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 PROCEDURE5.1 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:
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
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