical structure of (A) Glibenclamide, (B) Metformin hydrochloride and (C) Rosiglitazone maleate. Figure 1 Open in new tabDownload slide Chemical structure of (A) Glibenclamide, (B) Metformin hydrochloride and (C) Rosiglitazone maleate. Rosiglitazone maleate (Figure 1B) (+)-5-[[4-[2-(methyl-2-pyridylamino)ethoxy]phenyl] methyl]-2,4-thiazolidinedione,(Z)-2-butenedioate (1:1) (ROSI) is a thiazolidinedione oral antidiabetic agent. It is a highly selective and potent agonist for the nuclear peroxisome proliferation-activated receptors γ which are found in key target areas for insulin action, such as adipose tissue, muscle and liver. It thus improves glycemic control by reducing circulating insulin levels. Metformin hydrochloride (Figure 1C) [3-(diaminomethylidene)-1, 1-dimethylguanidine] (MET) is a biguanide antidiabetic agent. It decreases hepatic glucose production as well as its intestinal absorption and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. MET also enhances the binding of insulin to its receptors and stimulate insulin-mediated glucose disposal (1). As per literature review, GLIBEN, ROSI and MET were analyzed by Ultra-Violet (UV) spectrophotometric method either alone (3) or in combination with other drugs (4–7). Most quality control laboratories make use of expensive and elaborate high-performance liquid chromatographic (HPLC) methods to carry out the analysis of these drugs. Many HPLC methods for the analysis of these individual drugs (8–12) or in combination with other drugs (13–25) were reviewed. There is only one HPLC method available in which GLIBEN, ROSI and MET were analyzed simultaneously from a dosage form (26). A method using capillary zone electrophoresis for the determination of MET and ROSI is also available (27). There are few thin-layer chromatographic (TLC) and high-performance thin-layer chromatographic (HPTLC) methods reported for the analysis of these individual drugs (28–30), for MET and ROSI combination (31, 32) and for combination with other antidiabetic drugs (33). However, the literature survey did not reveal any method for the simultaneous estimation of Glibenclamide, Rosiglitazone maleate and Metformin hydrochloride using HPTLC technique i.e. RP-HPTLC in particular. The aim of this research project was to devise a new, simple and reliable sensitive method, validated using ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) Q2 (R1) guidelines (34) for the quantitative analysis of Glibenclamide, Rosiglitazone maleate and Metformin hydrochloride as bulk drugs as well as in combined dosage forms for in vitro studies. HPTLC is a good alternative to other analytical methods for routine quality control analysis because of the several advantages it offers. The HPLC method developed by Havele et al. for the analysis of the MET, ROSI and GLIBEN (26) has a run time of 10 min for the analysis of a single sample solution. The major advantage of the developed HPTLC method over the HPLC method is that several samples can be run simultaneously using a small quantity of mobile phase thus reducing the analysis time and cost per analysis. In HPTLC, the chromatograms can be re-evaluated several times if required by proper storage of the developed plates. There are also no limitations on the use of the solvents as diluents or mobile phase as these do not hamper the packing of the stationary phase, since it is an open system. Materials and Experimental Conditions Chemicals, reagents and materials The working standards Glibenclamide API was obtained from Sanofi Aventis, Goa, India; Rosiglitazone maleate API was obtained from Cipla Pvt. Ltd, Goa, India; and Metformin hydrochloride API was obtained from Kare Laboratories, Goa, India, as gift samples. The Triglucored tablets manufactured by Atoz Life Sciences, Thavalakuppam, Pondicherry, India, were purchased from the local market. Methanol (GR grade, for Analysis), Tetrahydrofuran (GR, for Analysis) and glacial acetic acid (GR grade, for Analysis) were purchased from Merck (Mumbai, India). Water (HPLC grade) was purchased from S.D. Fine Chemicals Limited (Mumbai, India). The stationary phase used was 20 × 20 cm RP-18 F254s TLC aluminum sheets (1.05559.0001) purchased from Merck KGaA, 64271 Darmstadt, Germany. These RP-18 precoated TLC sheets were cut into pieces of dimensions 20 × 10 cm and 10 × 10 cm as per the number of bands to be applied on it. Instrumentation Sample application as narrow bands was done using the spray-on technique of the Linomat 5 Sample applicator with the help of a Hamilton syringe. The development of the plates was done in the CAMAG Twin trough chamber with stainless steel lids. The CAMAG UV Detection Chamber was used for inspecting high-performance thin-layer chromatograms in an undarkened room. CAMAG TLC Scanner 3 (spectral range: 190 to 800 nm) with a deuterium lamp controlled by winCATS software was used for the densitometric evaluation of the chromatograms. Reprostar 3, a DigiStore 2 Documentation system with high-resolution 12 bit CCD camera and winCATS was used for archiving of all images of a HPTLC plate in one file together along with all analysis data. Mettler Toledo AG285 analysis balance and Mettler Toledo MX5 microbalance were used for weighing. A Leelasonic 200 bath sonicator was used to dissolve the solute in the solvent and for mixing the various solvents of the mobile phase. A Beckman coulter bench top centrifuge apparatus was used for the sample preparation. Chromatographic procedure The chromatographic separation was done using 20 × 10 cm precoated RP-18 F254s TLC aluminum sheets (Merck) as the stationary phase. The standard and sample solutions from a 100-μL Hamilton syringe were applied as 8 mm bands on to the HPTLC plates with the spray-on technique using nitrogen gas. This was done at a constant application rate of 150 nL/s using CAMAG Linomat 5 sample applicator. The bands were air dried for 2 to 3 min. The plates were developed in the 20 × 10 cm sized CAMAG Twin trough chambers previously saturated with methanol–tetrahydrofuran (THF)–water–glacial acetic acid (16: 3.6: 4: 0.4) mobile phase for 10 min at R.T. (25°C) in the ascending mode. The plates were removed from the chamber after attaining a solvent front at 80% of the plate length (development time 10 min). The solvent front was marked, and the plates were air-dried for 10–15 min. Densitometric analysis was carried out using CAMAG TLC Scanner 3 at 237 nm for Glibenclamide, 324 nm for Rosiglitazone maleate and 240 nm for Metformin hydrochloride. The chromatograms were integrated using winCATS software, and the results were evaluated. Preparation of standard solutions Glibenclamide stock solution (1 μg/μL) was prepared by dissolving 10 mg of Glibenclamide with 10 mL of methanol in a 10-mL volumetric flask. Similarly 1 μg/μL of Rosiglitazone maleate stock solution and 1 μg/μL of Metformin hydrochloride solution were prepared. Standard mixture solution The standard mixture solution is prepared by adding 0.6 mL of Metformin hydrochloride stock solution, 1 mL of Glibenclamide stock solution and 1 mL of Rosiglitazone maleate stock solution in a 10-mL volumetric flask. To this is added 7.4 mL of methanol, and this is mixed well. The concentration of Metformin hydrochloride is 0.06 μg/μL, Glibenclamide is 0.1 μg/μL and Rosiglitazone maleate is 0.1 μg/μL in this standard mixture solution. Method validation The method was validated as per the ICH Q2 (R1) guidelines. System suitability System suitability is based on the concept that the samples to be analyzed, analytical operations, equipments and the electronics form an integral system (34). System suitability was determined by applying six replicate bands of volume 6 μL of the standard mixture solution on chromatographic plate (10 × 10 cm). The chromatographic plate was then scanned at 237 nm for GLIBEN, 324 nm for ROSI and 240 nm for MET. Table I Results of Method Validation and Assay Parameter . Glibenclamide . Rosiglitazone . Metformin . System suitability % RSD = 1.36 (peak area) % RSD = 0 (Rf) % RSD = 1.27 (peak area) % RSD = 0 (Rf) % RSD = 1.49 (peak area) % RSD = 0 (Rf) Linearity (coefficient of correlation) 0.9991 0.9993 0.9988 Range 200–1000 ng/band 200–1000 ng/band 120–600 ng/band Accuracy 97.453% 99.343% 97.446% Precision (intra-day) (%RSD) 0.44 1.004 0.362 Precision (inter-day) (%RSD) 0.368 0.651 0.358 LOD 80 ng/band 80 ng/band 48 ng/band LOQ 200 ng/band 200 ng/band 120 ng/band Robustness (%RSD) 1.218 1.356 0.973 Assay 101.82% 101.40% 99.14% Parameter . Glibenclamide . Rosiglitazone . Metformin . System suitability % RSD = 1.36 (peak area) % RSD = 0 (Rf) % RSD = 1.27 (peak area) % RSD = 0 (Rf) % RSD = 1.49 (peak area) % RSD = 0 (Rf) Linearity (coefficient of correlation) 0.9991 0.9993 0.9988 Range 200–1000 ng/band 200–1000 ng/band 120–600 ng/band Accuracy 97.453% 99.343% 97.446% Precision (intra-day) (%RSD) 0.44 1.004 0.362 Precision (inter-day) (%RSD) 0.368 0.651 0.358 LOD 80 ng/band 80 ng/band 48 ng/band LOQ 200 ng/band 200 ng/band 120 ng/band Robustness (%RSD) 1.218 1.356 0.973 Assay 101.82% 101.40% 99.14% Open in new tab Table I Results of Method Validation and Assay Parameter . Glibenclamide . Rosiglitazone . Metformin . System suitability % RSD = 1.36 (peak area) % RSD = 0 (Rf) % RSD = 1.27 (peak area) % RSD = 0 (Rf) % RSD = 1.49 (peak area) % RSD = 0 (Rf) Linearity (coefficient of correlation) 0.9991 0.9993 0.9988 Range 200–1000 ng/band 200–1000 ng/band 120–600 ng/band Accuracy 97.453% 99.343% 97.446% Precision (intra-day) (%RSD) 0.44 1.004 0.362 Precision (inter-day) (%RSD) 0.368 0.651 0.358 LOD 80 ng/band 80 ng/band 48 ng/band LOQ 200 ng/band 200 ng/band 120 ng/band Robustness (%RSD) 1.218 1.356 0.973 Assay 101.82% 101.40% 99.14% Parameter . Glibenclamide . Rosiglitazone . Metformin . System suitability % RSD = 1.36 (peak area) % RSD = 0 (Rf) % RSD = 1.27 (peak area) % RSD = 0 (Rf) % RSD = 1.49 (peak area) % RSD = 0 (Rf) Linearity (coefficient of correlation) 0.9991 0.9993 0.9988 Range 200–1000 ng/band 200–1000 ng/band 120–600 ng/band Accuracy 97.453% 99.343% 97.446% Precision (intra-day) (%RSD) 0.44 1.004 0.362 Precision (inter-day) (%RSD) 0.368 0.651 0.358 LOD 80 ng/band 80 ng/band 48 ng/band LOQ 200 ng/band 200 ng/band 120 ng/band Robustness (%RSD) 1.218 1.356 0.973 Assay 101.82% 101.40% 99.14% Open in new tab Figure 2 Open in new tabDownload slide UV spectrum of (A) Metformin hydrochloride, (B) Rosiglitazone maleate and (C) Glibenclamide. Figure 2 Open in new tabDownload slide UV spectrum of (A) Metformin hydrochloride, (B) Rosiglitazone maleate and (C) Glibenclamide. Specificity Specificity is the ability to assess unequivocally the analyte in the presence of the components that may be expected to be present along with the analyte in the sample solution (34). Specificity was determined by applying the diluent, mobile phase, placebo, MET standard solution, ROSI standard solution, GLIBEN standard solution, standard mixture solution and tablet solution as bands on a RP-18 F254s chromatographic plate (10 × 10 cm). The plate was developed using the developed method till the level marked on the plate. The plate was dried completely. The chromatographic plate was viewed in the CAMAG TLC scanner 3 at 254 nm to inspect for bands at the retardation factor (Rf) of the three analytes. The plate was then scanned at 240 nm for MET, 237 nm for GLIBEN and 324 nm for ROSI in the densitometer to confirm for any interference at the Rf of the analytes at their respective detection wavelength. Linearity and range The linearity of an analytical method is its ability to elicit test results that are directly, or by means of well-defined mathematical transformations, proportional to the concentration of the analytes present in the samples within a given range (34). The linearity was determined by applying the standard mixture solution as bands of volume 2 μL, 4 μL, 6 μL, 8 μL and 10 μL. This would give a concentration range of 120–600 ng/band for MET, 200–1000 ng/band for GLIBEN and 200–1000 ng/band for ROSI. Limit of detection and limit of quantitation The limit of detection (LOD) is defined as the lowest concentration of an analyte in the sample that can be detected, but not necessarily quantitated. The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. LOD and LOQ were determined by the signal-to-noise ratio method (34). Accuracy The accuracy of an analytical method is the extent to which test results generated by the method and the true value agree. Accuracy was determined by standard addition technique and by performing recovery studies (34). Due to the vast differences in the strengths of Glibenclamide (5 mg), Rosiglitazone maleate (2 mg) and Metformin hydrochloride (500 mg) in the tablet formulation, two solutions were prepared for the recovery studies. One solution for the recovery study of GLIBEN and ROSI while the other, for the recovery study of MET. A standard mixture solution I containing 0.1 mg/mL of GLIBEN and 0.1 mg/mL of ROSI was prepared using GLIBEN and ROSI stock solutions. A known amount of this standard mixture solution I was added to the tablet powder to prepare 80, 100 and 120% with respect to the target assay concentration. The volume flasks were sonicated for 30 min, and the solution was centrifuged. The centrifugate was used for the Recovery study of GLIBEN and ROSI. Similarly, a 0.06 mg/mL standard solution of MET was prepared. This was added to the tablet powder and processed in a similar manner as that done for GLIBEN and ROSI. On one chromatographic plate, accuracy samples of GLIBEN and ROSI of three different concentrations were applied (triplicate bands of each concentration) along with the standard mixture solution (in various concentrations). Hence, linearity and accuracy for GLIBEN and ROSI were assessed on this plate by quantitating at 237 nm for GLIBEN and 324 nm for ROSI. Similarly on another plate, accuracy samples of MET of three different concentrations were applied (triplicate bands of each concentration) along with the standard mixture solution (in various concentrations). The bands on this plate were quantitated at 240 nm to assess the linearity and accuracy for MET. Precision Precision expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision was considered at three levels: repeatability, intermediate precision and reproducibility. The precision of the method was expressed as the percent relative standard deviation or percent coefficient of variation of a series of measurements (34). Figure 3 Open in new tabDownload slide HPTLC chromatograms of (A) Rosiglitazone maleate, (B) Metformin hydrochloride and (C) Glibenclamide (in a standard mixture solution) quantitated at their respective λmax. Figure 3 Open in new tabDownload slide HPTLC chromatograms of (A) Rosiglitazone maleate, (B) Metformin hydrochloride and (C) Glibenclamide (in a standard mixture solution) quantitated at their respective λmax. Repeatability was assessed by applying the homogeneous solution containing target assay concentration six times on a plate and developing it according to the above method. Intermediate precision was determined by the performing intraday and interday precision studies. These were carried out by preparing a standard solution of GLIBEN, ROSI and MET and a sample solution using the tablet powder at the different time intervals. Reproducibility also known as ruggedness of the method was determined by another analyst who performed the precision of the method in a different laboratory. Robustness Robustness of the method was determined by making small deliberate changes in the method. Changes in the saturation time (15 min) and the mobile phase composition (methanol: THF: water: glacial acetic acid in the ratio of 16: 3.6: 4: 0.5, v/v) were made to check for variations in the results. Figure 4 Open in new tabDownload slide System suitability for Glibenclamide, Rosiglitazone maleate and Metformin HCl. Figure 4 Open in new tabDownload slide System suitability for Glibenclamide, Rosiglitazone maleate and Metformin HCl. Figure 5 Open in new tabDownload slide Specificity for Glibenclamide, Rosiglitazone maleate and Metformin hydrochloride. D is diluent, P is placebo, Mob is mobile phase, M is Metformin, R is Rosiglitazone, G is Glibenclamide, Mix is standard mixture solution and T is the tablet sample solution. Figure 5 Open in new tabDownload slide Specificity for Glibenclamide, Rosiglitazone maleate and Metformin hydrochloride. D is diluent, P is placebo, Mob is mobile phase, M is Metformin, R is Rosiglitazone, G is Glibenclamide, Mix is standard mixture solution and T is the tablet sample solution. Figure 6 Open in new tabDownload slide A developed RP-18 F254s HPTLC precoated plate depicting bands for the linearity and accuracy of Glibenclamide and Rosiglitazone maleate. S1–S5 is the standard mixture solution (in varying concentrations) for linearity and R1, R2 and R3 are samples for recovery studies of Glibenclamide and Rosiglitazone maleate. Figure 6 Open in new tabDownload slide A developed RP-18 F254s HPTLC precoated plate depicting bands for the linearity and accuracy of Glibenclamide and Rosiglitazone maleate. S1–S5 is the standard mixture solution (in varying concentrations) for linearity and R1, R2 and R3 are samples for recovery studies of Glibenclamide and Rosiglitazone maleate. Figure 7 Open in new tabDownload slide A developed RP-18 F254s HPTLC precoated plate depicting bands for the linearity and accuracy of Metformin hydrochloride. Figure 7 Open in new tabDownload slide A developed RP-18 F254s HPTLC precoated plate depicting bands for the linearity and accuracy of Metformin hydrochloride. Assay of the marketed formulation (Triglucored tablets) Procedure Ten tablets were weighed, and the average weight was determined. The tablets were crushed to prepare a homogeneous mixture. Because of the vast differences in the strength of Metformin hydrochloride (500 mg), Glibenclamide (5 mg) and Rosiglitazone maleate (2 mg) in the tablet formulation, two separate solutions were prepared, one for determination of the label claim of Metformin hydrochloride and the other for determination of the label claim of Glibenclamide and Rosiglitazone maleate. For the determination of label claim of Metformin hydrochloride in the tablets A tablet powder equivalent to 10 mg MET was accurately weighed and transferred into a 10-mL volumetric flask. To this, 5 mL methanol was added and this was sonicated for 30 min. The volume was made up to 10 mL, and then the flask was sonicated for about 5 min. This solution (1 μg/μL) was centrifuged and used to determine the label claim of MET in the tablets. A 0.06-μg/μL sample solution was prepared from it. This sample solution along with a standard mixture solution was applied on a precoated RP-18 F254s chromatographic plate. The plate was developed as per the validated chromatographic method, dried completely and then scanned at 240 nm in the densitometer. For the determination of label claim of Glibenclamide and Rosiglitazone maleate in the tablets A tablet powder equivalent to 2.5 mg GLIBEN and 1 mg ROSI was accurately weighed and transferred into a 10-mL volumetric flask. This was further processed similarly like MET, and from it a sample solution containing 0.25 μg/μL of GLIBEN and 0.1 μg/μL of ROSI was prepared. This sample solution along with a standard mixture solution was applied on a precoated RP-18 F254s chromatographic plate. The plate was developed as per the validated chromatographic method, dried completely and then scanned at 237 nm for GLIBEN and 324 nm for ROSI in the densitometer. Results The trio anti-diabetic combination of MET, GLIBEN and ROSI when eluted on RP-18 F254s TLC aluminum sheets using methanol–THF–water–glacial acetic acid (GAA) (16: 3.6: 4: 0.4) as the mobile phase showed a good resolution in a short development time. The Rf obtained was 0.54 for GLIBEN, 0.62 for ROSI and 0.80 for MET. The UV Spectra of MET, ROSI and GLIBEN with their respective λmax are shown in Figure 2. The HPTLC chromatograms of GLIB, ROSI and MET from a standard mixture solution quantitated at their respective λmax of 237 nm, 324 nm and 240 nm are shown in Figure 3. The results of all the method validation parameters performed as per the ICH Q2 (R1) guidelines are given in Table I. Figures 4A and Figure 4B depict the developed plate for the system suitability of GLIBEN, ROSI and MET. The plate was photo-documented at 254 nm (short-wave UV) and at 366 nm (long-wave UV). Figure 4A shows the bands of GLIBEN, ROSI and MET at 254 nm. The bands have a good resolution from each other, and the % RSD for the peak area and for Rf is less than 2%. Figure 4B at 366 nm shows the presence of ROSI as fluorescent spots since its λmax is at 324 nm. Figures 5A and Figure 5B at 254 and 366 nm (bands of ROSI are seen), respectively, show no bands and hence no peaks of diluent, placebo or mobile phase at the respective Rf of GLIBEN, ROSI and MET as interferences. Hence, the method is specific for GLIBEN, ROSI and MET. The calibration curve of GLIBEN (y = 3.5069x + 401.11), ROSI (y = 2.9304x + 173.37) and MET (y = 8.5492x + 361.16) showed good linearity within the tested concentration range of 200–1000 ng/band for GLIBEN, 200–1000 ng/band for ROSI and 120–600 ng/band for MET. Hence, Beer–Lambert’s law was obeyed (results are given in Table I). The calibration curves of GLIBEN, ROSI and MET are shown as Figures S1–S3. The limit of detection and limit of quantitation were 80 and 200 ng/band for GLIBEN, 80 and 200 ng/band for ROSI and 48 and 120 ng/band for MET, respectively. Hence, the method is very sensitive. The percent RSD for repeatability or system precision was 1.36% for GLIBEN, 1.27% for ROSI and 1.49% for MET. The results for the intraday and interday precision studies were also within the acceptance limits (%RSD should be less than 2%) (results are given in Tables SI and SII). Hence, the method is precise. The recovery studies proved that the method is accurate as the results were within the acceptance limits of 80–120% (results are given in Table SIII). Figures 6 and 7 depict the developed plates for the linearity and accuracy of GLIBEN, ROSI and MET, respectively. The proposed method was found to be robust as the percent RSD was found to be 1.218% for GLIB, 1.356% for ROSI and 0.973% for MET. The above validated method was successfully used to perform the assay of Triglucored tablets from the local market. The percent assay was found to be 101.82% for Glibenclamide, 101.40% for Rosiglitazone maleate and 99.14% for Metformin hydrochloride. Discussion Optimization of the HPTLC method The logP value (distribution coefficient of the drug in 1-octanol and water) of MET, GLIBEN and ROSI is −1.3, 2.4 and 4.2, respectively. Since the drugs are highly soluble in water (MET), or slightly soluble (GLIBEN) to soluble (ROSI) in solvents like ethanol, methanol, acetone and chloroform, they would be better separated by reversed-phase chromatography. The stationary phase used was non-polar (RP-18 F254s TLC aluminum sheets) as it would show better elution of the drugs with help of the polar mobile phase. Methanol and water were used as the neat solvents in the proportion 16:4. Methanol and acetonitrile (ACN) combination in the ratio 16:4 was also tried. THF was added to give compact bands, as addition of water caused spreading of the bands. Small amount of GAA was used as a modifier to enhance the separation efficiency of mobile phase. Trials with various ratios of the above solvents were tried to adjust the strength of the mobile phase, example ACN–water–THF–GAA (16:1:1:1), methanol–ACN–water–THF–GAA (14: 1: 2: 2: 0.4), methanol–THF–water–GAA (16: 4: 4: 0.8). The mobile phase was finally optimized to methanol–THF–water- GAA (16: 3.6: 4: 0.4) as it gave compact, separated bands and a short development time. The Twin trough chamber was saturated with the mobile phase for 10 min as this resulted in the decrease in the volume of the mobile phase required to develop the plate, decrease in the development time and no ‘edge effect’. Due to the vast difference in the strengths of MET (500 mg), GLIBEN (5 mg) and ROSI (2 mg) in the Triglucored tablets, two sample solutions had to be prepared for precision, recovery and assay studies. One solution was prepared considering GLIBEN and ROSI as the analytes under analysis. Moreover, only the peaks obtained from these were quantitated, since the concentration of MET in this solution was very high. While another sample solution was prepared which was used for the quantitation of only MET, since the concentration of GLIBEN and ROSI in it was very less. Furthermore, ROZI showed three absorption maxima, first at 200 nm, second at 243 nm and third at 324 nm (which was λmax). A common wavelength for quantitation (240 nm) could be considered for all three drugs, but at this wavelength ROSI showed less sensitivity. Hence, three wavelengths were considered for quantitation viz. 237 nm for GLIBEN, 324 nm for ROSI and 240 nm for MET (Figures 2A–C give the UV spectra depicting the λmax for MET, ROSI and GLIBEN, respectively). Method validation The results depict that the HPTLC system along with the electronics, winCATS software, the developed analytical method and the three antidiabetic drugs viz. GLIBEN, ROSI and MET form an integral system. Hence, the HPTLC system was suitable for the developed method of GLIBEN, ROSI and MET. Conclusion The developed HPTLC method was found to be a very sensitive, rapid, accurate and a precise method. It has proved to be an efficient tool for the simultaneous analysis of Glibenclamide, Rosiglitazone maleate and Metformin hydrochloride. The method can be readily opted as an alternative to the expensive and time-consuming HPLC methods to separate and analyze the above antidiabetic trio from bulk drugs as well as from their multicomponent dosage forms. Acknowledgments The authors are very grateful to Sanofi Aventis, Goa, India; Cipla Pvt. Ltd, Goa, India; and Kare Laboratories, Goa, India, for providing pure drugs of Glibenclamide, Rosiglitazone maleate and Metformin hydrochloride, respectively, as gift samples. The authors are also immensely grateful to Anchrom Enterprises Pvt. Ltd, Mumbai, India, for providing excellent facilities for carrying out this research work. References 1. Tripathi , K.D. ; Essentials of medical pharmacology, 6th ed., Chapter 19; Jaypee Brothers Medical Publishers (P) Ltd. , New Delhi , ( 2008 ); Pg. 259 , 266 – 270 . OpenURL Placeholder Text WorldCat 2. Petersons , C.J. ; Second steps in managing type 2 diabetes ; Australian Prescriber , ( 2018 ); 41 ( 5 ): 141 – 144 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Arayne , M.S. , Sultana , N. , Zuberi , M.H. , Siddiqui , F.A. ; Spectrophotometric quantitation of Metformin in bulk drug and pharmaceutical formulations using multivariate technique ; Indian Journal of Pharmaceutical Sciences , ( 2009 ); 71 ( 3 ): 331 – 335 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Puranik , M. , Wadher , S.J. , Yeole , P.G. , Thakur , S. ; Simultaneous estimation of Metformin HCl and Rosiglitazone maleate in solid dosage form by ultra-violet spectrophotometry ; Indian Drugs , ( 2005 ); 42 ( 7 ): 428 – 431 . OpenURL Placeholder Text WorldCat 5. Mullani , A.K. , Bhatia , N.M. , Bhatia , M.S. ; Derivative spectrophotometric estimation of Rosiglitazone maleate and Metformin hydrochloride ; Asian Journal of Chemistry , ( 2008 ); 20 ( 1 ): 236 – 240 . OpenURL Placeholder Text WorldCat 6. Lalhriatpuii , T.C. , Kawathekar , N. ; Derivative spectrophotometric estimation of Pioglitazone and Metformin HCl ; Indian Drugs , ( 2005 ); 42 ( 11 ): 740 – 743 . OpenURL Placeholder Text WorldCat 7. Doredla , N.R. , Shanmugasundaram , P. , Hemant , V. ; Method development and validation of simultaneous estimation of Metformin hydrochloride, Pioglitazone hydrochloride and Glibenclamide in pure and tablet dosage form by spectrophotometric multi component method ; International Journal of ChemTech Research , ( 2010 ); 3 ( 4 ): 2011 – 2017 . OpenURL Placeholder Text WorldCat 8. Wanjari , M. , Sahu , D. , There , A. , Jawale , N. , Tajne , M.R. , Chopde , C. , et al. ; Determination of Metformin in rat plasma by RP-HPLC method with UV detection ; Indian Journal of Pharmaceutical Sciences , ( 2008 ); 70 ( 2 ): 198 – 202 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Bhavesh , D. , Chetan , G. , Bhat , K.M. ; Shivprakash; estimation of pharmacokinetics of Metformin in human volunteers ; Indian Journal of Pharmaceutical Education and Research , ( 2007 ); 41 ( 2 ): 135 – 139 . OpenURL Placeholder Text WorldCat 10. Chen , X. , Gu , Q. , Qiu , F. , Zhong , D. ; Rapid determination of Metformin in human plasma by liquid chromatography-tandem mass spectrometry method ; Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences , ( 2004 ); 802 ( 2 ): 377 – 381 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Wang , Y. , Tang , Y. , Gu , J. , Fawcett , J.P. , Bai , X. ; Rapid and sensitive liquid chromatography- tandem mass spectrometric method for the quantitation of Metformin in human plasma ; Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences , ( 2004 ); 808 ( 2 ): 215 – 219 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Niopas , I. , Daftsios , A.C. ; A validated high performance liquid chromatographic method for the determination of Glibenclamide in human plasma and its application to pharmacokinetic studies ; Journal of Pharmaceutical and Biomedical Analysis , ( 2002 ); 28 : 653 – 657 . Google Scholar Crossref Search ADS PubMed WorldCat 13. Onal , A. ; Spectrophotometric and HPLC determinations of anti-diabetic drugs Rosiglitazone maleate and Metformin hydrochloride in pure form and in pharmaceutical preparations ; European Journal of Medicinal Chemistry , ( 2009 ); 44 : 4998 – 5005 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Kolte , B.L. , Raut , B.B. , Deo , A.A. , Bagool , M.A. , Shinde , D.B. ; Simultaneous determination of Metformin in combination with Rosiglitazone by reversed-phase liquid chromatography ; Journal of Chromatographic Science , ( 2004 ); 42 : 70 – 73 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Ali , A.R. , Duraidi , I.I. , Saket , M.M. , Abu-Nameh , E.S. ; Column high-performance liquid chromatographic method for the simultaneous determination of Rosiglitazone and Metformin in a pharmaceutical preparation ; Journal of AOAC International , ( 2009 ); 92 ( 1 ): 119 – 124 . Google Scholar Crossref Search ADS PubMed WorldCat 16. Sultana , N. , Arayne , M.S. , Shafi , N. , Siddiqui , F.A. , Hussain , A. ; Development and validation of new assay method for the simultaneous analysis of Diltiazem, Metformin, Pioglitazone and Rosiglitazone by RP-HPLC and its applications in pharmaceuticals and human serum ; Journal of Chromatographic Science , ( 2011 ); 49 : 774 – 779 . Google Scholar Crossref Search ADS PubMed WorldCat 17. SaiThanuja , V. , Chandan , R.S. , Tengli , A.R. , Gurupadayya , B.M. , Prathyusha , W. ; Stability indicating RP-HPLC method for the simultaneous estimation of Metformin hydrochloride, Pioglitazone hydrochloride and Glibenclamide in bulk and pharmaceutical dosage forms ; IOSR Journal of Pharmacy and Biological Sciences , ( 2014 ); 9 ( 1 ): 124 – 133 . Google Scholar Crossref Search ADS WorldCat 18. Porwal , P.K. , Talele , G.S. ; Development of validated HPLC-UV method for simultaneous determination of Metformin, Amlodipine, glibenclamide and atorvastatin in human plasma and application to protein binding studies ; Bulletin of Faculty of Pharmacy, Cairo University , ( 2017 ); 55 ( 1 ): 129 – 139 . Google Scholar Crossref Search ADS WorldCat 19. Febrina , A.S. , Anisahtul , A. , Ayu Brilliany , F. , Sandra , M. , Arif Satria , W.K. , Taofik , R. , et al. ; Development and validation of simple simultaneous analysis for Amlodipine and Glibenclamide by nonderivatization high-performance liquid chromatography-fluorescence ; Journal of Advanced Pharmaceutical Technology & Research , ( 2018 ); 9 ( 4 ): 124 – 129 . Google Scholar Crossref Search ADS PubMed WorldCat 20. Dubey , A. , Shukla , I.C. ; Microgram determination of Glipizide and Metformin hydrochloride in pharmaceutical preparation by HPLC method ; Indian Drugs -Bombay , ( 2002 ); 39 ( 8 ): Pg. 446 – 448 . OpenURL Placeholder Text WorldCat 21. Armağan , O. ; Spectrophotometric and HPLC determinations of anti-diabetic drugs Rosiglitazone maleate and Metformin hydrochloride in pure form and in pharmaceutical preparations ; European Journal of Medicinal Chemistry , ( 2009 ); 44 ( 12 ): 4998 – 5005 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Lad , N.R. , Bhoir , S.I. , Bhoir , I.C. , Sundaresan , M. ; Concurrent assay of Metformin and Glimepiride in tablets using RP-HPLC with wavelength programming ; Indian Journal of Pharmaceutical Sciences , ( 2003 ); 65 ( 6 ): 650 – 653 . OpenURL Placeholder Text WorldCat 23. Ali , A.R. , Duraidi , I.I. , Saket , M.M. , Nameh , E.A. ; Column high-performance liquid chromatographic method for the simultaneous determination of Rosiglitazone and Metformin in a pharmaceutical preparation ; Journal of AOAC International , ( 2008 ); 92 ( 1 ): 119 – 124 . Google Scholar Crossref Search ADS WorldCat 24. Gayatri , S. , Shantha , A. , Vaidhyalingam , V. , Aruna , A. , Niraimathi , V. ; Simultaneous estimation of Gliclazide and Rosiglitazone from its pharmaceutical dosage form by HPLC method ; Indian Drugs , ( 2004 ); 41 ( 6 ): 374 – 375 . OpenURL Placeholder Text WorldCat 25. Vasudevan , M. , Ravi , J. , Ravisankar , S. , Suresh , B. ; ION-pair liquid chromatography technique for the estimation of Metformin in its multicomponent dosage forms ; Journal of Pharmaceutical and Biomedical Analysis , ( 2001 ); 25 : 77 – 84 . Google Scholar Crossref Search ADS PubMed WorldCat 26. Havele , S.S. , Dhaneshwar , S.R. ; Determination of Glibenclamide, Metformin hydrochloride and Rosiglitazone maleate by reversed phase liquid chromatographic technique in tablet dosage form ; Chemical Industry and Chemical Engineering Quarterly , ( 2014 ); 20 ( 1 ): 39 – 47 . Google Scholar Crossref Search ADS WorldCat 27. Yardimci , C. , Özaltin , N. ; Method development and validation for the simultaneous determination of Rosiglitazone and Metformin in pharmaceutical preparations by capillary zone electrophoresis ; Analytica Chimica Acta , ( 2005 ); 549 ( 1 ): 88 – 95 . Google Scholar Crossref Search ADS WorldCat 28. Sane , R.T. , Francis , M. , Moghe , A. , Khedkar , S. , Anerao , A. ; High-performance thin-layer chromatographic determination of Rosiglitazone in its dosage form ; Journal of Planar Chromatography- Modern TLC , ( 2002 ); 15 ( 3 ): 192 – 195 . Google Scholar Crossref Search ADS WorldCat 29. Hassan , M.H. , Salem , M.S. , Sallam , E. , Al-Hindiwi , M.K. ; Preparation and characterization of a new polymorphic form and solvate of Glibenclamide ; Acta Pharmaceutica Hungarica , ( 1997 ); 7 : 81 – 88 . OpenURL Placeholder Text WorldCat 30. Lazaric , K. , Tomaic , J. , Fistic , J. , Galekovic , A. , Rodin , V. ; Glibenclamide in tablets: Comparison of HPTLC and HPLC ; Journal of Planar Chromatography- Modern TLC , ( 1997 ); 10 : 286 – 289 . OpenURL Placeholder Text WorldCat 31. Susheel , J.V. , Paul , D. , Ravi , T.K. ; Development and validation of high-performance thin-layer chromatography method for the simultaneous densitometric determination of Metformin and Rosiglitazone in tablets ; Austin Journal of Analytical and Pharmaceutical Chemistry , ( 2016 ); 3 ( 3 ): 1071 . OpenURL Placeholder Text WorldCat 32. Mahgoub , H. , Youssef , R.M. , Korany , M.A. , Khamis , E.F. , Kamal , M.F. ; Development and validation of spectrophotometric and HPTLC methods for simultaneous determination of Rosiglitazone maleate and Metformin hydrochloride in the presence of interfering matrix excipients ; Drug Development and Industrial Pharmacy , ( 2014 ); 40 : 1190 – 1198 . Google Scholar Crossref Search ADS PubMed WorldCat 33. Gayatri , S. , Shantha , A. , Vaidyalingam , V. ; Simultaneous HPTLC determination of Gliclazide and Rosiglitazone in tablets ; Indian Journal of Pharmaceutical Sciences , ( 2003 ); 65 ( 6 ): 663 – 665 . OpenURL Placeholder Text WorldCat 34. Harmonized Tripartite Guideline , I.C.H. ; Validation of analytical procedures: text and methodology Q2 (R1); International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use . Geneva , Switzerland , ( November 2005 ), pp. 1 – 13 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - A Sensitive HPTLC Method for the Estimation of Glibenclamide, Rosiglitazone Maleate and Metformin Hydrochloride from a Multicomponent Dosage Form
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmz124
DA - 2020-04-25
UR - https://www.deepdyve.com/lp/oxford-university-press/a-sensitive-hptlc-method-for-the-estimation-of-glibenclamide-JCQE7kriJ0
DP - DeepDyve
ER -