Back to the Top
The following message was posted to: PharmPK
hello everybody
i am studying methotrexate (mtx) kinetics in rheumatoid arthritis
patients
i have following questions:
1. can anybody help me in establishing methotrexate curve on HPLC
i have been collecting the reference on pubmed but stil struggling
in HPLC
2. so also i wanted to know from where i can get 7-OH methotrexate a
metabolite
of mtx
3. what should be the internal standard used for mtx
thanks in advance
regards
yogita ghodke
interdisciplinary school of health sciences
pune university
pune
Back to the Top
The following message was posted to: PharmPK
Hi.
The best way to measure methotrexate (as well as 7OH-MTX and the
methotrexate polyglutamates if you want) by HPLC would be by
post-column UV-induced oxydation. You can find a very complete and
excellent discusion of methotrexate detection strategies in:
- Rubino FM. J Chromatogr B Biomed Sci Appl. 2001 Nov
25;764(1-2):217-54. Separation methods for methotrexate, its structural
analogues and metabolites.
Also, for the specific detection with UV-induced oxydation, you can
check:
- Fotoohi K, Skarby T, Soderhall S, Peterson C, Albertioni F. J
Chromatogr B Analyt Technol Biomed Life Sci. 2005 Mar 25;817(2):139-44.
Interference of 7-hydroxymethotrexate with the determination of
methotrexate in plasma samples from children with acute lymphoblastic
leukemia employing routine clinical assays. (which focus on the
important issue of interference of 7OH-MTX in the antibody-based assays
for methotrexate for clinical samples)
(if you don't have the post-column reactor, and can't afford one, you
could build your own for a little money).
Regarding the internal standard, most people have used aminopterin,
with the inconvenient that it takes quite a long time to come out of
the column, which would prolong your assay time from 8 minutes to 20.
Personally, I think that you can use methotrexate polyglutamate-2 (or
3), which would come out close from methotrexate (maybe 2 minutes
apart) and, if you are measuring plasma, should not be in detectable
amounts in the patient sample (assuming that you are planing to measure
patients' samples, i.e. after high dose methotrexate treatment for
leukemia).
You can buy 7-oh-MTX from Schircks lab (www.schircks.com)a, as well as
many other pteridines (including methotrexate-polyglutamate-2).
Hope this helps.
Facundo Garcia Bournissen.
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)