title: Quantification of the HIV-integrase inhibitor raltegravir and detection of its main metabolite in human plasma, dried blood spots and peripheral blood mononuclear cell lysate by means of high-performance liquid chromatography tandem mass spectrometry.
abstract: For the quantification of the HIV-integrase inhibitor raltegravir in human plasma, dried blood spots and peripheral blood mononuclear cell (PBMC) lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. The assay also allowed detection, but no quantification due to absence of reference substance, of the main metabolite, raltegravir-glucuronide. Raltegravir was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50microL plasma. Extraction from dried blood spots was performed with a simple one-step extraction with a mixture of methanol, acetonitrile and 0.2M zincsulphate in water (1:1:2, v/v/v) and extraction from cell lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. The analytical run time was 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 50-10,000ng/mL in plasma and dried blood spots and a range of 1-500ng/mL in PBMC lysate. Dibenzepine was used as the internal standard. The method was proven to be specific, accurate, precise and robust. Accuracies ranged from 104% to 105% in plasma, from 93% to 105% in dried blood spots and from 82% to 113% in PBMC lysate. Precision over the complete concentration range was less than 6%, 11% and 13% in plasma, dried blood spots and PBMC lysate, respectively. The method is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with raltegravir.
authors.affiliation: Slotervaart hospital, Department of Pharmacy and Pharmacology, Louwesweg 6, 1066EC Amsterdam, Netherlands. rob.terheine@slz.nl
authors.forename: R
authors.initials: R
authors.lastname: Ter Heine
authors.forename: M J X
authors.initials: MJ
authors.lastname: Hillebrand
authors.forename: H
authors.initials: H
authors.lastname: Rosing
authors.forename: E C M
authors.initials: EC
authors.lastname: van Gorp
authors.forename: J W
authors.initials: JW
authors.lastname: Mulder
authors.forename: J H
authors.initials: JH
authors.lastname: Beijnen
authors.forename: A D R
authors.initials: AD
authors.lastname: Huitema
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000998:Antiviral Agents / Q000097:blood* / Q000737:chemistry; D001681:Biological Assay; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D003890:Desiccation; D016903:Drug Monitoring; D004355:Drug Stability; D004356:Drug Storage; D005615:Freezing; D015658:HIV Infections / Q000097:blood; D019428:HIV Integrase Inhibitors / Q000097:blood* / Q000737:chemistry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D000432:Methanol / Q000737:chemistry; D015394:Molecular Structure; D010316:Particle Size; D011760:Pyrrolidinones / Q000097:blood* / Q000737:chemistry; D000068898:Raltegravir Potassium; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014867:Water / Q000737:chemistry; D019287:Zinc Sulfate / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D000998:Antiviral Agents; D019428:HIV Integrase Inhibitors; D011760:Pyrrolidinones; D012996:Solutions; D014867:Water; D000068898:Raltegravir Potassium; D019287:Zinc Sulfate; D000432:Methanol; C032159:acetonitrile