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Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface

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Abstract

We describe a capillary electrophoresis-mass spectrometry (CE-MS) method for newborn screening of a representative amino acid metabolic disease, namely, phenylketonuria (PKU). Underivatized phenylalanine and tyrosine in a dried blood spot (DBS) were simultaneously determined by CE-MS equipped with an ionophore membrane-packed sheathless electrospray ionization interface, which was developed by our group. The method was optimized for rapid determination of the underivatized amino acids, phenylalanine and tyrosine extracted from a DBS. Under the optimized conditions, the limit of detection of phenylalanine and tyrosine (signal-to-noise ratio, 3) was 0.03 and 0.07 mg/L in DBS, respectively, with a CE run time of less than 3 min. For repeated runs of a sample, coefficients of variation (CVs) for migration time were less than 3.7 %, whereas CVs for the area ratio under the curve were 2.1 and 2.9 % for 20 consecutive runs of 49.5 mg/kg Phe and 36.2 mg/kg Tyr, respectively. However, the relative standard deviations of intra- and interday assays for DBS samples were <6.2 and <5.8 %, respectively, which were substantially due to sample extraction from DBS. The analytical method was applied to real clinical samples of Korean neonates, and results were compared with those of conventional methods for PKU diagnosis, which required reference analytical methods such as isotope dilution CE-MS or high-performance liquid chromatography-mass spectrometry for quality assurance of the conventional kit-based assays. The distinct advantages of high sensitivity and extremely low sample volume, as well as a simple, easy, and economic sample pretreatment, were demonstrated for the proposed method.

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References

  1. Clague A, Thomas A (2002) Neonatal biochemical screening for disease. Clin Chim Acta 315(1–2):99–110

    Article  CAS  Google Scholar 

  2. Senk P, Kozak L, Foret F (2004) Capillary electrophoresis and mass spectrometry for screening of metabolic disorders in newborns. Electrophoresis 25(10–11):1447–1456

    Article  CAS  Google Scholar 

  3. Nagy K, Takats Z, Pollreisz F, Szabo T, Vekey K (2003) Direct tandem mass spectrometric analysis of amino acids in dried blood spots without chemical derivatization for neonatal screening. Rapid Commun Mass Spectrom 17(9):983–990

    Article  CAS  Google Scholar 

  4. Kok WT, Brinkman UA, Frei RW (1983) Rapid determination of phenylalanine and tyrosine in urine and serum by HPLC with electrochemical detection. J Pharm Biomed Anal 1(3):369–372

    Article  CAS  Google Scholar 

  5. Jeong JS, Sim HJ, Lee YM, Yoon HR, Lee DH, Hong SP (2009) Determination of phenylalanine in blood by high-performance anion-exchange chromatography-pulsed amperometric detection to diagnose phenylketonuria. J Chromatogr A 1216(30):5709–5714

    Article  CAS  Google Scholar 

  6. Chace DH, Sherwin JE, Hillman SL, Lorey F, Cunningham GC (1998) Use of phenylalanine-to-tyrosine ratio determined by tandem mass spectrometry to improve newborn screening for phenylketonuria of early discharge specimens collected in the first 24 hours. Clin Chem 44(12):2405–2409

    CAS  Google Scholar 

  7. Tuchman M, McCann MT (1999) Phenylalanine and tyrosine quantification by stable isotope dilution liquid chromatography–mass spectrometry from filter paper blood spots. Clin Chem 45(4):571–573

    CAS  Google Scholar 

  8. Guthrie R, Susi A (1963) A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 32:338–343

    CAS  Google Scholar 

  9. Li W, Tse FL (2010) Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules. Biomed Chromatogr 24(1):49–65

    Article  Google Scholar 

  10. Liu G, Ji QC, Jemal M, Tymiak AA, Arnold ME (2011) Approach to evaluating dried blood spot sample stability during drying process and discovery of a treated card to maintain analyte stability by rapid on-card pH modification. Anal Chem 83(23):9033–9038

    Article  CAS  Google Scholar 

  11. Rago B, Liu J, Tan B, Holliman C (2011) Application of the dried spot sampling technique for rat cerebrospinal fluid sample collection and analysis. J Pharm Biomed Anal 55(5):1201–1207

    Article  CAS  Google Scholar 

  12. Chace DH, Adam BW, Smith SJ, Alexander JR, Hillman SL, Hannon WH (1999) Validation of accuracy-based amino acid reference materials in dried-blood spots by tandem mass spectrometry for newborn screening assays. Clin Chem 45(8 Pt 1):1269–1277

    CAS  Google Scholar 

  13. Slazyk WE, Phillips DL, Therrell BL Jr, Hannon WH (1988) Effect of lot-to-lot variability in filter paper on the quantification of thyroxin, thyrotropin, and phenylalanine in dried-blood specimens. Clin Chem 34(1):53–58

    CAS  Google Scholar 

  14. Tamara T (1995) Inaccuracy in phenylalanine measurements—a problem towards acceptance of common screening approaches. Screening 4:5

    Google Scholar 

  15. Abdulrazzaq YM, Ibrahim A (2001) Determination of amino acids by ion-exchange chromatography on filter paper spotted blood samples stored at different temperatures and for different periods: comparison with capillary and venous blood. Clin Biochem 34(5):399–406

    Article  CAS  Google Scholar 

  16. Adam BW, Alexander JR, Smith SJ, Chace DH, Loeber JG, Elvers LH, Hannon WH (2000) Recoveries of phenylalanine from two sets of dried-blood-spot reference materials: prediction from hematocrit, spot volume, and paper matrix. Clin Chem 46(1):126–128

    CAS  Google Scholar 

  17. Hanley WB, Demshar H, Preston MA, Borczyk A, Schoonheyt WE, Clarke JT, Feigenbaum A (1997) Newborn phenylketonuria (PKU) Guthrie (BIA) screening and early hospital discharge. Early Hum Dev 47(1):87–96

    Article  CAS  Google Scholar 

  18. Schulze A, Mayatepek E, Hoffmann GF (2002) Evaluation of 6-year application of the enzymatic colorimetric phenylalanine assay in the setting of neonatal screening for phenylketonuria. Clin Chim Acta 317(1–2):27–37

    Article  CAS  Google Scholar 

  19. Allard P, Cowell LD, Zytkovicz TH, Korson MS, Ampola MG (2004) Determination of phenylalanine and tyrosine in dried blood specimens by ion-exchange chromatography using the Hitachi L-8800 analyzer. Clin Biochem 37(10):857–862

    Article  CAS  Google Scholar 

  20. Klampfl CW (2009) CE with MS detection: a rapidly developing hyphenated technique. Electrophoresis 30(Suppl 1):S83–91

    Article  Google Scholar 

  21. Zamfir AD (2007) Recent advances in sheathless interfacing of capillary electrophoresis and electrospray ionization mass spectrometry. J Chromatogr A 1159(1–2):2–13

    CAS  Google Scholar 

  22. Shi LH, Jin YX, Moon DC, Kim SK, Park SR (2009) A sheathless CE/ESI-MS interface with an ionophore membrane-packed electro-conduction channel. Electrophoresis 30(10):1661–1669

    Article  CAS  Google Scholar 

  23. Jeong JS, Kim SK, Park SR (2012) Capillary electrophoresis mass spectrometry with sheathless electrospray ionization for high sensitivity analysis of underivatized amino acids. Electrophoresis 33(14):2112–2121

    Article  CAS  Google Scholar 

  24. Youhnovski N, Bergeron A, Furtado M, Garofolo F (2011) Pre-cut dried blood spot (PCDBS): an alternative to dried blood spot (DBS) technique to overcome hematocrit impact. Rapid Commun Mass Spectrom 25(19):2951–2958

    Article  CAS  Google Scholar 

  25. Jeong JS, Lim HM, Kim SK, Ku HK, Oh KH, Park SR (2011) Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry. J Chromatogr A 1218:6596–6602

    Article  CAS  Google Scholar 

  26. Turgeon C, Magera MJ, Allard P, Tortorelli S, Gavrilov D, Oglesbee D, Raymond K, Rinaldo P, Matern D (2008) Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots. Clin Chem 54(4):657–664

    Article  CAS  Google Scholar 

  27. Jeong JS, Sim HJ, Lee YM, Yoon HR, Kwon HJ, Hong SP (2011) Chromatographic diagnosis of maple syrup urine disease by measuring the l-alloisoleucine/l-phenylalanine ratio in dried blood spots. J Chromatogr B Analyt Technol Biomed Life Sci 879(22):2171–2174

    Article  CAS  Google Scholar 

  28. Dhondt JL, Loeber J, Elvers LH, Paux E (1998) Preparation of the first European working standard for phenylalanine determination in dried blood spots. J Med Screen 5(2):63–66

    Article  CAS  Google Scholar 

  29. Dhondt JL, Paux E, Farriaux JP (1996) Need for a standardized procedure in the preparation of phenylalanine calibrators. Early Hum Dev 45(3):277–285

    Article  CAS  Google Scholar 

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Acknowledgment

This work was supported by the Korea Research Institute of Standards and Science under the project “Development of Protein Measurement Standards,” Grant 13011017.

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Authors and Affiliations

  1. Center for Bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Yuseong-gu, Daejeon, 305-340, Republic of Korea

    Ji-Seon Jeong, Sook-Kyung Kim & Sang-Ryoul Park

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  1. Ji-Seon Jeong
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  2. Sook-Kyung Kim
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  3. Sang-Ryoul Park
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Correspondence to Sang-Ryoul Park.

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Published in the topical collection Amino Acid Analysis with guest editor Toshimasa Toyo'oka.

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Jeong, JS., Kim, SK. & Park, SR. Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface. Anal Bioanal Chem 405, 8063–8072 (2013). https://doi.org/10.1007/s00216-013-6999-6

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  • DOI: https://doi.org/10.1007/s00216-013-6999-6

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