Journal of Chromatography A, 1216 (2009) 5709–5714 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Determination of phenylalanine in blood by high-performance anion-exchange chromatography–pulsed amperometric detection to diagnose phenylketonuria Ji-Seon Jeong a , Hee-Jung Sim a , Yong-Moon Lee b , Hye-Ran Yoon c , Dong Hwan Lee d , Seon-Pyo Hong a,∗ a Department of Oriental pharmaceutical sciences and Kyung Hee East-West Pharmaceutical Research Institute, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemoon-gu, Seoul 130-701, South Korea b College of Pharmacy, CBITRC, Chungbuk National University, Chongju 361-763, South Korea c Biomedical and Pharmaceutical Analysis Laboratory, College of Pharmacy, Duksung Women’s University, 419 Ssangmoon-dong, Dobong-gu, Seoul 132-714, South Korea d Department of Pediatrics, Soonchunhyang University Hospital, 657 Hannam-dong, Yongsan-gu, Seoul 140-743, South Korea article info Article history: Received 3 March 2009 Received in revised form 28 May 2009 Accepted 2 June 2009 Available online 7 June 2009 Keywords: Phenylketonuria Dried blood spot Phenylalanine Rapidity High throughput Pulsed amperometric detection abstract We have developed a high-performance anion-exchange chromatography with pulsed amperometric detection method for the detection of phenylalanine (Phe) and diagnosis of phenylketonuria (PKU). Sam- ple pretreatment steps were simplified without derivatization. The analyte was separated within 5min. The detection limit (S/N = 3) for Phe was 50 pg. Linear dynamic range was 1.23–14.43 mg/dL (r 2 = 0.9999) for a dried blood spot. The mean recoveries of Phe for intra- and inter-day assays were found to be 96.87–104.16%. This method clearly differentiated PKU-positive groups from normal groups, and proved to be a practical procedure for rapid screening and follow-up monitoring of PKU. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Phenylketonuria (PKU, OMIM 261600) is an autosomal reces- sive genetic disorder characterized by an enzyme deficiency in phenylalanine hydroxylase (OMIM 612349). PKU has critical effects on brain development leading to progressive mental retardation, seizures and albinism [1]. PKU is one of the few genetic diseases that can be controlled by diet or treatment with supplements. If an early diagnosis is made, a high tyrosine diet with low Phe or supplementation can be an effective treatment resulting in nor- mal mental and physical growth in PKU patients. Therefore, early confirmation of a diagnosis of PKU is crucial for the normal develop- ment of PKU patients in order to prevent irreversible brain damage. Phenylalanine hydroxylase deficiency increases the blood Phe con- centration, which has been used as a biomarker for PKU screening [2]. PKU is one of the representative amino acid metabolic disorders with a high frequency in Europe (approximately 1 in 4500–1 in 9000) [3] and in Japan (1 in 110000) [3,4] and South Korea (1 in 55 000) [4] in Asia. Based on blood Phe levels, PKU is divided into three types: classical PKU (>20 mg/dL Phe), mild PKU (10–20 mg/dL ∗ Corresponding author. Fax: +82 2 966 3885. E-mail address: seonhong@khu.ac.kr (S.-P. Hong). Phe) and non-PKU hyperphenylalaninemia (HPA) (2–10mg/dL Phe) [3,5]. The reported cut-off value for PKU is 3 mg/dL for newborn screening by tandem mass spectrometry (MS/MS) [6]. A bacterial inhibition assay (BIA) [7], fluorometry [8], enzy- matic colorimetry (ECM) [9], HPLC [10,11] and MS/MS [6,12] are among the methods that have been used for Phe measurement in blood or a dried blood spot for PKU diagnosis. Among these, BIA and ECM have been widely used owing to their simplicity and low cost, but they have the drawback of low accuracy. MS/MS has been used worldwide due to its rapidity and high-throughput capability for newborn screening, although the initial implementation cost is extremely high. Generally, wide use of HPLC methods requires multi-step derivatizations for their mechanical properties. Here we report a new method that overcomes this disadvantage of HPLC methods. Previously, we have reported that several high-performance anion-exchange chromatography with pulsed amperometric detec- tion (HPAEC–PAD) methods can be used for the diagnosis of galactosemia [13–15]. The HPAEC–PAD method has been used to quantify amino acids or carbohydrates in biological samples and other natural sources [16–18]. The properties of strong anion-exchange columns allow them to efficiently separate structurally similar carbohydrates or polar amino acids. PAD is a kind of electrochemical detector that detects only those compounds having an oxidizable functional 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.06.004