INTRODUCTION:

Metformin, [Mt] chemically [1, 1-dimethyl biguanide hydrochloride] (Figure 1) [1]. It acts by suppressing excessive hepatic glucose production and improving glucose clearance, its predominant effect is to decrease fasting plasma glucose. It is the most well-known member of the biguanide group, regarded asthemain compound in mixed therapies, and is always used in high doses of about 500 or 850mg. Nateglinide [Ng] is an oral hypoglycaemic agent which lowers the blood glucose level by stimulating the pancreaticb-cells to secrete insulin [2].

Figure No. 1: Structure of Metformin Hydrochloride

Chemically, it is1-(3-azabicyclo [3.3.0] oct-3-yl)-3-(p-tolylsulfonyl) urea (Figure 2). Pioglitazone hydrochloride (Pg) ischemically designated as5-[[4-[2-(5-Ethyl-2-pyridinyl) ethoxy] phenyl] methyl]-2, 4-thiazolidinedione (Figure 3). It is a member of the thiazolidinedione group. The combination of Metformin hydrochloride, Nateglinide, and Pioglitazone is used in pharmaceutical preparations. This combination however, is not present in any official pharmacopoeia. In this respect, a method for the analysis of this combination is needed. In the scientific literature, analysis of Mt, Ng and Pg has been reported as individual ingredientsand in combination with other compounds. Analytical methods have included estimation of Mt [3-8]. Ng [9-11], Pg individually [12]. And in two component formulations of Pg and Mt have been analyzed incombination by [13-20]. Simultaneous HPLC analysisof Metformin with gliclazide [21] in combinations with other drugs have also been reported [22-31]. Because no chromatographic method for simultaneous analysis of Mt, Pg, and Ng in a combined dosage form has yet been reported, it was essential to develop a chromatographic method for simultaneous estimation of all the three drugs in a tablet formulation. The method described is rapid, economical, precise, and accurate and can be used for routine analysis of tablets. It was validated as per ICH norm [32-34].

Figure No. 2: Structure of Nateglinide

Figure No. 3: Structure of Pioglitazone Hydrochloride

Figure 4: A Chromatogram of Metformin Hydrochloride, Nateglinide and Pioglitazone Hydrochloride

MATERIALS AND METHODS:

Experimental:

Pharmaceutical grade working standards Pioglitazone [Pg] (batch no. 2088581), Nateglinide [Ng] (batch no.2980007), Metformin HCl [Mt] (batch no. 1997418) were obtained from Sunpharma Laboratories, Dewas, India. All chemicals and reagents were of HPLC gradeand were purchased from Merck Chemicals, Mumbai, India.

Instrumentation:

The LC system consisted of a pump (model jasco PU1580, intelligent LC pump) with auto injecting facility (AS-1555 sampler) programmed at 20μl capacity per injection was used. The detector consisted of a UV–Vis (Jasco UV 1575) model operated at a wave length of 220nm. The software used was jasco borwin version 1.5, LC-Net II/ADC system. The column used was HiQSil C₁₈HS (250mm×4.6mm, 5.0μm) Kya Technologies Corporation. Different mobile phases were tested in order to find the best conditions for separation of Mt, Ng and Pg. The mobile phase contained methanol: 20mM potassium dihydrogen phosphate (85:15, v/v) and the flow rate was maintained at 1.2ml/min UV detection was carried out at 227nm. The mobile phase and samples was filtered using 0.45μm membrane filter. Mobile phase was degassed by ultrasonic vibrations prior to use. All determinations were performed at ambient temperature.

Standard Solutions and Calibration Graphs for Chromatographic Measurement:

Mt, Ng and Pg were weighed accurately and separately transferred to 10mL volumetric flasks. All the drugs were dissolved in HPLC-grade methanol to prepare 1000μg/ml standard stock solutions. Calibration standards at five levels were prepared by appropriately mixed and further diluted stock standard solutions in the concentration range of 50-250μg/ml for Mt and 3-15μg/ml for Ng and 2-10μg/ml Pg. Samples in triplicate were made for each concentration and peak areas were plotted against the corresponding concentrations to obtain the calibration graphs.

Sample Preparation:

For the analysis of a tablet dosage form, 20 tablets were weighed individually and their average mass was determined. Then, the tablets were crushed to a fine powder. The powder amount equivalent to 500mg of Mt, 30mg of Ng and 15 of Pg were transferred to a 25mL volumetric flask and dissolved in 25mL ofHPLC-grade methanol, sonication was done for 15min with swirling. After sonication, the solution was filtered through a Whatman filter paper (#41). Beforethe assay of tablet formulations, 6 replicate aliquots (each 20mL in volume) of the appropriately diluted tablet stock solution were sonicated for 15min, the ninjected into the chromatographic system, and analyzed quantitatively. The analysis was repeated sixtimes. The possibility of excipient interference with the analysis was examined.

Optimization of HPLC Method:

The HPLC procedure was optimized with a view todevelop a simultaneous assay method for Mt, Ng and Pg respectively. The mixed standard stock solution (200 μg/ml of Mt, 12μg/ml of Ng, 6μg/ml of Pg) injected in HPLC. Different ratios of methanol and potassium dihydrogen phosphate buffer at different pHand molarities were tried.

Method Validation:

The method was validated according to the ICHguidelines [19]. The following validation characteristics were addressed: linearity, accuracy, precision, and specificity, limits of detection and quantitation and robustness.

Linearity and Range:

Linearity of the method was studied by injecting the mixed standard solutions in the concentration range of 50-250μg/ml for Mt and 3-15μg/ml for Ng and 2-10μg/ml Pg injected six times into the LC system keepingthe injection volume constant. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs.

Precision:

The precision of the proposed method was evaluated by carrying out six independent assays of test sample. RSD (%) of six assay values obtained was calculated. Intermediate precision was carried out by analyzingthe samples by a different analyst on another instrument.

Limit of Detection and Quantification:

The limit of detection (LOD) and limit of quantitation (LOQ) for the procedure were performed on samples containing very low concentrations of analytes underthe ICH guidelines. By applying the visual evaluation method, LOD was expressed by establishing the minimum level at which the analyte can be reliably detected. LOQ was considered as the lowest concentration of analytes in standards that can bereproducibly measured with acceptable accuracy and precision.

Robustness and System Suitability:

The robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. The conditions studied were flow rate (altered by ±0.1ml/min), mobile phase composition (methanol±5ml). These chromatographic variations were evaluated for resolution between Mt, Ng and At.

Solution Stability:

To assess the solution stability, three different concentrations of Mt, Ng and Pg (2, 4 and 6μg/ml) were prepared from sample solution and kept at room-temperature for 8 days. These solutions were compared with freshly prepared standard solutions.

System Suitability:

The system suitability parameters with respect to theoretical plates, tailing factor, repeatability and resolution between Mt, Ng and Pg peaks were defined.

Specificity:

Injections of the extracted placebo were performed to demonstrate the absence of interference with the elution of the Mt, Ng and Pg. For determining selectivity of the method, a powder blend of typical tablet excipients containing lactose monohydrate, mannitol, maize starch, povidone K₃₀, citric acidanhydrous granular, sodium citrate, natural lemon and lime flavor and magnesium stearate was prepared and analyzed. All chromatograms were examined to determine if compounds of interest co-eluted with each other or with any additional excipients peaks.

Accuracy:

Accuracy of the method was carried out by applying the method to drug sample (Mt, Ng and Pg combination tablets) to which known amounts of Mt, Ng and Pg standard powder corresponding to 80, 100 and 120% of label claim had been added (standard addition method), mixed and the powder wasextracted and analyzed by running chromatograms inoptimized mobile phase. These mixtures were analyzed by the proposed method. The experiment was performed in triplicate and recoveries (%), RSD (%) were calculated.

Analysis of Marketed Formulation:

The marketed formulation was assayed as described above. The peak areas were measured at 227nm and concentrations in the samples were determined using multi level calibration developed on the same LC-system under the same conditions using linearregression analyzed for Mt, Ng and Pg in the same way as described earlier.

RESULTS AND DISCUSSION:

Method Development and Optimization:

The HPLC procedure was optimized with a view to develop a suitable LC method for the analysis of Mt, Ng and Pg in fixed dose combined dosage form. Initially methanol and water in different ratios were tried. But Mt gave broad peak shape, so water was replaced by potassium dihydrogen buffer (20mM), and mixture of methanol and potassium dihydrogen phosphate buffer in different ratios were tried. It was found that methanol: potassium dihydrogen phosphate buffer (20mM) in ratio of 85: 15, v/v gave acceptable retention time (RT 2.15 min for Mt, 3.78 min for Ng and 4.575 min for Pg), plates, and good resolution for Mt, Ng and Pg at the flow rate of 1.2ml/min) Illustration 4.

Validation:

Linearity:

Linearity was evaluated by analysis of working standard solutions of Mt, Ng and Pg of five different concentrations. The range of linearity was from 50-250 μg/ml for Mt and 3-15μg/ml for Ng and 2-10μg/ml Pg. The regression data obtained are represented in Table 1. The result shows that within the concentration range mentioned above, there was an excellent correlation between peak area and concentration of each drug.

Table 1: Linear regression data for the calibration curves ^a

Compound	Linearity (μg/ml)	y = A + Bx		r²
Compound	Linearity (μg/ml)	A	B	r²
Mt	50-250	94169	97790	0.999
Ng	3-15	6186.x	-250.0	0.999
Pg	2-10	36611	39561	0.999

^an = 6; r², coefficient of correlation.

Precision:

The results of the repeatability and intermediate precision experiments are shown in Table 2. The developed method was found to be precise, with RSD values for repeatability and intermediate precision

LOD and LOQ:

The LOD and LOQ values were found to be 0.03 and 0.09μg/ml for Mt. 0.22 and 0.67μg/ml for N, and 0.008 and 0.02μg/ml for Pg.

Table 2: Intra and Inter Day Precision of HPLC method^a

Compound	Repeatability		Intermediate
Compound	Mean% assay	% R.S.D.	Mean% assay	% R.S.D.
Mt	101.07	0.27	100.90	0.11
Ng	100.81	1.72	99.15	0.80
Pg	100.05	0.47	99.09	1.38

^an = 6, (2, 6, 10 µg/ml for Pg 3, 9, 15 µg/ml for an Ng 10, 30, 50 µg/ml for Mt)

Specificity:

Injections of the extracted commonly used excipients were performed to demonstrate the absence of interference with the elution of the drugs. These results demonstrate that there was no interference from other materials in the tablet formulation; therefore, confirm the specificity of the method Table 3.

Table 3: Statistical Analysis of Parameters Required for System Suitability Testing of the Proposed HPLC Method

Parameters	Mt	Ng	Pg
Theoretical plates	39625.65	2390.16	3765.46
Resolution	-	6.43	2.41
Peak asymmetry	1.44	1.34	1.27
% R.S.D.	0.08	0.03	0.06

System Suitability:

System suitability parameters such as the number of theoretical plate, HETP and peak tailing are determined. The results obtained are shown in Table 4.

Robustness of the Method:

To ensure the insensitivity of the developed HPLC method to minor changes in the experimental conditions, it is important to demonstrate its robustness. None of the alterations caused a significant change in resolution between Mt, Ng and Pg, peak area, R.S.D., tailing factor and theoretical plates Table 5.

Table 5: Stability of drugs in sample solutions^a

Parameters	Mt	Ng	Pg
% R.S.D	0.74	0.04	1.10

^a(n = 6) Average of three concentrations (2, 6, 10 µg/ml for Pg 3, 9, 15 µg/ml for Ng and 10, 30, 50 µg/ml for Mt).

Solution Stability Studies:

Three different concentrations of Mt, Ng and Pg, 2, 4and 6 μg/ml were prepared from sample solution andstored at room temperature for 8 days. They were then injected into the HPLC system and no additional peakwas found in the chromatogram indicating the stabilityof Mt, Ng and Pg in the solution Table 6.

Table 4: Robustness Testing ^a

Chromatographic Factors^a	Level	Retention time, t_R(min)			Resolution (Rs)			Asymmetry (As)
Chromatographic Factors^a	Level	Mt	Ng	Pg	Mt	Ng	Pg	Mt	Ng	Pg
Flow rate (ml/min)	1.1	2.27	3.93	5.01	0	6.01	2.39	1.15	1.46	1.38
	1.2	2.15	3.78	4.57	0	6.43	2.41	1.04	1.34	1.27
	1.3	2.01	3.70	4.00	0	6.49	2.48	1.01	1.30	1.25
% of methanol	83	2.23	3.98	4.89	0	6.04	2.40	1.19	1.66	1.18
	85	2.15	3.78	4.57	0	6.43	2.41	1.04	1.34	1.27
	88	2.08	3.10	4.13	0	6.67	2.86	1.09	1.50	1.55

^an = 6

Recovery Studies:

Good recoveries of the Mt, Ng and Pg were obtained atvarious added concentrations for the tablets as shownin Table 7.

Table 7: Applicability of the HPLC method for the analysis of the pharmaceutical formulations

Sample	Label claim (mg)	Drug Content (%)	% R.S.D.
Pg	15	99.03	0.15
Ng	30	99.19	0.72
Mt	500	99.88	0.08

Analysis of a Commercial Formulation:

Experimental results of the amount of Mt, Ng and Pg in tablets, expressed as a percentage of label claims were in good agreement with the label claims there by suggesting that there is no interference from any of the excipients which are normally present in tablets. Fixeddose combination tablets were analyzed using the proposed procedures Illustration 12. The data of summary of validation parameters arelisted in Table 8.

Table 8: Summary of Validation Parameters

Parameter	Mt	Ng	Pg
Linearity range (µg/ml)	50-100	3-1 5	2-10
Correlation coefficient	0.999 ± 0.09	0.999 ± 0.18	0.999 ± 0.06
Limit of detection (µg/ml)	0.03	0.22	0.008
Limit of quantitation (µg/ml)	0.09	0.67	0.02
Recovery (n = 6)	100.03	99.84	99.40
Precision (% R.S.D.) Repeatability Inter day	0.27 0.11	1.72 0.80	0.47 1.38
Robustness	Robust	Robust	Robust

CONCLUSION(s):

The new HPLC method described in this paperprovides a simple, convenient and reproducible approach for the simultaneous identification and quantification that can be used to determine Metformin hydrochloride, Nateglinide, Pioglitazone hydrochloride inroutine quality control.

REFERENCES:

1. Klepser TB, Kelly MW. Metformin hydrochloride: an antihyperglycemic agent. Am J Health System Pharm.1997; 54: 893-903.

2. Campbell DB, Lavielle R, Nathan C. The mode ofaction on clinical pharmacology of gliclazide a review. Diab Res Clin Pract. 1991; 14: S21–S36.

3. Sane RT, Banavalikar VJ, Bhate VR, Nayak VG. Gas chromatographic determination of metformin hydrochloride from pharmaceutical preparations. Indian Drugs. 1989; 26(11): 647-648.

4. El-Khateeb S Z, Assaad H N, El-Bardicy M G, Ahmad A S Determination of metformin hydrochloridein tablets by nuclear magnetic resonance spectroscopy. Anal Chim Acta. 1988; 208(1-2): 321-324.

5. Koseki N, Kawashita H, Niina M, Nagae, Y MasudaN, Development and validation for high selective quantitative determination of metformin in human plasma by cation exchanging with normal-phase LC/MS/MS. J Pharm Biomed Anal. 2005; 36:1063-1072.

6. Wang Y, Tang Y, Gu J, Fawcett JP, Bai X, Rapidand sensitive liquid chromatography-tandem mass spectrometric method for the quantitation of metformin in human plasma. J Chromatogr B. 2004; 808: 215-219.

7. Heinig K, Bucheli F, Fast liquid chromatographic-tandem mass spectrometric (LC–MS–MS) determination of metformin in plasma samples. J Pharm Biomed Anal. 2004; 34: 1005-1011.

8. Kar M, Choudhury PK, HPLC method for estimation of metformin hydrochloride in formulated microspheres and tablet dosage form. Indian J Pharm Sci. 2009; 71: 318-320.

9. El Kousy NM. Stability-indicating densitometric determination of some antidiabetic drugs in dosage forms, using TLC. Mikrochim Acta. 1998; 128: 65–68.

10. The European Pharmacopoeia. 2005; I: 1660–1662.

11. Noguchi H, Tomita N, Naruto S. Determination of Gliclazide in serum by High Performance Liquid Chromatography used solid-phase extraction. J Chromatogr Biomed Appl. 1992; 583: 266–269.

12. Yamashita K, Murakami H, Okuda T, MotohashiM.High-performance liquid chromatographic determination of pioglitazone and its metabolites inhuman serum and Urine. J Chrom A. 1996; 677 (1):141-146.

13. John-Lin Z, Karieger W. JiDD, Shum L, Simultaneous determination of pioglitazone and its two active metabolites in human plasma by L-MS-MS. JPharm Biomed Anal. 2003; 33: 101.

14. Kolte BL, Raut BB, Deo AA, Bagool MA, Shinde DB, Simultaneous High-Performance Liquid Chromatographic Determination of Pioglitazone and Metformin in Pharmaceutical-Dosage Form. J Chrom Science. 2004; 42: 27-31.

15. Sane RT, Menon SN, Mote M, Gundi G, Simultaneous determination of pioglitazone and glimpiride by high-performance liquid chromatography. Chromatographia. 2004; 59: 451.

16. Davison G, Beckette AH, Stenlake JB, Practical Pharmaceutical Chemistry, CBS Publishers and distributors, New Delhi, 1997.

17. T.C. Lalhariatpulli and N. Kawathekar, Indian Drugs. Derivative spectrophotometric estimation of pioglitazone and metformin hydrochloride. 2005;42(11): 740.

18. Kolte BL, Raut BB, Deo AA, Bagool MA, ShindeDB. Simultaneous High-Performance Liquid Chromatographic Determination of Pioglitazone and Metformin in Pharmaceutical-Dosage Form. J Chrom Science. 2004; 42: 27-31.

19. Shankar Madhira B, Modi Vaibhav D, Shah Dimal A, Bhatt Kashyap K, Mehta, Rajendra S, Geetha Madhira. Estimation of Pioglitazone hydrochloride and Metformin hydrochloride in tablets by Derivative Spectrophotometry and Liquid Chromatographic Methods. J J AOAC International. 2005; 88: 1167-1172.

20. Sahoo PK, Sharma R, Chaturvedi SC, Simultaneous Estimation of Metformin hydrochloride and Pioglitazone hydrochloride by RP-HPLC method from combined tablet dosage form. Ind J Pharm Sci. 2008; 70: 383-386.

21. Bhanu R, Kulkarni SK, Kadam AB. Simultaneous estimation of gliclazide and metformin in pharmaceutical dosage by reverse phase HPLC. Indian Drugs. 2006; 43(1): 16.

22. Aburuz S, Milership J, McElnay J. The development and validation of liquid chromatography method for the simultaneous determination of metformin and glipizide, glicazide, glibenclamide or glimeperide in plasma. Journal of Chromatography B. 2005; 817: 277–286.

23. Chaturvedi PK, Sharma R. Development and Validation of an RP-HPLC Method for Simultaneous Analysis of a Three-Component Tablet Formulation Containing Metformin Hydrochloride, Pioglitazone Hydrochloride, and Glibenclamide. Acta Chromatographica. 2008; 20(3): 451–461.

24. Mistri H N, Jangid A G, Shrivastav P S, Liquid chromatography tandem mass spectrometry method for simultaneous determination of antidiabetic drugs metformin and glyburide in human plasma. J. Pharm. Biomed. Anal. 2007; 45: 97-106.

25. Georgita C, Albu F, David V, Medvedovici AJ, Simultaneous assay of metformin and glibenclamide in human plasma based on extraction-less sample preparation procedure and LC/(APCI)MS, J Chromatogr B Analyt Technol Biomed Life Sci. 2007; 854: 211-218.

26. Zhang L, Tian Y, Zhang Z, Chen Y. Simultaneous determination of Metformin and rosiglitazone in human plasma by liquid chromatography/tandem Mass spectrometry with electrospray ionization: application to a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 2007; 854: 91-98.

27. Gupta VD. Quantitation of glipizide and glyburide (glibenclamide) in tablets using high performance liquid chromatography. J Liq Chromatogr. 1986; 9(16):3607-3615.

28. Nadkarni DR, Merchant RN, Sundaresan M, Bhagwat AM. Isocratic separation and simultaneous estimation of four anti-diabetic members of the sulphonyl urea family by reverse phase HPLC. Indian Drugs. 1997; 34(11): 650-653.

29. Aruna A, Nancey K. Simultaneous estimation of glipizide and metformine in solid dosage form by UV-spectrophotometry. Indian Drugs. 2000; 37(10):533-536.

30. Duby A, Shukla IC. Microgram determination of glipiozide and metformine HCl in pharmaceutical preparation by HPLC method. Indian Drugs. 2002;39(8): 446-449.

31. Ding CG, Zhou Z, Ge QH, Zhi XJ, Ma LL. Simultaneous determination of metformin and glipizide in human plasma by liquid chromatography-tandem mass spectrometry, Biomed Chromatogr. 2007; 21:132-138.

32. Code Q2(R1)-Text on Validation of Analytical Procedure Step-3 Consensus Guideline, ICH Harmonised Tripartite Guideline. 2005.

33. Code Q2A-Text on Validation of Analytical Procedure Step-3 Consensus Guideline, ICHHarmonised Tripartite Guideline. 1994.

34. Code Q2B-Validation of Analytical Procedure Methodology Step-4 Consensus Guideline, ICH Harmonised Tripartite Guideline. 1994.

Received on 25.12.2020 Modified on 10.01.2021

Asian J. Research Chem. 2021; 14(1):7-12.

DOI: 10.5958/0974-4150.2021.00002.X

Label claim	Amount of drug added (%)	Total amount mg	Amount recovered, mg ± % RSD	% Recovery
Pg 15 mg	80	27	26.74 ± 0.09	99.04
	100	30	29.85 ± 0.06	99.51
	120	33	32.88 ± 1.02	99.66
Ng 30 mg	80	54	53.78 ± 1.75	99.61
	100	60	59.94 ± 1.97	99.90
	120	66	66.01 ± 0.08	100.02
Mt 500 mg	80	900	900.18 ± 0.12	100.03
	100	1000	1004 ± 0.11	100.41
	120	1100	1098.24 ± 0.04	99.84