Quantification of β-hydroxymethylbutyrate and leucine by ultrahigh performance liquid chromatography tandem mass spectrometry at different situations and stages of a rodent life

• The natural evolution of HMB and leucine during the life of a rodent is studied.
• UHPLC-MS/MS is used to measure these compounds in different biological matrices.
• The method is validated in terms of selectivity, accuracy and sensibility.
• Important information regarding the metabolic pathway of HMB and Leu is obtained.
• Perinatal status, different ages and strains of animals are evaluated.

The main objective of this work was to develop a method to measure Leucine (Leu) and β-hydroxymethylbutyrate (HMB) at basal levels in serum, urine, milk and brain microdialysates in rats. Ultrahigh performance liquid chromatography–electrospray-tandem mass spectrometry (UHPLC-ESI-MS/MS) was used as analytical technique. The sample treatment was simple and consisted of dilution with methanol and centrifugation for serum and urine, dilution with water and filtration with an Amicon filter for milk, and treatment with formic acid with no further dilution for microdialyzates. The procedures for sampling and the UHPLC-MS/MS parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. For chromatographic separation, an Acquity UPLC BEH Amide column using acetonitrile-water gradient with formic acid as additive was used. The total run time was 4 min. The analytical characteristics (accuracy, selectivity and sensitivity) of the proposed method were evaluated. The limits of detection (LODs) obtained ranged from 0.4 to 7 ng mL−1 and the limits of quantification (LOQs) from 1 to 22 ng mL−1. Precision, expressed as relative standard deviation (% RSD), was lower than 15% in all cases, and the determination coefficient (R2) was equal or higher than 99.0% with a residual deviation for each calibration point lower than ±25%. Mean recoveries were between 85 and 115%. The method was successfully applied to these matrices being able to detect significant differences between physiological situations, strains and stages of life.