Chromatography Forum

I am perform assay Methocarbamol in tablet by HPLC as USP 35. The assay based on the C18 (250 mmx 4.6 mm, 5 microm) with caffein as internal standard. But when I jected samples for many replicate, the peak area of caffein and methocarbamol per injection vary from injection to injection but the peak area ratio (methocarbamol/caffein) is constant between injections. Pls help me to clarify this problem. is the RSD of each caffein and methocarbamol between replicate injections of same solution is < 2.0% or only care to the peak area ratio?

Thanks you very much!

If you are working with Internal Standard, the data you use for calculation is the area ratio. So small variations in individual areas should not affect the result.
I have never used IS in HPLC techniques but it is mandatory in GC because is near impossible to obtain repetitive areas from a hot needle syringe.

that is the whole point to using internal standards: to cancel out correlated errors.