Optimization and validation of a GC-MS method for determination of free D-amino acids in human urine in a diabetes type 1 study

Type 1 Diabetes Mellitus is one of the most common chronic disorders of childhood. Amino acids play a major role in energy metabolism, neurotransmission, and lipid transport. In humans, D-AAs originate primarily from bacterial metabolism and food intake. As D-AA elimination proceeds mainly by renal excretion and its easy collection, urine samples were selected. Amino acids were isolated from 1 ml of urine or standard solution using a cation exchanger Discovery DSC-SCX tube (500 mg/3 ml) and converted into their N(O)-pentafluoropropionyl amino acid 2-propyl esters. Enantiomers were separated and quantified by GC-IT-MS on a Chirasil-L-Val (N-propionyl-L-valine-tert-butylamidepolysiloxane) capillary column. The isolation (i), the derivatization process (ii), and the separation conditions (iii) were optimized for both urine and standards. Conditions such as cartridge sorbent mass/column volume for SPE (100 mg/1 ml and 500 mg/3 ml), pH (1-7), dilution of the loaded urine sample (0, 2, 5, 10 times diluted) (SPE) and solvent volumes (methanol, 10-2 M HCl and 4 M ammonia in methanol) were optimized for the isolation process (i). Volume of reagents and solvents (40-200 µl) and reaction time (30-60 min) were tested for the derivatization (ii). Regarding to the separation conditions (iii), split ratio (1:1-1:70), temperature program and mass range were optimized. The final method was validated in terms of linearity, accuracy and precision for D-Ala, D-Pro, D-Ser, D-Met, D-Phen, D-Glu, D-Orn and D-Lys. Identification of the amino acid was based on retention time and mass spectra. The results for the pool urine, obtained with healthy volunteers, were confirmed with previous literature. Urine samples from seven type 1 diabetic children and seven control, 6 to 11 years old, for boys and girls were analyzed. All of them were collected in La Paz Hospital, Madrid (Spain). D- Relative amounts (%D-AA) were determined for D-Ala, D-Val, D-Ser, D-Leu, D-Asp, D-Glu, D-Orn and D-Lys. Statistically significant differences were found for D-Ala, D-Val and D-Glu.