Assay Calculation in HPLC

1.0 OBJECTIVE 

To lay down the procedure for Assay Calculation in HPLC

2.0 SCOPE

This SOP is applicable to Assay Calculation in HPLC in company.

3.0 BACKGROUND 

NIL

4.0 RESPONSIBILITY

4.1 QC- Chemist 

5.0 PROCEDURE

5.1 Chromatographic conditions:

HPLC with UV detector at wavelength 210nm, Column Intersil ODS-3V, 250x4.6nm, 5 micron equivalent, Flow rate of 1.0mL per minute, injection volume of 20 microliters, column temperature of 25 degrees and run time of 20 min.

5.2 mobile phase is with buffer(phosphate 6.5pH), methanol and acetonitrile with 60:20:20 v/v, standard and samples are prepared at the concentrations of 1.5mg/ml.

5.3 Injection 20 microliters of blank(1injection), standard(5 injections) and sample(2 injections).

5.4 Calculation:

 

5.5 Before going into the actual calculation, we may need to know few things

1. WC is water content (%) in pregabalin sample and standard (NMT 0.5%)

Here i have presented a typical chromatogram of Pregabalin

Assay calculation for Pregabalin

1. Standard areas of Five injections

Injection	Area
1	749853
2	743554
3	740760
4	746618
5	747748
Sum	3728533
Average	745706.6

2. Sample Injections

Injection	Area
1	744709
2	740760
sum	1485469
Average	742734.5

Weight of the standard =149.8900 mg

Weight of the sample= 149.9600 mg

Water content of the sample=0.8900 w/w

Water content of the standard = 0.9916 w/w

Assay of the standard = 99.98

After applying the above formula for these values Assay is 99.43

6.0 ANNEXURES 

Nil