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Lysosomal storage disorders comprise a group of rare genetic diseases in which a deficit of specific hydrolases leads to the storage of undegraded substrates in lysosomes. Impaired enzyme activities can be assessed by MS/MS quantification of the reaction products obtained after incubation with specific substrates. In this study, a column-switching HPLC-MS/MS method for multiplex screening in dried blood spot of the lysosomal enzymes activities was developed. Mucopolysaccharidosis type I, Fabry, Gaucher, Krabbe, Niemann-Pick A/B and Pompe diseases were simultaneously assayed. Dried blood spots were incubated with substrates and internal standards; thereafter, supernatants were collected with minor manipulations. Samples were injected, trapped into an online perfusion column and, by a six-port valve, switched online through the C18 analytical column to perform separation of metabolites followed by MS/MS analysis. A total of 1136 de-identified newborn screening samples were analyzed to determine references for enzymes activity values. As positive controls, we analyzed dried blood spots from three patients with Pompe, one with Fabry, one with Krabbe disease and two with MPS I, and in all cases the enzyme activities were below the cutoff values measured for newborns, except for an MPS I patient after successful hematopoietic stem cell transplantation., (Copyright © 2014 John Wiley & Sons, Ltd.)

Enzyme deficiencies in amino acid (AA) metabolism affecting the levels of amino acids and their derivatives in physiological fluids may serve as diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and more than ever now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, detection, and quantification of some amino acids needed to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-electrospray ionization (ESI)-MS/MS method previously described by Qu et al. (Anal Chem 74: 2034-2040, 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatised amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step allowed us to achieve some important advantages: fast and simple sample preparation and exclusion of artefacts and interferences. The use of this technique is highly sensitive, specific, and allows monitoring of 40 underivatized amino acids, including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the Newborn Screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way, we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, feasibility of high sample throughput, and small amount of sample required make this method very suitable for routine analysis in the clinical setting.

Background: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine., Objectives: The aim of this work was to present a simple, fast and accurate UPLC-tandem-MS-based method for the simultaneous determination and quantification of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline. This method is designed for high sample throughput of only 10 μL of human plasma, serum or urine., Methods: The analysis time is reduced to 1.9 min by an ultrahigh-performance liquid chromatography run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection., Results: The method was validated in plasma, serum and urine. Correlation coefficients (r(2)) of the calibration curves in all matrices considered ranged from 0.9810 to 0.9993. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated for validation. The LOD was 0.01 μM for all compounds in water, plasma and serum and 0.1 μM in urine. The LOQ was 0.05 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit in water, plasma and serum; while in urine was 0.1 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit. The precision was ranged from 1% to 15% expressed as CV% and the accuracy (bias %) was <±7% for all added concentrations with the exception of NMMA (-10%). ADMA mean plasma levels, measured in healthy adults and newborns, were in accord with literature data published: (M±SD) 0.56±0.10 μM and 0.84±0.21 μM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54±0.18 μM and 1.14±0.36 μM), while in neonatal urine ADMA was 11.98±7.13 μmol mmol(-1) creatinine., Conclusions: Data from calibration curves and method validation reveal that the method is accurate and precise. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Deuterium (2H) in water and urine can be measured by off-line and, more recently, on-line techniques using isotope ratio mass spectrometry (IRMS). We describe a new simple on-line pyrolysis method for the analysis of 2H/1H in water and urine samples by continuous flow IRMS, normally used for 2H/1H measurements in organic compounds. A deactivated column connected the split injector to a high-temperature conversion reactor (TC HD), and 0.5 microL of sample was injected. Accuracy and precision were determined with Vienna Standard Mean Ocean Water (VSMOW), Standard Light Antarctic Precipitation (SLAP), and Greenland Ice Sheet Precipitation (GISP). The range of linearity was measured with a calibration curve of enriched water from 0 up to 0.1 atom percent excess (APE) (i.e. -72 up to 6323 delta per mil (deltaD per thousand)) with a precision of <5 per thousand and accuracy ranging between 1 and 55 per thousand. Blinded reanalysis of urine samples by an equilibration device (Gas Bench) and by a dedicated pyrolysis system (TC/EA) was performed and results compared by the Bland-Altman test. Enrichments ranged between 600 and 2400 per thousand deltaD(VSMOW) with a precision of +/-5 per thousand. Urine enrichments described by our method were strongly correlated with values obtained by Gas Bench and TC/EA (p < 0.0001). There was a significant memory effect that was reduced by injecting the sample 15 times and discarding the first 10 injections, together with accurate furnace conditioning and appropriate cleaning of the syringe. Data indicate that the method is accurate, and that it can be used for water and urine deuterium determination when a Gas Bench or TC/EA instrument is not available and the amount of sample is limited.