title: Optimization and validation of a chiral GC-MS method for the determination of free D-amino acids ratio in human urine: application to a gestational diabetes mellitus study.
abstract: Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance with onset or first recognition during pregnancy. It is affecting approximately up to 14% of all pregnancies with an increasing tendency. GDM has been related to relevant short-term and long-term health complications for both mother and offspring. Recent studies strongly emphasized the role of several essential amino acids in the pathogenesis of obesity and highlighted their strong correlation with insulin resistance, but there are no references related to modifications in D-AAs in biological fluids. As D-AA elimination proceeds mainly by renal excretion, urine was the selected sample to evaluate the alterations in free D-AAs ratio in a GDM study. Only 1 mL of first void urine or standard solution was required for purification, by using a Discovery DSC-SCX SPE cartridge (500 mg/3 mL) and derivatization into their N(O)-pentafluoropropionyl amino acid 2-propyl esters. Enantiomeric separation was carried out by GC-MS on a Chirasil-L-Val N-propionyl-L-valine-tert-butylamide polysiloxane fused-silica capillary column (25 m×0.25 mm I.D., 0.12 μm film thickness, Agilent Technologies, Waldbronn, Germany), under programmed temperature elution. Detection was performed with an ion trap mass analyzer, operating in the full scan mode in the m/z 50-350 range. 14 pairs of derivatives of D-and L-AAs were separated. The steps of sample preparation, derivatization and GC-MS conditions were optimized for both urine and standards. Several conditions affecting the SPE procedure, such as sorbent mass/volume ratio of the cartridge, sample dilution and pH, were optimized. Volume of reagents and solvents and reaction temperature and time were also tested for the derivatization. Regarding the GC-MS parameters, split ratio, temperature program and mass range were optimized. The final method was validated in terms of linearity, sensitivity, accuracy and precision for D-Ala, D-Pro, D-Ser, D-Met, D-Phe, D-Glu, D-Orn and D-Lys. Identification of AAs in urine samples was based on retention time and mass spectra. Urine from 20 women with GDM and 20 pregnant women with normal glucose tolerance (after 2-h 75-g oral glucose tolerance test), matched according to the week of gestation and age (22-28 week of gestation and age 24-37 years), were enrolled into the study. %D-Relative amounts were determined for Ala, Val, Thr, Ser, Leu, Asx (Asp+Asn), Glx (Glu+Gln), Met, Phe, Tyr, Orn and Lys. Statistically significant differences (p<0.05) were observed only for D-Phe and higher values were found in the GDM group. It is possible that D-Phe could be involved in metabolic/signaling pathways to compensate early stages of insulin resistance, although further work is necessary to confirm this hypothesis.
authors: Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, Madrid, Spain.
authors: Ma Paz
authors: MP
authors: Lorenzo
authors: Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, Madrid, Spain; Clinical Department of Perinatology, Public Clinic Hospital, Medical University of Bialystok, Bialystok, Poland.
authors: Danuta
authors: D
authors: Dudzik
authors: Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, Madrid, Spain.
authors: Elena
authors: E
authors: Varas
authors: Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 183, 41125 Modena, Italy.
authors: Manuel
authors: M
authors: Gibellini
authors: Clinical Department of Perinatology, Public Clinic Hospital, Medical University of Bialystok, Bialystok, Poland.
authors: Mariusz
authors: M
authors: Skotnicki
authors: Department of Pharmacology, Medical University of Bialystok, Bialystok, Poland.
authors: Marcin
authors: M
authors: Zorawski
authors: Clinical Department of Endocrinology, Diabetology and Internal Diseases, Public Clinic Hospital, Medical University of Bialystok, Bialystok, Poland.
authors: Wieslaw
authors: W
authors: Zarzycki
authors: Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 183, 41125 Modena, Italy.
authors: Federica
authors: F
authors: Pellati
authors: Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, Madrid, Spain. Electronic address: antogar@ceu.es.
authors: Antonia
authors: A
authors: García
mesh_terms: D000328:Adult; D000596:Amino Acids / Q000737:chemistry* / Q000652:urine*; D016640:Diabetes, Gestational / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005951:Glucose Tolerance Test / Q000379:methods; D006801:Humans; D053858:Metabolic Networks and Pathways / Q000502:physiology; D011247:Pregnancy; D015398:Signal Transduction / Q000502:physiology; D055815:Young Adult
chemical_list: D000596:Amino Acids
keywords: Chiral analysis; Gestational Diabetes Mellitus; SPE; Urine; d-Amino acids