Acetylcarnitine

Methoxyacetylfentanyl hydrochloride (purity ≥ 98%), U-51754 hydrochloride (purity 98%), and U-47931E were purchased from LGC Standards (Wesel, Germany). Isocitrate, Isocitrate dehydrogenase, superoxide dismutase, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), S-(5′-adenosyl)-L-methionine (SAM), dithiothreitol (DTT), reduced glutathione (GSH), acetyl carnitine, and acetyl coenzyme A (AcCoA) were all purchased from Sigma (Taufkirchen, Germany), NADP⁺ from Biomol (Hamburg, Germany), and acetonitrile (LC-MS grade), ammonium formate (analytical grade), formic acid (LC-MS grade), methanol (LC-MS grade), glucuronidase (EC No. 3.2.1.32)/arylsulfatase (EC No. 3.1.6.1) from Helix pomatia L, and all other chemicals and reagents (analytical grade) from VWR (Darmstadt, Germany). phS9 (20 mg protein/mL, from 30 individual donors), UGT reaction mix solution A (25 mM UDP-glucuronic acid), and UGT reaction mix solution B (250 mM Tris–HCl, 40 mM MgCl₂, and 0.125 mg/mL alamethicin) were obtained from Corning (Amsterdam, The Netherlands). After delivery, the enzyme preparations were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

One hundred micro-litres of each thawed 24 h urine sample were diluted with 900 μL of milliQ water/acetonitrile (90/10, v/v). For plasma deproteinization, 200 µL of fasting samples were mixed with 200 µL of cold methanol and stored at −20 °C overnight. The samples were then centrifuged and 100 µL of the supernatant was diluted with 900 μL of milliQ water/acetonitrile (90/10, v/v). All plasma and urine samples were then filtered using 0.2 nm Phenex-RC Syringe Filters with a cellulose membrane (Phenomenex, Le Pecq, France), transferred to a LC vial and either kept at −20 °C or stored at 4 °C if used within 1–2 days until analysis. Blank and quality control (QC) samples were injected every 10 analytical samples in order to control the possible instrument deviation. The blank samples contained acetonitrile and water (10/90, v/v) and QC samples contained the aliquots of all analyzed samples. All solvents used were liquid chromatography mass spectrometry (LC-MS) grade (Sigma-Aldrich; Saint Quentin Fallavier, France) and the aqueous solutions were prepared using purified Milli-Q water. The authentic standards of carnitine, acetyl-carnitine, propionyl-carnitine, butyryl-carnitine, 2-methylbutyroyl-carnitine, uric acid and 1-methylhistidine were purchased from Sigma-Aldrich.

All water used was purified using a Milli-Q™ water system from MilliPore (Bedford, MA, USA). Formic acid (LC–MS grade) and ammonium formate (LC–MS grade) was obtained from Fisher Scientific (Zurich, Switzerland). Acetonitrile (LC–MS grade) was purchased from Fisher Scientific. Acetonitrile used for protein precipitation of biological samples was kept on ice throughout the sample preparation procedure. Analytical standards of acetylcholine and serotonin were purchased from VWR International AB (Stockholm, Sweden) and 2-aminobenzoic acid, acetylcarnitine, caffeine, diclofenac, phenylalanine, theophylline, theobromine were purchased from Sigma-Aldrich (Steinheim, Germany). Pooled human blood plasma (Li-Hep) from anonymous healthy donors for the validation experiments was purchased from 3H Biomedical AB (Uppsala, Sweden).

Acetonitrile, methanol, formic acid and ammonium formate, all LC-MS grade were purchased from Fisher Scientific (Loughborough, UK). Ammonium hydroxide (25%, LC-MS grade) was bought from Merck (Darmstadt, Germany) and chloroform (HPLC-grade) was bought from BDH chemicals (Poole, UK). The isotopically labeled standards, guanine (¹³C, 2*¹⁵N, 98%), hypoxanthine (3*D, 98%), inosine (4*¹⁵N, >95%), tryptophan (5*D, 98%) and glutamine (5*D, 97%) were purchased from Cambridge Isotope laboratories (Andover, MA, USA) while valine (8* D, 98%) was obtained from Aldrich (St Louis, MO, USA). The standard compounds butyrylcarnitine (≥97%), hypoxanthine (≥99%), choline (≥99%), adenine (≥99%), L-tryptophan (≥99.5%), acetylcarnitine (≥99%), L-proline (≥99.5%), L-carnitine (≥98%) and L-arginine (≥98%) were obtained from Sigma (Steinheim, Germany). Phenylalanine was purchased from Merck. Creatine (99.5%) was bought from Chem Service (West Chester, PA, USA) and NADH (>95%) was bought from Appli Chem (Darmstadt, Germany). Betaine (>99%), taurine (>99%) and thiamine (99.9%) were purchased from Fluka (Steinheim, Germany). All water was purified using a Milli-Q purification unit (MilliporeMerck, Darmstadt, Germany).

Gradient grade water and LC-MS grade methanol for liquid chromatography plus the non LC-MS grade rinsing solvents 2-propanol and acetonitrile as well as formic acid (puriss.) and ammonia fluoride (puriss.) were purchased from Carl Roth (Carl Roth, Rothenfels, Germany). A collection of all l-amino acids and carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, valerylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine and stearoylcarnitine, were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The deuterated internal standards (IS) Cortisol ²H₄, Progesterone ²H₉, 17-Hydroxyprogesterone ²H₈, 11-Deoxycortisol ²H₅, 21-Deoxycortisol ²H₈, Androstenedione ¹³C₃, Corticosterone ²H₄, 11-Deoxycorticosterone ¹³C₃ were also products of Sigma-Aldrich (St. Louis, MO, USA). The deuterated amino acids and acylcarnitines were obtained from the MassChrom^® Amino Acids and Acylcarnitines from Dried Blood kit from Chromsystems (Munich, Germany). Dried blood spot cards (TFN filter paper) were provided by CAMAG (Muttenz, Switzerland). Fresh whole blood was obtained from the local blood donation center (Basel, Switzerland).

HPLC-grade methanol was obtained from Thermo Fisher Scientific (MA, United States). Ultrapure-grade water was purified using a Milli-Q system (Millipore, MA, United States). Ammonium acetate, acetylcarnitine, and formic acid were purchased from Sigma Aldrich (St. Louis, MO, United States). Oxyneurine and PC(17:0/0:0) were from ChromaBio (Chengdu, China).