INTRODUCTION:

Gemfibrozil, chemically 5-(2, 5-dimethylphenoxy)-2, 2-dimethylpentanoic acid belong to hypolipidamic group. It is of activation of the nuclear transcription receptor “peroxisomes proliferator- activated receptor alpha (PPAR-𝛼) and decrease plasma triglyceride level and increase plasma HDL¹. It is used as lipid regulating agent to treat very high cholesterol and triglyceride level in people with pancreatitis and is also used to lower risk of stroke, heart attack or other heart complications in people with cholesterol and triglyceride¹.

Figure 1: Chemical structure of Gemfibrozil

Quality intentionally may be a concept first out-line by documented quality expert Joseph M. Juran. QbD is "a systematic approach to pharmaceutical development that starts with predefined objectives and emphasizes the understanding of products and processes and process control, supported solid science and quality risk management"². Quality-by-design (QbD) has become a crucial paradigm within the pharmaceutical industry since its introduction by the US Food and Drug Administration. Various experimental designs are available. CCD is one among the experimental design techniques^3-6. Many benefits of this design like Facilitate continuous improvement and technological innovation through more advanced regulatory approaches to managing change, it's more effective use of industry and regulatory resources, also enhanced method robustness and ruggedness through the merchandise life cycle and fewer researches associated with the performance of the analytical method. Knowledge transfer of the more robust method following a far better understanding of the analytical method also as a far better knowledge management⁷.

The currently available literature reveals various analytical methods such as UV spectrophotometry⁸, HPLC^9-11, RP-LC¹², HPTLC¹³, LC-MS in plasma¹⁴, GC (plasma and urine)¹⁵ and Spectro-fluorimetry^16-17. However, no QbD based method is developed so far. A QbD based method provide Method Operable Design Region (MODR), which could function a source for Robust and price effective method, Within MODR, Normal Operating Range (NOR) and Analytical Control Space (ACS) are going to be identify. Once the Multiple rectilinear regression Analysis (MLRA) information collected consistent with chosen designs, the results are often analyzed using statistical method^18-25. Thus it is work while to developed a RPHPLC method for Gemfibrozil by applying CCD which provides understanding and control, Reduction in Variability in Analytical Attributes for method robustness, also applicable throughout the life cycle of the merchandise, then Regulatory flexibility Movements within “Design Space” aren't considered a change in method and thus no requirement of Re-Validation within MODR²⁶.

MATERIAL AND METHODS:

Chemical substances:

Gemfibrozil, AR grade acetonitrile has been used by Finar Pvt. From Molychem Pvt Ltd, AR grade potassium dihydrogen phosphate, AR grade orthophosphoric acid, AR grade potassium hydroxide has been used. Ltd, milliQ Water has been used by Loba Chemie,

Instruments:

High performance liquid chromatography (LC-10 Shimadzu), UV/VIS spectrophotometer (UV-1800 Shimadzu), FT-IR spectrometer (NICOLET iS10 Thermo-Scientific), melting point device (Janki Impex Pvt. Ltd), pH meter (Digital instrument DP-505), analytical balance (citizen CX-220), Sonicator (D-100 / IH Trans-O-Sonic).

Experimental work:

Development of the chromatographic method:

Preparation of the solution:

Preparation of the standard solution:

Carefully weigh about 10milligram of standard Gemfibrozil in a 100ml volumetric flask, add about 50 ml of diluent to be dissolved with the help of ultrasound for about 5 minutes and make up the volume with diluent. Dilute 1.0ml of this solution in 10ml of volumetric flask and make up to the mark with the mobile phase.

Preparation of sample solution:

Transfer 158.8milligram of sample (equivalent to 10 milligram of Gemfibrozil) into 100ml of volumetric flask, add about 50ml of diluent to dissolve with the help of ultrasound for about 5 minutes with and make up the volume with diluent. Filter the solution through Whatman filter paper. Dilute 1.0ml of this filtering solution in 10ml of volumetric flask and carry out the mobile phase.

Experimental Design:

The CCD has been widely used to adapt to a second order model and to require a minimum number of experiments to be performed. This design explored the wider process space while the star points set new extremes for low and high settings for all factors. The% of organic phase (X1), the pH of the mobile phase (X2) and the flow rate (X3) were selected as independent variables, while the retention time (Y1) and the theoretical plate (Y2) were selected as variables .employee. The mathematical model for CCD is expressed as follows:

Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3 + b23X2X3 + b11X12 + b22X22 + b33X32 + E

Where Y is the estimated response, b0 is the intersection and b1, b2, b3, b12, b13, b23, b11, b22 and b33 are regression coefficients. X1, X2, X3 represents the main effect and X12, X22, X32 represent the quadratic terms. And it represents a mistake. The detailed experimental design drawing for Gemfibrozil is shown in the following table. For the optimization of Gemfibrozil, all paths were formed and characterized to obtain the optimized condition.

Table No. 1 Coded levels of variables and their values

Variables	3 Levels
Variables	-1	0	+1
% ACN in Mobile phase	50	60	70
Ph	3	4	5
Flow rate	0.80	1.00	1.20

Table No. 2 Detail design layout of central composite design

Run	Coded values			Uncoded value
Run	% ACN in Mobile phase	pH	Flow rate	% ACN in Mobile phase	pH	Flow rate
1	0	0	0	60	4	1.0
2	+1	-1	+1	70	3	1.2
3	-1	+1	+1	50	5	1.2
4	0	0	0	60	4	1.0
5	-1	-1	-1	50	3	0.8
6	+1	+1	-1	70	5	0.8
7	0	0	0	60	4	1.0
8	+1	-1	-1	70	3	0.8
9	+1	+1	+1	70	5	1.2
10	-10	0	+1	50	4	1.2
11	0	-1	0	60	3	1.0
12	-1	+1	-1	50	5	0.8
13	0	0	0	60	4	1.0
14	-1.68	-1	0	+43.8	3	1.0
15	0	+1	-1.68	60	5	0.6
16	0	0	+1.68	60	4	1.34
17	0	-1	0	60	3	1.0
18	0	+1	0	60	5	1.0
19	0	0	0	60	4	1.0
20	+1.68	-1	0	76.5	3	1.0

Validation of the method^27-38

Linearity:

The linearity response was determined by analyzing 5 independent levels of the calibration curve in the range of 4-20µg/ml for Gemfibrozil. From the standard stock solution of Gemfibrozil (100µg/ml).

Accuracy (n = 3)

The accuracy of the method was carried out in triplicate by solid spiking three different concentration levels 50, 100 and 150% (8µg/ml, 10µg/ml and 12µg/ml) for Gemfibrozil drug.

Precision:

Repeatability:

Repeatability was determined by analyzing the solution containing Gemfibrozil at a concentration of 10µg/ml. The peak area of the same concentration was measured six times and the RSD% was calculated.

Intraday Precision:

The solution containing Gemfibrozil such as 8µg/ml, 10µg/ml and 12µg/ml was analyzed 3 times on the same days, the peak areas were determined and the RSD% was calculated.

Interday Precision:

The solution containing Gemfibrozil such as 8µg/ml, 10µg/ml and 12µg/ml was analyzed on 3 different days, the peak areas were determined and the % of RSD was calculated.

Detection of Limit:

LOD is calculated using the formula:

LOD = 3.3 * σ / s

Quantification of Limit:

LOQ is calculated using the formula:

LOQ = 10 * σ / s

In LOD and LOQ formula,

Where is it, s = slope of the calibration curve,

σ = Standard deviation of the intersection and of the regression line

Specificity:

The specificity of the method was determined by analyzing the diluent and the standard drug to examine the interference of the diluents with the peak of the analyte. The specificity of the proposed method was determined by comparing the blank chromatogram with the standard Gemfibrozil.

Response Surface Plots:

The relationship between dependent and independent variables was further obtained using the response surface graphs, as shown in Figures 2 and 3.

Table No.4 Accuracy of the assay for Gemfibrozil (n=3)

Conc. Level	Placebo added (milligram)	Amount added (milligram)	Area observed ± SD	Amount Recovered (µg/ml)	% Recovery
NA	NA	10	660809.67±8124.26	9.86	98.60
80%	12.66	8	535392.33±4077.99	8.02	100.25
100%	12.66	10	663546±8781.94	9.90	99.00
120%	12.66	12	810938.67±7355.62	12.06	100.50

Precision:

Repeatability (n=6)

Table No.5 Repeatability Data (n=6)

Conc. (µg/ml)	Mean of Peak area (mAU)	SD (n=6)	% RSD
10	662650.5	10411.4	1.57

Table No.6 Intraday and Interday precision data (n=3)

Level	Intra-day precision		Inter-day precision
Level	Mean peak area ± SD	% RSD	Mean peak area ± SD	%RSD
80%	531058.6 ± 9461.0	1.78	520749.2 ± 1652.1	0.32
100%	656621.8 ± 6966.7	1.06	662245 ± 9390.4	1.42
120%	805079.6 ± 10609.9	1.32	792744.9 ± 1476.9	0.19

Limit of Detection (LOD) and Limit of Quantitation (LOQ)(n=5)

Table No.7 LOD and LOQ of Gemfibrozil (n=5)

Drug	Gemfibrozil
Limit of Detection (µg mol)	0.33
Limit of Quantitation (µg mol)	0.99

Specificity:

Check the interference from blank by comparing the chromatogram of blank with standard Gemfibrozil.

Figure 6: Chromatogram of blank

Figure 7: Chromatogram of Gemfibrozil drugs

CONCLUSION:

A new, simpler, more robust and improved Gemfibrozil RP-HPLC analytical method has been successfully developed using an experimental design approach (design of main compounds) in pharmaceutical dosage forms. The central composite design one of the experimental design techniques, was used statistically to calculate the optimal conditions necessary for the desired responses. The simple preparation of the sample, the short analysis time, the high sensitivity, the robust and resistant nature make it suitable for high-performance analysis of marketed formulations, as well as for bulk drugs in quality control and routine analysis laboratories in pharmaceutical companies.

ACKNOWLEDGEMENT:

I would like to express my deep gratitude to Dr. Jayant R. Chavda, principal at B. K. Mody government pharmacy college, Rajkot who has provide us excellent atmosphere for research work thank you sir, for your kind support and motivational thoughts. I also take the opportunity to write my gratitude to Dr Ajay I. Patel, Dr. Ashok B. Patel, Dr. Amitkumar J. Vyas, Dr. Nilesh K. Patel, Dr. Sunny R. Shah and Dr. Ramesh D. Parmar for their valuable suggestion and kind support during research work.

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Received on 20.05.2020 Modified on 13.07.2020

Research J. Pharm. and Tech. 2021; 14(6):3009-3014.

DOI: 10.52711/0974-360X.2021.00527

Conc.	Peak Area
4	253754
8	557947
12	783385
16	1083286
20	1357189