Validated assay for the simultaneous quantification of total vincristine and actinomycin‐D concentrations in human EDTA plasma and of vincristine concentrations in human plasma ultrafiltrate by high‐performance liquid chromatography coupled with tandem mass spectrometry |
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| Authors: | Carola W. N. Damen Trijn Israëls Huib N. Caron Jan H.M. Schellens Hilde Rosing Jos H. Beijnen |
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| Affiliation: | 1. Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands;2. Department of Paediatric Oncology, Queen Elizabeth Central Hospital, College of Medicine, Box 360, Blantyre, Malawi;3. Department of Paediatric Oncology and Haematology, Emma Children's Hospital, Academic Medical Centre, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands;4. Science Faculty, Department of Pharmaceutical Sciences, Division of Biomedical Analysis, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands;5. Department of Medical Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands |
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| Abstract: | A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the simultaneous determination of total vincristine and actinomycin‐D concentrations in human plasma and an assay for the determination of unbound vincristine are presented. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and heated electrospray ionization (H‐ESI) were tested as ionization interfaces. For reasons of robustness ESI was chosen followed by tandem mass spectrometry (ESI‐MS/MS). For the plasma assay a 30 µL aliquot was protein precipitated with acetonitrile/methanol (50:50, v/v) containing the internal standard vinorelbine and 10 µL volumes were injected onto the HPLC system. To determine unbound vincristine, ultrafiltrate was produced from plasma using 30 kDa centrifugal filter units. The plasma ultrafiltrate was mixed with methanol (50:50, v/v), internal standard vinorelbine was added and 20 µL aliquots were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C18 column using 1 mM ammonium acetate/acetonitrile (30:70, v/v) adjusted to pH 10.5 with ammonia, run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies in plasma vincristine from 0.25 to 100 ng/mL and actinomycin‐D from 0.5 to 250 ng/mL using plasma sample volumes of only 30 µL. Vincristine in plasma ultrafiltrate can be quantified from 1 to 100 ng/mL. Validation results demonstrate that vincristine and actinomycin‐D can be accurately and precisely quantified in human plasma and plasma ultrafiltrate with the presented methods. The assays are now in use to support clinical pharmacological studies in children treated with vincristine and actinomycin‐D. Copyright © 2009 John Wiley & Sons, Ltd. |
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