Figure 1. UPLC-MS/MS identification of AcAc, βOHB, and βOHB structural isomers in serum extracts.

(A) Summary of compound names, precursor → fragment transition in Parallel Reaction Monitoring (PRM) negative mode, retention time (RT) in minutes, compound structures, and predicted fragments to quantify AcAc and βOHB ketone bodies (green boxes, top left and right), synthetized AcAc standards (gray box, lower left) after reduction with NaBD₄, and structural isomers of βOHB (orange box, lower right) using Cortecs UPLC T3 column. Stable isotope internal standard (I.S.) used for the quantification of AcAc was [U-¹³C₄]AcAc, whose concentration was externally quantified prior to its spiking into biospecimens by reduction to [3-D₁,U-¹³C₄]βOHB, and comparison against [3,4,4,4-D₄]βOHB. Stable isotope I.S. for quantification of βOHB was [3,4,4,4-D₄]βOHB, which was spiked into biospecimens. Lines indicate where the molecules are fragmented upon higher-energy collisional dissociation [HCD using 30 N(CE)], while dots indicate ¹³C atomic positions. (B) Ionizations of βOHB, 2-OHB, 4-OHB, and 3-HIB are more efficient in negative mode, as determined by the ratio of the area of Full MS scan signal of 20 μM standards detected in negative versus positive mode under same chromatographic conditions (n=3/group). (C) Utilization of MS/MS mode to quantify relative contributions of structural isomers βOHB in mouse serum. In extracted serum from mice fasted for 24h, m/z 103.0401 is comprised of 85% βOHB in control mice, but only 35% in ketogenesis insufficient mice (n>10/group). Data are expressed as the mean standard error of the mean (SEM).