Chromatography Forum

We have developed HPLC method for cleaning validation. However, area repeatability cannot be achieved. We checked the wavelength, the injector, solubility, the filter, peak shape and hlpc system. RSD % about 12% for three injections. what could be the reason for this?
Critical parameters
Wavelength: 205 Nm
Injection volume: 100 Microliter
Diluent: 15% acetic acid solution

Thank you

We have developed HPLC method for cleaning validation. However, area repeatability cannot be achieved. We checked the wavelength, the injector, solubility, the filter, peak shape and hlpc system. RSD % about 12% for three injections. what could be the reason for this?
Critical parameters
Wavelength: 205 Nm
Injection volume: 100 Microliter
Diluent: 15% acetic acid solution

Thank you

What peak are you measuring? Could you post example chromatograms? Could you explain the cleaning validation procedure? There is a lot of information critical besides those 3 parameters listed to try and troubleshoot this issue.

I haven't added it because we have checked the other critical parameters. The peak area of high concentrations is RSD% suitable but there is area variation at low concentrations. We measured mean peak.

Std 0.01 mg/mL-(10 µl) Std 0.01 mg/mL-(40µl) Std 0.00006 mg/mL
1 77696441 308059373 299775
2 77501942 307824455 253088
3 77742206 307325639 217954
4 77548165 307396267 236314
5 77638970 307081505 no peak
6 77658995 307359836 no peak
RSD: 0.11% 0.12% 13.9% (for 4 injections)

By the way no cleaning validation procedure is required. There is problem in standard solution.

Standard Solution: Transfer accurately weighed equal to 10.0 mg of XXXX into a 125.0 mL volumetric flask. Dissolve with diluent in an ultrasonic bath for 2 min. Dilute to volume with diluent and mix. Transfer 2.5 mL of this solution into a 200.0 mL volumetric flask and dilute to volume with diluent and mix. Transfer 6.0 mL of this solution into a 100.0 mL volumetric flask and dilute to volume with diluent and mix. Filter through 0.45 µm teflon filter.(C:0.00006 mg/mL)

Diluent: 15% acetic acid solution

What is the signal-to-noise ratio for the 0.00006 mg/mL standard (peak height vs. peak-to-peak noise)? Is it higher than 10? What are the plate number and the USP tailing factor of the peak? Is there any small interfering peak present on the blank chromatogram?
Also the low concentration standard may be unstable. The substance can degrade or adsorb on the vial wall. Try using plastic vials.