Application of dried blood spots combined with high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry for simultaneous quantification of vincristine and actinomycin-D

Abstract

A sensitive, specific and efficient high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of vincristine and actinomycin-D in human dried blood spots is presented. Dried blood spots were punched out of a collection paper with a 0.25-in.-diameter punch. The analytes were extracted from the punched-out disc using sonication during 15 min in a mixture of acetonitrile-methanol-water (1:1:1, v/v/v) containing the internal standard vinorelbine. Twenty-microlitre volumes were injected onto the HPLC system. Separation was achieved on a 50×2.1 mm ID Xbridge C 18 column using elution with 1 mM ammonium acetate-acetonitrile (70:30, v/v) adjusted to pH10.5 with ammonia and run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies vincristine from 1 to 100 ng/mL and actinomycine-D from 2 to 250 ng/mL using a blood sample obtained by a simple finger prick. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human dried blood spots with the presented method. The assay can now be used to support clinical pharmacologic studies with vincristine and actinomycin-D.

Dried blood spot sampling in combination with LC‐MS/MS for quantitative analysis of small molecules

“…In contract, no apparent impact of blood volume has been seen in several recent LC-MS/MS applications for quantitative analysis of small molecules, e.g. acetaminophen (Spooner et al, 2009), tacrolimus (Hoogtanders et al, 2007), vincristine and actinomycin-D (Damen et al, 2009), dextromethorphan and dextrorphan (Liang et al, 2009) and some antiviral drugs (ter Heine et al, 2008Heine et al, , 2009a, in DBS samples. Nonetheless, during developing a DBS-LC-MS/ MS method, the relationship between dried blood spot area/ weight and the amount of blood spotted on the paper/card should be examined by spotting increasing volumes of blood onto the paper/card (at least in duplicate) and measuring the areas of the obtained spots (ter Heine et al, 2008) or weighing the obtained spots (Hoogtanders et al, 2007).…”

“…After drying, a single punch (e.g. 3 mm) is taken from the center of each DBS sample and analyzed along with a set of calibration standards, for which the spotted blood volume could be the same as one of the above QCs, or at a different volume (Damen et al, 2009;Liang et al, 2009, Spooner et al, 2009ter Heine et al, 2008ter Heine et al, , 2009a. As long as the obtained QC sample results by LC-MS/MS are within ±15% of the nominal values, precise sample pipetting may not be necessary.…”

“…The analyte of interest is then extracted with gentle shaking or vortexing. Sonication may be necessary for better extraction effi ciency (Damen et al, 2009;Wilhelm et al, 2009;van der Heijden et al, 2009). After centrifugation, the resulting extracts are transferred manually or by an automated liquid handler to new tubes or microtiter plates.…”

“…The samples are then extracted without internal standard. The response of any cross interfering peak should be ≤20% of that of the analyte at the LLOQ level (Barfi eld et al, 2008;Damen et al, 2009;Hoogtanders et al, 2007;Liang et al, 2009;Spooner et al, 2009;ter Heine et al, 2008ter Heine et al, , 2009avan der Heijden et al, 2009). …”

“…The intra-and inter-day accuracy, the bias (%) from the nominal concentration values, should be within ±15% for all QC samples except the LLOQs, for which a bias of within ±20% is considered acceptable. The intra-and inter-day precision, assessed by the standard deviation divided by the mean (CV%) from the replicate analysis, should be ≤15% for the results of all QCs except the LLOQs, for which a ≤20% CV is considered acceptable (Damen et al, 2009;Koal et al, 2005;Liang et al, 2009;Spooner et al, 2009;ter Heine et al, 2008ter Heine et al, , 2009a.…”