TY - JOUR
T1 - 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
AU - ter Heine, R.
AU - Hillebrand, M. J.X.
AU - Rosing, H.
AU - van Gorp, E. C.M.
AU - Mulder, J. W.
AU - Beijnen, J. H.
AU - Huitema, A. D.R.
PY - 2009/2/20
Y1 - 2009/2/20
N2 - 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 50 μL plasma. Extraction from dried blood spots was performed with a simple one-step extraction with a mixture of methanol, acetonitrile and 0.2 M 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 (150 mm × 2.0 mm, particle size 5 μm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. The analytical run time was 10 min. 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 50-10,000 ng/mL in plasma and dried blood spots and a range of 1-500 ng/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.
AB - 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 50 μL plasma. Extraction from dried blood spots was performed with a simple one-step extraction with a mixture of methanol, acetonitrile and 0.2 M 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 (150 mm × 2.0 mm, particle size 5 μm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. The analytical run time was 10 min. 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 50-10,000 ng/mL in plasma and dried blood spots and a range of 1-500 ng/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.
KW - Dried blood spots
KW - HIV
KW - LC-MS/MS
KW - Pharmacology
KW - Raltegravir
UR - http://www.scopus.com/inward/record.url?scp=59049096346&partnerID=8YFLogxK
U2 - 10.1016/j.jpba.2008.11.025
DO - 10.1016/j.jpba.2008.11.025
M3 - Article
C2 - 19128911
AN - SCOPUS:59049096346
SN - 0731-7085
VL - 49
SP - 451
EP - 458
JO - Journal of Pharmaceutical and Biomedical Analysis
JF - Journal of Pharmaceutical and Biomedical Analysis
IS - 2
ER -