INTRODUCTION:

An outstanding role is played by the Pharmaceutical analysis in the testing of pharmaceutical formulations regarding quality control and assurance ^[1].For the identification of biological and pharmaceutical products HPLC plays a significant role ^[2]. The principle behind RP-HPLC is hydrophobic interaction ^[3]. When there are inadequate methods new methodologies are being progressed for evaluation of the novel product ^[4]. Method development is helpful to reduce the cost beside time for better results ^[5]. Analytical method validation is used during drug development and drug manufacturing ^[6].

It is most recognized and vital parameter of cGMP ^[7]. Biotin (Vitamin H) member of B vitamin group, water soluble and is widely distributed in nature ^[8]. It act as cofactor responsible for carbon dioxide transfer in several carboxylase enzymes ^[9]. Chemical name is Hexahydro2-oxo-1-H-thiemo (3, 4-dimidazole-4-pentanoic acid^[10]. Steady blood sugar level is maintained by it ^[11].Nutraceutical first coined by Stephen Defelice in 1979^[12]. Today people are very much concerned about food habits, health and life style consumption of junk food ^[13]. As nutraceutical are nutrabioactive, enriches the food helpful for better health their demand increased. In other term known as premixes. Premix (solid blend of multi vitamin) usually refers to a substance which is mixed in an early stage in the manufacturing and distribution process. They are the blend of vitamins and minerals. As per the Recommended Dietary Allowances (RDA) these micronutrients must be provided at specific doses for ensure the amount. There is no such method for the estimation of biotin from premixes. The proposed work will provide an effective method to estimate biotin from premixes.

Background:

The RP-HPLC method was developed for biotin in premixes (solid blend of multi – vitamin) has been validated in Piramal Enterprises Mahad Pvt. Ltd. Analytical method validation for the biotin in premixes (solid blend of multi – vitamin) by RP-HPLC has been conducted as per the protocol, to ensure that the performance characteristics of the method meet the requirements for its intended application. This work is done by following the general ICH Q2 (R1) Guideline.

MATERIALS AND METHOD:

Materials

Table No.:1

Reagent	Batch Details	Manufacturer
Sodium perchlorate monohydrate	A0568464	MERK
Ortho phosphoric acid	CF8C680349	MERK
Acetonitrile	270071117JR	FINAR
Instruments/ Equipments	Make And MODEL	-
HPLC	Agilent technology 1260 infinity series	-
Weighing Balance	Mettler Toledo	-
UV	JASCO V 630 Spectrophotometer	-
Ultra Sonicator	Life Care	-
Sample	Batch No.	-
D-Biotin	Standard	-
Solid Blend of multi-vitamin PLACEBO	Sample-1	-
Solid Blend of multi-vitamin	Sample-2	-
Column	Make: Thermo scientific	Dimensions:ODS C₁₈150× 4.6 (mm) 3µ

Chromatographic conditions:

Column: Thermoscientific, C18, 150 X 4.6 mm, 3 μ, Flow rate: 1.2 ml/min, Detection Wavelength:200 nm,Injection Volume: 100µl, Oven temperature: 40°C, Mobile phase: Buffer: Acetonitrile (91.5:8.5)

Preparation of Buffer solution:

2 g of sodium perchlorate monohydrate in 1000 ml of water was dissolved, to it added 2.0 ml of orthophospheric acid and diluted with water to 2000ml.

Preparation of Mobile phase:

A Mobile phase of Buffer: Acetonitrile in the ratio of 91.5:8.5 was prepared, mixed well and degased.

Preparation of Placebo:

Weighed accurately quantity 2 gram of placebo in 100ml volumetric flask, to it added 60ml of water and heat with intermediate shaking in a water bath at 65°C for 20min.Sonicated for 5min,shaked for 15 min and cooled to room temperature. Added 20ml of Acetonitrile, diluted with water to volume (Solution 1). Diluted further with mobile phase. Filtered the sample through 0.45 micron nylon filter. Filled the vial. The placebo solution was run.

Preparation of Standard Solution:

Weighed accurately about 10.0 mg of biotin working standard into a clean 100 ml volumetric flask. Sonicated to dissolve in diluent and make up the volume with diluent (solution-1). Diluted 1ml of this (solution-1) to 100ml with mobile phase. Filled the vial. Standard solution was run. (1ppm). Vial was filled. The standard solution was run.

Preparation of Sample Solution:

Weighed accurately quantity 2 gram of sample in 100ml volumetric flask, to it added 60 ml of water and heated with intermediate shaking in a water bath at 65°C for 20min.Sonicated for 5min, shacked for 15 min and cooled to room temperature. To it added 20 ml of acetonitrile, diluted with water to volume (Solution). 10ml of solution diluted to 20 ml wit mobile phase (1 ppm). The sample was filtered through 0.45 µ nylon filter. Filled the sample. The sample solution was run.

Calculation:

% Assay of biotin:

A_T × Std conc. (mg)×100× P × Sample conc.×LC

--------------------------------------------------------------------

A_s×100

= % of biotin

Where, A_T:Area of sample,A_s:Area of standard,P: Potency of stdandard,LC: Label Claim

RESULTS:

1. Specificity:

For the demonstration of the ability of analytical method to determine the specificity for the analyte in the presence of components that may be expected to be present in the sample matrix.

Chromatogram No.:1 for Placebo

Chromatogram No.:2 for Standard

Chromatogram No.:3 for Sample

RESULT:

No any interference was found from blank, placebo to analyte in the test solution.

Sample solution and result:

1. Blank:

1. No Retention time with no Area, 2. Placebo : No Retention time with no Area, 3. Standard: 10.28 Retention time with Area 12734714,4. Standard: 10.23 Retention time with Area 12342453.

2. Linearity:

1. It is expressed in terms of variance around the slope of the regression line calculated in accordance to established mathematical relationship between the concentration of solution and area obtained. Prepared different concentrations of standard in between 50% to 150%.

Preparation of solution:

1. For level 50% Dilute 0.5ml of standard stock solution to 100ml with mobile phase.(0.5 ppm), 2. For level 80% Dilute 0.8ml of standard stock solution to 100ml with mobile phase.(0.8 ppm), 3. For level 100% Dilute 1.0ml of standard stock solution to 100ml with mobile phase. (1.0 ppm), 4. For level 120% Dilute 1.2ml of standard stock solution to 100ml with mobile phase. (1.2 ppm), 5. For level 150% Dilute 1.5ml of standard stock solution to 100ml with mobile phase.(1.5 ppm)

Result: Table No.:2

Standard concentration (ppm)	Area	Mean
0.5	6779319, 6623225, 6720013	06707519
0.8	11117559, 11099031, 11229177	11048589
1.0	12744735, 12709152, 12748353	12734080
1.2	15375885, 15301433, 15207070	15294796
1.5	19258744, 19271764, 19203165	19244557

Correlation Coefficient

Fig No. 4 Correlation Coefficient Graph For Biotin

Results:

Correlation Coefficient was found to be 0.996

3. Precision:

The method precision was performed by analyzing a sample solution of premix (solid blend of multi-vitamin) as per the test method (six replicate sample preparation).

A) Reproduciability:

Variations are studied including analyst, equipment, day etc. Prepare standard and sample solution as per the procedure.

Prepare 1 Blank solution,1 standard with 5 replicant injections and 6 test batch samples.

Table No.:3

Replicate No.	Area	Retention Time
1	12097791	10.267
2	12074386	10.273
3	12019659	10.253
4	12192523	10.260
5	12142789	10.227
AVG	12105430	10.256
Standard Deviation	65877.98	0.018
%RSD	0.54	0.176

Result Sample:

The Assay (%) of Replication No. 1 -105.05,The Assay (%) of Replication No. 2 -106.06,The Assay (%) of Replication No. 3 -104.04,The Assay (%) of Replication No. 4 -105.05,The Assay (%) of Replication No. 5-109.09, The Assay (%) of Replication No. 6-103.03,Average -105.39%, Standard Deviation –2.09, % RSD-1.98

Results of System suitability:

1. %RSD of Assay (%)- 1.98 (NMT5.0%)

2. %RSD of RT for Standard-0.18 (NMT 1.0%)

3. %RSD of RT for Standard-0.54 (NMT 2.0%)

B) Intermediate Precision

Repeatibility:

As per the test method (six replicate sample preparation) and injected each solution in triplet on different day. The results of were compared with the results of precision.

Table for standard:

Table No.:4

Replicate No.	Area	Retention Time
1	11769163	11.113
2	11522559	11.100
3	11645823	11.107
4	11729500	11.107
5	11631567	11.093
AVG	11659722	11.104
Standard Deviation	95715.56	0.008
%RSD	0.82	0.07

Result for samples:

The Assay (%) of Replication No. 1 -105.12,The Assay (%) of Replication No. 2 -105.93,The Assay (%) of Replication No. 3 -103.69,The Assay (%) of Replication No. 4 -106.20,The Assay (%) of Replication No. 5 -102.88,The Assay (%) of Replication No. 6 -100.96,Average -104.13%, Standard Deviation –2.02, % RSD-1.94

Results of System suitability:

1. %RSD of Assay (%)- 1.94 (NMT5.0%)

2.%RSD of RT for Standard-0.07 (NMT 1.0%)

3.%RSD of RT for Standard-0.82 (NMT 2.0%)

4. Accuracy:

1. It is the measurement of exactness of the analytical method.

2. three different concentrations of sample at 50%,100% and 150% to be prepared.

3. at each level calculate the % recovery.

RESULT:

Table No.:5

Sr. No	Conc.	Weight of sample	Actual Qty added	Mg/gm	Mean recovery	% Mean recovery
1	50%	1.0000	1.0266, 1.0266, 1.2066	0.108, 0.104, 0.106	0.106	101. 92
2	100%	2.0000	2.0008, 2.0008, 2.0008	0.105, 0.104, 0.104	0.104	100. 00
3	120%	3.0000	3.0144, 30144, 3.0144	0.101, 0.101, 0.104	0.102	98.08

DISCUSSION:

Before applying the technique on RP-HPLC the effective working condition was completed for biotin. For better results the mobile phase used was of Buffer: Acetonitrile in the ratio 91.5:8.5. Peak obtained was sharp. The retention time was found to be between 10 & 12 min. The use of diluent water: ACN in final dilution in the ratio of 4:1 was not able to give the proper peak. The use of mobile phase as final diluent gave the required result. The column of 3µ was used for better elution. The use of column of dimension 150 ×4.6 gave the better peak. The retention time of drug was found to be within the limits of 0-15 minutes. These parameters represent the specificity of method. Particular concentration range was determined from the linearity studies. It was experimentally observed that biotin was in the extent of 50% - 150% for the aim concentration. The analysis was done in triplet form for the better results that is 3 injections at each level. The result were linear with correlation coefficient 0.9960. By precision and intermediate precision the validation for the proposed method was verified. The % recovery value of 95.0% - 105.00% for biotin was studied.By recovery studies the validation of the proposed method was verified. Throughout the process every parameters counting flow-rate, temperature, detector, wavelength and sensitivity were maintained constant. The method was found to be specific, linear, accurate and precise. The analytical method validation was carried as per the ICH Q2 (R1) guidelines.

AKNOWLEDGEMENT:

My Sincere thanks to my college Rajgad Dnyanpeeth’s College of Pharmacy Bhor-Pune, guide, principal sir, Industry Piramal Enterprises Ltd Mahad Raigad, Production Site head and industrial Guide for the contribution in my project work.

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Received on 31.07.2019 Modified on 20.09.2019

Research J. Pharm. and Tech 2020; 13(3):1314-1318.

DOI: 10.5958/0974-360X.2020.00242.5