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3 protocols using acetyl d3 l carnitine hydrochloride

1

Carnitine Quantification by LC-MS

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Glacial acetic acid and HPLC grade water were purchased from Fisher Scientific (Agawam, MA, USA). Optima LC-MS grade acetonitrile, methanol, ethyl acetate and formic acid were also purchased from Fisher Scientific. The following unlabeled standards were available and purchased from Sigma (St. Louis, MO, USA): acetylcarnitine (C2:0-CN), propionylcarnitine (C3:0-CN), butyrylcarnitine (C4:0-CN), valerylcarnitine (C5:0-CN), hexanoylcarnitine (C6:0-CN), octanylcarnitine (C8:0-CN) and decanoylcarnitine (C10:0-CN). As shown in Table S1 in the supplementary materials, the following stable isotope standards were available and purchased from Sigma (St. Louis, MO, USA), as well: acetyl-d3-L-carnitine hydrochloride (d3-C2:0-CN), butyryl-L-carnitine-N-methyl-d3 (d3-C4:0-CN), octanoyl-L-carnitine-N-methyl-d3 (d3-C8:0-CN), and decanoyl-L-carnitine-N-methyl-d3 (d3-C10:0-CN).
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2

HPLC-SRM Spiking Sample Preparation

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High-performance liquid chromatography-grade methanol, water, and acetonitrile were purchased from J.T. Baker (NJ, USA). Formic acid (mass spectrometry grade) was purchased from Fluka Analytical (Buchs, Switzerland). Four small molecules [sulfamethoxazole, ketoprofen, MES (4- morpholineethanesulfonic acid), glyphosate], and 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) were used for the spiking sample, and Acetyl-d3-L-carnitine hydrochloride used as the internal standard in selected reaction monitoring (SRM) analysis was purchased from Sigma-Aldrich (St. Louis, USA). Acetylcarnitine was purchased from Santa Cruz Biotechnology (TX, USA) and used for assay standard solution.
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3

Comprehensive Metabolite Profiling Protocol

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Acetylcholine, acetyl-d3-L-carnitine hydrochloride, acetyl-Lcarnitine, adrenic acid, L-carnitine, Lcitrulline, dodecanoic acid, (-)-epicatechin, gallic acid, glycochenodeoxycholic acid, glycocholic acid, glycocholic acid-(glycyl-1-13C) monohydrate, α-hydroxyisobutyric acid, indole-3-acetic-2,2-d2 acid, Lisoleucine, 7-ketocholesterol, L-leucine, leukotriene B4, margaric acid, palmitic acid, Lphenylalanine, stearic acid, syringic acid, L-tryptophan, uric acid, and L-valine were purchased from Sigma-Aldrich (St. Louis, MO). 4-hydroxyhippuric acid was purchased from PhytoLab GmbH and Co KG (Vestenbergsgreuth, Germany), naringenin from Extrasynthèse (Genay, France), and arachidonic acid from Cymit Quimica (Barcelona, Spain). UHPLC-MS-grade methanol, acetone, formic acid, and HPLC-grade acetonitrile were purchased from Scharlau Chemie S.A. (Barcelona, Spain). Ultrapure water (Milli-Q) was obtained from a Milli-Q Gradient A10 system (Millipore, Bedford, MA).
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