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

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 microL aliquot was protein precipitated with acetonitrile/methanol (50:50, v/v) containing the internal standard vinorelbine and 10 microL 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 microL aliquots were injected onto the HPLC system. Separation was achieved on a 50x2.1 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 microL. 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.

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

“…The method was based on a previously developed assay, describing the simultaneous analysis of vincristine and actinomycin-D in plasma [28]. In brief, a Finnigan TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with an electrospray ionisation (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA) operating in the positive ion mode was used as a detector.…”

“…Stability of all analytes in stock solutions has been investigated and reported [28]. The stability of the internal standard solution was determined during 6 h at ambient temperatures and during longterm storage at −20°C.…”

“…In our department, we previously developed a highperformance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantification of vincristine and actinomycin-D in plasma [28]. This method was used to support pharmacokinetic studies in children with a Wilms' tumour in Malawi.…”

Antineoplastic drugs and their analysis: a state of the art review

“…354,355 For the same compound, a ten-fold increase in sensitivity was observed in LC-MS. 361 Due to the concomitant administration of vincristine and actinomycin D in the treatment of various pediatric cancers, several LC-MS methods have been developed for the simultaneous determination of these two anticancer drugs in biological matrices. [356][357][358][359] An LOD of 0.007 ng mL −1 and LOQ of 0.05 ng mL −1 were reached for the analysis of actinomycin D in plasma using a SPE-LC-MS procedure. 358 A CE method coupled to chemiluminescence detection was also reported for the determination of mitoxantrone in injectable solutions and biological samples.…”

“…LC was generally used for the determination of mitoxanthrone and actinomycin D. Developed methods were based on C18 stationary phase, coupled to UV 354,355 and MS detection. [356][357][358][359][360][361] An LOQ of 5 ng mL −1 was obtained for the LC-UV analysis of mitoxantrone in plasma. 354,355 For the same compound, a ten-fold increase in sensitivity was observed in LC-MS. 361 Due to the concomitant administration of vincristine and actinomycin D in the treatment of various pediatric cancers, several LC-MS methods have been developed for the simultaneous determination of these two anticancer drugs in biological matrices.…”