Chromatography Forum

Hello

We're experiencing some problems with an HPLC assay method for morphine sulphate. The retention time for the major peak changes from approx. 6.5 min to 13 min during a 10-15 samples run. We have also seen the opposite, i.e. from 13 min to 6.5 min.

We have tested different HPLC systems (Waters Alliance 2695 with Empower) with similar results.

We allow the HPLC to mix channel A and B of the mobile phases. However, we will try to pre-mix the mobile phase (I don't think this will help since we experienced similar problems on several systems).

Parameters:
Pre-column: LiChrocart 4-4, LiChrospher 100 RP-18, 5 um
Column: LiChrocart 250-4, LiChrospher 100 RP-18, 5 um
Pump flow: 1.0 ml/min
Mobile phase A: Acetonitrile
Mobile phase B: Ammonium acetate buffer (containing acetic acid, triethylamine and 1-heptanesulfonate monohydrate, pH 4.85)
Mobile phase ratio: A:B, 10:90
Injection volume 75 ul
Column temp.: 30°C
Degasser: On
Wavelength: 285 nm

The samples are prepared in water pH adjusted to 3.6 with phosphoric acid.
Approximate sample conc. is 0.096 mg/ml.
All solutions used have been vaccuum filtered.

My questions: Could this problem arise from anything but a bad column? Have I missed something?

I would really appreciate your input on this matter.
/Peps

Premixing should do it. Sometimes check valves don't seal properly when you try to pump 100% ACN through them - the ruby balls sort of float instead of seating properly. The easiest way to see this (aside from the variance in RT) is to monitor the system pressure. If the mean and delta are different when you're getting 13' morphine peaks than when you're getting 6.5' peaks, it's time to premix or switch to ceramic check valve cartridges.

Another (less likely) possibility:

Your column may just be taking a long time to equilibrate as sometimes happens with TEA. If this is the case, make a small batch of B phase (~100mL) but with double the TEA, add the appropriate amount of ACN and allow this to recirculate in your system for several hours. It may help to raise the column temp. ~10°C while you do this.

An even more likely culprit is the hexane sulfonate. It is supposed to ion-pair with the morphine. If your column is not equilibrated consistently with the hexane sulfonate, the morphine peak will move around like crazy.

In order to get good equilibration with the ion-pair reagent, you need to preequilibrate the column for an extended (!!!) period of time with the mobile phase. In order to avoid having the same problem every day when you start up, I usually recommend to store the column in mobile phase and not to bother with any washing protocol.

Dr. Neue, if the mobile phase contains too much aqueous solution like this case and my case in cilastatin-imipenem, should we add preservative to the mobile phase before storing the column as your suggestion?

Thanks for the suggestions so far. We will try with longer equilibration times and maybe also store the column in the mobile phase.

/Peps

Syx: at 10% acetonitrile in the mobile phase, I have serious doubts that bugs will grow, especially with the acetic acid around. No problem.

I do not know exactly, which bugs die at which concentration of organic, but I am not familiar with one that would use acetonitrile as a nutrient.

So, I think we finally made it. It seems as if the problem has been solved with over-night equilibration of the column before the analysis.

Thanks for your help.

/Peps

Thanks for the feedback. It is good to hear what suggestions have worked.

It is work on Peps'!! I will try it to our cilastatin and imipenem injection, just wait the schedule.

We use USP method for injection morphine sulphate, may be useful for you

Mobile phase— Dissolve 0.73 g of sodium 1-heptanesulfonate in 720 mL of water, add 280 mL of methanol and 10 mL of glacial acetic acid

Standard preparation— Dissolve an accurately weighed quantity of USP Morphine Sulfate in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.24 mg per mL. Prepare a fresh
solution daily.

System suitability preparation— Dissolve suitable quantities of USP Morphine Sulfate RS and phenol in Mobile phase to obtain a solution containing about 0.24 and 0.15 mg per mL,

Assay preparation— Transfer about 24 mg of Morphine Sulfate, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography á 621 ñ ) — The liquid chromatograph is equipped with a 284-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation and the System suitability preparation, and record the peak responses as directed for Procedure: the tailing factor for the morphine sulfate peak is not more than 2.0; the resolution, between the phenol and morphine sulfate peaks is not less than 2.0; and the relative standard deviation for replicate injections of the Standard preparation is not more than 2.0%. The relative retention times are about 0.7 for phenol and 1.0 for morphine sulfate.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of (C 17H 19NO 3) 2•H 2SO 4 in the portion of Morphine Sulfate taken by the formula:
100C(r U / r S), in which C is the concentration, in mg per mL, of anhydrous morphine sulfate in the Standard preparation

If I want to store the column for a long time (more than one night) before next usage I should keep it using 10% acetonitrile in ion-pair solution without buffer… Is it right, Dr. Neue?

Syx, I did not say without buffer. Actually, you may be better off with the buffer. Are you using the exact same conditions as posted above by Peps?

Also for hwo long do you need to store the column? If you plan to staore the column for half a year, put it in acetonitrile. If you want to reuse the column after two days, keep it in the mobile phase.