AbstractThis work describes a novel liquid chromatography tandem MS (LC-MS/MS) method for the determination of ratios of acylcarnitines arising from acyl-CoA substrates and products that reflect metabolic disturbances caused by marginal biotin deficiency. The urinary ratios reflecting reduced activities of biotin-dependent enzymes include the following: 1) the ratio of 3-hydroxyisovalerylcarnitine: 3-methylglutarylcarnitine (3HIAc :MGc) for methylcrotonyl-CoA carboxylase; 2) the ratio ofpropionylcarnitine:methylmalonylcarnitine (Pc :MMc) for propionyl-CoA carboxylase (PCC); and 3) the ratio of acetylcarnitine: malonylcarnitine (Ac :Mc) for acetyl-CoA carboxylase. To demonstrate the suitability of the LC-MS/MS method for biomonitoring,we measured the 3 ratios for 7 healthy adults at various time points (d 0, 14, and 28) during the induction of marginal biotin through the consumption of egg white. The mean change in the Pc :MMc ratio relative to d 0 was 5.3-fold by d 14 (P = 0.0049) and 8.5-fold by d 28 (P = 0.0042). The mean change in the 3HIAc :MGc ratio was 2.8-fold by d 14 (P = 0.0022) and 3.8-fold by d 28 (P = 0.0001).The mean change in the Ac :Mc ratio was 2.9-fold by d 14 (P = 0.03) and 4.7-fold by d 28 (P = 0.02). The results suggest that simultaneous assessment of ratios of multiple biotin-dependent pathways offers insight into the complex metabolic disturbances caused by marginal biotin deficiency. We hypothesize that one or a combination of the ratios might be more sensitive or robust with respect to other nutrient deficiencies or confounding metabolic processes.