The Development and Validation of a Quantitative Liquid Chromatography-Tandem Mass Spectrometry Method for the Detection of Cremophor EL in Human Plasma

Cremophor EL (CrEL) is an excipient widely used in the pharmaceutical industry, which is derived from the castor oil plant (Ricinus communis). CrEL is produced by reacting castor oil with ethylene oxide at a molar ratio of 1:35. Patients who have advanced or multiple cancer sites are commonly treated using chemotherapeutic agents. Such mixtures are comprised of a drug compound and an excipient carrier which enable the agent to reach its intended site more efficiently. In high concentration, there is evidence to suggest that CrEL may be toxic to those undergoing chemotherapy. Therefore, CrEL should be monitored in order to reduce the incidence of adverse reactions. The measurement of CrEL in human plasma has been previously reported using a number of different analytical techniques ranging from the simple colorimetric dye assays to High Performance Liquid Chromatography with Ultraviolet detection. Throughout many of these techniques CrEL proved particularly difficult to measure using labour intensive sample preparation, which lacked sensitivity, reproducibility and linearity.

The purpose of this study was to develop and validate a Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) method for the determination of CrEL in human plasma, which could be applied to a Phase 1 Pharmacokinetically (PK) guided, dose escalation study for patients with advanced solid tumours. CrEL was extracted from plasma by liquid-liquid extraction using a mixture of Chloroform: Methanol (2:1 v/v) and butylated hydroxy toluene (BHT). Sodium hydroxide was added to the samples to release Ricinoleic acid. The resulting extracts were then analysed using the Waters Ultra Performance Liquid Chromatography and the Quattro Premier Tandem Mass Spectrometry. Separation of CrEL in the LC was achieved using an HSS T3 column 1.8 µm (hybrid particle) 2.1 mm x 100 mm and ionised using electrospray in negative mode. Multiple reaction monitoring (MRM) was carried out with a transition of m/z 279.3 ? 182.9. The assay performed well with no evidence of ion suppression and enhancement or carry over.

The lower limit of quantification was 0.07 µl/mL and the calibration curve in plasma was linear over the range 0.07 to 10.0µg/L with an excellent correlation coefficient (r²) >0.99. The assay precision was also excellent, intra assay and day to day co-efficient of variation (CV) reporting 17% for the low level and 10% on the high level.

This assay is now in routine use in a Hampshire laboratory where a PK study of patients with advanced solid tumours is being investigated. Patients are dosed with Dexanabinol a synthetically altered cannabinoid drug in combination with the excipient CrEL. Plasma samples are then analysed to evaluate the pharmacokinetics and assess the safety of the prescribed formulation.