JPH09133685A - Serum or plasma containing no vitamin k and method for determining vitamin k - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a serum or plasma which contains no vitamin K and can be used for detecting vitamin K and a method for determining vitamin K by which vitamin K contained in a serum or plasma can be determined accurately without receiving any influence from intrinsic vitamin K by using high- precision liquid chromatography. SOLUTION: In a method for determining vitamin K, a serum or plasma which contains no vitamin K and is obtained by decomposing and removing intrinsic vitamin K from a serum or plasma by irradiating the serum or plasma with ultraviolet rays while the serum or plasma is refrigerated and high- precision liquid chromatography are used. In the method, a standard serum or plasma is prepared by adding vitamin K to be determined to the serum or plasma containing no vitamin K and a calibration curve drawn by using the standard serum or plasma is used.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for quantifying vitamin K in serum or plasma using serum or plasma free of vitamin K, which can be used for quantifying vitamin K, and high performance liquid chromatography.

[0002]

2. Description of the Related Art Vitamin Ks are 2-methyl-1,4
-It has naphthoquinone as a basic skeleton, and consists of phylloquinone (PK) called vitamin K1 and menaquinone (MK) called vitamin K2. MK depends on the number (n) of isoprenoid groups that are substituents.
It is known that MK-1 to MK-14 exist in the natural world.

These vitamin Ks are one of blood coagulation factors, and PK and MK are contained in serum or plasma of healthy persons.
It is known that -7 and the like are contained in an amount of several ng / ml, and in recent years, a highly sensitive quantitative method has been demanded.

A method using high performance liquid chromatography (HPLC) is widely used as a highly sensitive quantitative determination method of vitamin Ks.

In the determination of vitamin Ks by HPLC, an absorbance detector, a fluorescence photodetector, an electrochemical detector, etc. can be used for the detection of vitamin Ks, but a method using a fluorescence photodetector (HPLC-fluorescence Method) is most suitable for highly sensitive quantification and can detect vitamin Ks of several ng / ml to several hundred pg / ml (Langenberg et al., Journal of).
Chromatography, 305, 61-72, 1984).

In the determination of vitamin Ks by HPLC, a calibration curve is usually used to calculate the concentration of vitamin Ks in a sample. Methods using a calibration curve include an absolute calibration curve method and a relative calibration curve method using an internal standard substance.To create a calibration curve, either method is used to quantify the target substance in the same matrix as the test substance. Vitamin Ks are added to prepare a standard sample of known concentration, and this standard sample is subjected to the same pretreatment (described later) as to the specimen, and then H
It is prepared by injecting it into a PLC device and obtaining a chromatogram.

[0007] For example, PK in serum or plasma
When quantifying by the LC method, PK is usually added to serum or plasma, which is the same matrix as the sample to be measured, to prepare standard serum or plasma of known concentration consisting of multiple concentration levels, preferably 5 to 8 concentration levels. To do. The PK concentration in this standard serum or plasma is plotted on the horizontal axis (X axis), and the peak intensity (height, area, etc.) corresponding to PK obtained from the chromatogram is plotted on the vertical axis (Y axis), and calibration is performed by the least squares method. A line is created and the PK concentration in the sample is determined using this calibration curve.

At this time, if the substance to be quantified is present in the matrix, the obtained calibration curve does not pass through the origin, and the accuracy and precision near the lower limit of quantification are remarkably reduced, so in the matrix used here. Is preferably vitamin K-free serum or plasma that does not contain vitamin K that is a substance to be quantified.

However, serum or plasma obtained from healthy adult persons usually always contains endogenous vitamin Ks. For example, human serum or plasma contains PK and M.
K-7 is present at about 0.5 to several ng / ml. When standard serum or plasma is prepared using this serum or plasma and a calibration curve is created, the Y-intercept of the calibration curve deviates from the origin depending on the amount of endogenous vitamin Ks. The deviation from the origin at this time is about 0.5 to several ng / as the X intercept corresponding to the deviation from the origin when Y = 0.
Therefore, when the limit of quantification of vitamin Ks using HPLC is set to 0.1 to 1 ng / ml, using such a calibration curve, the accuracy and precision near the limit of quantification are of course sufficient. There is the problem of not being guaranteed.

To solve the above problems, there is a method of screening for serum or plasma that does not contain endogenous vitamin Ks, but the possibility is extremely low.

There is also a method of preparing an alternative matrix other than serum or plasma, but it is difficult to completely match the matrix effect with serum or plasma.

Japanese Unexamined Patent Publication (Kokai) No. 64-15653 discloses an invention of an analyzer for vitamin Ks, but it does not describe the influence of endogenous vitamins K and its solution.

[0013]

The object of the present invention is to provide serum or plasma free of vitamin Ks that can be used for the determination of vitamins K, and to accurately measure the effects of endogenous vitamins K. Vitamin K in serum or plasma
An object of the present invention is to provide a method for quantifying vitamin Ks using high performance liquid chromatography, which is capable of quantifying compounds.

[0014]

The vitamin K-free serum or plasma of the invention according to claim 1 (hereinafter referred to as the present invention 1) is endogenous by irradiating the serum or plasma with ultraviolet rays under refrigeration. It is characterized in that it is obtained by decomposing and removing vitamin Ks of.

The type of ultraviolet ray source used in the present invention 1 is as follows.
It is not particularly limited as long as it can be irradiated with ultraviolet rays, and examples thereof include an ultraviolet lamp, a fluorescent lamp, and sunlight, and the ultraviolet lamp is preferable. In the case of an ultraviolet lamp, a lamp capable of irradiating ultraviolet rays having a wavelength of 365 to 254 nm is particularly preferable.

The irradiation time of ultraviolet rays depends on the endogenous vitamin K.
The shortest time that can decompose and remove the compounds is preferable, and it is preferably 15 minutes to 8 hours, depending on the wavelength, the discharge tube wattage, the distance from the light source, and the amount of serum or plasma to be treated.

When using light with low energy, such as white fluorescent lamps, ultraviolet lamps having a long wavelength, ultraviolet lamps having a low discharge tube wattage, it is necessary to prolong the irradiation time, and the irradiation is longer than necessary. Vitamin K over time
It causes denaturation of serum or plasma in addition to the decomposition of other species. Also, when using light with strong energy, for example, an ultraviolet lamp with a short wavelength, an ultraviolet lamp with a high discharge tube wattage, etc., the irradiation time can be shortened. In addition to degradation, it causes denaturation of serum or plasma.

It is essential that the ultraviolet irradiation is performed under refrigeration in order to reduce the denaturation of serum or plasma.
When the temperature becomes lower, the serum or plasma freezes, and when the temperature becomes higher, the serum or plasma heat-denatures.
° C is preferred, and 2-8 ° C is more preferred.

The method of ultraviolet irradiation is not particularly limited as long as serum or plasma can be exposed to ultraviolet rays under refrigeration, and for example, serum or plasma is placed in a glass beaker under refrigeration and an ultraviolet lamp is applied from above. How to irradiate,
Examples include a method in which serum or plasma is put in a Petri dish and placed on a UV transilluminator.

The obtained vitamin K-free serum or plasma can be cryopreserved if necessary.

The vitamin K-free serum or plasma of the present invention 1 is a method for quantifying vitamin K in serum or plasma, for example, a method for quantifying vitamin K in serum or plasma using high performance liquid chromatography. In (1), vitamin Ks to be quantified are added to prepare standard serum or plasma, and this can be effectively used for preparing a calibration curve.

The method for quantifying vitamin K according to the second aspect of the present invention (hereinafter referred to as the present invention 2) is a method for quantifying vitamin K in serum or plasma using high performance liquid chromatography. The method is characterized in that the standard serum or plasma is prepared by adding the vitamin Ks to be quantified to the serum or plasma free of vitamin Ks of 1 above, and the calibration curve obtained using this is used.

The method for quantifying vitamin Ks in serum or plasma using the above-mentioned high performance liquid chromatography is the same as the conventional method except that the above-mentioned vitamin Ks-free serum or plasma is used for preparing a calibration curve. The method is similar.

FIG. 1 is a flow diagram showing an example of the constitution of an apparatus for a liquid chromatography method used in the present invention 2. An injection sample prepared from a specimen or standard serum or plasma by a pretreatment method described later is automatically injected from the autosampler 2 into the mobile phase 6 which is delivered by the solution delivery pump 1. Vitamin K is separated into each component in the separation column 3 and introduced into the reaction coil 4. A reaction liquid 7 for reducing vitamin Ks is fed to the reaction coil 4 by a liquid feed pump 9, and the separated vitamin Ks are reduced from naphthoquinone to fluorescent hydroquinone in the reaction coil 4, Fluorescence detector 5
And is detected by the recorder 8 and is obtained as a chromatogram by the recorder 8.

As the analysis conditions, a reversed phase separation mode, a normal phase separation mode or the like is selected as the separation. In the reversed phase separation mode, a reversed phase type packing material for liquid chromatography (for example, manufactured by Shimadzu Corporation, " shim-pack CL
C-ODS ") is used, and as the mobile phase, for example, an aqueous methanol solution is used.

In the present invention 2, vitamin Ks to be quantified are added to the serum or plasma free of vitamin Ks obtained in the present invention to prepare a standard addition sample of known concentration, which is used for calibration. Create a line.

The procedure for measuring vitamin Ks in serum or plasma using serum or plasma free of vitamin Ks of the present invention 2 will be described below by taking PK as an example. First,
Vitamin K-free serum or plasma is used to prepare a standard serum or plasma used for preparing a calibration curve.

The concentration range and number of concentration levels of the calibration curve are not particularly limited, but for example, the final concentration of PK is 0.1 ng / m.
1, 0.4 ng / ml, 1 ng / ml, 4 ng / ml,
PK is added to serum or plasma at 10 ng / ml, 40 ng / ml, and 100 ng / ml to prepare standard serum or plasma at 7 concentration levels, and this is used as a pretreatment for concentration and purification.

The pretreatment method is not particularly limited as long as PK can be selectively extracted from serum or plasma and concentrated, and examples thereof include a solvent extraction method using an organic solvent.

As the organic solvent, ethyl acetate, dichloromethane, n-hexane, diethyl ether and a mixture thereof are preferable. The solvent extraction method is performed by adding 1 to 10 times the volume of the extraction solvent to standard serum or plasma, and then stirring and permeating the mixture, whereby PK is distributed to the organic phase.
The obtained organic phase can be further purified by solid phase extraction. In this case, for example, a solid phase such as silica, alumina or florisil can be used.

The organic phase obtained by extraction was dried under reduced pressure and redissolved in a small amount of an eluent or a solvent such as ethanol to obtain an injection sample for HPLC.
Inject into the LC instrument.

In the determination of vitamin K's by HPLC, an absorbance detector, a fluorescence detector, an electrochemical detector, etc. can be used for the detection of vitamin K's, but the fluorescence detector is sensitive and practical. Method (HPLC-
The fluorescence method) is preferable as a highly sensitive quantitative method.

When the vitamin Ks are measured by the HPLC-fluorescence method, since the vitamin Ks do not have fluorescence in their naphthoquinone form, they are reduced to a hydroquinone form having fluorescence and then introduced into a fluorescence detector for detection. As a reduction method,
For example, a method using a reducing agent such as NaBH ₄ , a method using a platinum column, a method of electrochemically reducing, and the like can be mentioned.

A calibration curve is prepared from the obtained chromatogram by using the absolute calibration curve method or the relative calibration curve method. In the case of the absolute calibration curve method, for example, the PK concentration in the standard serum or plasma is plotted on the horizontal axis (X axis), the PK peak height is plotted on the vertical axis (Y axis), and linear regression is performed by the least squares method. The line is obtained. In the case of the relative calibration curve method, for example, an internal standard substance is added to standard serum or plasma in advance and pretreated, and then H
Inject into PLC. From the obtained chromatogram, the horizontal axis (X axis) is the concentration of PK in standard serum or plasma, and the vertical axis (Y axis) is the ratio of the PK peak height to the peak height of the internal standard substance. If a linear regression is performed with, a more accurate PK calibration curve can be obtained. As the internal standard substance, various kinds of vitamin K derivatives can be used, and vitamin K having less endogenous properties such as MK-3 can also be used.

The peak area can be used instead of the peak height in the preparation of the above calibration curve.

The calibration curve obtained as described above has almost no deviation from the origin, and the X-intercept corresponding to the deviation from the origin when Y = 0 is a fraction of the target quantitative limit. The PK concentration in the sample can be quantified accurately and accurately by using the following calibration curve.

To quantify vitamin Ks in a serum or plasma sample, the sample is pretreated in the same manner as described above and then injected into an HPLC apparatus to obtain a chromatogram,
The concentration may be obtained from the above calibration curve.

The method described above is applicable to vitamin K other than PK.
It can be used in the same manner for the measurement of other species, and can also be used for serum or plasma samples of animals other than humans.

Thus, by using serum or plasma free of vitamin K, endogenous vitamin K can be obtained.
An accurate calibration curve can be prepared without being affected by the types of vitamins, and a method for quantifying vitamins K having excellent accuracy and precision near the limit of quantification can be obtained.

[0040]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention and comparative examples will be described. (Example 1) 5 ml of plasma obtained from healthy volunteers
In a petri dish made of quartz and the ultraviolet wavelength is 312 nm.
It was placed on 8 W × 5 UV transilluminators (manufactured by Funakoshi Co., Ltd.) and irradiated with ultraviolet rays at 4 ° C. Every 15 minutes to 1 hour, 0.5 ml is sampled over time,
Vitamin Ks were measured by the following procedure. 2.5 ml of ethanol was added to 0.5 ml of the sampled plasma to remove proteins, and then extracted twice with 5 ml of hexane. The obtained organic phase was dried under reduced pressure and redissolved in 0.2 ml of ethanol, which was used as an HPLC inject sample.
This was injected into the HPLC apparatus shown in FIG. 1 and measured by the HPLC-fluorescence method under the following analysis conditions.

Analysis conditions-Separation column shim-pack CLC-ODS (manufactured by Shimadzu Corporation) (inner diameter 6 mm x length 150 mm) -Mobile phase methanol: distilled water = 90:10 (volume ratio) -Flow rate 1 ml-Column temperature 40 ° C・ Reaction solution 0.1 (weight / volume)% NaBH ₄ ethanol solution ・ Reaction coil inner diameter 0.6 mm x length 1 m ・ Reaction temperature 40 ° C ・ Fluorescence detection Excitation wavelength: 325 nm, fluorescence wavelength: 435 nm

From the obtained chromatogram, the vertical axis (Y
The peak heights of PK and MK-7 are plotted on the axis, and the ultraviolet irradiation time is plotted on the horizontal axis (X axis). From FIG. 2, it can be seen that endogenous vitamin Ks PK and MK-7 can be decomposed and removed in about 2 hours, and vitamin K-free plasma can be obtained.

Example 2 5 ml of plasma obtained from a healthy volunteer was placed in a petri dish made of quartz, placed on a UV transilluminator having an ultraviolet wavelength of 254 nm, and irradiated with ultraviolet rays at 4 ° C. Every 15 minutes to 1 hour, 0.5 ml was sampled with time and measured by HPLC in the same procedure as in Example 1. From the obtained chromatogram, FIG. 3 shows the peak heights of PK and MK-7 on the vertical axis (Y axis) and the ultraviolet irradiation time on the horizontal axis (X axis). Further, the same procedure as shown in FIG. 3 was performed except that the UV transilluminator having an ultraviolet wavelength of 365 nm was used. From FIG. 3, PK and MK-7 which are endogenous vitamin K are 2
About 15 minutes when using 54 nm UV light, 365n
When using m of ultraviolet light, it can be decomposed and removed in about 6 hours,
It can be seen that vitamin K-free plasma can be obtained.

Example 3 Vitamin K was prepared in the same manner as in Example 1 except that the ultraviolet irradiation time in Example 1 was 4 hours.
Genus-free plasma was prepared. PK (manufactured by Sigma) and MK-7 are added to this vitamin K-free plasma in an appropriate amount,
Final concentration of PK or MK-7 was 0.1 ng / ml (quantitation limit), 0.4 ng / ml, 1 ng / ml, 4 ng / m
Standard plasma having 6 concentration levels of 1, 10 ng / ml and 40 ng / ml was prepared. These standard plasmas were used in Example 1.
The measurement was carried out by HPLC in the same procedure as in. From the obtained chromatogram, the vertical axis (Y axis) shows the peak height of PK or MK-7, and the horizontal axis (X axis) shows PK or MK- in the standard plasma.
7 prepared concentrations were taken and a calibration curve of PK or MK-7 was prepared using the least squares method (weight 1 / Y).

The regression equation of the obtained calibration curve, X intercept (Y = 0
And the correlation coefficient is shown in Table 1.
It can be seen that there is almost no deviation from the origin, and both PK and MK-7 are several times smaller than the quantification limit of 0.1 ng / ml.

[0046]

[Table 1]

(Comparative Example 1) 5 ml of plasma obtained from a healthy volunteer was placed in a petri dish made of quartz and kept at 4 ° C.
It was stored in the dark for 4 hours. Vitamin Ks were measured in the obtained plasma by the same procedure as in Example 1. The chromatogram obtained at this time is shown in FIG. 4, and the chromatogram obtained from the plasma when irradiated with ultraviolet rays for 4 hours in Example 1 is shown in FIG. Figure 4 clearly shows endogenous vitamin K.
Although it can be seen that the groups, especially PK and MK-7 are detected, in FIG. 5, these endogenous vitamins K are decomposed and removed, and factors that affect the separation of vitamins K such as the appearance of denatured peaks. It turns out that is not particularly recognized.

Comparative Example 2 The plasma obtained in Comparative Example 1 had P.
K (manufactured by Sigma) and MK-7 were added in appropriate amounts so that the final concentration of PK or MK-7 was 0.1 ng / ml (quantitation limit),
0.4 ng / ml, 1 ng / ml, 4 ng / ml, 10
Six concentration levels of standard plasma were prepared at ng / ml and 40 ng / ml. These standard plasmas were measured by HPLC in the same procedure as in Example 1. From the obtained chromatogram, a calibration curve for PK or MK-7 was prepared in the same manner as in Example 3.

The regression equation of the obtained calibration curve, X intercept (Y = 0
And the correlation coefficient is shown in Table 1.
The deviation from the origin is large, and both PK and MK-7 are several times higher than the quantification limit of 0.1 ng / ml, and the measurement accuracy and accuracy in the vicinity of the quantification limit are higher than those of the calibration curve obtained in Example 3. It turns out that it is clearly inferior.

[0050]

The structure of the present invention 1 is as described above.
In the method for quantifying vitamin Ks in serum or plasma using high performance liquid chromatography, standard serum or plasma is prepared by adding the vitamin Ks to be quantified, By using, it is possible to prepare an accurate calibration curve without being affected by endogenous vitamin Ks.

The constitution of the present invention 2 is as described above, and in the method for quantifying vitamin K in serum or plasma using high performance liquid chromatography, the serum or plasma free of vitamin K of the present invention 1 is Standard serum or plasma is prepared by adding vitamin Ks to be quantified, and a calibration curve is prepared using this, so that vitamin Ks in serum or plasma can be accurately quantified without the influence of endogenous vitamin Ks. You can In particular, accuracy and precision near the limit of quantification can be improved.

[Brief description of the drawings]

FIG. 1 is a flow diagram showing an example of the configuration of an apparatus for a liquid chromatography method used in the present invention 2.

2] From the chromatogram obtained in Example 1, the vertical axis (Y-axis) shows the peak heights of PK and MK-7, and the horizontal axis (X-axis).
It is a graph in which the UV irradiation time is plotted on the (axis).

3 shows from the chromatogram obtained in Example 2 that the vertical axis (Y axis) is the peak height of PK and MK-7, and the horizontal axis (X).
It is a graph in which the UV irradiation time is plotted on the (axis).

FIG. 4 is a chromatogram obtained using the plasma obtained in Comparative Example 1.

FIG. 5 is a chromatogram obtained using plasma in the case of being irradiated with ultraviolet rays for 4 hours in Example 1.

[Explanation of symbols]

 1 Liquid Delivery Pump 2 Autosampler 3 Separation Column 4 Reaction Coil 5 Fluorescence Detector 6 Mobile Phase 7 Reaction Liquid 8 Recorder 9 Liquid Delivery Pump

Claims (2)

[Claims] 1. A serum or plasma free of vitamin Ks, which is obtained by decomposing and removing endogenous vitamin Ks by irradiating serum or plasma with ultraviolet rays under refrigeration. 2. A method using high performance liquid chromatography,
In the method for determining vitamin K in serum or plasma,
Vitamin K characterized in that standard serum or plasma is prepared by adding vitamin Ks to be quantified to the serum or plasma free of vitamin Ks according to claim 1, and a calibration curve obtained using this is used. Method for determination of the class.