1. INTRODUCTION:

Pharmaceutical Analysis plays a vital role in quality assurance and quality control of bulk drugs and their formulations. Pharmaceutical analysis is a particular branch of analytical chemistry, which includes isolating, identifying and determining the relative amounts of compounds in a sample matter. It is concerned with chemical characterization of matter both quantitative and qualitative. In recent years many analytical techniques have been developed. Analytical method is a particular utilization of a procedure to solve a problem. Analytical instrumentation assumes an imperative part in the production and evaluation of new products and protection of Consumers and the environment. This instrumentation provides the lower detection limits required to assure safe foods, medications, water and air.

Validation of an analytical method is the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications. There are two important reasons for validating assays in the pharmaceutical industry. The first, and by for the most important, is that assay validation is an integral part of the quality control system. The second is that current good manufacturing practice regulation requires assay validation.

Metformin Hydrochloride is chemically known as (S)-Isopropyl 2-((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy)-(phenoxy) phosphorylamino) propanoate. It has a molecular formula of C₂₂H₂₉FN₃O₉P and a molecular weight of 529.45 (Fig. 1).

Metformin Hydrochloride is a white to off-white powder with a solubility of ≥ 2 mg/ml across the pH range of 2-7.7 at 37°C. The partition coefficient (log P) for Metformin Hydrochloride is 1.62 and the pKa is 9.3 (European Medicines Agency, 2014). Metformin Hydrochloride is a pangenotypic inhibitor of the HCV NS5B RNA-dependent RNA polymerase, which is essential for viral replication (Keating GM, 2014).

Alogliptin (LDV); is chemically known as Methyl [(2S)-1-{(6S)-6-[5-(9,9-difluoro-7- {2-[(1R,3S,4S)-2-{(2S)-2-[(methoxycarbonyl)amino]-3-methylbutanoyl}-2-azabicyclo[2.2.1] hept-3-yl]-1H-benzimidazol-6-yl}-9H-fluoren-2-yl)-1H-imidazol-2-yl]-5-azaspiro[2.4] hept-5-yl}-3-methyl-1-oxobutan-2-yl] carbamate. It has a molecular formula of C₄₉H₅₄F₂N₈O₆ and a molecular weight of 889.00 (Fig. 2). Alogliptin is a white to tinted (off-white, tan, yellow, orange, or pink), slightly hygroscopic crystalline solid. Alogliptin is practically insoluble (<0.1 mg/mL) across the pH range of 3.0-7.5 and is slightly soluble below pH 2.3 (1.1 mg/ml). The partition coefficient (log P) for Alogliptin is 3.8 and the pKa1 is 4.0 and pKa2 is 5.0 (European Medicines Agency, 2014).

Metformin Hydrochloride in human plasma was determined by UPLC-MS/MS method (Rezk MR et al., 2016). Quantification of Metformin Hydrochloride and its metabolite, GS-331007, in human plasma has been determined by UPLC-ESI-MS/MS method (Rezk MR et al, 2015). Simultaneous quantification ofet al., ribavirin, Metformin Hydrochloride and its metabolite in rat plasma by UPLC-MS/MS has been reported (Shi X et al., 2015). MET in pure form (Vikas PM et al., 2016), in bulk and tablet dosage form was determined by RP-HPLC (Ravikumar Vejendla et al., 2016). Finally, Metformin Hydrochloride (MET) was used as an internal standard (IS) in an UPLC-MS/MS method for the determination of daclatasvir (DAC) in human plasma (Rezk MR et al., 2016). While for LDV, only two methods have been published for its individual determination in bulk drug form by simple UV spectrophotometry (Ranjana S et al., 2016) and by RP-HPLC (Devilal J et al., 2016). Both Metformin Hydrochloride and Alogliptin in human plasma were determined by UPLC-MS/MS method (Rezk MR et al, 2015) and besides some antiviral agents (Ariaudo A et al., 2016). Alogliptin, Metformin Hydrochloride and its metabolite in rat plasma were also, determined by UPLC-MS/MS (Pan C et al., 2016).

According to the best of our knowledge, only three HPLC methods (Bakht Zaman et al., 2016; Rezk M.R et al., 2016) have been published, during the preparation of the present work for publishing. The present study aimed to develop a simple, sensitive, short retention time and accurate RP-HPLC method for the simultaneous determination of both Metformin Hydrochloride and Alogliptin together in pure and tablet dosage forms with high sensitivity, selectivity that can be used for the routine analysis of production samples.

Fig. 1 Structure of Metformin Hydrochloride, Fig. 2 Structure of Alogliptin

2. MATERIALS AND METHODS:

2.1 Materials and Reagents:

The analysis of the drug was carried out on Youngline (S. K.) Gradient System UV Detector. Equipped with reverse phase (Grace) C₁₈column (4.6mm x 250mm; 5µm), a SP930D pump, a 20µl injection loop and UV730D Absorbance detector and running autochro-3000 software. Metformin Hydrochloride and Alogliptin were procured from R.S.I.T.C Jalgaon. Orthophopsphoric acid (OPA) (Avantor Performance material India Ltd. Thane, Maharashtra) and methanol, acetonitrile, (HPLC grade Merck Specialties Pvt. Ltd. Shiv Sager Estate ‘A’ Worli, Mumbai.), water, 0.45µm filter (Millipore, Bangalore). A combination of Metformin Hydrochloride (400mg) and Alogliptin (90mg) in tablet formulation was procured from Hetero drugs Ltd. Mumbai (ALOfos brand).

2.2 Chromatographic Conditions:

Column C₁₈ (250mm× 4.6mm); particle size packing 5µm; detection wavelength of 283nm; flow rate 0.9ml/min; temperature ambient; sample size 20µl; mobile phase Acetonitrile: water (OPA 0.1% PH 2.5 with TEA) (80:20); run time of 12 mins.

2.3 Preparation of standard stock solution:

40 mg of Metformin Hydrochloride and 10mg of Alogliptin were weighed accurately and transferred to a 10ml volumetric flask dissolved in methanol and diluted to 10ml with the mobile phase Acetonitrile + 0.1% OPA water with TEA (80 + 20% v/v) to give a stock solution of 4000µg/ml Metformin Hydrochloride and 1000µg/ml Alogliptin (Table 1 and Fig. 3).

Fig. 3 Chromatogram of standard combination of Metformin Hydrochloride and Alogliptin

Table 1 Details of chromatogram of standard combination containing MET and ALO

Sr. No.	Name of drug	RT [min]	*Area [mVs]**	Area%	TP	TF	Resolution
1	Metformin ydrochloride	6.483	3883.2092	73.98	7934.8	1.333	0.0000
2	Alogliptin	8.7500	1365.7129	26.02	10613.9	1.2273	9.7647
Sum			5248.9219

2.4 Method development and validation:

Serial dilutions were done to prepared various concentration stock (Standard solution and diluted to get required concentration for calibration plot and which was injected (ICH, 1996; ICH Harmonised Tripartite Guideline, 2005; ICH, 2002, 2005, 2003, 1996; US DHHS, 2013, 2001; FDA, 1996, 2001; Smith, 2012; Hassan and Bahrani, 2014; Harona et al., 2019).

2.5 Assay preparation for commercial formulation:

For analysis of the tablet dosage form, weigh 20 Metformin Hydrochloride and Alogliptin combination tablets and calculated the average weight, accurately weigh and transfer the sample equivalent to 12.2mg MET and ALO into 10 ml volumetric flask. Add about 10ml ACN of diluent and sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45µm nylon membrane filter. Then volume was made up to the mark with Acetonitrile + 0.1% OPA water with TEA (80 + 20% v/v). The simple chromatogram of test MET and ALO shown in (Fig. 4). The amounts of MET and ALO per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated five times with tablet formulation. Tablet Assay for % Label claim for % RSD Calculated, Result was shown in (Table 2).

Fig. 4 Chromatogram for marketed formulation

Table 3: Analysis of marketed formulation

Assay	Drug	Label claimed	Amt. Found	% Label claim	SD	%RSD
RP-HPLC Method	MET	80	80.31	100.39	0.02	0.01
	ALO	20	20.00	100.00	0.01	0.01
	MET	80	80.28	100.35	0.28	0.01
	ALO	20	19.99	99.95	0.00	0.01

3. RESULTS:

3.1 Linearity and Range:

The data obtained in the calibration experiments when subjected to linear regression analysis showed a linear relationship between peak areas and concentrations in the range 20-100µg/ml for MET and 5-25µg/mL for ALO (Table 3 and 4) depict the calibration data of MET and ALO. The respective linear equation for MET was y = 38.01x + 80.60 and ALO equation y = 54.47x +7.385 where x is the concentration and y is area of peak. The correlation coefficient was 0.999. The calibration curve of MET and ALO shown (Fig. 5 and 6).

Table 3 Linearity data for Metformin Hydrochloride

Method	Conc. µg/ml	Peak area (µV.sec)		Average peak area (µV.sec)	S. D. of Peak Area	% RSD of Peak Area
Method	Conc. µg/ml	1	2	Average peak area (µV.sec)	S. D. of Peak Area	% RSD of Peak Area
RP-HPLC Method	20	849.7955	850.6942	850.24	0.64	0.07
	40	1598.8525	1599.3652	1599.109	0.36	0.02
	60	2340.5071	2380.40	2339.454	1.49	0.06
	80	3132.3569	3135.1005	3133.729	1.94	006
	100	3883.2092	3885.1035	3885.156	1.34	0.03
	Equation		y = 38.011x + 80.60
	R²		0.999

Fig. 5 Calibration curve of Metformin Hydrochloride

Table 4 Linearity data for Alogliptin

Method	Conc. µg/ml	Peak area (µV.sec)		Average peak area (µV.sec)	S.D. of Peak Area	% RSD of Peak Area
Method	Conc. µg/ml	1	2	Average peak area (µV.sec)	S.D. of Peak Area	% RSD of Peak Area
RP-HPLC Method	5	276.2312	277.2356	276.7334	0.71	0.26
	10	552.436	552.213	552.3245	0.16	0.03
	15	829.4583	830.2341	829.8462	0.55	0.07
	20	1097.2723	1098.1311	1097.702	0.61	0.06
	25	1365.7129	1365.1014	1365.907	0.27	0.02
	Equation		y = 54.47x + 7.385
	R²		0.999

Fig. 6 Calibration curve of Alogliptin, Fig. 7 Chromatogram of Accuracy 80%

3.2 Accuracy:

Recovery studies were performed to validate the accuracy of developed method. To a pre-analysed tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed. The % recovery was found to be within 98-101%. Statistical validation of recovery studies are shown in (Table 5, 6 and Fig. 7, 8 and 9).

Fig. 8 Chromatogram of Accuracy 100%,, Fig. 9 Chromatogram of Accuracy 120%

Table 5 Result of recovery data for Metformin Hydrochloride and Alogliptin

Method	Drug	Level (%)	Amt. taken (μg/ml)	Amt. Added (μg/ml)	Absorbance *Mean ± S.D.**	*Amt. recovered Mean ±S.D.**	% Recovery *Mean ± S.D.**
RP-HPLC Method	MET	80%	20	16	1453.68± 0.06	16.12±0.06	100.77±0.34
		100%	20	20	1604.77± 0.02	20.58±0.02	100.50±0.09
		120%	20	24	1755.25±0.03	24.05±0.03	101.58±0.11
	ALO	80%	5	4	496.06±0.01	3.97±0.01	99.46±0.12
		100%	5	5	547.8± 0.06	4.91±0.06	98.42±1.33
		120%	5	6	604.93±0.01	5.97±0.01	101.58±0.18

*mean of each 3 reading for RP-HPLC method

Table 6 Statistical validation of recovery studies Metformin Hydrochloride and Alogliptin

Method	Level of Recovery (%)	Drug	Mean % Recovery	S. D.*	% RSD
RP-HPLC Method	80%	MET	100.77	0.34	0.34
	80%	ALO	99.46	0.12	0.12
	100%	MET	100.50	0.09	0.09
	100%	ALO	98.42	1.33	1.35
	120%	MET	101.58	0.11	0.11
	120%	ALO	101.58	0.18	0.18

*Denotes average of three determinations for RP-HPLC

3.3 System suitability parameters:

To ascertain the resolution and reproducibility of the proposed chromatographic system for estimation of MET and ALO system suitability parameters were studied. The result shown (Fig. 10 and Table 7).

Fig. 10 Chromatogram of system suitability -1

Table 7 Repeatability studies on RP-HPLC for Metformin Hydrochloride and Alogliptin

Method	Conc. of MET and ALO (mg/ml)	Peak area	Amount found (mg)	% Amount found
RP-HPLC Method for MET	60	1419.4238	61.52	102.53
	60	2418.9814
		Mean	61.52
		SD	0.31
		%RSD	0.01
RP-HPLC Method for ALO	15	828.8961	15.09	100.60
	15	830.4302
		Mean	1.08
		SD	0.13
		%RSD

3.4 Precision:

The method was established by analyzing various standards of MET and ALO. All the solution were analyzed thrice in order to record any intra-day and inter-day variation in the result. The result obtained for interday and intraday variation are shown in the (Table 8 and Fig.11).

Fig. 11 Chromatogram of Precision

Table 8 Result of Intra day and Inter day Precision studies on RP-HPLC method for MET and ALO

Method	Drug	Conc. (µg/ml)	Intraday Precision		Intraday Precision
Method	Drug	Conc. (µg/ml)	Mean± SD	%Amt Found	Mean± SD	%Amt Found
Rp- HPLC Method	MET	20	849.39±1.52	101.40	846.66±1.93	100.75
		60	2340.47±1.11	99.53	24.08.27±9.80	102.05
		100	3794.04±67.83	97.69	3882.20±1.48	100.01
	ALO	5	278.54±0.81	99.56	279.08±0.96	98.00
	ALO	15	828.83±1.01	100.53	830.67±0.50	100.73
		25	1356.12±0.15	99.04	1356.46±0.91	99.04

*Mean of each 3 reading for RP-HPLC method

3.5 Robustness:

To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. The effect of changes in mobile phase composition and flow rate on retention time and tailing factor of drug peak was studied. The results indicate that less variability in retention time and tailing factor were observed (Table 9 and 10).

Table 9 Result of Robustness study of Metformin Hydrochloride

Parameters	Conc. (µg/ml)	Amount of detected (mean ±SD)	%RSD
Chromatogram of flow change 0.8ml	60	2232.99±32.73	1.47
Chromatogram of flow change 1.0 ml	60	2425.59±0.36.82	1.52
Chromatogram of comp change 79ml ACN+21ml water	60	2288.50±10.69	0.47
Chromatogram of comp change 81mlACN +19ml water	60	2401.26±15.20	0.63
Chromatogram of comp change wavelength change 282nm	60	2248.80±7.70	0.34
Chromatogram of comp change wavelength change 284nm	60	2478.70±4.81	0.19

Table 10 Result of Robustness study of Alogliptin

Parameters	Conc. (µg/ml)	Amount of detected (mean ±SD)	% RSD
Chromatogram of flow change 0.8ml	15	766.25±4.08	0.53
Chromatogram of flow change 1.0 ml	15	829.12±1.83	0.22
Chromatogram of comp change 79ml ACN +21ml water	15	790.40±1.19	0.15
Chromatogram of comp change 81mlACN +19ml water	15	828.50±1.17	0.14
Chromatogram of comp change wavelength change 282nm	15	759.10±0.40	0.05
Chromatogram of comp change wavelength change 284nm	15	860.32±2.68	0.31

4. DISCUSSION:

The proposed methods for simultaneous estimation of MET and ALO in tablet dosage forms were found to be simple, accurate, economical and rapid. The method was validated as per the ICH Q2 (R1) guidelines. Standard calibration yielded correlation coefficient (r²) 0.999 for both MET and ALO at all the selected wavelengths. The values of % RSD are within the prescribed limit of 2 %, showing high precision of methods and recovery was close to 100% for both drugs. Results of the analysis of pharmaceutical formulations reveal that the proposed method is suitable for their simultaneous determination with virtually no interference of any additive present in pharmaceutical formulations. Hence, the above methods can be applied successfully for simultaneous estimation of MET and ALO in formulations.

5. CONCLUSION:

The developed HPLC methods in that linearity, precision, range, robustness were found to be more accurate, precise and reproducible. The methods were found to be simple and time saving. All proposed methods could be applied for routine analysis in quality control laboratories.

6. ABBREVIATION USED:

HPLC: High performance liquid chromatography; UV: Ultraviolet; ICH: International Conference on Harmonization; LOQ: Limit of quantitation; LOD: Limit of detection; RSD: Relative standard deviation; RT: Retention time; OPA: Orthophosphoric acid; MET: Metformin Hydrochloride; ALO: Alogliptin; FDA: Food and Drug Administration; SD: Standard deviation.

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Received on 22.01.2021 Modified on 11.02.2021

Research Journal of Science and Technology. 2021; 13(2):111-118.

DOI: 10.52711/2349-2988.2021.00017