M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Approaches to analytical method development. Exp.No.:Roll No:Date:Page No.

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Aim: Linearity establishment of Hydrochlorthiazide by HPLC. References: 1) United States pharmacopoeia30-NF-25, Page No.1225. 2) Practical HPLC method development, 2nd edition, Snyder Page no.644-45. Requirements:

Sr.no A) Apparatus: 1.

Requirements

Quantity

Volumetric flask:100ml 10ml

2 5 1 2 1

2.

Pipettes

:10ml 1ml

3. B) Instrument: 1. 2. C) Chemicals: 1.

Injection

HPLC instrument Column (Inertsil-ODS-3) no. 8DI86210

1 1

Acetonitrile : Water

65:35

________________________________________________________________________ School of Pharmacy and Technology Management

M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Approaches to analytical method development. Exp.No.:Roll No:Date:Page No.:-

Theory: The linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analytes in samples within a given range or proportional by means of welldefined mathematical transformations. Linearity should be established across the range of analytical procedure. Linearity is determined by a series of 3 to 6 injections of 5 or more standards whose concentrations span 80120 percent of the expected concentration range. The response should be directly proportional to the concentrations of the analytes or proportional by means of a welldefined mathematical calculation. A linear regression equation applied to the results should have an intercept not significantly different from 0. Frequently, the linearity is evaluated graphically, in addition to or as an alternative to mathematical evaluation. The evaluation is made by visually inspecting a plot of signal height or peak area as a function of analyte concentration. The linearity of the method is the measure of how well a calibration plot of response Vs concentration approximates a straight line, or how well the data fit to the linear equation: y = mx + b where, y is the response (peak area) x is the concentration m is the slope b is the intercept of a line fit to the data. Ideally a linear relationship (with b0) is preferred because it is more precise, easier for calculations and can be defined with fewer standards. Also, UV detector response for a dilute sample is expected to follow Beers law and be linear (with b0). Therefore a linear calibration curve gives evidence that the system is performing properly throughout the concentration range of interest. In addition a method that is linear (with b 0) permits a quick, convenient check to confirm calibration accuracy.

________________________________________________________________________ School of Pharmacy and Technology Management

M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Approaches to analytical method development. Exp.No.:Roll No:Date:Page No.:-

Procedure: 1) Weigh 100mg of Hydrochlorthiazide . 2) Transfer it into a clean 100ml volumetric flask. 3) Dilute it with water upto 100ml.This forms the solution of 1mg/ml that is 1000ppm. (Stock solution). Stock Solution:1000ppm . 4) 1ml of 1000ppm solution diluted to 100ml 10ppm solution. Stock Solution: 10ppm. 5) 0.5ml of 10ppm solution diluted to 10ml 0.5ppm solution. 6) 1ml of 10ppm solution diluted to 10ml 1ppm solution. 7) 2ml of 10ppm solution diluted to 10ml 2ppm solution 8) 3ml of 10ppm solution diluted to 10ml 3ppm solution. 9) 4ml of 10ppm solution diluted to 10ml 4ppm solution. 10) Inject the prepared solutions in triplicates take the chromatograms. 11) Calculate the average peak areas from the obtained chromatograms for each concentration. 12) Determine the Standard deviation and Relative standard deviation for the data. 13) Plot a graph of Peak area Vs Concentration and determine the Correlation Co-efficient of the plot.

Observation Table: CONCENTRATION CHROMATOGRAM No. 1 0.5 PPM 2 3 1 1 PPM 2 3 1 2 PPM 2 3 1 3 PPM 2 3 1 4 PPM 2 3 AREA 44815.77 44553.96 44817.26 81004.17 82292.47 80295.24 178458 178316.4 176183.9 226451.5 228415.7 230183.7 315495.5 318042.5 316840.6 AVERAGE AREA 44729 STDV RSD

151.588

0.3389

81197.29

1012.524

1.2469

177652.7

1274.066

0.7171

228350.3

1866.969

0.8175

316792.9

1274.17

0.4022

________________________________________________________________________ School of Pharmacy and Technology Management

M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Approaches to analytical method development. Exp.No.:Roll No:Date:Page No.:-

Graph for Linearity:

Regression Co-efficient (R2) = 0.992 Result: Since the Regression Co-efficient was found to be 0.992 it can be concluded that the Linearity of Hydrochlorthiazide is established.

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