A chemical derivatization based UHPLC-LTQ-Orbitrap mass spectrometry method for accurate quantification of short-chain fatty acids in bronchoalveolar lavage fluid of asthma mice.

Recent studies have demonstrated the important role of short-chain fatty acids (SCFAs) in the maintenance of homeostasis of respiratory immunity. However, there is still no report focus on the determination of SCFAs level in bronchoalveolar lavage fluid (BALF), the most common sample used for screening biomarkers of the pulmonary diseases. Herein, an ultra-high-performance liquid chromatography with LTQ-Orbitrap mass spectrometer (UHPLC-LTQ-Orbitrap) oriented 3-nitrophenylhydrazine (3-NPH)-based derivatization method was developed for the quantification of SCFAs in BALF. To achieve accurate quantitation, d4-acetate was used as internal standard to compensate for the matrix effects. Method validation showed a good linearity (R2 > 0.9992) with wide concentration range, and the intra-day and inter-day precision for determination of eight SCFAs in BALF samples was ≤ 14.79%. The quantitation accuracy, assessed by relative recoveries, ranged from 90% to 110% for target SCFAs at three concentration levels. Matrix effects ranged from 85% to 115%, and the lower limits of quantification of these targeted SCFAs were varied from 3 to 24 nmol/L. The SCFAs-targeted method was then applied to determine the changed levels in BALF samples from OVA-induced asthma mice and normal mice. In addition, the universality of our developed method was also demonstrated by determining the SCFAs concentrations in feces, serum and lung tissue samples from asthma and normal mice. These results indicate that 3-NPH derivatization based UHPLC-LTQ-Orbitrap provides accurate view of global SCFAs alternation in different samples, giving a support to deduce the origin of SCFAs in lung. The present study is of great importance for understanding the role of SCFAs in modulation of host metabolism and immunity.