title: Determination of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction: application for the assessment of their transplacental passage at delivery.
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.
authors.affiliation: Division de Pharmacologie Clinique, Laboratoire BH 18-218, Département de Médecine, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne CHUV, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Marzolini
authors.forename: A
authors.initials: A
authors.lastname: Béguin
authors.forename: A
authors.initials: A
authors.lastname: Telenti
authors.forename: A
authors.initials: A
authors.lastname: Schreyer
authors.forename: T
authors.initials: T
authors.lastname: Buclin
authors.forename: J
authors.initials: J
authors.lastname: Biollaz
authors.forename: L A
authors.initials: LA
authors.lastname: Decosterd
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D017320:HIV Protease Inhibitors / Q000097:blood*; D006801:Humans; D061466:Lopinavir; D008431:Maternal-Fetal Exchange; D019829:Nevirapine / Q000097:blood*; D011247:Pregnancy; D011744:Pyrimidinones / Q000097:blood*; D018894:Reverse Transcriptase Inhibitors / Q000097:blood*; D012680:Sensitivity and Specificity
chemical_list: D017320:HIV Protease Inhibitors; D011744:Pyrimidinones; D018894:Reverse Transcriptase Inhibitors; D061466:Lopinavir; D019829:Nevirapine