Hi,

it's me again with peptide ointment.

I don't know if my 'method validation scheme' is proper, so i will write down what i plan to do. Please tell me if something is wrong.


0.5mg of peptide in 1 g od vaseline

Extraction procedure:
a) 0.25 g of ointment dissolved in 2ml of n-hexane
b) 2ml of 30% (ACN +0,1%TFA) is added
c) 30 minute stirring
d) 100ul injected on HPLC

So the peptide concentration in 2 ml of 30% (ACN +0,1%TFA) is 62,5 ug/ml (125 ug/2ml)

1) Specificity -> Analysis of blank vaseline + analysis of vaseline with peptide

2) Linearity

5 blank vaseline samples spiked with 100ul peptide solutions before dissolving in n-hexane.

Spike solution Concentrations (range 80%-120%):

a) 1,5 mg/ml (120%)
b) 1,25 mg/ml (100%)
c) 1,125 mg/ml (90 %)
d) 1,0625 mg/ml (85%)
e) 1 mg/ml (80 %)

(if it is stability testing is rang 80-120% ok?)
3) Accuracy

no idea -> maybe described by recovery? :

Spiked vaseline vs peptide ointment?
or
Standard addition ?

4)Precission

Linearity will be made with 5 samples/3 replicates each so repeatability will be assessed from those analysis.

5) LOD and LOQ from SD and Slope


Is robustness necessary ?

Thank you in advance for response,

Dorian