JP2003139753A - Method for simultaneous analysis for 18 components in bile acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stable method for separation-determining highly sensitively a multi-component bile acid such as bile acid in a human serum directly and easily in a short time without requiring a technique such as solid phase extraction. SOLUTION: In this simultaneous analytical method for the multi-component bile acid by an LC-MS method, respective bile acid components are separation- determined by changing continuously and/or stepwisely a composition of a mobile phase from (i) at least an initial composition A of an aqueous solution of pH 9-11 containing 10-25 wt.% of water-soluble organic solvent and 0.05-0.2 M of salt to (ii) at least an initial composition B of an aqueous solution of pH 9-11 containing 35-50 wt.% of water-soluble organic solvent and 0.05-0.2 M of salt, using gradient elution by two liquid or more.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for analyzing bile acids. Specifically, it is a simultaneous analysis method for multi-component bile acids by high performance liquid chromatography-mass spectrometry (hereinafter abbreviated as LC-MS method).

[0002]

BACKGROUND OF THE INVENTION In human bile and blood, bile acids such as cholic acid, chenodeoxycholic acid, deoxycholic acid,
Ursodeoxycholic acid and lithocholic acid exist in free form, glycine-conjugated form and taurine-conjugated form, respectively, in a total of 15 forms. Separation and quantification of each of these bile acid components is particularly useful in the field of clinical examination for diagnosis of hepatobiliary diseases, and various analytical methods have been studied and put into practical use.

As existing methods, there are methods such as gas chromatography and gas chromatography / mass spectrometry. However, these methods can be analyzed only as free bile acids and require complicated pretreatment such as trimethylsilylation. It is not an effective method because it has many problems.

Next, a method by high performance liquid chromatography (hereinafter sometimes abbreviated as HPLC) using a 3α-hydroxysteroid dehydrogenase-immobilized enzyme column has been disclosed (Japanese Patent Laid-Open No. 4-365500).
Two types of columns are required, a separation column for each bile acid component and an enzyme reaction column, and there are drawbacks such as pretreatment for about 3 hours and analysis per sample for about 70 minutes, which is long. Furthermore, in order to analyze bile acids with high sensitivity, it is necessary to perform an extraction operation using a solid phase filled with silica gel having an octadecylsilyl group, and a pretreatment technique is required.

The above-mentioned measuring method is an indirect fluorescence detection utilizing an enzymatic reaction, in which a reaction solution containing coenzyme NAD ⁺ is added in a post column to each bile acid separated in an analytical column,
NADH generated by enzymatic reaction in an immobilized 3α-HSD column
Is measured by the intensity of the emitted fluorescence. Therefore, as shown in FIG. 1, there is a case in which there is a difference in detection intensity between the components, and a difference in column lot due to a difference in enzyme immobilization may be observed.

[0006]

It is known that the analysis of bile acids is particularly useful in the field of clinical examination, particularly for the diagnosis of hepatobiliary diseases. In order to analyze bile acids rapidly and utilize them for treatment, a simple method with a short analysis time is desired, but existing methods require time and technique. Also,
A steady and stable measurement method is desired, but existing methods have concerns about reproducibility of pretreatment and sensitivity.

An object of the present invention is to provide a stable method for separating and quantifying multicomponent bile acids such as bile acids in human serum directly and simply and in a short time without requiring a technique such as solid phase extraction. To provide.

[0008]

Means for Solving the Problems As a result of earnest research, the present inventors have completed a simultaneous analysis method for multi-component bile acids such as human bile acids by the LC-MS method.

That is, the gist of the present invention is 1. A simultaneous analysis method for multi-component bile acids by LC-MS method, comprising: (i) 10-25% by volume of at least water-soluble organic solvent and 0.05-
From the initial composition A, which is an aqueous solution containing 0.2 M and having a pH of 9 to 11, (ii) an organic solvent at least 35 soluble in water 35 to 5
PH 9-containing 0% by volume and 0.05-0.2 M salt
A simultaneous analysis method for bile acids, which comprises separating and quantifying each bile acid component by continuously and / or stepwise changing the composition of the mobile phase to the initial composition B which is the aqueous solution of 11. 2. The above-mentioned simultaneous analysis method for bile acids, wherein the water-soluble organic solvent is acetonitrile. Is. The present invention introduces a sample solution containing free-form, glycine-conjugated and taurine-conjugated bile acids into a separation column together with an eluent to separate each bile acid component by HPLC, and then electrospray
This is a method for detection by mass spectrometry using an ionization method.By using gradient elution of two or more eluents containing triethylamine, reducing the column inner diameter, and using mass spectrometry for detection, multicomponent bile acids can be detected. The feature is that they can be separated and have high selectivity and high detection sensitivity.

According to the analysis method of the present invention, cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid and isolesodeoxycholic acid, which are contained in human bile acids and the like, are respectively free and glycine-conjugated. And a total of 18 types of taurine-conjugated bile acids, simply and in a short time without pretreatment such as conjugation fractionation or solid phase extraction.
It can be directly separated and quantified.

The analytical method of the present invention will be described in more detail below. The present invention first separates each bile acid component by continuously and / or stepwise changing the composition of a mobile phase from a specific initial composition A to a final composition B by gradient elution of two or more liquids in HPLC. To do.

The mobile phase in the initial composition A is formed by mixing an aqueous solution containing at least a water-soluble organic solvent and a salt having a pH of about 9 to 11, preferably about pH 10. Examples of the water-soluble organic solvent include lower alcohols and lower fatty acid nitriles, and lower fatty acid nitriles are preferable, and the content is about 10 to 25% by volume. Further, as the lower fatty acid nitrile, acetonitrile,
Propionitrile or the like is used, and acetonitrile is preferable. As the salt, a volatile salt such as ammonium acetate or ammonium formate is used in an amount of about 0.05 to 0.2M, preferably about 0.1M. The pH of the salt solution is preferably around 10. If the pH is less than 9, the separation of each component will be insufficient, and if the pH exceeds 11, the column will deteriorate rapidly and is not practical. The pH of the aqueous solution is adjusted by adding an alkaline solution (solvent) such as triethylamine or ammonia, preferably triethylamine, to the aqueous solution of these salts. Triethylamine adjusts the pH,
It not only separates each component, but also improves the ionization in mass smectometry.

When the lower fatty acid nitrile is used as the water-soluble organic solvent, the initial composition A should contain the lower fatty acid nitrile in an amount of about 20% by volume and a salt of about 0.1M. When the content of the water-soluble organic solvent is increased in the initial composition of the mobile phase, the elution output of the mobile phase becomes excessive, and bile acids are eluted simultaneously without separation. On the other hand, if the content of the water-soluble organic solvent is decreased, the elution force is decreased and the retention of bile acid is strengthened.
Elution will take a long time. Further, if the salt content in the initial composition of the mobile phase is increased, it interferes with repeated analysis by mass spectrometry, and if the content is decreased, the separation of each bile acid component becomes poor.

The mobile phase in the final composition B is a mixture of an aqueous solution containing at least a water-soluble organic solvent and a salt adjusted to have a pH of about 9 to 11, preferably about 10. Examples of the water-soluble organic solvent include lower alcohols and lower fatty acid nitriles, and the lower fatty acid nitriles are preferable, and the content is about 35 to 50% by volume. Further, as the lower fatty acid nitrile, acetonitrile, propionitrile and the like are used, and acetonitrile is preferable. As the salt, a volatile salt such as ammonium acetate or ammonium formate is about 0.05 to 0.2 M,
Preferably about 0.1 M is used. The pH of the salt solution is preferably around 10. If the pH is less than 9, the separation of each component will be insufficient, and if the pH exceeds 11, the column will deteriorate rapidly and is not practical. The pH of the aqueous solution is adjusted by adding an alkaline solution (solvent) such as triethylamine or ammonia, preferably triethylamine, to the aqueous solution of these salts. Triethylamine not only regulates the pH and separates the components, but also improves the ionization in mass smectometry. In the final composition B, for example, when a lower fatty acid nitrile is used as the water-soluble organic solvent, it is necessary that the lower fatty acid nitrile contains about 40% by volume and the salt contains about 0.1M. The water-soluble organic solvent and salt in the final composition B are usually the same as those used in the initial mobile phase A.

For the continuous and / or stepwise compositional change from the initial composition A to the final composition B of the mobile phase, the mixing ratio of two or more eluents is adjusted by a gradient device or the like so that a predetermined composition gradient is obtained. It is achieved by The gradient method may be, for example, a gradient of 2 liquids from an eluent 100% adjusted to composition A to an eluent 100% adjusted to composition B, and 3 liquids combining solutions having different compositions. The above gradient may be used.

Other analytical conditions are appropriately selected depending on the type of the packing material of the column, the composition of the mobile phase, the mixing ratio and the like.

The sample in the present invention is not particularly limited as long as it contains multiple components of bile acid, and examples thereof include biological samples such as human bile, blood and urine. These biological samples are subjected to a pretreatment of deproteinization with an organic solvent such as methanol, ethanol, or acetonitrile, preferably methanol, if necessary, and free and conjugated bile acids are recovered in the organic solvent, and thereafter, Refined. Examples of the internal standard substance include hyocholic acid, 23-nordeoxycholic acid, 3α, 7β-hydroxy-12-oxo-5β-cholanic acid, 3α-hydroxy-7,12-dioxo-5β.
-Colanic acid, 3α, 7β, 12α-trihydroxy-5
β-cholanic acid, 3α-hydroxy-7-oxo-5β-
Cholanic acid, 3α-hydroxy-12-oxo-5β-cholanic acid, 3α, 12α-dihydroxy-7-oxo-5
β-cholanic acid and one or more selected from these glycine conjugates and taurine conjugates, preferably hyocholic acid or 23-nordeoxycholic acid are used.

A typical apparatus for carrying out the present invention is shown in FIG. Reference numeral 1 is an eluent tank, and 2 is an eluent pump. The sample injected from the sample injector 4 is introduced into the separation column 5 together with the mobile phase prepared in a desired mixing ratio by the gradient mixer 3, and separated into each component.

As the packing material for the separation column, a reverse phase type chemical bonding packing material such as silica gel or porous polymer modified with functional groups such as octadecyl group, octyl group, phenyl group and cyanopropyl group is used. . The particle size of the filler is about 2.5 to about 10 μm, more preferably about 5 μm.
m, the pore size is about 50 to about 300Å, more preferably about 1
Use a 20Å one. As the size of the separation column, a diameter of about 1.0 to about 2.0 mm and a length of about 15 to about 25 cm can be used, and a diameter of about 1.5 mm and a length of about 15 cm are preferable. The column temperature can be analyzed as long as it is above the freezing temperature of the mobile phase, but it is preferably 35 to 50 ° C, more preferably about 40 ° C.

Next, the separated liquid is introduced into the detector 6 and ionized by electrospray, then detected by the mass number of each component, the result is taken into the computer 7, and the concentration is analyzed by the chromatogram analysis program. It is calculated.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The analytical method of the present invention will be described below with reference to Examples, but the present invention is not limited thereto.

Example 1. Analytical Method (1) Sample Solution A total of 18 kinds of human bile acid components, free type, glycine-conjugated type and taurine-conjugated type described below, were dissolved in methanol to prepare a 1 mM standard solution. Mix the solutions and dilute with a mixture of acetonitrile and water to 50
To 5000 μM, and these solutions were used as they were, or after adding to serum, deproteinized samples with methanol were measured. In addition, parenthesized after each bile acid name shows the abbreviation of each bile acid in this specification.

I Free cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDC)
A), lithocholic acid (LCA), isoulsodeoxycholic acid (isoUDCA)

Ii Glycine-conjugated glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycoursodeoxycholic acid (GUDCA), glycolitocholic acid (GLCA), glycoisolesodeoxycholic Acid (GisoUDCA)

Iii Taurine-conjugated taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurolsodeoxycholic acid (TUDCA), taurolithocholic acid (TLCA), tauroysoursodeoxycholic acid (T
isoUDCA)

(2) HPLC device HP1100series (made by HEWLETT PACKARD)

(3) Separation column Capcell Pak C ₁₈ UG120 (5 μm) 1.5φ × 150 mm (made by Shiseido)

(4) Column temperature 40 ° C.

(5) Mobile phase 5 mM ammonium acetate aqueous solution with triethylamine to pH 1
0 solution (80% by volume) and acetonitrile (20% by volume)
(Volume%) mixed solution (solution A), a 5 mM ammonium acetate aqueous solution adjusted to pH 10 with triethylamine (60 volume%) and acetonitrile (40 volume%) mixed solution (B
Liquid) was used.

(6) Flow rate 0.2 mL / min

(7) Gradient condition: 100% of solution A is flown for 1 minute, and then 19 minutes, from 100% of solution A to 10% of solution B.
It was changed linearly to 0. Immediately after solution B reaches 100%, solution A 100
% And run for 10 minutes.

(8) Mass spectrometer Quatro LC (made by micromass)

(9) Ionization mode electrospray ionization method-negative ion detection

(10) Measurement Mode Single Ion Monitoring Method The chromatogram obtained in this example is shown in FIG. The separation of each bile acid component was good.

2. Specificity Since each bile acid component is an endogenous substance, it was confirmed that there was no interfering peak in serum-derived measurement during the elution time of the internal standard substance, hyocholic acid (HCA). The chromatogram of the blank serum of a healthy person is shown in FIG.

3. Calibration curve A standard solution of each bile acid corresponding to a serum concentration of 50 to 5000 nM was measured to prepare a calibration curve. It was also confirmed that there was no difference in the slope between the standard solution and the serum addition calibration curve. Table 1 shows the correlation coefficient of the calibration curve of the standard solution. The free form, the glycine conjugate and the taurine conjugate showed good linearity with a correlation coefficient of 0.98 or more.

[0037]

[Table 1]

4. Lower limit of quantification Regarding the confirmation of the lower limit of quantification, since each bile acid is an endogenous substance and the lower limit of quantification cannot be confirmed by the addition of serum, the accuracy and precision were confirmed using a standard solution. The accuracy is the standard deviation (S.
It was calculated by dividing D.) by the average value.

Table 2 shows the S / N ratio of the lower limit of quantification, and Table 3 shows the accuracy and precision of the lower limit of quantification. The accuracy of the lower limit of quantification is within ± 20%,
Since the accuracy was 20% or less and the S / N ratio was 5 or more, the lower limit of quantification was set to 50 nM.

[0040]

[Table 2]

[0041]

[Table 3]

5. Reproducibility The intraday and day reproducibility was confirmed for a medium concentration of 500 nM and a high concentration of 4000 nM. As with the lower limit of quantification, the standard solution was used for 500 nM, and serum was added for 4000 nM.

The intra- and inter-measurement variations of 500 nM (trueness and precision) are shown in Table 4, and the intra- and inter-measurement variations of 4000 nM (trueness and precision) are shown in Table 5. The reproducibility was good with accuracy of ± 15% or less and accuracy of 15% or less at any concentration.

[0044]

[Table 4]

[0045]

[Table 5]

[0046]

EFFECT OF THE INVENTION The analytical method of the present invention has excellent resolution and detectability, and can easily prepare multi-component bile acids such as human bile acid without pretreatment of the conjugated fraction for about 1.5 hours. Each bile acid component can be directly separated and quantified in a short time of about 25 minutes for the analysis per one subject.

That is, according to the analysis method of the present invention, cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid and isolesodeoxycholic acid in free form, glycine-conjugated form and taurine-conjugated form, respectively. It is possible to separate and quantify a total of 18 kinds of bile acids, and it can also be used for in vivo bile acid analysis of animals of other species.

[0048]

[Brief description of drawings]

FIG. 1 is a typical chromatogram of 15 components of bile acid and 23-nordeoxycholic acid used as an internal standard substance by an HPLC method using an existing immobilized enzyme column.

FIG. 2 is an explanatory view showing an example of an apparatus used for the bile acid analysis method of the present invention.

FIG. 3 is a representative chromatogram of 18 components of bile acid and HCA used as an internal standard substance in Examples.

FIG. 4 is a representative chromatogram of human blank serum in Examples.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Naomi Takehan             2-6 Nihonbashihonmachi, Chuo-ku, Tokyo             Mitsubishi Wel Pharma Co., Ltd. Tokyo head office

Claims (2)

[Claims] 1. A simultaneous analysis method for multi-component bile acids by high performance liquid chromatography-mass spectrometry, which comprises (i) at least 10 to 25 volumes of an organic solvent soluble in water by gradient elution of two or more solutions. % And salt 0.05 to
From the initial composition A, which is an aqueous solution containing 0.2 M and having a pH of 9 to 11, (ii) an organic solvent at least 35 soluble in water 35 to 5
PH 9-containing 0% by volume and 0.05-0.2 M salt
A simultaneous analysis method for bile acids, which comprises separating and quantifying multi-component bile acids into individual bile acid components by continuously and / or stepwise changing the composition of the mobile phase to the initial composition B which is the aqueous solution of 11. 2. The simultaneous analysis method for bile acids according to claim 1, wherein the water-soluble organic solvent is acetonitrile.