Gas chromatography–mass spectrometry method for determination of phenylalanine and tyrosine in neonatal blood spots

doi:10.1016/S1570-0232(02)00632-3

Journal of Chromatography B

Volume 780, Issue 2, 25 November 2002, Pages 407-413

https://doi.org/10.1016/S1570-0232(02)00632-3 Get rights and content

Abstract

In this paper we developed a simple, rapid and sensitive method for the quantitative analysis of phenylalanine (Phe) and tyrosine (Tyr) in dried blood spots of newborns by gas chromatography–mass spectrometry (GC–MS). Phe and Tyr in blood samples were reacted with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide at 120 °C for 30 min and their corresponding single derivatives were obtained. Phe and Tyr were determined by measurement of their derivatives by GC–MS in the selected ion monitoring mode. Contents of Phe and Tyr in blood spots were calculated by external standard method. The ratio of Phe to Tyr was used for neonatal screening for phenylketonuria. The present method only took a few minutes to perform and required minimal sample preparation. In addition it provided low detection limits of 1.2 μmol l⁻¹ for Phe and 1.6 μmol l⁻¹for Tyr.

Introduction

Phenylketonuria (PKU) is a fairly common autosomal recessive disease, usually caused by a deficiency of phenylalanine hydroxylase [1], [2], [3]. The normal catabolism of phenylalanine (Phe) in mammals requires its initial conversion to tyrosine (Tyr) in the liver. The enzyme defect leads to a specific pattern of plasma amino acids with increased Phe at normal or decreased Tyr. Newborn screening for PKU relies on the detection of Phe in the filter paper blood specimens obtained prior to discharge. In general, PKU was screened by a bacterial inhibition assay (BIA), which allowed for the easy, rapid screening of elevated blood Phe levels collected on newborn filter paper samples [4]. Since that time, high-performance liquid chromatography (HPLC), micellar electrokinetic chromatography and laser-induced fluorescence, flurometry and ion-exchange chromatography were used for screening neonatal PKU [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. Recently, tandem mass spectrometry (MS–MS) has been reported as a powerful diagnostic tool in patients with PKU [15], [16], [17], [18]. Chace and Millington [18] demonstrated that the reduction of false positive samples was achieved through higher accuracy of measurements of Phe and Tyr concentrations in patients with PKU.

Gas chromatography–mass spectrometry (GC–MS) is a simple and rapid technique with high-power resolution, which has been applied to screening for inborn errors of metabolism (IEM) including PKU by simultaneous determination of amino acids and organic acids in urine [19], [20]. Kaiser et al. [21] and Zoomezely et al. [22] reported that 20 protein amino acids in normal blood modified by n-butanol and trifluroacetic anhydride were analyzed by GC–flame ionization detection (FID). In our previous study, five amino acids with aminoacidemias in blood samples of patients modified by n-butanol and trifluroacetic anhydride were determined by GC–MS [23]. However the derivatization reactions included two steps (esterification+acylation), which were time-consuming and tedious. Recently bis-(trimethylsilyl)trifluoroacetamide (BSTFA) and N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSFA) were introduced to modify amino acids, which made amino and carboxyl groups of amino acids simultaneous silylated in a single step. BSTFA was used to modify amino acids and organic acids in urine in clinical diagnosis [19], [20]. MTBSFA could provide more stable and single amino acid derivatives under more convenient reaction conditions than BSTFA. Molnar-Perl and Katona investigated derivatization reactions of protein amino acids with three different silylation agents and the fragmentation patterns of the derivatives were achieved [24]. However the optimum derivatization conditions for the amino acids were not obtained.

In this paper, derivatization reactions of 10 protein amino acids including Phe and Tyr with MTBSFA were investigated and the optimum derivatization temperature and time were obtained. Phe and Tyr in blood samples were modified by MTBSTFA under the optimum derivatization conditions of 120 °C and 30 min. They were determined by measuring selected ion peak areas of their derivatives. PKU was diagnosed on basis of content ratio of Phe to Tyr in neonatal blood spots.

Section snippets

Chemicals, standards and samples

All chemicals were of analytical grade or better. MBSTFA was obtained from Merck. l-Phenylaline (Phe), l-tyrosine (Tyr), l-alanine (Ala), l-glycine (Gly), l-methionine (Met), l-serine (Ser), l-threonine (Thr), l-asparagine (Asp), l-glutamine (Gln) and l-glutmic acid (Glu) were obtained from Sigma. Standard and GC calibration solutions spanning the concentration range for Phe and Tyr from 5.0 to 160.0 μmol l⁻¹ were made by dissolving the appropriate amino acid in water. Standard solutions with a

Results and discussion

The total ion chromatogram of 10 protein amino acids derivatives obtained at reaction temperatures of 60, 120 and 160 °C with the same reaction time of 30 min is shown in Fig. 1. The retention times of Ala, Gly, Met, Ser, Thr, Phe, Asp, Glu, Gln and Tyr derivatives are 10.4, 10.5, 13.6, 13.7, 13.9, 14.4, 14.8, 15.5, 16.4, 17.6 min, respectively. Single derivatives of Met, Ser, Thr, Phe, Asp were obtained under reaction temperature of 160 °C with a reaction time of 30 min. While other amino acids

Conclusion

The optimum reaction conditions (120 °C, 30 min) were used to modify Phe and Tyr in blood samples, and the derivatization reactions were rapid, complete and single derivatives could be obtained. Phe and Tyr could be further determined by measuring their corresponding derivatives. The SIM mode was used to improve the detection limits and sensitivity. The present method for the quantitative analysis of Phe and Tyr in neonatal blood sample is simple, rapid and sensitive, which makes it suitable for

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Gas chromatography–mass spectrometry method for determination of phenylalanine and tyrosine in neonatal blood spots

Abstract

Introduction

Section snippets

Chemicals, standards and samples

Results and discussion

Conclusion

J. Chromatogr. B

J. Chromatogr. B

Clin. Chim. Acta

Clin. Chim. Acta

Early Hum. Dev.

J. Chromatogr. B

J. Chromatogr.

J. Chromatogr. B

Arch. Med. Res.

New. Engl. J. Med

Pediatrics

Clin. Chem.