Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids is a reliable tool for the diagnosis and follow-up of patients with inborn errors of metabolism (original) (raw)
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Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is used for the diagnosis of more than 30 inborn errors of metabolisms (IEMs). Accurate and reliable diagnosis of IEMs by quantifying amino acids (AAs) and acylcarnitines (ACs) using LC-MS/MS systems depend on the establishment of age-specific cut-offs of the analytes. This study aimed to (1) determine the age-specific cut-off values of AAs and ACs in Bangladesh and (2) validate the LC-MS/MS method for diagnosis of the patients with IEMs. A total of 570 enrolled healthy participants were divided into 3 age groups, namely, (1) newborns (1-7 days), (2) 8 days-7 years, and (3) 8-17 years, to establish the age-specific cut-offs for AAs and ACs. Also, 273 suspected patients with IEMs were enrolled to evaluate the reliability of the established cut-off values. Quantitation of AAs and ACs was performed on an automated LC-MS/MS system using dried blood spot (DBS) cards. Then the specimens of the enrolled clinically suspected patients were analyzed by the established method. Nine patients came out as screening positive for different IEMs, including two borderline positive cases of medium-chain acyl-CoA dehydrogenase deficiency (MCAD). A second-tier test for confirmation of the screening positive cases was conducted by urinary metabolic profiling using gas chromatography-mass spectrometry (GC-MS). Out of 9 cases that came out as screening positive by LC-MS/MS, seven cases were confirmed by urinary GC-MS analysis including 3 cases with phenylketonuria, 1 with citrullinemia type II, 1 with methylmalonic acidemia, 1 with isovaleric acidemia and 1 with carnitine uptake defect. Two borderline positive cases with MCAD were found negative by urinary GC-MS analysis. In conclusion, along with establishment of a validated LC-MS/MS method for quantitation of AAs and ACs from the DBS cards, the study also demonstrates the presence of predominantly available IEMs in Bangladesh.
Journal of Chromatography B: Biomedical Sciences and Applications, 2001
Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with 28 concentrations as low as 4310 M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.
Clinical Chemistry, 2002
Background: Detection of abnormal metabolites in urine is important for the diagnosis of many inborn errors of metabolism (IEM). Rapid, comprehensive screening methods are needed.Methods: We used electrospray ionization tandem mass spectrometry in positive- and negative-ion modes to detect selected metabolites in urine. For positive-ion analysis, samples were dried and butylated, whereas for negative-ion analysis, samples were merely diluted with the mobile phase. Analysis was by direct injection with multiple reaction monitoring for 32 metabolites in positive mode (amino acids and acylcarnitines) and 30 metabolites in negative mode (organic acids). Run time was 2.1 min in each mode.Results: Interbatch CVs ranged from 4.8% to 32%, enabling quantification of many metabolites. The procedure was applied to controls (278 and 120 in positive- and negative-ion mode, respectively) and 108 IEM individuals representing 37 different IEM. In 105 IEM individuals, representing 36 different IEM, ...
Id-GC/MS as a Tool for Diagnosis of Inborn Errors of Metabolism
Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Veterinary Medicine, 2007
Inherited metabolic diseases identified currently i n humans exceed 500, from which more than 100 are inborn errors in amino acids metabolism. Ph enylketonuria (PKU) and maple syrup urine diseases (MSUD) are autosomal recessive disorders caused by deficiency of enzymes implicated in the metabolism of phenylalanine (Phe) and branched chain aminoacids. PKU and MSUD lead to mental retardation unless the newborns are maintained on strict diet, free of Phe and, respectively of branched chain aminoacids. Th e early diagnosis of these two diseases is extremely import ant for the newborns life. An ID-GC/MS method is proposed for the quantitation of Phe, Tyr, Leu, Val and Tyr in blood spots. Aminoacids are extracted from blood spots (20 �l blood) prelevated from newborns on filter paper w ith methanol:HCl and derivatized as N-trifluoroacetyl b utyl esters. A Trace DSQ Thermo Finnigan quadrupole mass spectrometer in the EI mode coupled with a Trace GC and a capillary column Rtx-5MS was used. ...
Comparison of Amino Acid Concentrations in Plasma and Dried Blood Spot Samples Using LC-MS/MS
Iranian Journal of Pediatrics
Background: Dried blood spot samples are suitable for diagnosing some congenital errors of metabolism; however, they provide limited benefit in the regular monitoring of amino acids. Objectives: The present study aimed to evaluate alanine (Ala), arginine (Arg), citrulline (Cit), glutamic acid (Glu), glycine (Gly), isoleucine (Ileu), leucine (Leu), methionine (Met), ornithine (Orn), phenylalanine (Phe), tyrosine (Tyr), and valine (Val) amino acid concentrations in dried blood and plasma samples obtained simultaneously. Methods: Amino acid concentrations were determined in the plasma, and dried blood spot samples obtained simultaneously from 145 patients (50 females and 95 males). Amino acid concentrations in the plasma and dried blood spot samples were studied by LC-MS/MS using original kits. Results: There were significant differences between dried blood spots and plasma in all amino acid concentrations, except for Met and Val. Bland-Altman analysis revealed the highest mean differe...
Indian journal of clinical biochemistry : IJCB, 2002
Quantification of total and individual amino acids in biological fluids such as plasma, urine and cerebrospinal fluid has an important diagnostic implication in laboratory medicine. The present paper describes protocols for the assay of total amino acids by modified method based on dinitrophenyl and HPLC profile involving pre-column derivatization with o-pthalaldehyde (OPA) derivatization, respectively. The method, based on the alkylation of-SH groups prior to OPA derivatization of amino acids followed by reverse phase high performance liquid chromatography, provide a comprehensive profile of more than twenty amino acids (including-SH group containing) in a single run lasting about 45 minutes. The present study, apart from establishing the normal profile of amino acids in plasma of Indian sub population, also presents HPLC profile for some of the rare amino acidopathies.
Clinica Chimica Acta, 2019
Precise quantification of amino acids (AAs) is mandatory for successful diagnosis and monitoring of patients with metabolic diseases. We compared ion-exchange chromatography (IEC) and liquid chromatography with tandem mass spectrometry (LC-MS/MS), the two methods most commonly used in clinical laboratories for the quantification of AAs in physiological samples. Design & methods: 123 apparently healthy children were selected for the study. The plasma samples for LC-MS/ MS were prepared accordingly to the aTRAQ Kit for Physiological Fluids on Sciex 3200 Qtrap, for IEC according to the protocol from Pickering laboratories on the AA analyzer Pinnacle PCX. Results were interpreted using the Pearson correlation coefficient and the percent difference Bland-Altman test. Results: The Spearman correlation coefficients of the 14 AAs that we evaluated varied from 0.67 in Tau to 0.89 in Leu and Thr. The mean differences in measurements (IEC compared to LC-MS/MS) of 11 AAs complied with our acceptance criterion of < 15%, the differences of Ser and Tyr were higher (19.5% and −19.0%, respectively), and the measured concentrations of Cit were much lower in LC-MS/MS than IEC (31% difference). Conclusion: The two methods are sufficiently comparable for most AAs and the reference values for individual AAs did not have to be refined, with the exception of citrulline. For the monitoring of patients on therapy (e.g. patients with phenylketonuria), it is still advisable to always use the same analytical method for the quantification of AAs.