D4 citric acid

An internal standard stock mixture was prepared using the following compounds and concentrations: citric acid-d₄ (Cambridge Isotope Laboratories, DLM-3487), 225 µM; L-lysine-d₄ (Cambridge Isotope Laboratories, DLM-2640), 112.5 µM; L-Tryptophan-(indole-d₅) (Cambridge Isotope Laboratories, DLM-1092), 5.625 µM; stearic acid-d₃₅ (Cambridge Isotope Laboratories, DLM-379), 225 µM; succinic acid-d₄ (Sigma 293,075), 112.5 µM; ¹³C₆-carbamazepine (Sigma, C-136), 2.25 µM; Leucine-d₁₀ (Sigma 492,949), 22.5 µM and methionine-d₄ (Cambridge Isotope Laboratories, DLM-2933), 22.5 µM.
Fig. 1

Incubation of cells in serum for metabolomics analysis to determine transporter substrates. Following incubation of cells in serum, spent serum is collected after centrifugation, followed by extraction using methanol. The remaining cell pellet is washed with PBS (at 37 °C), followed by quenching and extraction of intracellular metabolites using 80% methanol. The spent medium and intracellular extracts are subsequently lyophilised (with a mixture of internal standards spiked in prior to lyophilisation) and reconstituted in water ready for analysis by LC-HRMS/MS

Extraction buffer was prepared by adding 2:2:1 methanol:acetonitrile:water to internal standards at 1 μg/mL each (D4-Citric Acid, ¹³C5-Glutamine, ¹³C5-Glutamic Acid, ¹³C6-Lysine, ¹³C5-Methionine, ¹³C3-Serine, D4-Succinic Acid, ¹³C11-Tryptophan, and D8-Valine; Cambridge Isotope Laboratories). Plasma volume was included in the calculations for the water portion of the buffer. A total of 720 µL of extraction buffer was then added to 40 μL of each plasma sample. Samples were placed on a rotating platform at –20 °C for 1 h and centrifuged at 4 °C for 10 min at 21,000× g. A total of 300 µL of the metabolite extracts was transferred into fresh microcentrifuge tubes. An equal volume of each extract was pooled to serve as a quality control (QC) sample, which was analyzed at the beginning, end, and after every tenth sample throughout the instrument run. Extraction buffer alone was analyzed as a processing blank sample to account for carryover or background contamination from the sample extraction process. Metabolite extracts, the QC sample, and the processing blank were evaporated to dryness using a speed-vacuum. All samples were reconstituted in 30 μL of acetonitrile/water (1:1, v/v), vortexed for 10 min, and incubated at –20 °C for 18 h. Samples were then centrifuged at 4 °C for 2 min at 21,000× g and the supernatant was transferred into autosampler vials for analysis.