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Determination of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction: application for the assessment of their transplacental passage at delivery
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
ID
3704494
Author(s)
Marzolini, C.; Béguin, A.; Telenti, A.; Schreyer, A.; Buclin, T.; Biollaz, J.; Decosterd, L. A.
Determination of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction: application for the assessment of their transplacental passage at delivery
Journal
Journal of Chromatography B
Volume
774
Number
2
Pages / Article-Number
127-40
Mesh terms
Calibration; Chromatography, High Pressure Liquid, methods; Female; HIV Protease Inhibitors, blood; Humans; Lopinavir; Maternal-Fetal Exchange; Nevirapine, blood; Pregnancy; Pyrimidinones, blood; Reverse Transcriptase Inhibitors, blood; Sensitivity and Specificity
Abstract
An adaptation of the HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz after solid-phase extraction is proposed here for the separate analysis of the newer PI lopinavir (LPV) and the NNRTI nevirapine (NVP). After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl), with clozapine added as internal standard, is diluted 1+1 with phosphate buffer pH 7 and subjected to a solid-phase extraction on a C(18) cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H(3)PO(4) neutralised with NaOH to pH 7. LPV and NVP are eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl MeOH 50%. A 40-microl volume is injected onto a Nucleosil 100, 5 microm C(18) AB column. LPV and NVP are analysed separately using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.07 and containing 0.02% sodium heptanesulfonate. LPV and NVP are detected by UV at 201 and 282 nm, respectively. The calibration curves are linear up to 10 microg/ml. The mean absolute recovery of LPV and NVP is 91% and 88%, respectively. The method is precise with mean inter-day C.V.s within 2.1-6.6% and 0.9-1.7% for LPV and NVP, and accurate (range of inter-day deviations -1.1 to +2.4%, and -1.9 to +0.8%, for LPV and NVP, respectively). The method has been validated and is currently applied to the monitoring of LPV and NVP in HIV patients, and has been notably applied in a study aimed at assessing the extent of transplacental passage of nevirapine and PIs, notably lopinavir, at the time of delivery in pregnant HIV-infected women.
