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MS quantitation of AQC-derivatized amino acids for detection of inborn errors of metabolism
140
Abstracts
existing assay. The procedure utilizes reversed phase liquid chromatography with a C18 column which is capable of quantitatively determining the levels of l-carnitine along with thirteen other acylcarnitines in human serum. The separation power of the LC method allows for baseline resolution of isobaric biological acylcarnitines such as butyrylcarnitine and isobutyrylcarnitine, along with other potential interferences such pivaloylcarnitine, a drug-carnitine conjugate which is isobaric to isovalerylcarnitine, to greatly increase the overall selectivity of the method. Preliminary validation has shown analytical figures of merit equal to or better than the current flow injection method and has shown promise as a complimentary, second-tier confirmatory test for inborn errors of fatty acid metabolism.
doi:10.1016/j.clinbiochem.2014.07.045
Homocysteine analysis in dried blood spots by LC–MS/MS as a second tier test for homocystinuria Amy E. Chambers a,*, Nathan McIntosh a, Osama Y. Al-Dirbashi a, b, Pranesh Chakraborty a, b a Newborn Screening Ontario, Ottawa, Ontario, Canada b Department of Pediatrics, University of Ottawa, Ottawa, Ontario, Canada Newborn screening methods that use the quantification of methionine as the sole screening marker for homocystinuria are subject to an increase in false negative results. Most newborn screening protocols accept samples that are drawn from newborns at and after 24 h of life. It has been noted that the methionine in individuals affected by homocystinuria may not elevate until 7 days after birth. A better marker for the detection of homocystinuria caused by cystathionine betasynthase deficiency, known as classical homocystinuria, is the quantification of the total homocysteine present. We developed a quick and easy 96-well plate method that requires a run time of 3 min per sample. The method involves liquid chromatography tandem mass spectrometry and a deuterium labeled homocystine internal standard. Dithiothretol was used as a reducing agent to cleave the disulfide bonds converting the homocystine to homocysteine. 3 controls and 8 calibrants were prepared in dried blood spots. The average within day reproducibility had a coefficient of variation (CV) of 7.28% and the average between day CV was 5.45%. The eight calibrants prove to be linear over a range of 0 to 75 μmol/L. The method was validated using samples from confirmed patients (n = 8) and results were compared with those obtained from a reference lab in Heidelberg, DE. Reference range was obtained using over 500 normal samples. The method we developed is simple, sensitive and reproducible. We anticipate the implementation of this method as a second tier test to improve our screening algorithm for homocystinuria.
doi:10.1016/j.clinbiochem.2014.07.046
UPLC MS/MS quantitation of AQC-derivatized amino acids for detection of inborn errors of metabolism Lorne E. Seargeant a, b,*, Catherine J. Mallory b a University of Manitoba, Pediatrics & Child Health, Canada b Diagnostic Services Manitoba, Canada Methods Reagent: 6-Aminoquinolyl-N-hydroxysuccinimidyl (AQC) was obtained from Chemos GmbH.
carbamate
Equipment: Waters Acquity UPLC TQD ESI-MS/MS. Column: BEH C18 column (Waters 186002353) at 40 °C. Mobile phase A: 10 mmol/L ammonium bicarbonate; B: 60% acetonitrile. Gradient: 5–41% B in 16.5 min. Amino acid standards: Sigma physiological acidics and neutrals and basics, plus homocitrulline, sulfocysteine, allo-isoleucine, glutamine and argininosuccinate. Sample prep: 25 microliters (μL) of sample (plasma, CSF, urine diluted to creatinine of 1 mmol/L) 25 μL of internal standard mixture (29 deuterated amino acids) 200 μL acetonitrile (centrifuge at 13,000 rpm for 5 min). Derivatization: Add 175 μL 0.2 M borate buffer pH 8.8 to 25 μL sample supernatant followed by 25 μL AQC. Incubate at 55 °C for 10 min. Add 1 mL of water. Results Derivatization of CSF, plasma, or urine amino acids was complete within minutes. Derivatives were stable for N1 week. Concentrations were linear to N2× ULN. Peak shapes were symmetrical and iso-mass amino acids (ala, B-ala; leu, ile, allo-ile) were separated chromatographically. Within run precision for replicates (n = 10) was b5% for all amino acids. Correlations with Biochrom 30 AAA (where possible) were very good. Between run precision (n = 12) averaged b10% for all amino acids. Conclusions Improved analysis time, reagent stability, accurate quantitation, specificity, reproducible chromatography, freedom from drug and reagent interference, low reagent costs, capability for adding additional analytes, and lack of need for a dedicated instrument all support physiological AQC-amino acid analysis by UPLC MS/MS.
doi:10.1016/j.clinbiochem.2014.07.047
Novel methylated Gb3 isoform biomarker analysis using UPLC–MS/MS for Fabry disease patients Mona Abaoui*, Michel Boutin, Pamela Lavoie, Christiane Auray-Blais Service of Genetics, Department of Pediatrics, Université de Sherbrooke, Québec, Canada
Objectives: Fabry disease is an X-linked lysosomal storage disorder, caused by a deficiency in alpha-galactosidase A. Patients present an accumulation of glycosphingolipids in biological fluids and tissues. Two biomarkers are used for screening and diagnosis: globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). A recent metabolomic study revealed the detection of five new categories of Gb3-related isoforms/analogs. One of these categories of biomarkers, the methylated Gb3 isoforms, are intermediate compounds in the metabolic pathway involving the deacylation of Gb3 to generate the lyso-Gb3 molecule. The objectives are to: 1) Develop and validate a liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the relative quantification of methylated Gb3 isoforms; 2) evaluate urinary and plasma levels in 150 Fabry patients and 50 controls; and 3) establish correlations between excretion levels of these methylated Gb3 isoforms with disease severity and progression. Method: Samples were processed by liquid–liquid extraction and injected into the Acquity UPLC I-Class system coupled to a Xevo TQ-S mass spectrometer (Waters Corp.). The methodology was validated. Results: Seven methylated Gb3 isoforms were evaluated in patients and controls. The isoform at m/z 1122 is highly excreted in Fabry patients compared to controls. Plasma profiles of Gb3 and methylated Gb3 isoforms were different. All isoform levels were
Abstracts
existing assay. The procedure utilizes reversed phase liquid chromatography with a C18 column which is capable of quantitatively determining the levels of l-carnitine along with thirteen other acylcarnitines in human serum. The separation power of the LC method allows for baseline resolution of isobaric biological acylcarnitines such as butyrylcarnitine and isobutyrylcarnitine, along with other potential interferences such pivaloylcarnitine, a drug-carnitine conjugate which is isobaric to isovalerylcarnitine, to greatly increase the overall selectivity of the method. Preliminary validation has shown analytical figures of merit equal to or better than the current flow injection method and has shown promise as a complimentary, second-tier confirmatory test for inborn errors of fatty acid metabolism.
doi:10.1016/j.clinbiochem.2014.07.045
Homocysteine analysis in dried blood spots by LC–MS/MS as a second tier test for homocystinuria Amy E. Chambers a,*, Nathan McIntosh a, Osama Y. Al-Dirbashi a, b, Pranesh Chakraborty a, b a Newborn Screening Ontario, Ottawa, Ontario, Canada b Department of Pediatrics, University of Ottawa, Ottawa, Ontario, Canada Newborn screening methods that use the quantification of methionine as the sole screening marker for homocystinuria are subject to an increase in false negative results. Most newborn screening protocols accept samples that are drawn from newborns at and after 24 h of life. It has been noted that the methionine in individuals affected by homocystinuria may not elevate until 7 days after birth. A better marker for the detection of homocystinuria caused by cystathionine betasynthase deficiency, known as classical homocystinuria, is the quantification of the total homocysteine present. We developed a quick and easy 96-well plate method that requires a run time of 3 min per sample. The method involves liquid chromatography tandem mass spectrometry and a deuterium labeled homocystine internal standard. Dithiothretol was used as a reducing agent to cleave the disulfide bonds converting the homocystine to homocysteine. 3 controls and 8 calibrants were prepared in dried blood spots. The average within day reproducibility had a coefficient of variation (CV) of 7.28% and the average between day CV was 5.45%. The eight calibrants prove to be linear over a range of 0 to 75 μmol/L. The method was validated using samples from confirmed patients (n = 8) and results were compared with those obtained from a reference lab in Heidelberg, DE. Reference range was obtained using over 500 normal samples. The method we developed is simple, sensitive and reproducible. We anticipate the implementation of this method as a second tier test to improve our screening algorithm for homocystinuria.
doi:10.1016/j.clinbiochem.2014.07.046
UPLC MS/MS quantitation of AQC-derivatized amino acids for detection of inborn errors of metabolism Lorne E. Seargeant a, b,*, Catherine J. Mallory b a University of Manitoba, Pediatrics & Child Health, Canada b Diagnostic Services Manitoba, Canada Methods Reagent: 6-Aminoquinolyl-N-hydroxysuccinimidyl (AQC) was obtained from Chemos GmbH.
carbamate
Equipment: Waters Acquity UPLC TQD ESI-MS/MS. Column: BEH C18 column (Waters 186002353) at 40 °C. Mobile phase A: 10 mmol/L ammonium bicarbonate; B: 60% acetonitrile. Gradient: 5–41% B in 16.5 min. Amino acid standards: Sigma physiological acidics and neutrals and basics, plus homocitrulline, sulfocysteine, allo-isoleucine, glutamine and argininosuccinate. Sample prep: 25 microliters (μL) of sample (plasma, CSF, urine diluted to creatinine of 1 mmol/L) 25 μL of internal standard mixture (29 deuterated amino acids) 200 μL acetonitrile (centrifuge at 13,000 rpm for 5 min). Derivatization: Add 175 μL 0.2 M borate buffer pH 8.8 to 25 μL sample supernatant followed by 25 μL AQC. Incubate at 55 °C for 10 min. Add 1 mL of water. Results Derivatization of CSF, plasma, or urine amino acids was complete within minutes. Derivatives were stable for N1 week. Concentrations were linear to N2× ULN. Peak shapes were symmetrical and iso-mass amino acids (ala, B-ala; leu, ile, allo-ile) were separated chromatographically. Within run precision for replicates (n = 10) was b5% for all amino acids. Correlations with Biochrom 30 AAA (where possible) were very good. Between run precision (n = 12) averaged b10% for all amino acids. Conclusions Improved analysis time, reagent stability, accurate quantitation, specificity, reproducible chromatography, freedom from drug and reagent interference, low reagent costs, capability for adding additional analytes, and lack of need for a dedicated instrument all support physiological AQC-amino acid analysis by UPLC MS/MS.
doi:10.1016/j.clinbiochem.2014.07.047
Novel methylated Gb3 isoform biomarker analysis using UPLC–MS/MS for Fabry disease patients Mona Abaoui*, Michel Boutin, Pamela Lavoie, Christiane Auray-Blais Service of Genetics, Department of Pediatrics, Université de Sherbrooke, Québec, Canada
Objectives: Fabry disease is an X-linked lysosomal storage disorder, caused by a deficiency in alpha-galactosidase A. Patients present an accumulation of glycosphingolipids in biological fluids and tissues. Two biomarkers are used for screening and diagnosis: globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). A recent metabolomic study revealed the detection of five new categories of Gb3-related isoforms/analogs. One of these categories of biomarkers, the methylated Gb3 isoforms, are intermediate compounds in the metabolic pathway involving the deacylation of Gb3 to generate the lyso-Gb3 molecule. The objectives are to: 1) Develop and validate a liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the relative quantification of methylated Gb3 isoforms; 2) evaluate urinary and plasma levels in 150 Fabry patients and 50 controls; and 3) establish correlations between excretion levels of these methylated Gb3 isoforms with disease severity and progression. Method: Samples were processed by liquid–liquid extraction and injected into the Acquity UPLC I-Class system coupled to a Xevo TQ-S mass spectrometer (Waters Corp.). The methodology was validated. Results: Seven methylated Gb3 isoforms were evaluated in patients and controls. The isoform at m/z 1122 is highly excreted in Fabry patients compared to controls. Plasma profiles of Gb3 and methylated Gb3 isoforms were different. All isoform levels were