Gas chromatography coupled with mass spectrometry (GC-MS) is largely used for the quantitative analyses of the organic compounds from biological fluids. Metabolic fingerprinting, metabolite profiling or metabolite target analysis are strategies used in the metabolomics analysis for rapid diagnosis. Biological fluids are ideal candidates for diseases diagnosis because they can easily and inexpensively be isolated from the body. The study deals with possible diagnosis of neonatal diseases caused by inborn error of metabolism using gas chromatography - mass spectrometry (GC-MS). Phenylketonuria (PKU) is a metabolic disease usually caused by phenylalanine hydroxylase deficiency, which leads to an increase of phenylalanine and a decrease of tyrosine content in plasma. Enzyme deficiency results in high levels of blood phenylalanine and an accumulation of phenylketones in the urine. Partial deficiency of the enzyme results in hyperphenylalaninemia. Maple syrup urine disease (MSUD) is a metabolism disorder caused by a gene defect, in which the body cannot break down some branched-chain amino acids. In MSUD, the enzymes necessary to break down leucine, isoleucine, and valine are either absent, inactive, or only partially active. As a result of the enzyme deficiency, the branched-chain amino acids and ketoacids become elevated in the blood, resulting in an altered mental state and progressive neurodegeneration. PKU is general screened by a bacterial inhibition assay (BIA) of elevate blood phenylalanine levels on newborn filter paper samples of blood specimens. There are many chromatographic methods for screening PKU and also mass spectrometry as electrospray mass spectrometry, ESI-MS-MS. The main goal of the present work was to perform a rapid and precise analysis of volatile derivatives of amino acids from dry plasma and blood spots for diagnosis of two inborn errors diseases: PKU and MSUD. This minimum invasive method is based on profiling and quantitative determination of some amino acids in blood samples of 20 µl by using filter paper blood specimens and the GC-MS technique. The method is useful for the diagnosis of the PKU disease, by determination of phenylalanine (Phe) and tyrosine (Tyr) content in blood or for the diagnosis of the MSUD disease, by estimation of valine (Val), leucine (Leu) and proline (Pro) content.
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Chemicals and Samples
Acetyl chloride and ion exchange resin Dowex 50W-X8 50-100 mesh were purchased from Fluka, while acetone and trifluoroacetic anhydride were obtained from Merck (Darmstadt, Germany). Amino acids standards were purchased from Sigma. [15N]-isoleucine (99%) was produced by chemical synthesis. All the other chemicals were from Comchim (Bucharest). The blood and urine samples were obtained from patients and volunteers from the Pediatric Clinic III Cluj-Napoca. Written informed consents were obtained from each subject's parent prior to this study.
Extraction Procedure and Derivatization of Amino Acids
The blood was placed in a screw cap vial with 200 µl methanol/HCl 0.1% and extraction was performed either 1 h at 4 °C or by sonication for 1 min. 100 µl of the extract were placed in a vial and after the addition of the internal standard and removing of the reagent using a nitrogen stream, the amino acid extract was derivatized. Amino acids of the blood samples or standard samples were derivatized as trifluoroacetyl butyl ester derivatives. Derivatization was performed in screw-cap tubes, in two steps. Dry samples were esterified with 100 µl butanol: acetyl chloride, 4:1 (v/v) for 30 min at 100 °C. The excess reagent was removed with a stream of nitrogen. The amino group was acetylated with 100 µl trifluoacetic anhydride (TFAA) at 100 °C for 30 min. After cooling, the excess reagent was removed under nitrogen at ice temperature and ethyl acetate was added.
A Trace DSQ ThermoFinnigan quadrupole mass spectrometer in the EI mode coupled with a Trace GC was used. The capillary column Rtx-5MS had 30 m in length, 0.25 mm as diameter and a film thickness of 0.25 µmThe. experiments were performed by using a temperature program from 50 °C (1 min), then 20 °C min-1 to 260 °C, 100 °C min-1 to 300 °C, in the selected ion monitoring mode (SIM). Helium (5.5) carrier gas had a flow rate of 1 mlmin-1. The qualitative analysis was carried out in the 50-500 a.m.u. mass range. The following conditions were ensured: transfer line temperature 250 °C, injector temperature 200 °C, ion source temperature 250 °C, splitter 10:1, electron energy 70 eV and emission current 100 µA. In the SIM mode, the following ions were used: m/z 168 for valine, m/z 182 for leucine, m/z 166 for proline, m/z 91 and 148 for phenylalanine, m/z 203, 260, 316 for tyrosine. 25 µg ml-1 of the 15N-isoleucine (15N-Ile, m/z 183) internal standard was added at each sample. For Val, Leu and Pro amino acids, the important ions selected in the SIM experiment from the trifluoroacetyl butyl esters derivatives mass spectra correspond to the loss of butyl ester from the molecular ion [M - COOC4H9]+. Linearity of the method was calculated by representing the ratio of the selected ion peak area for each amino acid and the internal standard versus the amino acid standard concentrations (in µg ml-1). The regression curves were obtained by injecting standard solutions containing amino acids in concentration of 1, 5, 10, 20, 30 and 40 µg ml-1 with 25 µg ml-1 of 15N-Ile added to each standard solution per ml of blood sample. The regression curves obtained were: Val: y = 0.0912x + 0.0555, regression coefficient r = 0.999; Leu: y = 0.0515x - 0.0097, r = 0.998; Pro: y = 0.1835x - 0.1382, r = 0.998; Phe: y = 0.1119x - 0.0665, r = 988; Tyr: y = 0.08x - 0.1938, r = 0.984. The precision and the accuracy were studied by extracting four times the standard solutions of 1, 30 and 40 µg ml-1. The R.S.D. values obtained ranged between 9-12.9 % for 30 µg ml-1 and 6.7-18.6 % for 40 µg ml-1. For 1 µg ml-1, the precision was between 45-50 %. Accuracy values were between 4-30% for 30 µg ml-1 and lower than 5.5 for 40 µg ml-1 (n = 4). The limit of detection (L.O.D.) was lower than 0.1 µg ml-1. In our study, 13 cases of PKU children were found, Phe/Tyr value being significantly higher than control.
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Quantitative analysis of five amino acids, valine, leucine, proline, phenylalanine and tyrosine, in blood samples, by using blood spots or 0.5 ml blood gave similar results, the coefficient of regression obtained by comparing the amino acid values in the two extraction methods gave r = 0.91 (n = 4). Fig. 1. shows the chromatogram of separation of the five amino acids by using the minim invasive method, from 20 µl blood. The intraindividual amino acid values variation is shown in Table I. Table II shows the comparison of the results obtained for the amino acids average value in control blood samples (n = 53). Good amino acid precision in the same child was obtained, R.S.D. lower than 10.4 %. The results obtained by using only 20 µl of blood spots showed that the PKU diagnosis could be tested by calculating the Phe/Tyr ratio. Diagnosis of the MSUD disease should be obtained by calculating the ratio between aliphatic and aromatic amino acids in the blood samples. The results for some PKU patients are presented in Tables III and IV. The high benefits of the early diagnosis and treatment are strong arguments for the neonatal screening of metabolic disorder. The classical bacterial inhibition assay (BIA) used for the diagnosis of PKU is a semiquantitative method giving false positive results. More precise methods, such is the MS/MS technique, were developed, but they have the disadvantage of high price and less affordable equipment. By comparison, the proposed GC/MS method is simple, inexpensive, easily operated and high-speed technique. The internal standard used increased the precision of the method. The use of mass spectrometer as a detector for GC is important not only for its high sensitivity but also for selectivity and the identity of the analytes.
Measurements performed on amino acids from dried blood spots showed that GC-MS is a suitable method for PKU diagnosis in neonatal blood samples, from Phe/Tyr ratio and MSUD from the amino acid values1. The labelled internal standard used increased the precision of the method and simplified the samples injection. The method is a minim invasive, by using very small quantities of blood. Monitoring these diseases is important because once the diagnosis is made and treatment is started in the first few weeks of life, normal brain development is not disturbed or affected.