1. INTRODUCTION:

Metformin is an oral antidiabetic drug in the biguanide class. It is the first-line drug of choice for the treatment of type II diabetes mellitus, in particular, in overweight and obese people and those with normal kidney function. Metformin is now the most widely prescribed oral hypoglycemic agent. The main mechanisms of action include reduction of appetite and of intestinal carbohydrate absorption, of hepatic gluconeogenesis, and increased glucose uptake by peripheral tissues.

Piperine is the pungent alkaloid present in Piper nigrum has been shown to possess diverse biochemical and pharmacological activities and is known to modify the biotransformation of drugs. Piperine acts by suppressing P-glycoprotein and CYP450 enzymes, which counter act the metabolism of metformin via these proteins, thus enhancing the oral bioavailability of metformin^1-4

Literature survey may not reveal any RP-HPLC method or UV- spectrophotmetry method for the simultaneous estimation of metformin and piperine in combined tablet dosage form. Simultaneous estimation of metformin and piperine combined pharmaceutical dosage form is not official in any pharmacopoeia, hence no official method is available for their estimation. Hence an attempt has been made to develop a simple, accurate, sensitive, rapid and economic method for the simultaneous estimation of metformin and piperine in combined tablet dosage form using UV- Spectrophotmetry for which a combined tablet dosage form of metformin with piperine were prepared and developed analytical method was validated and subjected to ICH guidelines^5-10

Fig.1. N,N-dimethylimidodicarbonimidic diamide

Fig.2. Piperine (1-[(2E, 4E)-5-(1,3-benzodioxol-5-yl)-1- oxo-2,4- pentadienyl] piperidine)

2. EXPERIMENTAL:

2.1. Chemicals and reagents:

A Gift sample of metformin was obtained from Aurobindo pharmaceuticals, Hyd, gift sample of piperine was obtained from Aldrich Sigma, Hyderabad, HPLC grade methanol and water were purchased from S.D Fine Chemicals, Mumbai, India. All other chemicals/reagents were of research grade and used without further purification.

2.2. Instruments:

Instruments used were Shimadzu UV-1800 UV-Visible Spectrophotometer, Remik Tablet Punching Machine, Shimadzu BL-220H Digital Balance, PCI Analytics 6.5 li200H Ultra Sonic Bath Sonicator

2.3. Standard solutions:

Standard piperine of 1000μg/mL solution was prepared by dissolving 100mg of piperine working standard in 100mL of methanol and mixed well. From this 100 μg/ml solution was prepared by taking 10mL of piperine standard solution in 100mL methanol. Standard metformin of 1000μg/mL solution was prepared by dissolving 100mg of nateglinide working standard in 100mL methanol.

2.4 Selection of analytical wavelength:

10µg/mL solutions of piperine and metformin were prepared from the stock standard solutions of seperately using methanol as solvent and the solutions were scanned agianst blank (methanol) in the entire UV range to determine the λmax values. Clear peaks were observed at 237nm and 342nm for metformin and piperine respectively Hence these wavelengths were choosen as the λmax values for metformin and piperine respectively. By the overlay graph it was evident that both the drugs absorb at λmax of each other drug. So those particular wavelengths were choosen as λmax.

2.5. Calibration curve for piperine (2-10µg/ml):

From the working standard stock solution of piperine (100µg/ml), appropriate aliquots like 2, 4, 6, 8, 10ml solutions were transfered in 10ml graduated tubes. The solution in each tube was made up with methanol to obtain working standard concentration ranging from 2-10 µg/ml. The absorbance of these solutions was measured agianst blank (methanol) at 342nm.

2.6 Calibration curve for metformin (2-10µg/ml):

From the working standard stock solution of metformin (1000µg/ml) appropriate aliquots like 2, 4, 6, 8, 10ml solutions were transfered in 10ml graduated tubes. The solution in each tube was made up with methanol to obtain working standard concentration ranging from 2-10µg/ml. The absorbance of these solutions was measured agianst blank (methanol) at 237nm.

2.7 Validation of Simultaneous estimation method for estimation of piperine and metformin:

The method developed was validated according to the ICH Guidelines Q2 (R1): Validation of Analytical Procedures: Text and Methodology. Method was validated for various parameters such as Linearity and Range, LOD (Limit of Detection), LOQ (Limit of Quantification), Precision to ensure that the performance characteristic of the method meets the requirements for the intended analytical applications^11-12

2.8 Formulation of Combined Dosage Form Tablets:

Table 1. Ingredients for the formulation of combined dosage form tablets

S. No	Drugs and Excipients	Manufacturers	Quantity (mg)
1	Metformin (Drug)	Aurobindo pharmaceuticals, Hyd	100
2	Piperine (Drug)	Aldrich Sigma	10
3	Sodium starch glycollate	S d fine chem limited, Mumbai	5
4	Talcum powder	S d fine chem limited, Mumbai	3
5	Magnesium powder	S d fine chem limited, Mumbai	2
6	Dibasic calcium phosphate	NR Chem, Mumbai	20
7	Starch	S d fine chem limited, Mumbai	10%
8	Lactose	S d fine chem limited, Mumbai	40

2.9. Simultaneous Estimation of Piperine And Metformin Combined Tablet Dosage Form:

Sample preparation:^13-22

10 tablets were weighed; average weight was calculated and crushed into fine powder in a cleaned and dried mortar with a pestle. Tablet powder equivalent to 100mg of piperine and metformin was weighed and transferred into a 100ml volumetric flask, to this 10mL of methanol was added and sonicated for 15 minutes with intermediate shaking by obtaining a clear solution with maximum sediments settled at the bottom of the tube. Then it is diluted and made up with methanol after being achieved to room temperature and mixed well and sonicated further for 2 minutes. Then the graduated tube with the test solution is kept aside for 20min by closing it with a glass stopper or lid. This solution was filtered through Whatmann filter paper (no.41).

6μg/ml solution of metformin and 6μg/ml solution of piperine was prepared by transfering 0.6ml from the prepared tabet stock solution to 10ml volumetric flask and the volume was made upto mark with methanol the absorbance is checked at 237 nm and 342nm by using this value we calculated the %purity by using formula.

% purity of piperine and metformin tablets was calculated using the formula.

Concentration x Dilution factor x avg wt of the tablet

Assay = -------------------------------------------------- X 100

Weight of powder taken x label claim of the drug

3. RESULTS AND DISCUSSION:

New UV spectrophotometric method was developed and optimised by Shimadzu UV-1800 instrument using simultaneous equation method.

3.1. Development and Optimisation:

3.1.1. Absorption maximum of Piperine (10µg/mL):

The absorption maximum of 342nm was taken from absorption spectrum and selected as wavelength for spectrophtometric determinations. The difference absorption spectrum was shown in Fig.3

Fig.3 Absorption maximum of Piperine

3.1.2 Absorption spectrum of metformin(10µg/ml)

Absorption maximum of 237nm for metformin was taken from absorption spectrum and selected as wavelengeth for spectrophtometric determinations. The difference absorption spectrum was shown in Fig.4

Fig.4 Absorption spectrum of metformin

3.1.3. Overlay Spectrum of nateglinide and piperine:

Absorption maximum for metformin and piperine was found to be 237nm and 342nm respectively from the overlay spectrum as shown in Fig. 5

Fig. 5: Overlay Spectrum of nateglinide and piperine

3.1.4 Selection of analytical concentration range and verification of Beers law:

In the concentration range 2-10μg/ml of metformin and 2-10μg/ml of piperine, there was a linear relationship between concentration and absorbance values for piperine and metformin respectively. This range was selected for construction of calibration curve. The optimised instrumental conditions are shown in Table.2

Table 2. Optimized UV conditions

Drugs	Piperine	Metformin
λ_max	342nm	237nm
Beer’s law range	2-10μg/ml	2-10μg/ml
Regression equation(y)	y=0.0984x+0.0015	y =0.0678x+0.019
Slope	0.0984	0.0678
Intercept	0.0015	0.019
Coefficient of determination(r²)	0.999	0.998

3.1.5 Simultaneous Equation Method:

Standard solutions of metformin and piperine in combination were prepared in methanol and the absorbance of these solutions was measured at 237nm and 244.6nm. Calibration curves were plotted to verify the Beer’s law and absorptivity values calculated at the respective wavelengths for both the drugs. Two simultaneous equations as below were formed using these absorptivity values, A (1%,1cm)

Cx = A2ay1 – A1ay2 / ax2ay1 – ax1ay2

Cy= A1ax2 – A2ax1 / ax2ay1 – ax1ay2

Where,

Cx and Cy are the concentrations of metformin and piperin measured using sample solutions.

A1 and A2 are absorbance of the diluted sample at λ1 and λ2 respectively.

ax1, ax2 are the absorptivity values of X at λ1 and λ2 - ay1, ay2 are the absorptivity values of Y at λ1 and λ2

The obtained values: A1= 1.240; A2= 0.005; ax1= 0.124 ; ax2 = 0.55; ay1=5.5; ay2 = 24.9

By substituting the values in the above equations Cx and Cy were determined.

3.2 Method validation:

The developed method was validated for the following parameters

3.2.1 Linearity:

Linearity was obtained in the 2-10μg/ml and 2-10μg/ml concentration range for piperine and metformin respectively and the standard calibration curve data is shown in Table.3. calibration curve in fig.6 for piperine and fig.7 for metformin.

Fig.6 Standard calibration curve for Piperine

Fig.7 Standard calibration curve for Metformin

The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y= 0.0984x+0.0015, y= 0.0678+0.019 for piperine and metformin respectively and correlation coefficient was 0.999 for piperine and 0.998 for metformin respectively.

3.2.2 Limit of Detection (LOD) and Limit of quantitation (LOQ):

The LOQ and LOD reported as a means to analyse the sensitivity. LOD calculated by using standard deviation method was found to be for metformin0.030µg/ml, for piperin 0.0267µg/ml. LOD = 3.3σ/S LOQ calculated by using standard deviation method was found to be for metformin 0.0929µg/ml, for piperine 0.081µg/ml. LOQ = 10σ/S

3.2.3 Precision:

The precision of the developed analytical method was assessed by checking repeatability, intra-day precision and inter-day precision.

3.2.3.1 Repeatability:

The precision of the instrument was checked by repeatedly checking (n=6) solution of piperine (6μg/ml) and metformin (6μg/ml) and measuring respective absorbance. The results were reported in terms of relative standard deviation and were found to be not more than 2 %. The results were shown in table 4.

Table 3: Standard calibration curve data for piperine and nateglinide

S. No	Volume of stock solution transferred (ml)	Final Volume (mL)	Concentration (μg/ml)	Absorbanceat237nm(metformin)	Absorbanceat 342nm(piperine)
1	0.2	10	2	0.15	0.19
2	0.4	10	4	0.28	0.38
3	0.6	10	6	0.42	0.59
4	0.8	10	8	0.57	0.77
5	1.0	10	10	0.69	0.99

The developed method was precise as the absorbance values of six replicates are close. The calculated %RSD values are within the limits.

Intra-day precision:

The intra-day precision of the proposed method was determined by analyzing the corresponding concentration (80%, 120%, 150%) 3 times on the same day and the results were reported in terms of relative standard deviation. The results were shown in table 5.

Inter-day precision:

The inter-day precision of the proposed method was determined by analyzing the corresponding concentrations (80%, 120%, 150%) on 3 different days and the results were reported in terms of relative standard deviation. The results were shown in table 5.

Table 5 Inter-day and Intra-day precision data for Piperine and Metformin

S. No	Concentration (μg/mL)	Concentration (μg/mL)	%RSD Calculated at 237nm	% RSD Calculated at 342nm
1	4.8	4.8	1.03	1.92
2	6	6	1.27	1.05
3	7.2	7.2	0.85	1.03

* Average of 3 determinations

The results shows that all the calculated %RSD values are below 2, therefore the method may be precise.

3.2.4. Accuracy:

The analytical accuracy is the nearness of the results obtained against the real values. The results of obtained for accuracy studies for the drug substance and drug product were reported in terms of % RSD and % recovery respectively.

3.2.4.1. For drug substance:

Accuracy data for piperine and metformin drug substance was shown in table 6 and 7.

Table.6 Accuracy data for metformin drug substance

S. No	Accuracy level	Concentration (μg/ml)	Absorbance At 237nm	Mean ± SD	% RSD
1	80%	6+4.8	0189	0.191±0.003	1.02
			0.190
			0.195
2	100%	6+6	0.398	0.0393±0.005	1.27
			0.388
			0.395
3	120%	6+7.2	0.596	0.59±0.005	0.84
			0.589
			0.585

Table.7 Accuracy data for piperine drug substance

S. No	Accuracy level	Concentration (μg/ml)	Absorbance At 342nm	Mean ±SD	% RSD
1	80%	6+4.8	0.100	0.102±0.002	1.96
			0.103
			0.105
2	100%	6+6	0.285	0.285± 0.003	1.05
			0.289
			0.283
3	120%	6+7.2	0.489	0.484±0.005	1.03
			0.485
			0.479

From the above data it was found that the %RSD (acceptance criteria <2%) values are within the acceptance limits.

3.2.4.2. For drug product (Recovery study):

The recovery was assessed by determining the agreement between the measured standard concentration and added known concentration to the sample. The test was done by spiking the pre-analysed tablet powder with pure piperine and metformin at three different levels (80%, 100% and 120%) the recovery percentage values ranged 98%, 101%. 98 % and % for metformin and 99%, 98%, 101% for piperine respectively. The results were shown in Table 8.

From the above data it was found that the %recovery values are within the limits.

Table.8 Recovery data for metformin and piperine:

Tablet used	Amount of sample added (µg/mL) (metformin+ piperine)	Amount of standard added (µg/mL) (metformin+piperine)	Amount of standard added (metformin+piperine)	Total amount added (µg/mL)	Total amount added (µg/mL)	Amount recovered (µg/mL) (metformin)	Amount recovered (µg/mL) (piperine)	%Recovery ±Standard Deviation (metformin)	%Recovery ±Standard Deviation (piperine)
Metformin and piperine (180mg)	6+6	4.8	4.8	10.8	10.8	10.6	10.78	98.148± 0.691	99.81±0.491
	6+6	6	6	12	12	12.2	11.88	101.666± 0.950	98.33± 0.298
	6+6	7.2	7.2	13.2	13.2	12.98	13.4	98.787± 0.160	101.515± 1.75

* Average of 3 determinations

From the above data it was found that the %RSD (acceptance criteria <2%) values are within the acceptance limits.

Table 9. Assay values of Piperine and Metformin combined dosage form tablets

Tablet used	Label claim(mg)		Amount found(µg/mL)		% Assay
	Piperine	Metformin	Piperine	Metformin	Piperine	Metformin
Piperine and metformin combined tablet dosage form	10	100	6.3	6.2	100.8	99.2

3.2.5 Assay of Piperine and Metformin tablets:

The calculated percent purity of Piperine and Metformin combined dosage form tablets were found to be 100.8% and 99.2% for Piperine and Metformin respectively and the values were shown in Fig. 4.8.

4. CONCLUSION:

A new, simple and reproducible simultaneous equation method (Vierdott’s method) was developed and validated for the quantitative estimation of Metformin and piperine in combined tablet dosage form using Schimadzu UV1800 instrument with UV Probe 4.0 software. The analysis of samples was carried out by using quartz cuvettes as sample cells. The method was developed and optimised with instrumental conditions, absorbance mode as measurement mode. Wavelengths 258.6nm and 244.6nm selected as absorption maximum for Metformin and Piperine respectively.

Simultaneous Equation method was developed by considering the absorbance measurements of Metformin and Piperine in methanol.

The analytical methods were validated as per ICH Q2 (R1) guidelines with respect to various parameters - linearity, accuracy, precision, selectivity, LOQ and LOD. Simultaneous equation method was linear over a concentration range of 50-250μg/ml and 5-25μg/ml, for Metformin and Piperine respectively. Limit of Detection was found to be 3.3μg/mL and Limit of Quantification was found to be 10μg/mL for Metformin and Limit of Detection was found to be 1.98μg/mL and Limit of Quantification was found to be 6μg/mL for piperine. Relative standard deviation for intra-day precision and inter-day precision were within the acceptable limits. The mean recovery of the combined tablet dosage form of Metformin and piperine with the developed method was found to be 99.5% and 99.07%.

From the results, it can be concluded that the developed method was effective for quantitative determination of Metformin and Piperine in bulk and combined tablet dosage form without any interference of other constitute in the formulation. Tablets were prepared and analyzed by the proposed method and assay of the drug was calculated. The developed method could be readily used for the quantitative estimation of Metformin and Piperine in their combined dosage form.

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Received on 06.01.2022 Modified on 11.03.2022

Asian J. Pharm. Ana. 2022; 12(4):221-227.

DOI: 10.52711/2231-5675.2022.00036

S. No	Concentration metformin (µg/mL)	Concentration piperine (µg/mL)	Absorbance (237)	Absorbance (342)	% RSD	% RSD
1	6	6	0.398	0.289	0.21	0.38
2	6	6	0.397	0.289
3	6	6	0.398	0.288
4	6	6	0.396	0.289
5	6	6	0.398	0.288
6	6	6	0.398	0.286