KR100600900B1 - Simultaneous screening for multiple organic acids, amino acids and glycines using GC/MS - Google Patents

Abstract

According to the present invention, TMS-TFA derivatization of an organic acid and amino acid and glycine standard to amino group and carboxyl group, which is a polar group of organic acid and amino acid, is suitable for GC / MS analysis, followed by GC / MS selective ion monitoring. The present invention relates to an organic acid to be analyzed using the simultaneous analysis of amino acids and glycine and a kit used for the analysis.

Description

Simultaneous Screening for Multiple Organic Acids, Amino Acids and Glycines by GC / MS

1 is a GC / MS chromatogram of a blank.

2 is a GC / MS chromatogram of an organic acid mixture.

3 is a GC / MS chromatogram of an amino acid mixture.

4 is a GC / MS chromatogram of a glycine mixture.

5 is a GC / MS chromatogram of an organic acid, amino acid and glycine mixture.

6 is a GC / MS chromatogram of a normal person.

7 is a GC / MS chromatogram of a propionic aciduria patient.

FIG. 8 is a diagram of a patient analyzing propionic aciduria using GC / MS selective ion monitoring. FIG.

9 to 10 show mass spectra after TMS-TFA derivatization of organic acids, amino acids and glycines;

The present invention relates to the simultaneous analysis of organic acids and amino acids by GC / MS, and more particularly, to TMS-TFA derivatives of amino and carboxyl groups, which are polar groups of organic acids and amino acids, suitable for GC / MS analysis in organic acids and amino acid standards. The present invention relates to the simultaneous analysis of organic acids and amino acids and their kits for use in the analysis after derivatization and analysis using GC / MS selective ion monitoring.

In general, Inborn error of metabolism (hereinafter referred to as 'IEM') is largely referred to as organic acid metabolism (organic acidemia), amino acid metabolic disorders (aminoacidopathy), fatty acid metabolic disorders (fatty acid oxidation disorder), etc. It can be divided into. Organic acids or amino acids are the final product or intermediate metabolites of amino acids, sugars, lipids and other endogenous compounds. These diseases are inherent defects of carbohydrates and proteins we ingest, metabolites of organic acids and enzymes that metabolize amino acids. As we do not normally metabolize the food we eat in vivo, in most cases the balance of acid and base in vivo is broken. In severe congenital metabolic disorders, due to congenital enzyme defects, compounds in the body to be metabolized are not metabolized and accumulate and are excessively excreted in urine or blood. These metabolites are the basis for diagnosis as each disease overdischarges specific metabolites.

Enzyme deficiency is a single gene disorder caused by mutation of a gene. The amount of organic acid or amino acid that accumulates can cause severe or mild clinical symptoms or disease in early childhood or adulthood, depending on the activity of the defective enzyme. Diagnosis is made by identifying or quantifying toxic substances or certain organic acids or amino acids in the urine or blood that are excessively or underexposed in the body.

Depending on the disease, early diagnosis in newborns or infancy prevents the progression of the disease and allows for early treatment. Any delay in their diagnosis slows down the therapeutic effect of the disease rapidly [ Mass spectrum Review 1996, 15: 43-57; Padiatr Padol 1993, 28 (1): 3-8; Pediatr Res 1991, 30 (4): 315-21]. For example, if treatment for patients with phenylketonuia begins after 18 days of age, it can lead to irreversible psychosis.

In order to diagnose hereditary metabolic disorders, organic acids in urine and amino acids in serum or plasma should be analyzed. Since the Gas Chromatography-Mass Spectrometer (GC / MS) was first applied in 1966 to diagnose isovaleric acidemia, a congenital metabolic disorder, more than 40 organic and amino acid metabolic disorders have been diagnosed and treated to date. Tohoku J Exp Med 1999, 188: 317-34. Molecular diagnostics and enzymatic methods are also used to confirm IEM, but the analysis of abnormal metabolites excessively secreted from the body fluids of patients by GC / MS is still the most common method [New Engl J Med 1988; 319: 1308 -1313; Pediatr Res 1986; 19: 459-466; In; Clinical Aspects of Human Carnitine Deficiency, Borum PR (ed). New York: Pergamon 1985; 97-107. Yale University's Tanaka, who applied GC / MS for the first time in the diagnosis of hereditary metabolic diseases, discovered isovaleric aciduria, the first organic acid metabolic disorder, using GC / MS in 1967. The possibility of systematic research through various metabolite analysis was presented. Since the diagnosis using GC / MS, Jellum and Charmers [Organic Acids in Man. London: Chapman and Hall. 1982], Sweetman [Clin Chem 1996; 42: 345], etc., have developed a systematic diagnostic method.

GC / MS has contributed to the discovery and diagnosis of new metabolic disorders due to the rapid development of computer software since the 1980s and the increasing scope and role of its application. Tohoku J Exp Med 1999, 188: 317-34; New Engl J Med 1988; 319: 1308-1313.
On the other hand, in spite of many advantages, when the organic acid and amino acids are separately analyzed using GC / MS and amino acid autoanalyzer for diagnosis of IEM, sample pretreatment and analysis time are long. When analyzing organic acid using GC / MS, it takes approximately 8 hours of sample preparation time and 1.5 hours of analysis time. Amino acid analysis by an automatic amino acid analyzer takes one hour of sample preparation and 3.5 hours of analysis time to analyze one sample.

Therefore, establishing the accuracy of sample pretreatment and analysis and developing a rapid metabolic disease diagnosis method is very important to prevent the occurrence of serious diseases caused by congenital metabolic disorders, mental retardation, and the inability to retard. It is also indispensable for. At present, even if the occurrence of this disease in Korea is skeptical in some cases, it has been found that there are a large number of patients in the experience of the laboratory of the present inventors, so that the screening and overhaul nationwide by developing an effective precision inspection method as soon as possible We believe that at least accurate measures should be taken to identify and cope with them.

Currently, the most widely used derivatization method for analyzing traces of biological metabolites by GC is TMS (trimethylsilyl) derivatization [ Biomed Environ Mass Spectrom 1988, 15 (6): 311-22; J Chromatogr 1991, 2; 562 (1-2): 125-38; J Chromatogr B Biomed 199 9, 731: 141-7; Acta Chem Scan. 1969, 23 (3): 999-1006 .; J Chromatogr B Biomed Sci l 1997,6; 693 (2): 499-502]. However, the biggest disadvantage of the TMS derivatization method is that it is very unstable in water, so that TMS (trimethylsilyl) and TFA (trifluoroacetamide) can be derivatized at the same time while improving it [ J Chromatogr 1989, 12; 468: 43-53]. We developed a method to simultaneously perform derivatization and applied it to a real patient sample to confirm whether it can be diagnosed.

The present inventors have established a derivatization method for simultaneous analysis of organic acids and amino acids, and have developed this method to be useful in diagnosing hereditary metabolic disorders.

SUMMARY OF THE INVENTION The present invention solves the above problems and has been devised by the above necessity, and an object of the present invention is to provide a method for simultaneous and accurate analysis of organic acids and amino acids.

It is another object of the present invention to provide a kit for use in the assay and the assay.

In order to achieve the above object, the present invention is to add a methyl orange indicator to the sample, to which N-methyl-N- (trimethylsilyl) -trifluoroacetamide is added to induce the TMS derivatization reaction and It provides a simultaneous analysis of organic acids and amino acids comprising the step of inducing a TFA derivatization reaction by adding N-methyl-bis (trifluoroacetamide) to the reaction solution.

In addition, the assay of the present invention further includes the step of simultaneously separating the organic acid and the amino acid using GC / MSD chromatography after the two derivative reactions.

In addition, the addition of N-methyl-N- (trimethylsilyl) -trifluoroacetamide of the present invention preferably adds 10-30% of the injected amount after injecting to the point where the red solution of MSTFA becomes yellow. TMS derivatization or TFA derivatization reaction is preferably carried out at 40 ℃ to 90 ℃. Below 40 ° C., there is a disadvantage that the derivatization reaction time is long and the reaction is incompletely progressed. At 90 ° C. and above, the reaction proceeds abruptly, so that the resulting compound is not stable. This adverse effect can be problematic when performing actual sample screening. Assuming 20-30 samples are analyzed per day, GC / MS takes 48 minutes to analyze a sample. Therefore, the 30th sample of the automatic sample injector should be analyzed after about 940 minutes after the reaction, but there may be a disadvantage in that the resulting compound is decomposed.

Moreover, it is preferable to advance said TMS derivatization reaction or said TFA derivatization reaction for 10 to 60 minutes. If the reaction time is less than 10 minutes, the derivatization reaction proceeds incompletely and is easily decomposed.If the reaction time is longer than 60 minutes, many side reaction products other than the desired compound are generated and screened with a sample for actual patient diagnosis. Analytical disturbances are generated during the GC / MS selective ion monitoring, which lowers the sensitivity of the target compound and interferes with the analysis.

The present invention also provides a kit for the simultaneous analysis of organic acids and amino acids, including methyl orange indicator, N-methyl-N- (trimethylsilyl) -trifluoroacetamide, and N-methyl-bis (trifluoroacetamide) to provide.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

In this method, TFA derivatization reaction was carried out to have volatility for amino group, which is a polar group of amino acid, which is difficult for GC analysis, to simultaneously analyze volatile organic acid and nonvolatile amino acid by GC / MS. After TMS derivatization, profiles were analyzed and quantified using GC / MS selective ion monitoring.

In general, GC analysis requires compounds to be volatile. Derivatization methods for volatilization of polar compounds include silylation, acylation, esterification, alkylation, ketone-base condensation, and nitrate. Nitrophenyl derivatives, fluorescence derivatives, UV labeling reagents and the like are various. Among these, the silylation method, TMS (trimetylsylyl-) derivatization, is suitable for derivatization of all compounds including active hydrogen and is the most powerful and simple and widely used derivatization method. TMS derivatization, which is a series, is applied to polar compounds having -OH, -COOH, -SH, -NH ₂ , = NH, -POH, and -SOH functional groups, and derivatization using an acylation trifluoroacetyl- (TFA) Is applied to polar compounds having functional groups of -NH ₂ , -SH, -C ₆ H ₄ -OH.

In the present invention, the TMS group is introduced into the carboxyl group (-COOH) of the organic acid, the TMS group is introduced into the carboxyl group (-COOH) of the amino acid, and the amino group of the amino acid (-NH ₂ ) is used using methyl orange (MO) as an indicator. The quantification of the derivatization reaction was maximized by acylation by introducing a TFA group immediately before the strong TMS derivatization. The principle of the present invention is shown as follows.

In the above figure, TMS and TFA have the following structure.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. The subject sample of the present invention is urine, blood, or plasma, and the subject organic acid is linear organic acid, branched organic acid, OH acid, keto acid, phenolic acid, dicarboxylic acid, 3-OH dicarboxylic acid, and subject amino acid. Are aliphatic amino acids, aromatic amino acids, OH amino acids, carboxy amino acids, alpha-imino acids, sulfur-containing amino acids, amide amino acids (glycines).

Example

1) Preparation of reagents and standards

Twelve amino acid standards (Leucine, Isoleucine, Valin, Phenylalanine, Norleucine, Homocysteine, Methionine, Homocystine, Cystine, Ornithine, Lysine and Glycine, all from Sigma) were dissolved in high purity distilled water, and six glycine standards (propionylglycine, Isobutyrylglycine, isovalerylglycine, hexanoylglycine and phenylpropionylglycine were dissolved in high purity distilled water and 8 organic acid standards (2OH-isovaleric acid, Methylmalonic acid, 2OH-isocaproic acid, Uracil, Phenyllactic acid, Tropic acid, OH phenylacetric acid) , Most of which are manufactured by Sigma) was dissolved in methanol to prepare a stock solution. The methyl orange indicator was dissolved in a solution of CH ₃ CN: TFA = 6: 4 to prepare 200 ppm. Derivative reagents MSTFA (N-Methyl-N- (trimethylsilyl) -trifluoroacetamide), and MBTFA (N-Methyl- bis (trifluoro acetamide)) were purchased from Sigma.

2) appliance

The instrument used an HP 6890 GC and an HP 5973 Mass Selective Detector. Ultra2 (Crosslinked 5% PH ME Siloxane, 50 m × 0.2 mm × 0.33 μm) was used as a column, and a thermo vap (PIERCE Model 18780) was used for concentration. The oven temperature was programmed so that the temperature of the interface and injection port of the GC was 300 ° C., and the oven temperature was increased by 5 ° C. per minute from 80 ° C. to 290 ° C.

3) Simultaneous analysis experiment method of organic acid and amino acid

a. 500 µl of a working solution (1000 ppm) prepared from the standard of amino acids and organic acids was added to a 1 ml glass vial with methylene chloride (CH ₂ Cl ₂ ) and completely dried with nitrogen (amino acid and organic acid). 500 μg of standard product), add 5-10 μl of methyl orange indicator, close the stopper, and inject to the point where the red solution of MSTFA turns yellow, add 10-30% of the injected amount, and TMS derivatization was carried out for 20 minutes. After cooling the solution at room temperature, 5-10 μl of MBTFA was added and cooled at room temperature after cooling the TFA derivatization reaction at 60 ° C. for 20-30 minutes, and then transferred to an autosampler bail to scan GC / MSD. Chromatograms were obtained in scanning mode.

b. Spectra of each amino acid and organic acid were obtained and identified from the chromatogram obtained above.

c. The selected ion mode (SIM) program was completed through retention time and ion selection of each amino acid and organic acid. Retention time and selected ions were selected for each compound. Most of the selected ions used for the SIM mode were TMSylated molecular ions (M +) and M-15 ions in which the CH ₃ group was dropped (Table 2).

d. Chromatogram using SIM mode was obtained from a standard liquid mixture of 12 amino acids, 8 organic acids, and 6 glycines.

e. Target amino acids, organic acids, and glycines were quantified from the chromatogram obtained using the SIM mode.

f. We applied the method developed by the present inventors to the actual sample to see if it can be used as a screening method for diagnosing hereditary metabolic disorders.

The results of the present invention will be described with reference to the drawings.

1-5 are chromatograms of a mixture of glycine, organic acid, amino acid, and glycine, which are useful diagnostic markers of hereditary metabolic disorders, which are organic acid profiles, amino acid profiles, and amino acids in GC / MS, respectively. It became. The interferences in the blanks were very small (the ionic strength was very small) and the peaks were irrelevant to the ions selected for the target compound to be analyzed and had no effect on the analysis.

Analysis of the method developed by the present inventors with plasma of propionic aciduria (FIG. 7), a hereditary metabolic disorder, shows glycine (glycine) and two methylcitrate markers (diagnostic markers not seen in normal persons (FIG. 6). The peak of methylcitric acid was clearly seen, indicating that it was propionic acidemia.

Table 1 lists the target compounds and diseases in 22 hereditary metabolic disorders (organic acid metabolic disorders, Methylmalonic aciduria; amino acid metabolic disorders, Maple syrup urine disease, phenylketoneuria, hyperphenylalaniemia, homocystinuria, cystinuria, lysinuria, nonketotic-hyperglycinemia, etc.). An example is shown. Table 2 shows the molecular weights of organic acids and amino acids and glycines, retention times in GC / MS, and selected ions for using selective ion monitoring techniques.

The results analyzed by the amino acid analyzer and the results of the method developed by the present inventors were compared (Table 3), showing almost the same results, which proved the usefulness of the method. In addition, the results of quantifying glycine and two methylcitric acids in patients with propionic acidemia (Table 2) were significantly increased compared to normal. Figure 8 shows chromatograms of selected ion monitoring of GC / MS in the patient's plasma, showing glycine (m / z 228) and citric acid (m / z 389).

Among patients with hereditary metabolic disorders, we found positive patients with propionic acidemia, methylmalonic aciduria, citricullinemia, and phenylketonuria, an amino acid metabolic disorder. It has been found that the method is a useful method for rapidly screening organic acid metabolic disorders and amino acid metabolic disorders.

TABLE 1 Target compounds for diagnosis on disease

Table 2 List of selected ions for amino acids, organic acids, glycine

[Table 3] Comparison of Quantitative Results by Amino Acid Analyzer and GC / MS in Patients with Propionic Acidemia

(Unit: nmol / ml)

[Table 4] Amino acid and organic acid results detected in patients with propionic aciduria

The following figure is a structural formula for the amino acids mainly mentioned in the present invention.

The following figure is a structural formula for the organic acid mainly mentioned in the present invention.

The following figure is a structural formula for glycines mainly mentioned in the present invention.

The present invention having the above-described configuration was able to identify benign diseases as a result of applying the present method to patients with hereditary metabolic disorders, and thus it was confirmed that the present method is a useful method for screening organic acid metabolic disorders and amino acid metabolic disorders. .

Claims (6)

a) adding a methyl orange indicator to the sample, and adding N-methyl-N- (trimethylsilyl) -trifluoro acetamide to induce the TMS derivatization reaction; And b) Simultaneous analysis of organic acids and amino acids, comprising the step of inducing a TFA derivatization reaction by adding N-methyl-bis ( trifluoroacetamide ) to the reaction solution. The method of claim 1, Said analysis method further comprises the step of simultaneously separating the organic acid and amino acids and glycine using GC / MSD chromatography after step b) above. The method of claim 1, The addition of N-methyl-N- (trimethylsilyl) -trifluoroacetamide is carried out to the point where the red solution of MSTFA becomes yellow, and then 10-30% of the injected amount is added to the organic acid and amino acid. Simultaneous analysis of. The method according to claim 1 or 3, Said TMS derivatization reaction or said TFA derivatization reaction is carried out at 40-90 degreeC, The simultaneous analysis method of an organic acid and an amino acid. The method of claim 1, The TMS derivatization reaction or the TFA derivatization reaction is carried out for 10 to 60 minutes, characterized in that the simultaneous analysis of organic acids and amino acids and glycine. a) methyl orange indicator; b) N-methyl-N- (trimethylsilyl) -trifluoro acetamide; And c) Kit for simultaneous analysis of organic acids and amino acids comprising N-methyl-bis ( trifluoroacetamide ).

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