KR100565939B1 - Method for determination of nucleosides in urine with liquid chromatography-mass spectrometry - Google Patents

Abstract

The present invention relates to a method for detecting nucleosides in urine based on high performance liquid chromatography (HPLC), wherein the method for detecting nucleosides in urine according to the present invention comprises a first method for removing protein from urine, a sample. step; A second step of passing the sample from which the protein is removed through the first step through an analysis column; And a third step of analyzing the sample passing the analysis column using a mass spectrometer (MS).

The present invention is directly injected into a high performance liquid chromatography / mass spectrometer after a simple filtration process without pretreatment, ensuring a desirable analysis sensitivity even with a small amount of sample, and wastes time and money wasted due to extraction operation and derivatization operation. It has the advantage of shortening, simplifying the procedure and easy operation. Therefore, the present invention can satisfy simultaneous analysis, securing analysis sensitivity, accuracy, reproducibility, analysis speed and simplicity. As a result, the content of nucleosides in the urine can be accurately known, and thus it is possible to provide an effective and appropriate judgment indicator as a biomarker such as Alzheimer's and cancer.

Nucleoside, liquid chromatography, mass spectrometer, SRM mode

Description

   Method for determination of nucleosides in urine with liquid chromatography-mass spectrometry

      Figure 1 shows the chromatogram of the nucleoside extracted from the detection method according to the invention by liquid chromatography / mass spectrometry.

The present invention relates to a method for detecting nucleosides present in urine. More specifically, rapid analysis of urine directly through an analytical column using a high performance liquid chromatography / mass spectrometer without performing extraction operations such as solid-phase extraction or liquid-liquid extraction. It is possible to provide an excellent method for detecting nucleosides in urine that can increase the sensitivity of the assay while enabling it.

The nucleoside to be analyzed in the present invention includes a DNA adduct produced by oxygen free radicals.

O ² (singlet oxygen), H ₂ O ₂ (hydrogen peroxide), OH (hydroxyl radical), etc. are mainly produced by intracellular oxygen respiration process and are reactive oxygen species (ROS). It is called. Because of its high reactivity, it is easy to react with any substance around it, and thus, the active oxygen easily acts on lipids, proteins, and nucleic acids, which are the main biological components, and many reports about their oxygen damage have been reported. J Chromatography B., 2001, 754, 97-1006; Cancer Epidemiology, Biomarkers & Prevention., 2002, 11, 1072-75].

Oxidation of lipids has long been known as damage by free radicals, and the hydroperoxyl radicals of these lipids remove one hydrogen atom from the double bond chains of neighboring unsaturated lipids, resulting in hydroperoxide and alkyl. It becomes an alkyl radical. The alkyl radical, when combined with oxygen, becomes a new hydroperoxyl radical, which can cause a chain oxidation reaction. At this time, various by-products including unsaturated aldehydes are made, and they are active and may act as mutagens or inhibit enzymes, or they may cross-link with proteins or nucleic acids. Moreover, lipid peroxidation can reduce cell membrane fluidity and lead to membrane protein damage. When the nucleic acid is subjected to oxidative damage, various DNA adducts, including 8-hydroxy-2′-deoxyguanosine, are produced. Enzymatic or non-enzymatic defenses (catalase, peroxidase, superoxide dismutase, etc.) that process these free radicals are present in the cell, but free radicals that are not fully processed by these defenses react with various cellular components. May cause cell damage. This damage by free radicals is one of the major causes of various diseases such as cancer, adult disease, heart disease and degenerative diseases and causes aging of cells and body [J Neurosci Res., 2002, 70, 447-5; Carcinogenesis., 1998, 19, 1319-21; Neurosci Lett., 2003, 336, 105-8].

Quantification of the nucleoside to be analyzed in the present invention is very important in the study of biomarkers of cancer, adult disease, heart disease and degenerative disease.

The reported report of nucleosides in urine has been extracted and concentrated using solid phase extraction, followed by High Performance Liquid Chromatography (HPLC) and photo diode array detectors. ), Analysis using an electrochemical detector (ECD) or J Chromatogr B Biomed Sci Appl., 1999, 732, 307-13; J Chromatogr A., 1997, 763, 193-7; Pharmacol Res., 2002, 46 , 129-31] , most often after derivatization and analyzed using gas chromatography and mass spectrometry [Chem Res Toxicol, 1999, 12, 802-8; Chem Res Toxicol., 2000, 13, 541-9; Biochem Biophys Res Commun., 1999, 259, 374-8].

However, the method of detecting nucleoside concentration using gas chromatography and mass spectrometry had to undergo derivative reaction in order to reduce the polarity of the strong nucleoside and increase the volatility and the sensitivity of the analysis. In addition, there is a problem that the by-products of the processing by the temperature during the derivatization process interferes with the accurate quantification of nucleosides.

In addition to the above method, a method of analyzing by an electrochemical detector (ECD) of high performance liquid chromatography [Pharmacol Res., 2002, 46 , 129-31] has been reported, but in this method 8-oxodeoxyguanosine (8 -oxodeoxyguanosine) is limited to the simultaneous analysis of all nucleosides because of the limited analysis.

Therefore, it is necessary to develop a stable detection method that can analyze urine directly with high performance liquid chromatography / mass spectrometer and obtain simultaneous, rapid, economical and excellent analytical sensitivity without going through such complicated extraction operation or careful derivative reaction operation. there was.

      In order to solve the above problems, the method of detecting nucleosides in the urine according to the present invention, a small amount (about 50 μl) of urine after a simple filtration process without pretreatment directly injected into a high performance liquid chromatography / mass spectrometer In addition, it aims to secure desirable analytical sensitivity even with a small amount of sample, to reduce time and cost wasted due to extraction operation and derivatization operation, and to simplify operation by simplifying the procedure. In addition, the aim of the present invention is to provide an accurate and highly sensitive detection method by improving the sensitivity of analysis using only a small amount of urine by improving the method of using a large amount of samples (2 ml or more).

Nucleoside detection method according to the present invention in order to achieve the above object, the detection method of nucleosides in the urine using liquid chromatography, the first step of removing the protein from the sample urine; A second step of passing the analytical column using the ammonium acetate and methanol as a developing solvent for the sample from which the protein was removed through the first step; And a third step of analyzing the sample passing through the analytical column using a mass spectrometer, wherein the nucleoside is 4,6-diamino-5-formamidopyrimidine, sudouridine, and cytidine. , Uridine, 1-methyladenosine, 5-methylcytidine, guanosine, 7,8-dihydro-8-oxoadenosine, 3-methyluridine, adenosine, N ² , N ² -dimethylguanosine and 5- It is characterized by being deoxyadenosine.

delete

In the nucleoside detection method according to the present invention described above, the third step is characterized in that the quantification in SRM (Selective Reaction Monitoring) mode.

Hereinafter, a detection method according to the present invention will be described in detail.

First, the detection method according to the present invention is schematically as follows. Centrifuge to remove protein from the sample, urine, and then filter only the supernatant to remove protein from urine. Then, 1.8 to 2.2 mM aqueous ammonium acetate (pH) as a developing solvent on the analytical column. 5) Using a solution of 1.8 to 2.2 mM of ammonium acetate in 88-92% methanol, elute the nucleoside from the analytical column and apply it to a mass spectrometer to measure the concentration.

In the present invention, it is preferable to quantify the SRM in the MS-MS mode. Selected Ion Monitoring (SIM) is a method of using ions generated by colliding once in the source portion of an MS, whereas SRM selects a unique ion one more time from the broken ions and selects another MS source connected in series. It is a method of using the ions obtained from the collision after passing through it once more. Therefore, in SIM, there is a problem that the selected quantitative ion can interfere with the quantification when the ion is also detected in the urine.However, in case of using SRM, if the ion having the same mass is broken once more, the molecular structure is different again. Since broken ions show a different tendency, they can be used as quantitative ions to remove disturbing peaks in the background, resulting in a clearer baseline, resulting in better analytical sensitivity and much higher sensitivity to simultaneously correct nucleosides desired in the present invention. Can be analyzed.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example 1

1. First step: protein removal step

500 μl of human urine was taken, placed in a 1.5 ml polypropylene tube, and centrifuged at 2500 rpm for 5 minutes, and the supernatant was filtered through a Millex-GV type filter (Millipore). Only 50 μl of urine was taken and 100 nmol / ml and 5 μl of isocytosine (internal standard) were placed in an autosampler vial.

2. Second and Third Steps: Analytical Steps (Analysis of Nucleoside Concentrations Using a Mass Spectrometer)

      Instruments used to determine the concentration of nucleosides were Shiseido nanospace SI-2 high performance liquid chromatography (Shiseido Co., Tokyo, Japan) from Shiseido and LCQ Advantage mass spectrometer (ThermoFinnigan, version 1.0, San Jose, CA, USA). As the column used, Agilent's column having a length of 15 cm, an inner diameter of 2.1 mm, and a particle size of 5 μm was used. The developing solvent was 2 mM aqueous ammonium acetate (pH 5.0) (solvent A), a solution of 2 mM ammonium acetate in 90% methanol (solvent B), and the flow rate was 200 μl / min. Flowing solvent conditions in the analysis were as follows. In other words, start to flow into solvent A for 5 minutes, and then flow 10% of solvent B until 10 minutes, and then increase solvent B to 20% by 12 minutes, and then add 100% of solvent B to 26 minutes. Drifted to and stayed here for 4 minutes. Nucleosides that passed through the analysis column were analyzed by applying a mass spectrometer (MS, Mass Spectrophotometer). The mass spectrometer used for the analysis was equipped with an electrospary ionizer, and the conditions of the electrospray for ionizing the material were 20 au (arbitary unit) with an auxillary gas (nitrogen) rate and 5.1 KV with an ion spray voltage. The capillary voltage was 35KV, the tune lens offset was 5V, and the capillary temperature was 285 ° C. and analyzed in positive ionization mode. Quantitative ions were analyzed in SRM (Selective Reaction Monitoring) mode using these conditions. The chromatograms of the analyzed nucleosides are shown in FIG.

3. Quantitative step

The present inventors have presented a calibration curve for quantifying the concentration of nucleosides, through which the concentration of nucleosides was quantified. The curve is as shown in Table 1. This showed good linearity of R = 0.987 or more in the concentration range of 0.2 to 400 ng / ml. In addition, the coefficient of variation (Coefficient Variation) also showed good repeatability as the value was within 14.92% as shown in the following table. The results are summarized in Tables 2 and 3 below. Table 2 shows the daily variation and Table 3 relates to the day to day variation. The concentration range of each compound was determined in consideration of the concentration of each substance contained in urine.

Selected ions (m / z) Regression line Linearity ( r ) Slope, a Intercept, b 4,6-diamino-5-formamidopyrimidine 126 , 136, 154 1.031 -8.730 0.987 Pseudouridine 209 , 245, 179 0.991 2.831 0.992 Cytidine 112 , 244 1.015 -3.961 0.994 Uridine 113 , 245 1.004 -2.112 0.997 1-methyladenosine 150 , 282 1.008 -2.338 0.993 5-methylcytidine 126 , 258 0.993 1.916 0.992 Guanosine 152 , 284 1.000 -0.008 0.997 7,8-dihydro-8-oxoadenosine 328 , 346, 152 1.005 -1.179 0.997 3-methyluridine 127 , 259 0.991 2.373 0.991 Adenosine 136 , 268 0.991 2.645 0.990 N ^2, N ² - dimethyl-guanosine (N ^2, N ² -dimethylguanosine) 180 , 312 0.998 0.536 0.992 5-deoxyadenosine 136 , 252 0.996 1.025 0.996

As described above, the present invention enables the effective measurement of the concentration of nucleosides in urine, and can be performed without any extraction or derivatization using a high performance liquid chromatography-mass spectrometer. Can be significantly reduced. Therefore, when the present invention is used, it is possible to satisfy an analysis sensitivity, accuracy, reproducibility, analysis speed and simplicity. As a result, the presence or content of nucleosides necessary for diagnosing a disease can be accurately known, thereby providing an effective and appropriate judgment index suitable for diagnosing a specific disease.

Claims (3)

As a method for detecting nucleosides in urine using liquid chromatography, A first step of removing the protein from the sample urine; A second step of passing the analytical column using the ammonium acetate and methanol as a developing solvent for the sample from which the protein was removed through the first step; And And a third step of analyzing the sample passing through the analytical column using a mass spectrometer, wherein the nucleoside is 4,6-diamino-5-formamidopyrimidine, sudouridine, cytidine, Uridine, 1-methyladenosine, 5-methylcytidine, guanosine, 7,8-dihydro-8-oxoadenosine, 3-methyluridine, adenosine, N ² , N ² -dimethylguanosine and 5-de It is an oxiadenosine, The detection method of the nucleoside in urine. delete The method of claim 1, wherein the third step is quantified in a Selective Reaction Monitoring (SRM) mode.

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