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Lynn Hewson ☆ Uruguay, 2014-05-12 19:17 (4031 d 10:21 ago) Posting: # 12932 Views: 5,267 |
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Dear all, We are developing am HPLC/MS method to measure omeprazole enantiomers in human plasma. So far, we have tried two column brands but were not lucky with any of them. The first brand (Phenomenex Lux) showed lack of robustness: when switching from one column batch to another there was a shift in retention times, changes in resolution between enantiomer peaks and a significant decrease in the slope of the calibration curve. With one particular column we even had a lack of linearity in the calibration curve range. So we decided to try another brand, hoping for consistency in results. We decided to try a Chiralpack IC-3, based on published data. We employed chromatographic conditions as per recommendations of the manufacturer of the column and resolution between peaks was ok when using standard solutions (without plasma). But, as soon as we injected the calibration curve in plasma, the peaks did not resolve at all. The manufacturer claims that the column was not meant to be used with plasma samples. But, in this particular case, plasma samples were cleaned-up by SPE extraction prior to injection, and only 11 injections of plasma samples were performed! So we think that there is a problem with this column in particular. Does anyone have any experience (good/bad) with chiral columns? Any suggestions are welcomed. Chiral columns are very expensive and it has been very frustrating not to be able to find a suitable one. Thank you very much! Lyn |
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AngusMcLean ★★ USA, 2014-05-24 01:44 (4020 d 03:54 ago) @ Lynn Hewson Posting: # 13004 Views: 4,249 |
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Lyn: I have a lot of experience with chiral columns. For the analysis of mixtures of dextro and levoamphetamine in plasma. The plasma extract was an elaborate procedure involving extraction, back-extraction into acid, basifying and then extracting finally into organic solvent. The column we used was a normal phase chiral column so it was like old established normal phase chromatography eluting wiht organic solvents. The sample preparation as always with plasma is the crucial step. We ran lots of samples supporting NDA type studies. Hope this helps, Angus |
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Helmut ★★★   Vienna, Austria, 2014-05-24 02:51 (4020 d 02:47 ago) @ AngusMcLean Posting: # 13005 Views: 4,281 |
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Hi Angus & Lyn, repetitive extraction/back-extraction is indeed an excellent procedure to get very “clean” extracts. Unfortunately it requires the analyte to be stable in all pHs employed. Omeprazole has a half-life of a couple of seconds (!) in strong acidic pH. Remaining options:
— Dif-tor heh smusma 🖖🏼 Довге життя Україна! ![[image]](https://static.bebac.at/pics/Blue_and_yellow_ribbon_UA.png) Helmut Schütz ![[image]](https://static.bebac.at/img/CC by.png) The quality of responses received is directly proportional to the quality of the question asked. 🚮 Science Quotes |
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AngusMcLean ★★ USA, 2014-06-02 22:45 (4010 d 06:53 ago) @ Helmut Posting: # 13027 Views: 4,224 |
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Lyn: Helmut is indeed correct the above sample preparation procedure for a stable amine is not applicable to an acid labile drug like omeprazole. I think as Helmut suggest SPE may work or perhaps an extraction with a solvent of minimal polarity yet still cable of extracting the drug. Angus |
Ing. Helmut Schütz