Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry

Coleman T. Turgeon, Mark J. Magera, Carla D. Cuthbert, Perry R. Loken, Dimitar K. Gavrilov, Silvia Tortorelli, Kimiyo M. Raymond, Devin Oglesbee, Piero Rinaldo, Dietrich Matern

Research output: Contribution to journalArticle

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Abstract

BACKGROUND: Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY,MMA,andMCAin dried blood spots (DBSs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d3-MMA, d3-MCA, d 8-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening. RESULTS: The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 μmol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3% of all NBS samples led to the identification of 13 affected infants. CONCLUSIONS: Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations.

Original languageEnglish (US)
Pages (from-to)1686-1695
Number of pages10
JournalClinical Chemistry
Volume56
Issue number11
DOIs
StatePublished - Nov 2010

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Methylmalonic Acid
propionylcarnitine
Homocysteine
Tandem Mass Spectrometry
Mass spectrometry
Screening
Blood
Newborn Infant
Methionine
formic acid
Homocystine
Assays
Butanols
1-Butanol
Dithiothreitol
Propionates
Vitamin B 12
Liquid Chromatography
Liquid chromatography
Metabolism

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Turgeon, C. T., Magera, M. J., Cuthbert, C. D., Loken, P. R., Gavrilov, D. K., Tortorelli, S., ... Matern, D. (2010). Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry. Clinical Chemistry, 56(11), 1686-1695. https://doi.org/10.1373/clinchem.2010.148957

Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry. / Turgeon, Coleman T.; Magera, Mark J.; Cuthbert, Carla D.; Loken, Perry R.; Gavrilov, Dimitar K.; Tortorelli, Silvia; Raymond, Kimiyo M.; Oglesbee, Devin; Rinaldo, Piero; Matern, Dietrich.

In: Clinical Chemistry, Vol. 56, No. 11, 11.2010, p. 1686-1695.

Research output: Contribution to journalArticle

Turgeon, CT, Magera, MJ, Cuthbert, CD, Loken, PR, Gavrilov, DK, Tortorelli, S, Raymond, KM, Oglesbee, D, Rinaldo, P & Matern, D 2010, 'Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry', Clinical Chemistry, vol. 56, no. 11, pp. 1686-1695. https://doi.org/10.1373/clinchem.2010.148957
Turgeon, Coleman T. ; Magera, Mark J. ; Cuthbert, Carla D. ; Loken, Perry R. ; Gavrilov, Dimitar K. ; Tortorelli, Silvia ; Raymond, Kimiyo M. ; Oglesbee, Devin ; Rinaldo, Piero ; Matern, Dietrich. / Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry. In: Clinical Chemistry. 2010 ; Vol. 56, No. 11. pp. 1686-1695.
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abstract = "BACKGROUND: Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY,MMA,andMCAin dried blood spots (DBSs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d3-MMA, d3-MCA, d 8-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening. RESULTS: The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 μmol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3{\%} of all NBS samples led to the identification of 13 affected infants. CONCLUSIONS: Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations.",
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T1 - Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry

AU - Turgeon, Coleman T.

AU - Magera, Mark J.

AU - Cuthbert, Carla D.

AU - Loken, Perry R.

AU - Gavrilov, Dimitar K.

AU - Tortorelli, Silvia

AU - Raymond, Kimiyo M.

AU - Oglesbee, Devin

AU - Rinaldo, Piero

AU - Matern, Dietrich

PY - 2010/11

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N2 - BACKGROUND: Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY,MMA,andMCAin dried blood spots (DBSs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d3-MMA, d3-MCA, d 8-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening. RESULTS: The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 μmol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3% of all NBS samples led to the identification of 13 affected infants. CONCLUSIONS: Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations.

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