heparin and LMWH problem
Posted: Wed Nov 04, 2015 3:26 pm
Hi, everyone
Recently I performed the average molecular weight determination of heparin and LMWH with USP and EP conditions respectively(SEC method,not shown here), and the data aquisition and chromatograms are OK, however, the manupulation and interpretation of the chromatogram confused me, which are as following:
1. USP applies a broad molecular weight standard for calibration(both two articles) and the ceritificate of reference standard cites the cummulative distribution percentage of different fractions, so I can make the fragments of standard with those information(total 15 pieces in RS) and plot the calibration curve and deduct the regression equation, although there is no indication of peak slice number for peak from unknown distribution sample, how can I do it appropriately for make accurate average molecular weight of heparin and whether there is some information or literature, thanks!
2. EP(item 0828) uses Neilsen method, UV and RI detections in series, for determination of average molecular weight and distribution of low-molecular mass heparin. For calibration, both total and fragments of ratio between UV and RI responses of peak from CRS(Batch 3, labelled MW: 3800)are used, unlike reference standard in USP method, there is no fractions distribution percentage and only one MW number, which puzzled me greatly and resulted in inappropriate peak slice in standard solution and abnormal calibration and sample determination, I hope you give me some information or guides, and I'll appreciate it greatly, thanks in advance!
Best wishes
Austin
Recently I performed the average molecular weight determination of heparin and LMWH with USP and EP conditions respectively(SEC method,not shown here), and the data aquisition and chromatograms are OK, however, the manupulation and interpretation of the chromatogram confused me, which are as following:
1. USP applies a broad molecular weight standard for calibration(both two articles) and the ceritificate of reference standard cites the cummulative distribution percentage of different fractions, so I can make the fragments of standard with those information(total 15 pieces in RS) and plot the calibration curve and deduct the regression equation, although there is no indication of peak slice number for peak from unknown distribution sample, how can I do it appropriately for make accurate average molecular weight of heparin and whether there is some information or literature, thanks!
2. EP(item 0828) uses Neilsen method, UV and RI detections in series, for determination of average molecular weight and distribution of low-molecular mass heparin. For calibration, both total and fragments of ratio between UV and RI responses of peak from CRS(Batch 3, labelled MW: 3800)are used, unlike reference standard in USP method, there is no fractions distribution percentage and only one MW number, which puzzled me greatly and resulted in inappropriate peak slice in standard solution and abnormal calibration and sample determination, I hope you give me some information or guides, and I'll appreciate it greatly, thanks in advance!
Best wishes
Austin