KR20090027896A - Method for analysis of polyamines in urine or plasma using liquid chromatography/electronspray ionization-tandem mass spectrometry along with amine carbamylated derivatization - Google Patents

Abstract

A method for detecting polyamine in urine or serum sample is provided to analyze it using sample of amount less than those of an existing method, to improve sensitivity of the analysis, to detect 9~10 kind of polyamines including cetyl polyamine at the same time and to decrease time, cost and effort required for analysis. A method for detecting polyamine in urine or serum sample includes steps of: derivatizing a polyamine within the urine or the serum sample into amine carbamylated and extracting selectively polyamine from the urine or the serum sample; separating the extracted polyamine using a high performance liquid chromatography(HPLC); and determining concentration of the polyamine by analyzing the separated polyamine by a mass spectrograph.

Description

Method for analysis of polyamines in urine or plasma using liquid chromatography / electronspray ionization-tandem mass spectrometry using amine carbamylated derivatization and liquid chromatography / electrospray-double mass spectrometry along with amine carbamylated derivatization}

The present invention relates to polyamines (1,3-diaminopropane, putre) in human urine or serum samples using high performance liquid chromatography and electrospray ionization-tandem mass spectrometry. Cine, cadaverine, acetylputresin, acetylcadaverine, spermidine, acetylspermidine, spermine, ene ¹ -acetylspermine).

Since polyamines possess positive charges due to amino groups in the molecule, they bind to various molecules in the living body and are controlled by the activity of various enzymes involved in the biosynthesis process. Polyamines are known as tumor markers because they have important significance as cell proliferation factors in normal as well as malignant cells. Increasing concentrations of polyamines in urine samples of malignant cancer patients were reported in 1971 by D.H. First reported by Russell. Since then, many analyzes have been made of polyamines as markers for early diagnosis, progression, efficacy of chemotherapy, and predictive of disease recurrence.

As a conventional method for detecting polyamines in urine and tissue samples of cancer patients, gas chromatography-mass spectrometry, gas chromatography-nitrogen, phosphorus detector, liquid chromatography-ultraviolet detector, and the like have been widely used.

Among the above methods, gas chromatography-mass spectrometry (GC-MS) method has been reported to be the best for simultaneous analysis of acetyl polyamine, which is of increasing importance as a marker of cancer. However, the present inventors require complicated pretreatment when using a gas chromatography-mass spectrometer, and it takes a long time to experiment, making it difficult to process a large number of samples at once. Was found. In addition, an attempt was made to use liquid chromatography to solve this problem, but acetyl polyamine could not be analyzed.

In order to solve the above problems, the present invention simplifies the pretreatment process for selectively extracting analyte from analyte by using amine carbamylated derivatization in detecting analyte present in a biological sample It is an object of the present invention to provide a detection method that can save time, effort, and cost. It is also an object of the present invention to provide a detection method that can be more accurately and accurately analyzed using a high performance liquid chromatography-electrospray dual mass spectrometer.

In order to achieve the above object, the present invention provides a method for detecting a polyamine in a urine or serum sample, the step of selectively extracting a polyamine from the urine or serum sample by amine carbamylated derivatization of the polyamine in the urine or serum sample ; Separating the extracted polyamine by type using high performance liquid chromatography (HPLC); And analyzing each of the separated polyamines using a mass spectrometer (MS) to determine the concentration of each of the polyamines.

In the method for detecting a polyamine in the urine or serum sample of the present invention, the amine carbamylated derivatization is characterized in that it is performed using isobutyl chloroformate.

In the method for detecting polyamine in the urine or serum sample of the present invention, the step of selectively extracting the polyamine may be performed by liquid-liquid extraction using ethyl ether after amine carbamylated derivatization of the polyamine in the urine or serum sample. And it is characterized in that it is carried out by a method of collecting only the organic layer.

In the method of detecting a polyamine in the urine or serum sample of the present invention, the amine carbamylated derivatization is characterized in that the pH of the sample is carried out at a pH of 8-10 or at a pH of 9 .

In the method of detecting a polyamine in the urine or serum sample of the present invention, the step of separating the polyamine using high performance liquid chromatography (HPLC) is acetonitrile containing 0.2-0.3% acetic acid and 0.2-0.3% acetic acid aqueous solution It was carried out using the as a developing solvent, the developing solvent was maintained at a flow rate of 100μL / min, acetonitrile containing 0.2-0.3% acetic acid as the developing solvent initially: 0.2-0.3% acetic acid aqueous solution Beginning with a 50:50 volume ratio, acetonitrile: 0.2-0.3% acetic acid solution containing 0.2-0.3% acetic acid was mixed slowly at a volume ratio of 95: 5 until 12 minutes, containing 0.2-0.3% acetic acid. It is characterized by increasing the acetonitrile ratio.

After 5 minutes, acetonitrile containing 0.2-0.3% acetic acid was mixed with an aqueous solution of 0.2-0.3% acetic acid in a volume ratio of 95: 5, followed by 0.2-0.3% acetic acid for 11 minutes. Acetonitrile: 0.2-0.3% acetic acid aqueous solution is characterized in that it is carried out by a method using a mixture of 50:50 by volume ratio.

In the method for detecting polyamine in the urine or serum sample of the present invention, the step of determining the concentration of each polyamine is characterized in that it is carried out using an electrospray ionization (tandem mass spectrometry) .

In the method for detecting polyamine in the urine or serum sample of the present invention, the step of separating the polyamine by high performance liquid chromatography (HPLC), the polyamine in the urine or serum sample 1,3-diaminopropane , Fu tray sour cadaverine, acetyl Fu tray sour acetyl cadaverine, spermidine, acetyl spermidine, spermine and yen ¹ characterized in that the separated-acetyl spermine.

When the polyamine concentration in urine or serum is analyzed according to the detection method of the present invention, a smaller amount of sample can be used, and an analyte is removed from the analyte in the biological sample through an amine carbamylated derivative. Selective extraction can reduce time, cost and effort, and in the polyamine separation step using liquid chromatography, using a separate developing solvent suitable for each step and using a double mass spectrometer to secure excellent sensitivity, including acetyl polyamine 9 to 10 polyamines can be detected simultaneously.

By using the polyamine detection method, in order to monitor the cancer progression stage of the patient and use it as an indicator of cancer disease, it is easy and quick to compare the polyamine concentration of various cancer patients and normal people, and to observe the change in the concentration of polyamine in the body easily and quickly. can do.

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

FIELD OF THE INVENTION The present invention relates to a method for detecting polyamines in a urine or serum sample using a liquid chromatography-mass spectrometer, which is not known for use in detecting polyamines using a liquid chromatography-mass spectrometer. Millified derivatization is used to increase sensitivity to the analyte. In particular, amine carbamylated derivatization is carried out using isobutyl chloroformate, which is inexpensive and easily undergoes amine carbamylated derivatization. Amine carbamylated means that the compound having an amine group to which no group is attached causes carbamylation by SN2 reaction.

After the derivatization process is completed, liquid-liquid extraction is performed with ethyl ether, and only the organic layer is collected and dried. This process removes many of the analytes in the biological sample and selectively extracts only the substances to be analyzed.

After that, the analysis is performed by electrospray-double mass spectrometry combined with liquid chromatography. In the polyamine separation step using liquid chromatography, excellent sensitivity can be obtained by using a separate developing solvent suitable for each step. In addition, by using an electrospray-double mass spectrometer, the specific ion species to be analyzed are mass analyzed to select specific m / z as precursor ions, and then the product ions generated by collision-induced chipping Mass spectrometry is more accurate and more sensitive than a single mass spectrometer.

That is, unlike the conventional pretreatment process in which the polyamine was analyzed through a rather complicated pretreatment process, such as using a liquid phase extraction and a solid phase extraction, or performing acid hydrolysis and then liquid phase extraction, in the present invention, the amine carba easily in the water layer. The isobutyl chloroformate with milliated derivatization reaction can be used to shorten the pretreatment process using short derivatization reaction time (15 minutes) and reaction temperature (35 ° C.). This makes it easier and faster to analyze polyamines than pretreatment.

Hereinafter, the present invention will be described in detail with reference to Examples.

<Example 1. Extracting an analyte from an analyte in a urine or serum sample>

Urine or serum, which is a sample used in the present invention, has other analyte that has a similar structure in addition to the analyte (polyamine) to be analyzed as a biological sample. In the present invention, amine carbamylated derivatization was performed to selectively extract the analyte from the analyte, and amyl carbamylated derivatization was performed using isobutyl chloroformate. same.

In order to carry out amyl carbamylated derivatization using isobutyl chloroformate, 25 μL of isobutyl chloroformate was added to each 200 μL sample, followed by reaction at 35 ° C. for 15 minutes.

When derivatizing amine carbamylated with isobutyl chloroformate, the pH of the sample was adjusted to pH 9 for optimal derivatization. This is a result obtained through various pH tests, the pH test up to pH 7-12 due to the characteristics of the amine series. As a result, the polyamine having basic acid dissociation constant (pKa) around 8-10 showed the best derivative reaction efficiency at pH 9. After the derivatization process, liquid-liquid extraction was performed with ethyl ether, and then the organic layer was collected and dried with nitrogen.

<Example 2. Separation of the extracted analyte>

After the analyte was removed in the same manner as described above, the polyamine was separated by type using high performance liquid chromatography on the concentrated polyamine. A separate developing solvent suitable for each step was used; The developing solvent was maintained at a flow rate of 100 μl / min, and starting with mixing acetonitrile: 0.2-0.3% acetic acid aqueous solution containing 0.2-0.3% acetic acid as a developing solvent in a volume ratio of 50:50, 12 Slowly increase the ratio of acetonitrile containing 0.2-0.3% acetic acid so that the acetonitrile with 0.2-0.3% acetic acid: 0.2-0.3% acetic acid aqueous solution is mixed at a volume ratio of 95: 5 by minutes. After 5 minutes, acetonitrile containing 0.2-0.3% acetic acid was mixed with an aqueous solution of 0.2-0.3% acetic acid in a volume ratio of 95: 5, followed by 0.2-0.3% acetic acid for 11 minutes. Separation was carried out by the method using acetonitrile: 0.2-0.3% acetic acid aqueous solution mixed in a volume ratio of 50:50.

The separation column was a 15 cm long, 1.5 mm inner diameter and 5 μm particle sized Capcell Pak C18 MG column.The instrument for separating analytes was Shiseido nanospace SI-2 high performance liquid chromatography (SECID). Shiseido Co., Tokyo, Japan).

Figure 2 is a selected ion chromatogram of the polyamine appeared as a result of separating the analyte using high performance liquid chromatography, it can be seen that nine polyamines including acetyl polyamine separated over time. 1,6-diaminohexane is used as an internal standard, and each peak has a different m / z, and the precursor ions m / z and the generated ions m / z respectively correspond to each polyamine.

Example 3 Determining the Concentration of the Separated Polyamine

1. Analysis of polyamine concentration using mass spectrometer

After establishing the optimal conditions for mass spectrometry analysis through Examples 1 and 2, the concentration of polyamine was analyzed using LCQ Advantage mass spectrometer (ThermoFinnigan, version 1.0, Scan Jose, CA, USA) of Summer Finigangan. It was. The mass spectrometer was equipped with an electrospray-ionizer and the conditions of the electrospray when ionizing the material were as follows: Sheath gas (nitrogen) rate was 30 arb (arbitary unit), ion spray voltage was 6 KV, Capillary voltage 4 V, tube lens offset 40 V, capillary temperature 250 ° C.

Mass spectrometry was analyzed using MS's selected reaction monitoring (SRM) mode. [MH] ⁺ value of the polyamine was selected as the precursor ion m / z, and mass spectrometry of the generated ion [MH] ⁺ -74 through collision-induced chipping was performed for more accurate material analysis. The mass spectrum of the generated ion value obtained by the above method is shown in FIGS. 3A-B.

2. Quantification of Polyamine Concentration Using Calibration Curve

The present inventors used the calibration curves of Table 1 (urine sample) and Table 2 (serum sample) to quantify the concentration of polyamine in urine samples and serum samples of normal people.

The calibration curve showed good linearity over R ² = 0.99 and the coefficient of variation (Cofficient Variation) was within 20%, indicating good repeatability.

Quantitative results are shown in Table 3 (normal person's urine sample) and Table 4 (normal person's serum sample).

compound Regression equation Linearity (R ² ) 1,3-diaminopropane Y = 0.0058X + 0.0271 0.9952 Putrescine Y = 0.0034X + 0.0059 0.9994 Cadaverine Y = 0.0053X-0.0134 0.9959 Acetylputrescine Y = 0.0007X + 0.0082 0.9949 Acetylcadaverine Y = 0.0009X + 0.0135 0.9994 Spermidine Y = 0.0152X-0.0163 0.9991 Acetylspermidine Y = 0.0094X + 0.0189 0.9997 Spermine Y = 0.006X + 0.0045 0.9996 Yen ^1-acetyl spermine (N ¹ -acetylspermine) Y = 0.0044X-0.0034 0.9958

compound Regression equation Linearity (R ² ) 1,3-diaminopropane Y = 0.0245X-0.034 0.9963 Putrescine Y = 0.0518X + 0.1184 0.9978 Cadaverine Y = 0.0238X-0.0365 0.9966 Acetylputrescine Y = 0.0021X-0.0043 0.9968 Acetylcadaverine Y = 0.0034X-0.0059 0.9983 Spermidine Y = 0.065X-0.1586 0.9960 Acetylspermidine Y = 0.0409X-0.0197 0.9981 Spermine Y = 0.021X + 0.0058 0.9972 Yen ^1-acetyl spermine (N ¹ -acetylspermine) Y = 0.0181X + 0.0289 0.9939

compound Normal person (n = 104) 1,3-diaminopropane 0.06 ± 0.05 Putrescine 0.22 ± 0.21 Cadaverine 0.12 ± 0.10 Acetylputrescine 17.26 ± 12.81 Acetylcadaverine 2.19 ± 2.19 Spermidine 0.12 ± 0.10 Acetylspermidine 4.04 ± 3.12 Spermine 0.09 ± 0.08 Yen ^1-acetyl spermine (N ¹ -acetylspermine) 0.02 ± 0.02

compound Normal person (n = 30) 1,3-diaminopropane 3.00 ± 2.11 Putrescine 26.04 ± 19.75 Cadaverine 2.17 ± 0.58 Acetylputrescine 13.49 ± 12.53 Acetylcadaverine 1.77 ± 1.68 Spermidine 15.97 ± 14.15 Acetylspermidine 9.22 ± 2.69 Spermine 1.90 ± 1.61 Yen ^1-acetyl spermine (N ¹ -acetylspermine) 2.21 ± 0.056

1 is a schematic diagram illustrating the principle of derivatizing amine carbamylated polyamines in urine or serum samples with isobutyl chloroformate.

2 is a selected ion chromatogram of polyamines resulting from separation of analytes using high performance liquid chromatography.

3A-B are mass spectra of generated ion values used when quantifying analytes using a mass spectrometer.

Claims (11)

A method of detecting polyamines in urine or serum samples, Selectively extracting the polyamine from the urine or serum sample by amine carbamylated derivatization of the polyamine in the urine or serum sample; Separating the extracted polyamine by type using high performance liquid chromatography (HPLC); And Analyzing each of the separated polyamines using a mass spectrometer (MS) to determine the concentration of each of the polyamines. The method of claim 1 wherein the amine carbamylated derivatization is performed using isobutyl chloroformate. The method of claim 1 or 2, wherein the step of selectively extracting the polyamine A method for detecting polyamines in urine or serum samples, which is carried out by liquid-liquid extraction using ethyl ether after the polyamines in the urine or serum samples are derivatized with amine carbamylated derivatives. The method of claim 1 or 2, wherein the amine carbamylated derivatization is performed in a state in which the pH of the sample is pH 8-10. 5. The method of claim 4, wherein the amine carbamylated derivatization is performed while the pH of the sample is at pH 9. 6. The method of claim 1 or 2, wherein the separating the polyamine using high performance liquid chromatography (HPLC) A method for detecting polyamines in a urine or serum sample, characterized by the use of acetonitrile containing 0.2-0.3% acetic acid and 0.2-0.3% acetic acid aqueous solution as a developing solvent. The method of claim 6, wherein the separating of the polyamine using high performance liquid chromatography (HPLC) The developing solvent was maintained at a flow rate of 100 μL / min, As the developing solvent For 12 minutes from the beginning, acetonitrile containing 0.2-0.3% acetic acid: 0.2-0.3% acetic acid solution was mixed at a volume ratio of 50:50, and then 0.2-0.3% for 12 minutes so that the volume ratio was 95: 5. Using an increased acetonitrile ratio containing acetic acid, For 5 minutes thereafter, acetonitrile containing 0.2-0.3% acetic acid: mixed with 0.2-0.3% acetic acid in a volume ratio of 95: 5 was used, After 11 minutes, the polyamine in the urine or serum sample, characterized in that it is carried out by mixing acetonitrile: 0.2-0.3% acetic acid solution containing 0.2-0.3% acetic acid in a volume ratio of 50:50 How to detect. The method of claim 1 or 2, wherein determining the concentration of each polyamine is performed using an electrospray ionization (tandem mass spectrometry) polyamine in the urine or serum sample How to detect. 8. The method of claim 7, wherein determining the concentration of each polyamine is performed using electrospray ionization (tandem mass spectrometry). . According to claim 1 or 2, wherein the step of separating the polyamine by high performance liquid chromatography (HPLC), the polyamine in the urine or serum sample 1,3-diaminopropane, putrescine, carda papaverine, acetyl Fu tray sour acetyl cadaverine, spermidine, acetyl spermidine, spermine and ^yen - a method for detecting the polyamines in urine or serum sample which comprises separating acetyl spermine. According to claim 7, wherein the step of separating the polyamine by high performance liquid chromatography (HPLC), the polyamine in the urine or serum sample 1,3-diaminopropane, putrescine, cadaverine, acetylfu Tre sour acetyl cadaverine, spermidine, acetyl spermidine, spermine and ^yen - a method for detecting the polyamines in urine or serum sample which comprises separating acetyl spermine.

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