Determination of phenylalanine and tyrosine in dried blood specimens by ion-exchange chromatography using the Hitachi L-8800 analyzer

• Published in: Clinical biochemistry 2004-10, Vol.37 (10), p.857-862
• Main Authors: Allard, Pierre, Cowell, Lurley D., Zytkovicz, Thomas H., Korson, Mark S., Ampola, Mary G.
• Format: Article
• Language: English
• Subjects: Blood Specimen Collection; Blood spot; Case-Control Studies; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Difference plot; HPLC; Humans; Phenylalanine; Phenylalanine - blood; Phenylketonuria; Phenylketonurias - blood; Phenylketonurias - diagnosis; Plasma; Spectrometry, Mass, Electrospray Ionization; Tandem mass spectrometry; Tyrosine; Tyrosine - blood
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2004.06.004

Objectives: The treatment for phenylketonuria (PKU) includes monitoring blood phenylalanine (Phe) levels on a regular basis. To reduce inconvenience to the patient and family, blood specimens on filter paper can be obtained at home and mailed to the clinic or analytical laboratory. For this reason, we validated an 8-min isothermal and isocratic HPLC method using the Hitachi L-8800 analyzer for quantitation of Phe and tyrosine (Tyr) from dried blood specimens (DBS). Design and methods: The method was worked out using DBS fortified with Phe and Tyr. For method comparison, blood samples from 31 PKU patients and 5 non-PKU volunteers were analyzed as DBS by HPLC using the Hitachi L-8800 analyzer, and compared both to plasma analyzed by HPLC and DBS analyzed using tandem mass spectrometry (MS/MS). Results: For HPLC analysis of DBS, the within-run precision for Phe and Tyr was ≤5.1% and ≤4.5%, respectively, and total precision measured over a 3-month period was ≤7.2% and ≤8.7%, respectively. Correlation analysis was performed using results from fresh plasma analyzed by HPLC ( r = 0.988 for Phe, r = 0.964 for Tyr) and from DBS analyzed by MS/MS ( r = 0.960 for Phe, r = 0.942 for Tyr). Difference plots revealed good agreement between the HPLC and MS/MS methods. Conclusions: Determination of Phe and Tyr in DBS using this HPLC technique compares well with other methods. This technique with its short analytical time is convenient for monitoring patients with PKU and might be particularly useful in centers following many patients.