Chromatography Forum

i am conducting research on the morphine(MOR) metabolites specifically M3G and M6G. Because these two metabolites are isomers i have to rely on good separation to quant and evaluate each individual metabolite.

i am currently using a published method using a HILIC zorbax plus column, 10mM ammonium formate (pH=6.4)as mobile phase A and 90/10:: ACN to 10mM ammonium formate as my mobile phase B. the method includes an isocratic decrease in mobile phase B to 55% and a hold for 5.5min foloowed by about a 3 min re-equlibration time

i am getting alright separation of M3G, M6G and MOR but not great. M6G is eluting first then M3G and then morphine last.

i was wondering how i can optimize this method. i have already changed mobile phase A to a 95/5 mix but all analytes were retained longer and no increase in seperation was achieved. I also am using a positive MRM mode n the MS/MS interface and i appear to begetting a little ionization suppression of the morphine.

Any suggestions would be greatly appreciated

Since you have at least a minimally workable separation now, you may as well be empirical about optimizing it. Start with your existing conditions and then "tweak" the various parameters to see what happens. Your resolution will either improve, stay the same, or get worse. If resolution improves, then that parameter has a significant effect on selectivity and you can use it to "fine tune" the selectivity.If resolution gets worse, you can still fine-tune, but just go in the other direction. If resolution didn't change, then that parameter is not helpful and you can leave it as is.

My parameters of choice would be:
- gradient steepness (either double the time it or cut in in half)
- temperature (go up or down by 15 degrees C)
- pH (go up or down by half a unit).

You should be able to set up and do all four runs in less than a day. At that point, you'll have a good idea as to which parameter(s) to focus on.

A last comment: if you're close to what you need and can take the run-time increase, coupling two columns together should increase your resolution by 40% (the "brute force" approach! ).

The elution order you mention seems to be the reverse of what you should see in HILIC. The glucuronide metabolites are more polar than morphine and should elute after it.

Please provide a complete description of all chromatographic conditions.

increase your buffer to 50 or even 100 mM
or move to citric buffer

or go the oposite, use only water without any buffer, this sometimes also improves seperation

i am conducting research on the morphine(MOR) metabolites specifically M3G and M6G. Because these two metabolites are isomers i have to rely on good separation to quant and evaluate each individual metabolite.

i am currently using a published method using a HILIC zorbax plus column, 10mM ammonium formate (pH=6.4)as mobile phase A and 90/10:: ACN to 10mM ammonium formate as my mobile phase B. the method includes an isocratic decrease in mobile phase B to 55% and a hold for 5.5min foloowed by about a 3 min re-equlibration time

i am getting alright separation of M3G, M6G and MOR but not great. M6G is eluting first then M3G and then morphine last.

i was wondering how i can optimize this method. i have already changed mobile phase A to a 95/5 mix but all analytes were retained longer and no increase in seperation was achieved. I also am using a positive MRM mode n the MS/MS interface and i appear to begetting a little ionization suppression of the morphine.

Any suggestions would be greatly appreciated

I used to do some work on HILIC separation of morphines at Waters.
Maybe you could search on Waters website.

I am still not obtaining the correct elution order but i am getting a workable separation. Morphine is still eluting about a 1min after M3G.
i am wondering if anyone has run into a problem like this while using HILIC. Morphine should be eluting first because it is the least polar of my analytes

The literature being used for the chromatographic conditions is:
Kolmonen M, Leinonen A, Kuuranne T, Pelander A, Ojanpera I. Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine. J Chromatogr B 2010;878:2959-2966

Column is ZORBAX HILIC plus from agilent
mobile phase A is 10mM ammonium formate pH=6.4
mobile phase B is 90% ACN and 10% 10mM ammonium formate pH=6.4

Initial %B 100%
.5min %B 100%
2.0min %B 55%
7.5min %B 55%
8min %B 100%
11min %B off
i also noticed less ion suppression when using 95:5 mobile phase B but all retention times increased by about 45sec as excpected so the method was increase by 3min. this however did not change the elution order

I suspect that your running conditions were developed with convenience of mass spec analysis in mind and rather less concern for the chromatography. Your observed elution order is caused by electrostatic repulsion effects. Uncoated silica has a surprisingly high cation-exchange capacity at a pH high enough for the silanols to be ionized. At your pH 6.5, both they and the carboxyl- group of a glucuronide will have full negative charge. It takes about 20 mM salt in the mobile phase to form a complete electrical double layer of counterions that shields a charged analyte from the charged stationary phase. To see a good example of these effects, look up: Y. Takegawa et al., J. Sep. Sci. 29 (2006) 2533-40; specifically, Fig. 3, comparing the relative elution in HILIC of some glycans with sialic acid residues and the same glycan without sialic acid, in 5 mM and 20 mM ammonium acetate.

Here, you're starting with a mobile phase containing 10% of 10 mM ammonium formate. That means that the overall salt concentration is 1 mM. No wonder your electrostatic effects are so prominent! Try using 20 mM ammonium formate overall in both mobile phases and you'll get the elution order that would be anticipated in HILIC (cf. J.A. Boutin et al., Drug Metab. Disp. 21 (1993) 1157). Then, if you have to have a lower concentration of salt in the mobile phases, try 15 mM, then 10 mM, in the mobile phase overall until the usable range is evident.

If you want to read an overview of electrostatic effects in HILIC, look up: A.J. Alpert, Anal. Chem. 80 (2008) 62-76 [per the following link: http://pubs.acs.org/doi/pdf/10.1021/ac070997p]

Keep an eye on composition in the sample vial it has to be high organic.
Also I found that for HILIC it is sometime needed 97% organic in the starting gradient.
Sub-2 micron can also help to improve resolution.