Method for the determination of blood methotrexate by high performance liquid chromatography with online post-column electrochemical oxidation and fluorescence detection
Methotrexate (MTX) has been widely used at low dose for the treatment of different diseases including rheumatoid arthritis. MTX might be present in plasma in free form, and in blood cells in methotrexate polyglutamate (MTXPG). A rapid and sensitive HPLC method was developed for the determination of...
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Published in | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 845; no. 1; pp. 164 - 168 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Amsterdam
Elsevier B.V
2007
Elsevier Science |
Subjects | |
Online Access | Get full text |
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Summary: | Methotrexate (MTX) has been widely used at low dose for the treatment of different diseases including rheumatoid arthritis. MTX might be present in plasma in free form, and in blood cells in methotrexate polyglutamate (MTXPG). A rapid and sensitive HPLC method was developed for the determination of plasma MTX level, whole-blood MTX level, and whole-blood total MTX (MTX
+
MTXPG) level. To determine plasma MTX level or whole-blood MTX level, a 0.2-ml aliquot of plasma or whole blood (after a freeze–thaw cycle to break blood cells) was well mixed with 0.8
ml methanol and centrifuged. To determine whole-blood total MTX level, a 0.1-ml aliquot of whole blood (after a freeze–thaw cycle) was mixed with 80
μl ascorbic acid (114
mM) and incubated at 37
°C for 2
h to enzymatically convert the MTXPG to MTX. Then 20
μl NaOH solution (0.5
M) and 0.8
ml methanol were added and mixed well. After centrifugation, a 0.5-ml aliquot of the supernatant was evaporated to dryness and re-dissolved in 0.2
ml hydrochloric acid (10
mM). Methylene chloride (0.2
ml) was added and mixed well. After centrifugation, the top aqueous layer was injected to HPLC for analysis. After the MTX was eluted from the HPLC column, it was electrochemically oxidized and detected by a fluorescence detector. Recoveries of spiked MTX at ppb (ng/ml) level were between 87.9 and 118% with within-day relative standard deviation less than 5.2% and day-to-day relative standard deviation less than 9.8%. The limit of detection (LOD) and limit of quantitation (LOQ) of the described method were 1.2 and 2.6
ng/ml, respectively. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1570-0232 1873-376X |
DOI: | 10.1016/j.jchromb.2006.07.026 |