Simultaneous determination of β-hydroxybutyrate and β-hydroxy-β-methylbutyrate in human whole blood using hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry

•The LC–MS/MS method does not require chemical modifications of BHB and HMB.•Isotope dilution is used for quantification of both substances.•The estimated lower limits of quantification are 3 μM for BHB and 0.4 μM for HMB.•The mean true recoveries are greater than 95% for both substances.

ObjectivesFor the quantification of β-hydroxybutyrate (BHB) and β-hydroxy-β-methylbutyrate (HMB) in human whole blood, a method using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC–MS/MS) was developed, which does not require chemical modification of the analytes.Design and methodsSamples were deproteinised by a mixture of methanol and acetonitrile, and the extracts were cleaned-up using both polymeric strong cation exchange and strong anion exchange sorbents. The analytes and their structural isomers were separated using a column with a zwitterionic stationary phase. Isotope dilution of both analytes was used for quantitative analysis.ResultsSeparation of BHB from isobaric interferences was achieved through chromatography. The relative intra-laboratory reproducibility standard deviations were better than 10% for blood samples at concentration levels of 10–20 μM BHB and 1 μM HMB and better than 5% at concentration levels 10 times higher. The mean true extraction recoveries were close to 100%. The trueness expressed as the relative bias of test results was within ± 5% at concentration levels of 10–1000 μM BHB and 1–20 μM HMB. The lower limits of quantification were estimated to be 3 μM for BHB and 0.4 μM for HMB.ConclusionsA simple and highly sensitive and selective HILIC–MS/MS method was developed that is suitable for the quantification of BHB and HMB in whole blood.

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