Spectrophotometric Multi-Component Quantitation of Rosiglitazone, Glibenclamide and Metformin HCl in Pharmaceutical Dosages Form by using Cramer’s Matrix

Uttam Singh Baghel^1,2*, Abhay Sharma², Harshit Gautam², Mohammad Mukim³,

Deeksha Singh⁴, Shuchi Dave⁵, B. Srivastava⁶, Bhawani Singh⁷

¹Gurukul Pharmacy College, Ranpur-325003, Kota, Rajasthan, India.

²Department of Pharmacy, University of Kota, Kota-324005, Rajasthan, India.

³Kota College of Pharmacy, Ranpur-325003, Kota, Rajasthan, India.

⁴E. S. I. Hospital, Kota-324005, Rajasthan, India.

⁵Guru Ramdas Khalsa Institute of Science and Technology (Pharmacy),

Barela, Kukrikheda, Jabalpur-483001, Madhya Pradesh, India.

⁶School of Pharmaceutical Sciences, Jaipur National University, Jaipur, Rajasthan, India.

⁷Deparment of Pure and Applied Chemistry, University of Kota, Kota-324005, Rajasthan, India.

*Corresponding Author E-mail: drusb1985@yahoo.com

ABSTRACT:

Objective: This article illustrates the development of a simple, precise and accurate spectrophotometric analysis method for the determination of Rosiglitazone, Glibenclamide and Metformin hydrochloride in their pharmaceutical preparations by using Cramer's matrix. Method: The absorbance of the sample solution was measured at 315.6nm and 229.2nm for the estimation of rosiglitazone and glibenclamide, respectively and at 237.0nm for estimation of metformin hydrochloride by Shimadzu double beam UV-visible spectrophotometer. Results: The result of the analysis for method was tested and validated for various parameters according to ICH guidelines and found satisfactory. The method was successfully applied for the determination of these drugs in their tablet dosage forms. Conclusion: Simultaneous estimation of active ingredients in multicomponent pharmaceutical products normally requires the use of chromatographic separation techniques, like thin layer chromatography, high performance thin layer chromatography, high performance liquid chromatography or gas chromatography etc., followed by their quantitation. But this article illustrates direct spectroscopic estimation of multicomponent formulation by using Cramer's matrix.

KEYWORDS: UV Spectrophotometer, Rosiglitazone, Glibenclamide, Metformin hydrochloride, Cramer’s matrix, Validation.

INTRODUCTION: 

Diabetes is a metabolic disorder characterized by hyperglycemia, glycosuria, hyperlipemia, negative nitrogen balance and some ketonemia.  Diabetes is commonly classified as: Type I, insulin-dependent diabetes mellitus. Type II non-insulin dependent diabetes mellitus. Type II diabetes is a progressive and complex disease that is not easy to manage effectively in the long-term¹. In Type II diabetes the Insulin secretion capacity progressively worsens over time in most patients².

Many patients having Type II diabetes, monotheraphy with an oral antidiabetic agent is not sufficient to attain target glycaemic control aims. So to control this disease, now a day’s combination therapy is often used. A broad range of hypoglycemic agents are prescribed to control blood sugar levels³.

Due to severe side effects, drug monitoring during combination therapy is an important process for titrating the appropriate dosing control and diagnostic purpose⁴. These drugs provide the basis for the development of a quantitative multianalyte spectrophotometric method. This article illustrate the development of a novel, fast and simple spectrophotometric method for the simultaneous determination of Rosiglitazone (figure.1), (±)-5-{p-[2-(Methyl-2-pyridylamino)ethoxy]benzyl}-2,4-thiazolidinedione, is a thiazolidinedione derivative.

Figure 1: Typical overlain Spectra of a ROSI, GLIBE and MET in Methanol

It exerts its glucose lowering effects in Type II diabetic patients by improving the responsiveness to insulin⁵. Glibenclamide, 1-{4-[2-(5-chloro-2-methoxy benzamido)ethyl}benzene sulphonyl} -3-cyclohexylurea, is second generation sulphonylurea. It is used in the management of type-II diabetes mellitus⁶. Metformin Hydrochloride is 1,1-dimethylbiguanide  Hydrochloride. It is categorized as Biguanides. It increases the peripheral insulin sensitivity and reduced hepatic glucose production in type 2 diabetes⁷. Metformin is a biguanide with a guanidine and galegine connection⁸.

Literature survey reveals several methods and techniques have been developed to be used for the analysis of ROSI, GLIBE and MET such as high performance liquid chromatography (RP-HPLC)^9-11, HPLC-PDA¹², UHPL-CHRMS¹³, HPTLC^14-16, spectroscopic^17-21 etc. However, none of these methods were suitable for routine analysis. Some of them used solvent extraction in sample preparation. This is tedious and time-consuming involving complex sample preparation, such as equilibrium dialysis, ultra filtration, solid phase extraction and liquid–liquid extraction. Some hyphenated techniques for analysis of ROSI, GLIBE and MET like liquid chromatography/mass spectrometry or tandem mass spectrometry with improved sensitivity and efficiency has been published^22-24. However, there is no method reported for the simultaneous estimation of ROSI, GLIBE and MET. The complexity of the multicomponent dosage forms includes multiple entities and excipients poses considerable challenge to the analytical chemist during the development of assay procedure.

For the simultaneous estimation of the drugs present in multicomponent dosage forms, spectrophotometric method is considered to be most suitable since this is simple and economic. It is also extremely specific, linear, precise, accurate, sensitive and rapid. The result of the analysis for method was tested and validated for various parameters according to ICH guidelines²⁵.

MATERIALS AND METHODS:

Equipments:

A Shimadzu UV-1800, UV-Visible spectrophotometer with a 10mm quartz cell was used for all spectrophotometric measurement was used.

Materials:

Pure samples of Rosiglitazone and Metformin hydrochloride were procured from Glenmark pharmaceutical Pvt. Ltd. Mumbai.  Glibenclamide was procured from Dr. Reddies, Hyderabad. Methanol was used of spectroscopic grade and obtained from Merck chemicals, Mumbai and RGM (Micro Carsyon) tablet from local pharmacy.

Simultaneous equation method development:

Standard stock solutions (1000µg ml⁻¹) of ROSI, GLIBE and MET were prepared in methanol. Overlain spectra of standard solutions of ROSI, GLIBE and MET, were scanned in the wavelength range of 400-200nm with medium scan speed against methanol blank [Fig.1].  ROSI shows absorption maxima at 315nm, GLIBE shows at 229.2nm and MET at 237nm. Calibration curves were prepared by using working standard in concentration range of 5-70μg/ml for ROSI, 2-20μg/ml for GLIBE and 1-20μg/ml for MET. The absorptivity coefficients were determined for all the drugs at all the wavelengths and following equation were made.

A ₁ = 106.82 Cx…………………………………….. (1)

A ₂ = 447.81 Cx+555 C_Y+759.53 Cz……................... (2)

A ₃ = 414.98Cx+439.37Cy+969.27Cz….................... (3)

Where A ₁ and A ₂ are absorbances at 315.6nm and 229.2nm, respectively and A₃ is absorbance at 237.0nm.  C_x and C_y are concentrations of ROSI and GLIBE (mole/lit), respectively and C_Z  is concentration of MET ( mole /lit ). The content of the ROSI, GLIBE and MET was directly found from the Eqns. 4, 5 and 6 by using Cramer's rule.

Cx = Dx/D…………………………………………... (4)

Cy = Dy/D…………………………………………... (5)

Cz = Dz/D………………………………………….... (6)

Where D, Dx, Dy and Dz are

D = a_x1 (a_y2 a_z3 – a_y3 a_z2) - a_y1 (a_x2 a_z3 – a_x3 a_z2) + a_z1 (a_x2 a_y3 – a_y2 a_x3)……………….......................................... (7)

Dx = A₁ (a_y2 a_z3 – a_y3 a_z2) - a_y1 (A₂ a_z3 – A₃ a_z2) + a_z1 (A₂ a_y3 – A₃ a_y2)…………………………………………...(8)

Dy = a_x1 (A₂ a_z3 – A₃ a_z2) - A₁ (a_x2 a_z3 – a_x3 a_z2) + a_z1 (a_x2 A₃ – a_x3 A₂)…………………………………………...(9)

Dz = a_x1 (a_y2 A₃ – a_y3 A₂) - a_y1 (a_x2 A₃ – a_x3 A₂) + A₁ (a_x2 a_y3 – a_x3 a_y3)………………………………………….(10)

The validity of formed equation was checked by preparing 5 mixed standards measuring their absorbance at respective wavelength and comparing these with the absorbance calculated using above formed equations. 

Application of developed method to the RGM tablets:

The developed method was applied on RGM (Micro Carsyon) tablet.  Twenty tablets, each containing 500 mg ROSI, 5mg GLIBE and 500mg MET, were accurately weighed and finely powdered. A quantity of powder equivalent to 250mg ROSI, 2.5mg GLIBE and 250mg MET was weighed and transferred to a 250ml volumetric flask. To it 247.5mg of pure GLIBE was added and sonicated for 10 minutes with about 100ml of methanol. The solution was made up to the mark with methanol. Aliquot portion of this solution was further diluted to achieve final concentration of 10μg/ml for each drugs of tablet. The solution was filtered with a Whatman filter paper no.1. The absorbance was noted at respective wavelengths. The concentration of each drug in tablet formulation was determined using above developed method. The content of the ROSI, GLIBE and MET was directly found from equation using cramer’s rule.

RESULTS:

Optimization of method:

Careful investigations of chromophores were carried out for the selection of solvents. Polarity of analyte was predicted on the basis of functional group present in their chemical structure. After examining the polarity of all the three drugs in different solvents such as water, acetic acid, methanol, ethanol, 0.1N NaOH, 0.1N HCl etc. methanol was selected as the common and cheapest solvent. Overlain spectra of standard solutions of ROSI, GLIBE and MET, were scanned in the wavelength range of 400-200nm with medium scan speed [Fig.1]. 315nm and 229.2nm were selected as the optimum wavelength for ROSI and GLIBE respectively and 237nm was selected for MET against the reagent blank. Optical parameters were determined after optimization of the method. The wavelength, correlation coefficient, intercept, slope, molar absorptivity and Sandell’s sensitivity were calculated by aliquotes of working standard. Molar absorptivity were found to be 4.4207×10⁴ L/mol.cm and 2.9524×10⁴L/mol.cm for ROSI and GLIBE, respectively and 1.236×10⁴L/mol.cm for MET which shows that the drug used is rich in chromophores. The Sandell’s sensitivity found for ROSI and GLIBE was 0.0808 and 0.0166 respectively and for MET was 0.0104 which shows that the instrument used is sensitive for above method using the all three analytes.

Validation:

The method was validated according to the ICH guidelines for validation of analytical procedures in order to determine the linearity, precision, specificity, robustness and accuracy for developed spectrophotometric method. The results of validation studies were statistically analyzed.

(a) Linearity:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Aliquots of working standard solution were transferred into a series of 10ml stopper volumetric flasks. The volumes were made up with methanol. The absorbance of Rosiglitazone and Glibenclamide were measured at 315 and 229.2 respectively and of Metformin 237nm against methanol as the reagent blank, and the calibration curve was plotted. The results were incorporated in table 1.

Table 1: Optical Characteristics of ROSI, GLIBE and MET

Parameters	Rosiglitazone	Glibenclamide	Metformin
λmax (nm)	315	229.2	237
Beer’s Law Limit (µg/ml)	5-30	2-20	1-20
Molar absorptivity (L/mol.cm)	4.4207×10^3	2.9524×10^4	1.236×10^4
Sandell’s sensitivity (µg cm^-2/0.001 Absorbance unit)	0.0808	0.0166	0.0104
Regression equation (y = mx + c)	y = 0.013x - 0.016	y = 0.054x + 0.035	y = 0.050x + 0.091
Slope (m)	0.013	0.054	0.050
Intercept (c)	-0.016	0.035	0.091
Correlation coefficient (r^2)	0.998	0.997	0.999

Where, λmax = maximum absorbance

(b) Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

Repeatibility (Intraday Precision):

The repeatability precision of an analytical method was determined by assaying a sufficient number of aliquots of a homogeneous sample to be able to calculate statistically valid estimates of standard deviation or relative standard deviation. Repeatability was assessed by performing the determination, of six concentrations of working standard solution. The Coefficient of variation (RSD) of 6 determinations is less than 1% which shows that the developed method is precised and ready for quality control OF ROSI GLIBE and MET . The results are shown in table 2.

Intermediate (Interday Precision):

Intermediate precision expresses within laboratory variations on different days. It is assessed by taking 6 concentration of working standard solution. The Coefficient of variation (RSD) of 6 determinations is less than 1% which shows that the developed method is highly precised. The results are shown in table II.

Table 2: Precision of ROSI, GLIBE AND MET

Precision	Parameters
	ROSI(n=6)	GLIBEN(n=6)	MET(n=6)
Intraday precision	X =  498.428±1.9490 SD= 2.4358 RSD= 0.4887% S.EM= 0.994427	X = 499.519±2.0295 SD= 2.5364 RSD= 0.5077% SEM= 1.0355	X =497.769± 0.9860 SD= 1.2323 RSD= 0.2475 SEM= 0.5030
Interday precision	X = 498.192±1.9009 SD= 2.3757 RSD = 0.4768% SEM=  0.96985	X= 498.055±1.0217 SD= 1.2761 RSD= 0.2563% SEM= 0.5213	X = 498.403±1.233 SD= 1.5421 RSD= 0.3094% SEM= 0.6295

Where, n= no. of aliquotes; X= mean; SD= Standard deviation; RSD= Relative standard deviation and SEM= standard error of mean

(c) Reproducibility:

Reproducibility expresses the precision between laboratories. The reproducibility of an analytical method was determined by analysis of aliquots, from homogeneous lots in different Laboratory. The Coefficient of variation (RSD) of six determinations is less than 1% which implies that the developed method can be used in different environments. The results are given in table 3.

Table 3: Reproducibility and Statistical Analysis of ROSI, GLIBE and MET

No. of Aliquots	Active drug in mg
	Lab-I			Lab-II
	ROSI	GLIBE	MET	ROSI	GLIBE	MET
01	499.5283	499.3497	496.4714	497.6415	496.9140	498.639
02	498.5849	497.4272	497.6422	500.9434	495.0146	498.3468
03	494.8113	499.4783	497.7060	498.1132	496.6911	498.5900
04	498.1132	497.5951	498.8513	500.0000	496.4984	498.5420
05	499.0566	497.8598	498.4315	494.3396	499.8596	499.7472
06	500.4717	495.9875	498.5720	496.6981	499.0093	497.2672
SD	1.948679	1.307951	0.868504	2.361643	1.780607	0.790575
RSD( in %)	0.390965	0.262667	0.174417	0.474267	0.358032	0.158584
SEM	0.795541	0.533966	0.354564	0.964133	0.726927	0.32275
Percentage range of error (within 95% confidence limits)	1.559261± 498.4277	1.046574± 497.9496	0.694945± 497.9457	1.8897± 497.956	1.424776± 497.3312	0.632589± 498.522

Where, SD-standard deviation; RSD- Relative standard deviation and SEM- Standard error of mean

(d) Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method. The Robustness of the method was established by making the deliberate minor variation in the wavelength. Original method and changed method results deviates less than 1%. This indicates that the developed method is robust and unaffected by minor change in the environment condition as well as wavelength. The results are given in table 4.

(e) Specificity:

Specificity was done by spiking the drug substance or product with appropriate levels of impurities or excipients and demonstrating the assay result which was unaffected by the presence of these extraneous materials. The percentage deviation of significant absorbance was found to be less than 1 which implies that the developed method is specific for the analytes in the tablet dosage form. The results are given in table V.

Table 4: Specificity by Placebo Interference

No. of Aliquots	Conc. of ROSI ( in mg)	Conc. of GLIBE ( in mg)	Conc. of MET ( in mg)
01	0.4717	0.9204	0.9337
02	0.7547	0.3074	0.0027
Interference	0.6132	0.6139	0.4682

Table 5: Percent deviation for changed method

No. of Aliquots	Wavelength			Active drug in %			Percentage Deviation
	ROSI	GLIBE	MET	ROSI	GLIBE	MET	ROSI	GLIBE	MET
1	315	229.2	237	100.1416	99.5673	99.6181	0.8491	0.3460	0.4082
2	316	230.2	238	99.2925	99.2213	99.2099

Table 6: Summary of analytical method validation

Validation parameters	Acceptance criteria	Inferences
		Rosiglitazine	Glibenclamide	Metformin HCl
Intraday precision (%RSD)	NMT 2	0.4887	0.5077	0.2475
Interday precision  (%RSD)	NMT 2	0.4768	0.2563	0.3094
Reproducibility (%RSD)	NMT 2	0.3909	0.2626	0.1744
Specificity	NMT 1	0.6132	0.6139	0.4682
Robustness	NMT 1	0.8491	0.3460	0.4082
Accuracy(%recovery)	98-102	99.59	99.38	99.19

Where, NMT= Not more than

(f) Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.

Accuracy was calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample. The results are shown in table VI. Accuracy assayed by using a minimum of nine determinations over a minimum of three concentration levels, covering the specified range (i.e. 3 concentrations and 3 replicates of each concentration). The percentage recovery lied between 98-102%. The result indicates that the developed method is accurate.

DISCUSSIONS:

The developed method is found to be a suitable method for the determination of a combination of Rosiglitazone, Glibenclamide and Metformin hydrochloride in pharmaceutical dosage form. The validation data demonstrate good precision and accuracy, which prove the reliability of the method. Hence this UV spectrophotometric method can be used routinely for quality control of ROSI, GLIBE and MET in tablet dosage form.

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Received on 07.05.2022            Modified on 04.12.2022

Accepted on 12.06.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(12):5671-5676.

DOI: 10.52711/0974-360X.2023.00917