We are developing a HPLC potency method for hydromorphone hydrochloride and some its intermediates. We have used two standard/sample diluents. The first is water and the second is a 50 mM potassium phosphate buffer at pH 7.2. The mobile phase for each of these runs is 0.1% heptanesulfonic acid, 0.1% triethylamine, 5% methanol and 95% water pH'ed to 2.5 with phosphoric acid. The pH 7.2 buffer is necessary because it converts one the intermediates into a hydromorphone. The problem is that standards with the pH 7.2 diluent have a response 20% greater than the water diluent.

Is this due to a shift in the maximum absorbance or better recovery of the hydromorphone phosphate ion-pair moiety? Why wouldn't the large volume (~20 mL) of mobile phase negate the 30 microliters of the diluent in the injection?

Your compound should be separated from your sample diluent. You could look for shifts in lambda max with a DAD or perhaps collect fractions and analyze with a UV spectrophotometer.

A 20% low recovery for a sample prepared with water would be easy to spot with FIA, flow injection analysis. Just substitute a piece of restriction capilary tubing for the column and compare peak areas for injections with and without a column.

Your mobile phase composition seems a little strange, but I have never worked with your compound. Good luck.

The chromatographic system is not a mixer. In fact, the better design you have with respect to extra-column (void) volume the less mixing. Consequently, is this a conventional system or are you using small, micro or less ID column? An injection of 30 uL can be too large.

Secondly, it seems to me that you have different pH between sample, standard and mobile phase? That is prone to give problems, especially since you are using an ion-pairing agent. My experience from these is that one need to keep everything very constant to avoid goast peaks, noise and drift.

I agree with Tom that the mobile phase composition is strange and it indicates that you have low retention on a C18 column.

However, a similar method has been validated by;
AE. Smet, G. Van Der Weken, W.R.G. Baeyens and J.P. Remon.
A Validated HPLC Method for Assay of Morphine Hydrochloride and Hydromorphone Hydrochloride in Pharmaceutical Injections.
Chromatographia, 2001, 53, 35-39.

"A sensitive and rapid routine HPLC method is proposed for quantitative estimation of morphine hydrochloride and hydromorphone hydrochloride in pharmaceutical dosage forms. The drugs were chromatographed on a C 18 reversed-phase column; the mobile phase was acetonitrile-water, 35:65 (v/v), containing sodium dodecyl sulphate (0.5%, w/v), as ion pairing reagent, and acetic acid (0.4% v/v). Detection was at 230 nm.
The optimized method was validated, and linearity (r > 0.999), precision, and accuracy were found to be acceptable within the concentration ranges 86–124mg m L–1 for morphine hydrochloride and 60–180mg mL –1 for hydromorphone hydrochloride.

The method is being used to investigate the stability of morphine hydrochloride and hydromorphone hydrochloride in solutions used for intramuscular injection."

Else, I think ZIC™-HILIC is an alternative.

Maybe something else is going on - maybe the basic sample is adsorbed onto the sample vial, and the addition of the phosphate buffer ions remove it from the wall.

I've seen a number of such reports in IC work. The problem is usually connected to contamination of sample introduction components. For example, in cation IC work, introduction of "real" water samples apparently sometimes contaminates the sample loop with some sort of "Bio" material which renders the sample loop sensitive to sample pH and ionic strength, giving erroneous results with neutral pH samples. Presumably, the contaminating material is working as an in situ concentrator when the sample is at low ionic strength. The solution is generally to replace the sample loop but sometimes more extensive cleaning of the sample introduction system is necessary. Depending on the nature of the contaminant, this may require cleaning with acidic solutions, basic solutions or even peroxide treatment.

Respected Sir,
Will u send me complete information related to the working of Gas Liquid Chromatography And HPLC.Plz help bcauze this is related to my project work.