INTRODUCTION:

Sitagliptin is used to reduce blood sugar levels in patients with type 2 diabetes mellitus. In this condition, blood sugar is too high because it does not produce or use insulin usually. Sitagliptin belongs to a class of dipeptidyl peptidase-4 (DPP-4) inhibitors that are orally active, strong, and selective¹.

DPP-4 inhibitors increase glucose metabolism by inducing the hormone incretin, which stimulates insulin secretion and inhibits glucagon secretion in a glucose-dependent manner, thereby increasing glycemic control with the risk of hypoglycemia or lower weight gain^2-5. People with diabetes and high blood sugar can have severe or life-threatening complications over time, including heart failure, stroke, kidney problems, nerve damage, and eye problems. Taking Sitagliptin, making lifestyle changes, and regularly checking blood sugar can help manage diabetes and improve health.

To support the clinical study of Sitagliptin, several analytic methods were developed and validated. Previously, a simple, accurate and rapid analysis method was generated using the UV Spectrophotometer and RP-HPLC method for estimation of Sitagliptin phosphate from tablet formulation^6-8. The online extraction of high turbulence liquid chromatography (HTLC) and HILIC tandem mass spectrometry (MS) methods were then used in many other studies to measure sitagliptin in human plasma, urine and dialysate^9-11. These techniques are simple and have fast sample preparation that allows larger samples to be analyzed, and have been used to help many clinical studies. However, the transfer of methods between laboratories became quite challenging, including personal training for operators and instrumentation adjustments. Some previous studies have also combined the LC-MS approach with protein precipitation^9-11. Protein precipitation is very good for the quantitative determination of Sitagliptin. However, the samples resulting from the preparation are not always clear and leave residual precipitate that can affect the analyte and cause a significant matrix effect and positively affect the method's accuracy and precision value. Besides, the use of internal standards that are still difficult to obtain and relatively expensive is an obstacle in the pharmaceutical industry. According to Prashar (2011), laboratory control, including the analytical method used, is an important priority that must be validated in the pharmaceutical industry¹².

This study aims to develop and validate a simple LC-MS method, with easily obtainable materials, while still providing precise and accurate analysis results for application in the pharmaceutical industry in several countries, especially Indonesia. This study combines the method of separating liquid-liquid extraction (LLE) with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) that has never been done before to determining Sitagliptin in human plasma quantitatively. LLE can assist in producing spectroscopically clean samples and prevent the entry into the MS column and system of non-volatile material. Clean samples are important in LC-MS analysis to minimize ion suppression and matrix effects¹³. The LLE method has been carried out in previous studies that integrate it with the HPLC-MS method^13-15. Then we developed it using UPLC-MS, which has a faster analysis efficiency and the ability to analyze samples with smaller particle sizes, but still with the same and simple operating techniques as HPLC-MS. Also, further, development is carried out by using Nebivolol as an internal standard. Nebivolol is a hypertension drug that has been widely used in Indonesia. Nebivolol (NEB) is a, third-generation vasodilating cardioselective [beta]-blocking agent. Chemically, it is 1-(6-fluorochroman-2-yl) -2-[[2-(6-fluorochroman-2-yl)-2-hydroxy-ethyl] amino] ethanol¹⁶. The use of Nebivolol is based on the closeness of the structure to Sitagliptin (Figure 1), its commercial availability, and relatively cheap price coverage. The parameters of the validation method presented are selectivity, the lower limit of quantification (LLOQ), linearity, accuracy, precision with five different concentrations (LLOQ, low QC, medium QC, high QC, the upper limit of quantification (ULOQ)), Integrity of dilution, matrix effect, and test for stability. The trial results were then compared with the 2011 Medicines Agency (EMA) acceptance requirements in 2011¹⁷.

Figure 1 Chemical structure of Sitagliptin and Nebivolol

MATERIAL AND METHODS:

Chemicals and Reagents:

Sitagliptin and Nebivolol were obtained from the United States of Pharmacopeia (USP) with a purity of 96.60%, and 99.80 %. Other chemicals used are methanol HPLC grade, acetonitrile HPLC grade, isopropanol for HPLC, formic acid, and dichloromethane obtained from Sigma-Aldrich.

Instrumentation:

The main tools used are the Waters UPLC tandem mass spectrometry, the Xevo TQS Micro Acquity detector; and Poroshell 120 EC-C18 4.6 x 50mm column 2.7µm. Supporting equipment consists of a refrigerator, freezer, vortex mixer, ultramicro balance, universal centrifuge, EBA 20 macro, hot plate stirrer, turbo vap ® LV, 0.2 mmµm PTFE filter paper; microtube, ultrasonic water bath, micropipette, blue tip, yellow tip, and other glassware.

Samples for Standard Solutions and Quality Control (QC):

A standard solution of sitagliptin 0.1µg/mL was prepared from a stock solution of 100µg/mL sitagliptin in a 10mL measuring flask. The standard solution of sitagliptin is also made in concentrations (0.2; 0.5; 1.0; 2.0; 4.0; 8.0; 10) µg/mL in a measuring flask 5 mL. All standard series solutions in the 10mL and 5mL measuring flasks were then flushed with 50% methanol and homogenized. All solutions were put into 10mL screw tubes and stored in a refrigerator. Sitagliptin quality control (QC) stock solutions at concentrations (0.1; 0.3; 3, 7.5; 10) μg/mL, which were then bred with 50% methanol and homogenized, were prepared using separate weighing. All solutions were put into a screw tube and stored in a refrigerator.

Operational Conditions UPLC-MS:

The mixture of acetonitrile with formic acid (3:7) was used as the mobile phase using the gradient mobile phase flow technique. Poroshell 120 EC-C18 column 2.7 µm; 4.6 x 50mm is used at 40℃ for samples with a temperature of 5℃. The mobile phase flow rate is 0.4 mL/min, with a total injection volume of 5µL. In this condition, the whole run time is 5 minutes. Mass spectrometry measurements were performed with Waters UPLC-MS with ES⁺ionization mode. In UPLC conditioning tandem mass spectrometry parameters need to be set multiple reaction monitoring (MRM) parameters. The UPLC MRM parameter of mass spectrometry used in this experiment is ion transition m/z 408.23 → m/z 127.02 for Sitagliptin and m/z 406.25 → m / z 151.08 for IS Nebivolol.

System Suitability Test:

The solution for the system suitability test was made by mixed and homogenized 150μL sitagliptin solution at a concentration of 1000ng/mL, 300μL IS nebivolol concentration of 2μg/mL, and 450μL recons solution. An aliquot was inserted into the vial of the sample and injected into the UPLC-MS. The test was carried out six times on the same vial. The %RSD results obtained must be ≤ 5% for the area before using the instrument for further validation.

Human Plasma Sample Preparation:

The plasma containing 250µl sitagliptin was added with 50µL IS nebivolol concentration of 2µg/mL, then homogenized for 10 seconds. After that, the dichloromethane extracting solution was added as much as 3mL and shaken for 1 minute, followed by centrifugation at 4000rpm for 10 minutes to separate the organic solution layers. The organic solvent layer is removed, and the supernatant is extracted into a test tube, then evaporated using 45^oC nitrogen gas for 10 minutes. The dried extract produced was then added 150 µL of acetonitrile recons: 0.1% formic acid in water (1: 1) shaken for 30 seconds, then put into the microtube and centrifuged for 5 minutes at a speed of 14000 rpm. The aliquot (the resulting liquid to be tested) is then put into a vial.

Validation Procedure:

This method is validated according to the European Medicine Agency (EMA) guidelines¹⁷.

Selectivity Test:

Selectivity was determined by measuring blank plasma and plasma that containing Sitagliptin at a lower limit of quantification (LLOQ) concentrations (10ng/mL) using seven different plasmas with codes (A, B, C, D, E, F, G). Selectivity on the plasma blank was made by added 250µL plasma into a screw tube, then 50µL IS Nebivolol with 2µg/mL. Selectivity on the LLOQ is made by added 225µL plasma into a screw tube, then 25µL of LLOQ sitagliptin solution and 50µL IS of nebivolol concentration of 2µg/mL. The plasma blank and LLOQ are then extracted according to the sample preparation method. An aliquot was inserted into the vial of the sample and injected into the UPLC-MS.

The Lower Limit of Quantification (LLOQ) Test:

LLOQ test solution was made by added 225µL plasma into a screw tube, then 25µL sitagliptin solution LLOQ concentration of 10ng/mL and 50µL IS nebivolol 2 µg/mL. LLOQ solution is made five repetitions in the same way. The LLOQ solution is then extracted according to the way of sample preparation. An aliquot was put into a sample vial and injected into a UPLC-MS.

Linearity, Accuracy, and Precision Study:

Linearity is determined by measuring a standard series. Based on the data obtained, a standard calibration curve is made from the regression line equation and determined the correlation coefficient (r) with the terms of acceptance in the linearity measurement that is r ≥ 0.9900. The regression equation used is y = a + b x. The accuracy and precision test is determined by measuring the sitagliptin solution in plasma with five QC solutions (LLOQ, Low QC, Medium QC, High QC, and upper limit of quantification (ULOQ)). For determining the accuracy value, % differentiation from the measured average concentration of Sitagliptin, and the actual concentration of Sitagliptin is sought. As for the determination of the precision value, the difference in measured value is calculated with the standard deviation value, and the relative standard deviation value at each concentration of 7 sitagliptin samples.

Integrity Test for Dilution:

The dilution integrity test was started by adding 1.5 times the ULOQ concentration of 10µg/mL. The dilution solution of the ULOQ concentration that has been made then diluted back to a concentration of 2 and 4 times from the 1.5 times the ULOQ value. The 1000 ng/mL sitagliptin solution was pipetted 125µL and 62.5 µL respectively, with 125µL and 187.5 µL plasma tubes for concentrations of 2 and 4 times dilution, so that the total volume in the plasma was 250µL. Dilution integrity test solution was made five repetitions in the same way for each dilution concentration, then added 50 µL IS of nebivolol concentration of 2µg/mL. The dilution integrity solution is then extracted according to the way of sample preparation. The aliquot was put into a sample vial and entered into the UPLC-MS.

Matrix Effect:

The matrix effect was conducted to determine the impact of interference from the sample matrix on the analysis results. The matrix effect is carried out by making plasma samples with Low QC concentrations and High QC concentrations originating from seven different plasmas, including one plasma hemolysis lot. Test solutions for testing the matrix effect made first are QC in solution and plasma, namely Low and High QC.

Test for Stability:

The stability of human plasma analytes and IS under various temperatures and time conditions and their in-stock stability solutions have been tested. QC samples experience short-term ambient temperature conditions and long-term storage conditions (2 - 8°C). All stability studies were carried out at two concentration levels (30 and 7500ng/mL as low and high QC values) with seven repetitions.

Data Processing Stage:

The test results of each parameter were compared with the company acceptance criteria that refer to the European Medicines Agency (EMA) in 2011. Analysis data obtained were processed using Microsoft office excel and mass lynx ver. 4.2.

RESULT AND DISCUSSION:

Method development:

In this study, we developed a simple UPLC-MS approach to measure the concentration of Sitagliptin in human plasma, where Nebivolol is used as an IS with much availability and is relatively inexpensive still reduces experimental errors and ensures method accuracy. In order to boost detection sensitivity, peak shape and shorten the processing time, optimization of chromatographic conditions was carried out. The LLE method is used in sample preparation. This extraction uses extracting solvent, which is dichloromethane, followed by the drug's concentration to be analyzed. Analytes that have been extracted to the organic phase will quickly be recovered through the evaporation process.

It is optimized through several attempts to achieve resolution and symmetrical peak form, which is good for analytes and IS, and shorter term times, in chromatographic conditioning, specifically the composition of the mobile phase. It was found that this goal could be achieved by a mixture of 0.1% formic acid and acetonitrile (30:70, v/v) and then used as the mobile phase. Several organic solvents in different combinations and ratios have been evaluated to determine the correct strong wash and seal wash solvents in this method. Finally, for solid wash solvents and 10% methanol for seal wash, a variety of 2% formic acid in methanol, acetonitrile, water, and isopropanol (1: 1: 1: 1) was found to be optimal. For plasma blanks, it could generate clean chromatograms.

Selectivity Test:

To determine selectivity, the amount of analyte in the test matrix containing all potential components is compared with the amount of analyte in the solution only. Selectivity is an analytical parameter that describes to what degree the analyte can be determined by a process without interference from other compounds¹⁸. Selectivity tests were carried out on seven different plasmas with the same treatment at blank and LLOQ concentrations (10.32ng/mL). The results showed no significant disturbing peaks of endogenous plasma substances at the same retention time from IS and analyte (Figure 2). The retention times for Sitagliptin and IS were 1.99 and 2.69 minutes, respectively. The selectivity test results are also supported by the calculation of the value of % interference in Sitagliptin (Table 1.) that is equal to (0.00 - 0.38) %, while the results of % interference on IS nebivolol that is equal to (0.04 - 0.24) %. These results meet the acceptance requirements that refer to EMA.

Table 1: Selectivity test result

Human plasma sample	% Interference of Sitagliptin (% diff ≤ 20 %)	% Interference of IS Nebivolol (% diff ≤ 5 %)


A	0.00 %	0.07 %
B	0.38 %	0.06 %
C	0.37 %	0.08 %
D	0,38 %	0.06 %
E	0.00 %	0.04 %
F	0.00 %	0.05 %
G	0.00 %	0.24 %
EMA acceptance requirements	≤ 20 %	≤ 5 %

The Lower Limit of Quantification (LLOQ):

The LLOQ test is carried out using plasma which does not contain Sitagliptin as a blank and plasma with the lowest concentration of sitagliptin solution in a standard series, LLOQ concentration. In the analysis of drug levels in plasma, we need a sensitive method to measure up to the smallest concentration of drug in plasma. LLOQ test results can be seen in Table 2. Based on the LLOQ test results obtained % RSD value of 5.27 %. The results obtained show that at five repetitions, the proximity of the Sitagliptin measured area was measured at each measurement of the actual concentration of 10.32ng/mL.

Figure 2 Sitagliptin and IS Nebivolol Chromatogram from Plasma Blank and LLOQ

Table 2: LLOQ test result

*Actual concentrations of sitagliptin* (ng/mL)**	Repetitions	*Measured concentration of sitagliptin* (ng/mL)**


10.32	1	10.70
	2	10.48
	3	11.15
	4	11.29
	5	11.99
Mean		11.12
SD		0.59
% RSD		5.27 %
EMA acceptance requirements		≤ 20 %

Linearity, Accuracy, and Precision Study:

The linearity test of sitagliptin analysis in human plasma can be seen from the calibration curve made in the blank range, zero blank, and the standard series of concentrations (10.32 - 1032.34) ng/mL. The calibration curve determines the relationship between detector response and analyte concentration in the sample matrix. For some analytes, a sample calibration curve is generated for each analyte. Only if more than 67% (that is, four out of six) of the QCs are within 15% of the nominal value should the calibration curve be considered true (20 % for the LLOQ)¹⁹. The results of linearity measurements can be seen in Figure 3.

Figure 3 Calibration Curve

Based on the results of statistical calculations, the regression equation y = -0.0025 + 0.0014 x, with the correlation coefficient (r) = 0.9991; and the coefficient of determination (r²) = 0.9983. X is the concentration, and y is the response (comparison of Sitagliptin's peak area with IS nebivolol). The intercept value (a) of -0.0025 indicates that when the standard concentration of Sitagliptin is 0 mg/L, the resulting response is -0.0025. The slope (b) value of 0.0014 indicates that each one-unit change in standard sitagliptin concentration will produce a response change of 0.0014. The coefficient of determination (r²) obtained was 0.9983 or 99.83%, indicating that the standard concentration of Sitagliptin affected the tool's response by 99.83% and the remaining 0.17% (100 - 99.83)% was influenced by other factors outside the regression equation or error. The correlation coefficient (r) of 0.9991 shows a linear relationship between the standard concentration of Sitagliptin with the instrument signal's response in the concentration range of (10.32 - 1032.34)ng/mL. The sitagliptin calibration curve shows a positive correlation coefficient meaning that the relationship between the analyte concentration and the response is directly proportional. When the concentration of the analyte rises, the response will also increase.

Accuracy is the closeness of the value obtained by analytical methods with the actual value. Accuracy is determined by repeat analysis of samples containing known amounts of the analyte. Except for LLOQ, where it should not deviate by more than 20%, the mean value should be within 15% of the nominal value . The deviation of the mean from the nominal value acts as an accuracy measure²⁰. The accuracy test was carried out using a plasma containing Sitagliptin at five different concentrations: LLOQ, Low QC, Medium QC, High QC, and ULOQ concentrations with five repetitions each concentration. The accuracy test results can be seen in Table 3.

Table 3: Accuracy test result

Sample	K	k	Differentiation (%)	EMA acceptance requirements
Low QC	30.97	28.76	-7.14	≤ 15 %
Medium QC	309.70	291.36	-5.92	≤ 15 %
High QC	774.26	676.82	-12.58	≤ 15 %
ULOQ	1032.34	935.69	-9.36	≤ 15 %

*K = actual concentration of sitagliptin (ng/mL), k = measurable average concentration of sitagliptin (ng/mL)

Based on Table 3, the results of the % differentiation obtained by (7.77; -7.14; -5.92; -12.58; -9.32) % at the concentration of LLOQ, Low QC, Medium QC, High QC, ULOQ. This shows the closeness between the test result data with the actual level, which is indicated by the value of the measured difference in sitagliptin concentration and the actual concentration (% differentiation). The value of per cent differentiation obtained demonstrates that the method has good accuracy. This is indicated by a comparable level of compatibility between the test results with the actual analyte content and accurate results.

Concerning accuracy, this method's precision (coefficient of variation) is determined to describe the method's level of repetition in regular operation. Precision testing was carried out five repetitions at each concentration. The analysis results calculated the average concentration, standard deviation (SD), and relative standard deviation (RSD). The results of the precision test can be seen in Table 4.

Table 4 Precision test result

Sample	K	k	SD	% RSD	EMA acceptance requirements
LLOQ	10.32	11.12	0.59	5.27	≤ 20 %
Low QC	30.97	28.76	0.47	1.62	≤ 15 %
Medium QC	309.70	291.36	11.58	4.09	≤ 15 %
High QC	774.26	676.82	12.64	1.88	≤ 15 %
ULOQ	1032.34	935.69	49.79	5.32	≤ 15 %

Based on Table 4, the % RSD results obtained from the table are (5.27; 1.62; 4.09; 1.88; 5.32) % at concentrations of LLOQ, Low QC, Medium QC, High QC, ULOQ. This shows that this method provides measurement results at the level of meticulous repeatability at Low QC concentrations with an RSD value of 1.62%; repeatability with moderate accuracy at the Medium QC concentration with RSD value of 4.09 %; and the repeatability with accuracy is not good at ULOQ concentration with RSD value of 5.32%. Precision results show that the operating system of tools, solvents, the environment, and analysts have good precision values for the analysis method.

Integrity Tes for Dilution:

The dilution integrity test is done by adding analytes at 1.5 x ULOQ concentration. The dilution integrity test requirement is diluting 4 and 2 times of 1.5 x ULOQ concentration and doing five repetitions. The integration of dilution test results can be seen in Table 5. The % differentiation results obtained were -9.33% to -9.20% and the % RSD obtained were 5.58% to 2.57%. The dilution integrity test results prove that analyzes with concentrations greater than ULOQ can still be accepted with an appropriate repeatability level.

Matrix Effect:

The effect of the matrix is characterized as the effect of other matrix compounds, different from the analyte, on the quantification of the analyte. The leading cause of the ionization matrix effect is assumed to be the endogenous matrix portion and the analyte. A high level of measurement sensitivity requires ME testing to determine the sample matrix's interference effect on the results of the analysis¹⁸. The test is done by calculating the ratio of the peak area to the presence of a matrix in it (measured by analysis of samples that are loaded after extraction) to the peak area without a matrix (analytes in pure solvents). The RSD percentage from the IS normalized matrix factor (MF) is calculated from seven matrices ≤ 15%. The results of the matrix effect measurement are shown in Table 6. The % RSD results obtained were 6.49 % for Low QC and 2.20 % for High QC. These data indicate that the sample matrix's interference effect does not significantly impact the analysis results, so the research still shows a high level of measurement sensitivity.

Table 5 Dilution integrity test result

S	K	Ki	k	% Dif	SD	% RSD
4 x	1545.65	1397.25	1403.47	-9.20	78.26	5.58
		1526.10
		1369.89
		1312.74
		1411.38
2 x	1545.65	1445.50	1404.01	-9.33	36.08	2.57
		1350.30
		1423.82
		1390.76
		1409.68
EMA acceptance requirements				≤ 15 %		≤ 15 %

*S = dilution of human plasma sample, Ki = measured concentration of sitagliptin. Dif = differentiation

Table 6 Matrix effect test result

QC	Plasma	ME of Sitagliptin	ME of IS Nebivolol	IS Norm. MF	% RSD	EMA acceptance requirements
LOW	A	0.9778	0.9223	1.06	6.49	15 %
	B	0.9839	0.9479	1.04
	C	0.8969	0.9670	0.93
	D	0.9711	0.9587	1.01
	E	0.9771	1.0046	0.97
	F	0.9376	1.0020	0.94
	G	0.9039	1.0181	0.89
HIGH	A	0.9519	0.9892	0.96	2.20	15 %
	B	0.9906	1.0302	0.96
	C	0.9720	0.9968	0.98
	D	0.9808	1.0394	0.94
	E	0.9838	1.0218	0.96
	F	0.9734	1.0567	0.92
	G	0.9703	1.0499	0.92

Test for Stability:

The stability test is carried out to determine the standard solution's stability, and the sample analyzed during the analysis and in storage conditions. A stability test aims to detect any degradation of the interest's analyte during the entire period of sample collection, processing, storing, preparing, and analysis²¹. The stability test in this experiment consists of three types: the short-term temperature stability test, the long-term stability test, and the stock solution stability test. Tests were carried out on two concentrations: low QC concentration (30.97 ng/mL) and High QC concentration (774.37 ng/mL). The procedure was repeated five times. Then the solution is stored at room temperature for 0, 2, 4, and 6 hours. Based on the tests that have been done, the result of % differentiation is -6.25 % to -14.68 % for Low QC concentrations and -8.80 % to -13.69 % for High QC concentrations. The test results showed the stability of the sitagliptin solution in human plasma at room temperature. Long-term stability is carried out on days 0 and 4 of two concentrations, namely Low QC concentration (30.97ng/mL) and High QC concentration (774.37ng/mL). The solution is stored in a refrigerator (2 - 8)⁰C. Based on the testing that has been done, the result of % differentiation is -4.80 % to -9.54% for Low concentration and -8.43% to -10.23%, indicating that Sitagliptin is still stable for four days.

The stability of sitagliptin stock is tested to provide efficiency when working. If it is stable, then the leading solution used to validate it does not need to be made every new analysis. This will be very useful if active substances are available in limited quantities. Stock solution testing was carried out at 0 hours, 6 hours, day 0, and day 11. Tests at conditions of 0 and 6 hours were carried out on stock solutions of sitagliptin concentrations Low and High QC, as well as IS Nebivolol concentrations of 2.02 µg/mL. Tests on day 0 and day 11 conditions were carried out on Sitagliptin and IS Nebivolol stock solution with concentrations of 10.32 μg/mL and 2.02 μg/mL, respectively; then stored in the refrigerator (2 - 8) ⁰C for 11 days. Based on experimental results, the stock solution of Sitagliptin and IS Nebivolol remained stable at room temperature for 6 hours with % differentiation (-1.56 - 4.14) % at Low QC concentrations and (0.18 - (- 10.19)) % at the High QC concentration. Stock solutions at refrigerator temperatures remained stable until days 0 and 11, with a % differentiation of 0.00 % and 6.21 %. The test results show the sitagliptin solution's stability at room temperature in the range of 0 to 6 hours and storage in the refrigerator (2 - 8) ⁰C in the range of 0 to 11 days.

CONCLUSION:

The UPLC-MS method was successfully validated for quantitative determination of Sitagliptin in simple human plasma using IS nebivolol and the LLE extraction method. This method shows good linearity with a correlation coefficient of 0.9991. Based on all the results of other experiments that have been carried out on the parameters of selectivity test, LLOQ, accuracy, precision, dilution integrity, matrix effect, and stability test can meet the acceptance requirements that refer to EMA in 2011, so that the method of determining the levels of Sitagliptin in human blood plasma using UPLC-MS can be used in routine analysis of Sitagliptin in human plasma as a simple and relatively inexpensive analysis method for the pharmaceutical industry.

ACKNOWLEDGEMENT:

We are deeply grateful for Politeknik AKA Bogor and PT Equilab International for the support given during this study.

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Received on 01.02.2021 Modified on 06.05.2021

Research J. Pharm. and Tech 2022; 15(1):89-96.

DOI: 10.52711/0974-360X.2022.00016