Chromatography Forum

I use the USP method to analize Isotretinoin capsules by normal phase, the selectivity was perfect buy I got problems with reproducibility of samples, however I take special care with the preparation ( Samples are in hexanes). What can I do to obtaine a good RSD?

Thanks,

Nelly Gómez

Is the problem quantification or retention time?

Retinoids are sensitive to light and oxygen. Keep the lights turned down low, use amber glassware, refrigerate the samples, degas the solvents, prepare only a few samples at a time, work quickly.

Normal phase is touchy about residual water in the mobile phase, and the equilibration times are longer than for reversed-phase. Watch out for stabilizers in the solvents. If your pump has piston seal wash, change it to something compatible. If your pump is a gradient pump, make sure the proportioning valve does not leak from an unused channel.

If the pump vendor offers special seals for normal-phase work, get some. Seals for reversed-phase don't last long in normal-phase solvents.

We had problems with reproducability but found that slowing the injector down when drawing up sample to eliminate the possibility of air being drawn up cured our problem .
Most of the time por reproducability is caused by a bad injector system unless your chromatography is poor.