We have developed a solvent extraction procedure &hplc protocol (Internal STD method)to quantify trace residues of a nitrostyrene compound in chicken plasma.

The validation of the method involved:
Fortified plasma controls (with CPD & IS-1ppm) at 7 conc: 0.05 to 5 ppm over 2 days. Six replicates at each concentration. Due to limited test volume the 3rd day could not be executed. The fortified plasma extracts & aqueous stds of equiv concs were tested by HPLC_PDA. LOQ is 0.1PPM

WHICH METHOD SHOULD BE USED TO CALC RECOVERY?

#A Use the AQ_STD Line of best to derive the PPM Detected_Fortified plasma extracts and then divide by fortified ammount in PPM.

#B Divide the Peak height ratio(CPD/IS)_Fortified PL by the equivalent PK height ratio (CPD/IS)_Aqueous Standard. This would give Absolute Recovery?

Here are the %ave recoveries using the above:

DAY 1
CONCs 0.05 0.1 0.2 0.5 1.0 2.0 5.0
#A 84 88 84 102 105 103 102
#B ND 77 84 99 104 107 107

DAY 2
CONCs 0.05 0.1 0.2 0.5 1.0 2.0 5.0
#A 84 83 79 96 90 90 89
#B ND 71 76 93 89 94 89

ND=not detected
Note: Day 2 peak height data for CPD & IS were higher than Day 1, suggesting that sample volume changes ie evaporation. Our lab does not have const Temp & the caps for low vol inserts on the 96-vial carousel are not the re-sealable septa type.

Your thoughts please...

A This should help correct for any minor vial evap problems because the evap rate for stds and samples should be similar. It's also easier to explain if you simply use ppm found/ppm fortified.