Short-Term Stability of Amino acids and Acylcarnitines in the Dried Blood Spots Used to Screen Newborns for Metabolic Disorders

Abstract Background: Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. Methods: Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. Results: Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. Conclusions: The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

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Combined Newborn Screening for Succinylacetone, Amino Acids, and Acylcarnitines in Dried Blood Spots

Clinical Chemistry ◽

10.1373/clinchem.2007.101949 ◽

2008 ◽

Vol 54 (4) ◽

pp. 657-664 ◽

Cited By ~ 102

Author(s):

Coleman Turgeon ◽

Mark J Magera ◽

Pierre Allard ◽

Silvia Tortorelli ◽

Dimitar Gavrilov ◽

...

Keyword(s):

Amino Acids ◽

Newborn Screening ◽

False Negative ◽

Internal Standard ◽

Early Death ◽

Cost Effective ◽

Dried Blood Spots ◽

Type I ◽

Blood Spots ◽

Negative Results

Abstract Background: Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS). Methods: We extracted 3/16-inch DBS punches with 300 μL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 μL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155–137) and 13C5-SUAC (m/z 160–142). Analysis time was 1.2 min. Results: SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13–81 μmol/L), with Tyr concentrations ranging from 65 to 293 μmol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 μmol/L. Conclusion: The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.

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Detection of TPN contamination of dried blood spots used in newborn and metabolic screening and its impact on quantitative measurement of amino acids

Clinica Chimica Acta ◽

10.1016/j.cca.2011.04.009 ◽

2011 ◽

Vol 412 (15-16) ◽

pp. 1385-1390 ◽

Cited By ~ 11

Author(s):

Donald H. Chace ◽

Víctor R. De Jesús ◽

Timothy H. Lim ◽

W. Harry Hannon ◽

Reese H. Clark ◽

...

Keyword(s):

Amino Acids ◽

Quantitative Measurement ◽

Dried Blood Spots ◽

Blood Spots ◽

Metabolic Screening

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Long-term stability of I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 enzymes in frozen dried blood spots

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2016.11.281 ◽

2017 ◽

Vol 120 (1-2) ◽

pp. S110

Author(s):

Anna Potier ◽

Jason Cournoyer ◽

Joe Trometer ◽

Alyssa Vranish ◽

James DiPerna ◽

...

Keyword(s):

Dried Blood Spots ◽

Term Stability ◽

Blood Spots ◽

Long Term Stability

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Validation of Accuracy-based Amino Acid Reference Materials in Dried-Blood Spots by Tandem Mass Spectrometry for Newborn Screening Assays

Clinical Chemistry ◽

10.1093/clinchem/45.8.1269 ◽

1999 ◽

Vol 45 (8) ◽

pp. 1269-1277 ◽

Cited By ~ 58

Author(s):

Donald H Chace ◽

Barbara W Adam ◽

S Jay Smith ◽

J Richard Alexander ◽

Steven L Hillman ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Amino Acid ◽

Tandem Mass Spectrometry ◽

Newborn Screening ◽

Dried Blood Spots ◽

Tandem Mass ◽

Blood Spots ◽

Amino Acid Contents ◽

Amino Acid Concentrations

Abstract Background: Advances in technology and the earlier release of newborns from hospitals have pressed the demand for accurate calibration and improved interlaboratory performance for newborn screening tests. As a first step toward standardization of newborn screening aminoacidopathy tests, we have produced six-pool sets of multianalyte dried-blood-spot amino acid reference materials (AARMs) containing predetermined quantities of five amino acids. We describe here the production of the AARMs, validation of their amino acid contents, and characterization of their homogeneity and their stability in storage. Methods: To each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0–200 mg/L), Leu (0–200 mg/L), Met (0–125 mg/L), Tyr (0–125 mg/L), and Val (0–125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs. Results: The absolute mean biases across the analytic range for five amino acids were 2.8–9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P >0.05). Regression slopes (0 ± 0.01) for amino acid concentrations vs storage times and their P values (>0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 °C, or −20 °C during the intervals tested. Conclusion: The validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.

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