engleski

Shortened validation procedure for a method of quantitative analysis of 45 amino acids in plasma on tandem mass spectrometer

Introduction High performance liquid chromatography, combined with tandem mass spectrometry (LC-MS/MS), is a technology used for quantitative measurement of of numerous metabolites, including amino acids in plasma, urine and cerebrospinal fluid. For analysis, only 40 µL of sample is needed to quantify 45 amino acids in a very short time span. Aim Shortened validation procedure for a method of quantifying 45 amino acids in plasma. Materials and methods The validation was carried out using aTRAQ™ reagent manufactured by AB Sciex. Amino acids were chromatographically separated on the AB Sciex C18 column (5 µm, 4.6 mm x 150 mm) on UPLC-NEXERA instrument manufactured by Shimadzu, with binary gradient of water (mobile phase A) and methanol (mobile phase B) and addition of 0.1% of formic acid and 0.01% of heptafluorobutyric acid to both mobile phases. Amino acids were quantified on API 3200 mass spectrometer manufactured by AB Sciex. The following commercial standards were used for validation: AMI-01 produced by SKML (MCA Laboratory, the Netherlands), preparations of A6407 and A6282 amino acid standards produced by Sigma, and laboratory preparation of glutamine standard. Shortened validation procedure for the method included testing of between-run and within- run imprecision, inaccuracy, limit of detection, limit of quantitation and linearity. Results Calculated values of the coefficient of variation (CV) for within-run and between-run imprecision and inaccuracy for most amino acids were lower than 20%, with the exception of glutamine (CV=24%). Limit of detection (LoD) and limit of quantitation (LoQ) were lower than 2 µmol/L for most amino acids except for serine, glycine and ß- alanine which had somewhat higher LoQ. The method was linear up to 1000 µmol/L for most amino acids. Conclusion Based on results of shortened validation procedure, it was concluded that the method is acceptable for routine laboratory practice. Advantages of the method are significantly low sample volume needed and reduced analysis time

validation; amino acids; mass spectrometry

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