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Highlights: [•] Reversed phase HPLC coupled to MS is a powerful method to quantify almorexant and its primary metabolites in human plasma. [•] Liquid–liquid extraction is superior to protein precipitation when ng/mL or sub-ng/mL sensitivity needs to be achieved. [•] Sample preparation by SLE and LLE yield comparable results, LLE is easy to automate. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive liquid chromatography–tandem mass spectrometry method has been developed to quantify miglustat in mouse plasma and in human plasma. The method involved simple protein precipitation with methanol. N-(n-nonyl)deoxynojirimycin was used as internal standard. Separation was performed on a Gemini C18 column (2.1mm×50mm, particle size 5μm) with a binary gradient at a flow rate of 600μl/min. The mobile phases were methanol and water both containing 0.01% of a 25% ammonium hydroxide solution. The triple stage quadrupole mass spectrometer was operated in APCI mode using the transitions m/z 220.1≥158.0 for miglustat and m/z 290.1≥228.0 for the internal standard. The method was linear over a range of 10–10,000ng/ml. The intra-day coefficients of variation for mouse plasma were equal to or smaller than 14.1%. The intra- and inter-day accuracies were 84.5–107.2% and 90.9–104.0%, respectively. For human plasma the intra-day coefficients of variation were equal to or smaller than 13.5%, while accuracies ranged between 93.6% and 100.0%. The validated method offered increased sensitivity (10 times higher) and decreased cycle times compared to other methods. It was successfully applied to the pharmacokinetic assessment of miglustat during treatment of patients with cystic fibrosis. [Copyright &y& Elsevier]