A Rapid and Sensitive UPLC-MS/MS-Method for the Separation and Quantification of Branched-Chain Amino Acids from Dried Blood Samples of Patients with Maple Syrup Urine Disease (MSUD)
Abstract
: Newborn screening for MSUD is a special challenge since patients with MSUD can metabolically decompensate rapidly without adequate treatment within the first two weeks of life. However, the screening method does not detect the actual marker metabolite (alloisoleucine) specifically, but only as part of the group of the other isobaric amino acids leucine, isoleucine and hydroxyproline. We describe a sensitive and rapid second-tier UPLC-MS/MS method to determine branched-chain amino acids from the initial extraction of the screening sample. Quantification is based on a seven-point calibration curve. Reference ranges (mean ± SD in µmol/L) were determined from 179 normal, not pre-selected samples from the newborn screening: leucine: 72 ± 27; isoleucine: 37 ± 19; valine: 98 ± 46; hydroxyproline: 23 ± 13. The concentration of alloisoleucine was below the detection limit in about 55% of the cases, and the highest concentration was 1.9 µmol/L. In all 30 retrospectively studied screening samples from patients with confirmed MSUD the concentration of alloisoleucine was significantly increased. In 238 samples with false-positive newborn screening due to a significant increase in the combined concentration of leucine + isoleucine + alloisoleucine + hydroxyproline (400 to >4000 µmol/L), alloisoleucine was below 6.5 µmol/L (n = 57) or not detectable (n = 181). The application of this assay markedly reduces the false-positive rate and the associated anxiety and costs. It is also suitable for routinely monitoring blood spots of patients with MSUD.1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions
Author Contributions
Conflicts of Interest
Abbreviations
MSUD | Maple Syrup Urine Disease |
UPLC | Ultra Performance Liquid Chromatography |
MS/MS | Tandem Mass Spectrometry |
NBS | Newborn Screening |
CV | Coefficient of Variation |
TPN | Total Parenteral Nutrition |
FP | False Positive |
MRM | Multiple Reaction Monitoring |
C.I. | Confidence Interval |
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Compound | Parent [Da] | Daughter [Da] | Dwell Time [s] | Cone Voltage [V] | Collision Energy [eV] |
---|---|---|---|---|---|
Leucine * | 132.1 | 86.1 | 0.05 | 22 | 10 |
2H3-leucine # | 135.1 | 89.1 | 0.05 | 22 | 10 |
valine | 118.1 | 72.1 | 0.05 | 22 | 10 |
2H8-valine # | 126.1 | 80.1 | 0.05 | 22 | 10 |
Samples | n | xle * | Leucine * | Isoleucine * | Allo-Ile * | Valine * |
---|---|---|---|---|---|---|
normal | 179 | 137 ± 36 | 72 ± 27 | 37 ± 19 | n.d.–1.9 | 98 ± 46 |
MSUD | 30 | 247–3530 | 280–4000 | 79–643 | 10–400 | 157–1206 |
TPN/FP ‡ | 238 | 300 – >4000 | 400 – >4000 | 107 – >3000 | n.d.–7 | 397 – >3000 |
CV # | 29 | - | 10.3/10.8 | 7.1/12.9 | 12.2/17.5 | 15.3/15.9 |
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Fingerhut, R.; Röschinger, W.; Heck, M. A Rapid and Sensitive UPLC-MS/MS-Method for the Separation and Quantification of Branched-Chain Amino Acids from Dried Blood Samples of Patients with Maple Syrup Urine Disease (MSUD). Int. J. Neonatal Screen. 2016, 2, 2. https://doi.org/10.3390/ijns2020002
Fingerhut R, Röschinger W, Heck M. A Rapid and Sensitive UPLC-MS/MS-Method for the Separation and Quantification of Branched-Chain Amino Acids from Dried Blood Samples of Patients with Maple Syrup Urine Disease (MSUD). International Journal of Neonatal Screening. 2016; 2(2):2. https://doi.org/10.3390/ijns2020002
Chicago/Turabian StyleFingerhut, Ralph, Wulf Röschinger, and Markus Heck. 2016. "A Rapid and Sensitive UPLC-MS/MS-Method for the Separation and Quantification of Branched-Chain Amino Acids from Dried Blood Samples of Patients with Maple Syrup Urine Disease (MSUD)" International Journal of Neonatal Screening 2, no. 2: 2. https://doi.org/10.3390/ijns2020002
APA StyleFingerhut, R., Röschinger, W., & Heck, M. (2016). A Rapid and Sensitive UPLC-MS/MS-Method for the Separation and Quantification of Branched-Chain Amino Acids from Dried Blood Samples of Patients with Maple Syrup Urine Disease (MSUD). International Journal of Neonatal Screening, 2(2), 2. https://doi.org/10.3390/ijns2020002