JPH07159400A - Measurement of mevalonic acid - Google Patents

Abstract

PURPOSE:To analyze mevalonic acid at a low cost by performing acid treatment for the mevalonic acid in a specimen, guiding the acis into dehydromevalonolactone, whose detection with UV absorption and the like is easy, and thereafter performing the analysis by liquid chromatography. CONSTITUTION:When acid is added to mevalonic acid in a specimen and mevalonic acis is transformed into dehydroevalonolactone, strong acid, by which the specimen becomes acidic at pH of about 2, is added. After mevalonolactone is formed in the specimen by lactonization, the mevalonolactone is extracted with a solvent, and a sample for liquid chromatography analysis is prepared. The extracting solvent is a solvent which is liquid at the normal temperature and whose solubility in water is low. After the extraction, the extracing solvent is enriched and dried, and the dehydromevalonolactone is prepared. The efficiency of dehydration reaction from the mevalonolactone into the dehydromevalonolactone is improved. After the concentration and drying, the dehydromevalonolactone is dissolved again into the moving phase for the liquid chromatography.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring mevalonic acid, and more specifically, it is measured by liquid chromatography after dehydromevalonolactone, which is easily detected by UV absorption or the like, is introduced into mevalonic acid in a sample. The present invention relates to a method for measuring mevalonic acid.

[0002]

2. Description of the Related Art In recent years, due to westernization of diet, ischemic heart diseases such as angina and myocardial infarction tend to increase in Asian countries including Japan. Therefore, the importance of prevention or treatment is increasing year by year. It is increasing. From the viewpoint of prevention of these ischemic heart diseases, it is important to prevent or treat arteriosclerosis, which is the underlying underlying disease.

[0003] Today, hyperlipidemia, particularly hypercholesterolemia, is an important risk factor for the etiology of arteriosclerosis, based on studies in all aspects of pathology, physiology, biochemistry and epidemiology. There is no doubt. Therefore, the treatment of hypercholesterolemia is extremely important as a basis for prevention of arteriosclerosis.

Generally, cholesterol present in blood is composed of dietary cholesterol and cholesterol biosynthesized in the body. For the purpose of treating hypercholesterolemia, various cholesterol lowering drugs have been developed today, but the action mechanism of these drugs is diverse (for example, cholesterol biosynthesis inhibition, cholesterol absorption inhibition, etc.). Therefore, in order to obtain a therapeutic guideline for hypercholesterolemia (which drug and how to use it), it is extremely important to evaluate the amount of cholesterol biosynthesis in each case.

Conventionally, the evaluation of the amount of cholesterol biosynthesis has been carried out by
Was administered ¹⁴ C-cholesterol to the subject, ¹⁴ C in the blood
Turnover stud to track specific activity of
y), or a balance method for measuring neutral sterols in food, stool and urine, but these methods not only cause significant pain to the subject but also the labor of measurement. Therefore, it is extremely difficult to use these turnover methods or balance methods in daily clinical practice.

On the other hand, in recent years, it has been proposed to estimate the amount of cholesterol biosynthesis from the blood concentration or urinary excretion of an intermediate of cholesterol synthesis, and the measurement of squalene, methylsterol, lastosterol, and mevalonic acid is clinically significant. Is said to be. Of these, mevalonic acid (Proc. Natl. Aca
d. Sci., USA, 79 , 3037-3041, 1982) is not only a direct reaction product of HMG-CoA reductase (the rate-limiting enzyme of the cholesterol biosynthetic pathway), but also the synthesis of mevalonic acid. Is the rate-limiting step in cholesterol biosynthesis. Therefore, it is extremely important to measure mevalonic acid in the evaluation of cholesterol biosynthesis.

The quantification of mevalonic acid is currently carried out by GC-MS.
(Gas chromatograph mass spectrometer) method or radioenzyme method (Medical Science and Pharmacy, 27 (4) ,
939-945, 1992), it was reported that the blood mevalonic acid value thus obtained and the cholesterol synthesis amount obtained by the above-mentioned balance method show a good correlation (J. Clin. Invest., 74 , 795-80.
4, 1984).

[0008]

[Chemical 1]

In the above radioenzyme method, [γ
- ³² P] using ATP, mevalonic acid in to be measured specimen phosphorylated 5- [γ- ³² P] Phosphpomevalonate
And as an internal standard 5-Phosphpo [ ¹⁴ C] mevalonate
5-Ph separated by applying an ion exchange column
The mevalonic acid in the sample is quantified based on the ³² P / ¹⁴ C ratio of osphpomevalonate (Journal of Lipid Research
, 20 , 716-728, 1979).

On the other hand, in the above-mentioned GC-MS method, a special internal standard substance (β-hydroxy-β-ethyl-
δ-valerolactone), and a pH of 2.
After setting to 0, it is left to convert to mevalonolactone (Chemical Formula 2), which is extracted with an organic solvent, dried to dryness, trimethylsilylated, and then measured using a mass fragmentography method in GC-MS (medical science). And pharmacy, 27
(4) , 939-945, 1992).

[0011]

[Chemical 2]

[0012]

However, in the above radioenzyme method, not only is there a difficulty in using a radioactive substance, the pretreatment is complicated, and a high measured value tends to be given due to the interference of glycolic acid. There is.

Further, in the above-mentioned GC-MS method, it is necessary to invest in the GC-MS itself to be used (about tens of millions of yen per unit), and a dedicated operator is required for maintenance and actual measurement operation. Therefore, the facilities where the GC-MS can be actually installed are considerably limited. Therefore, this GC-MS method cannot be said to be a method that can be widely and generally used as a mevalonic acid measuring method.

Further, GC-MS not only has a tendency to lack sensitivity as a method for analyzing biological components, but also requires complicated pretreatment, and its accuracy and stability cannot be said to be sufficient. In addition, in the conventional GC-MS method, in order to obtain practical measurement accuracy, the recovery rate was increased by using the above-mentioned special internal standard substance (β-hydroxy-β-ethyl-δ-valerolactone). It is essential to correct it.

Therefore, an object of the present invention is to provide a method for measuring mevalonic acid which is easy to pretreat.

Another object of the present invention is to provide a method for measuring mevalonic acid which has good sensitivity and also good measurement accuracy and stability.

Still another object of the present invention is to provide a widely and generally available method for measuring mevalonic acid.

Still another object of the present invention is to provide a method for measuring mevalonic acid which has low equipment cost and running cost.

[0019]

Means for Solving the Problems As a result of earnest research by the present inventor, the mevalonic acid in a sample is subjected to a predetermined treatment to dehydromevalonolactone (dehydromevalonolactone, which is easily detected by UV absorption or the like). It has been found that it can be easily and efficiently converted into Chemical formula 3). As a result of further research, the present inventor has found that dehydromevalonolactone in the sample thus obtained can be easily measured by liquid chromatography.

[0020]

[Chemical 3]

The method for measuring mevalonic acid of the present invention is based on the above findings, and more specifically, after measuring mevalonic acid in a sample to dehydromevalonolactone, the dehydromevalonolactone is measured by liquid chromatography. It is characterized by doing.

[0022]

In the present invention, mevalonic acid (Chemical formula 1) in the sample
Can be lactonized and dehydrated by a simple operation (for example, acid treatment) and efficiently converted to dehydromevalonolactone (Chemical Formula 3).

Furthermore, this dehydromevalonolactone does not require any special pretreatment (for example, trimethylsilylation necessary for GC-MS treatment, purification by silica gel column), and is, for example, a liquid chromatograph using a UV detector. According to the present invention, mevalonic acid can be measured with good sensitivity and with good measurement accuracy and stability because it can be easily measured by means of chromatography.

The operating procedure in a typical example of the mevalonic acid measuring method of the present invention and the operating procedure in the conventional mevalonic acid measuring method by GC-MS are shown in the flow chart of FIG. As shown in the flow chart of FIG. 1, according to the present invention, purification by a silica column after extraction with an organic solvent is not essential, and pretreatment for chromatography measurement becomes simple. Therefore, according to the present invention, it is possible to perform mevalonic acid measurement which is extremely simple as compared with the conventional GC-MS method.

As the liquid chromatography in the present invention, high-performance liquid chromatography (High-performance liquid chromatography) is used.
Even if liquid chromatography (HPLC) is used, the equipment cost is about several million yen at most, and unlike the above-mentioned GC-MS, maintenance is simple and a dedicated operator for the actual measurement operation is required. Since it is not essential, the present invention provides a method for measuring mevalonic acid in which both the device cost and the running cost are suppressed.

The ultraviolet absorption of dehydromevalonolactone (Chemical Formula 3) analyzed by liquid chromatography in the present invention is very strong (molar extinction coefficient is several tens to several hundreds of times that of mevalonic acid), and the above-mentioned UV detection. When (for example, wavelength 2
Since it is possible to use the longer wavelength side (for example, a wavelength of 215 nm or more) with less interfering substances (compared to 10 nm), it is possible to measure mevalonic acid with higher sensitivity and accuracy.

Furthermore, in the present invention, the conventional GC-
Since it is not essential to correct the recovery rate using a special internal standard substance (β-hydroxy-β-ethyl-δ-valerolactone) as in the MS method, it is sufficiently accurate without performing such recovery rate correction. It is possible to obtain stable measurement values.

As described above, according to the present invention, there is provided a mevalonic acid measuring method which is low in both equipment cost and running cost and can be generally used.

The present invention will be described below in detail with reference to the drawings as necessary.

(Sample) As long as it is a sample that may contain mevalonic acid, body fluids of animals including humans (eg, blood, lymph, urine, etc.) can be used without particular limitation. When human blood is used as the sample, it is usually preferable to use plasma or serum. In the present invention, the sample can be used after diluting it with a liquid such as a buffer solution as necessary.

(Conversion to dehydromevalonolactone) Mevalonic acid (Chemical Formula 1) in the above-mentioned sample was treated at a constant efficiency (yield).
The means for converting mevalonic acid to dehydromevalonolactone is not particularly limited as long as it can be converted (lactonization / dehydration) into dehydromevalonolactone (Chemical Formula 3). Preferably converts mevalonic acid to dehydromevalonolactone by adding an acid to the sample.

As the above-mentioned acid, any of Bronsted acid, Lewis acid, solid acid (ion exchange resin, etc.) can be used, but from the viewpoint of easy post-treatment, Bronsted acid, especially strong acid is used. It is preferable. As such a strong acid, for example, hydrochloric acid, sulfuric acid, or perchloric acid can be preferably used. Perchloric acid is particularly preferably used from the viewpoint of dehydration efficiency and stability.

When the above-mentioned acid is used, it is preferable to add to the sample such an amount that the sample becomes acidic with a pH of 2 or less (further, 0.5 to 1.0). When plasma or serum is used as the sample, it is preferable to set the pH to a low level (for example, about 0.5) in consideration of deproteinization efficiency. On the other hand, when urine is used as the sample, the pH is raised (for example, 1.0
It's good). When using perchloric acid as the above acid, for example, 10% for 1.0 ml of the sample (serum, urine, etc.)
About 0.2-1 ml of perchloric acid aqueous solution (further 0.4-
It is preferable to use about 0.5 ml).

According to the experiments of the present inventor, although the lactonization of mevalonic acid is considerably advanced by such an acid treatment,
It has been observed that the dehydration of the lactone mainly proceeds during the dryness described below (for example, under N ₂ atmosphere).
It has also been observed that the presence of a small amount of acid during this dryness improves the dehydration efficiency.

(Treatment after lactonization / dehydration) After producing mevalonolactone in a sample by the above-mentioned lactonization (deproteinization), the mevalonolactone can be extracted with a solvent to prepare a sample for liquid chromatography analysis. ,
It is preferable from the viewpoints of improving the accuracy of mevalonic acid measurement and maintaining the equipment of the liquid chromatograph (sample inlet, prevention of contamination of the column, etc.).

As a solvent used in this extraction operation, an extraction solvent (eg, an organic solvent) which is liquid at room temperature (25 ° C.) and has low solubility in water can be used without particular limitation. is there. From the viewpoint of boiling point and the like, for example, diethyl ether, chloroform, methylene chloride, ethyl acetate and the like can be preferably used. Ethyl acetate is particularly preferably used from the viewpoint of solvent toxicity and flammability. Prior to this extraction, it is preferable to add a salt such as sodium sulfate or sodium chloride to the aqueous phase from the viewpoint of improving the extraction rate. According to the experiments by the present inventor, when sodium sulfate is used (in Example 1 described later), the extraction ratio is improved to about 2 times that when no salt is added, and about 1.5 times that when sodium chloride is added. Was observed.

In the present invention, it is preferable to carry out the extraction treatment as described above and then concentrate and dry the solvent phase for extraction to produce dehydromevalonolactone. The presence of a small amount of acid during the concentration and dryness improves the efficiency of the dehydration reaction from mevalonolactone to dehydromevalonolactone.

In the present invention, after the above concentration and dryness,
It is preferably redissolved in a mobile phase for liquid chromatography as described below. From the viewpoint of suppressing the denaturation of the measurement object, this concentration / drying operation is performed with an inert gas (for example,
Nitrogen) atmosphere is preferred.

If necessary, the solvent phase may be subjected to a centrifugal separation treatment before the above concentration and dryness operations. It is preferable to perform such centrifugation from the viewpoint of improving the accuracy of mevalonic acid measurement and maintaining the equipment of the liquid chromatograph. The centrifugation conditions in this case are 450 to 6
It is preferable to use an acceleration of about 50 G (a rotation speed of about 1500 to 1800 rpm when a rotor having a radius of about 18 cm is used).

(Centrifugal Separation Before Mevalonolactone Extraction) Before extraction of the mevalonolactone, a by-product of the lactonization / dehydration treatment (for example, an acid-denatured product of a protein) is carried out by subjecting a sample to centrifugation if necessary. ) And the like may be removed. Performing such centrifugation is
It is preferable from the viewpoint of improving the accuracy of mevalonic acid measurement and maintaining the equipment of a liquid chromatograph. In this case, the centrifugal separation condition is an acceleration of about 12,000 to 14,000 G (when a rotor with a radius of about 7 cm is used, 120
It is preferable to use a rotation speed of about 0 to 13000 rpm).

(Liquid Chromatography Measurement) In the present invention, so-called open column chromatography can be used as liquid chromatography, but from the viewpoint of measurement accuracy and measurement reproducibility, high performance liquid chromatography (HPLC) is used. ) Is preferably used.

(Column) The column used in liquid chromatography is not particularly limited as long as it can separate mevalonic acid (Chemical formula 1), mevalonolactone (Chemical formula 2) and dehydromevalonolactone (Chemical formula 3) from each other. . In the present invention, both columns for normal phase chromatography and columns for reverse phase chromatography can be used, but dehydromevalonolactone (other 2
It is preferable to use a column for reversed-phase chromatography from the viewpoint that a good separation can be easily obtained with respect to polarities lower than those of other people.

In the present invention, from the viewpoint of the uniformity or reproducibility of analysis, a so-called chemical bond type packing material is preferably used as the adsorption carrier constituting the column.
In particular, silica gel in which an alkylsilyl group such as octadecylsilyl (C ₁₈ , ODS) group is chemically bonded is particularly preferably used.

(Column switching) The method of liquid chromatography used in the present invention is not particularly limited, but from the viewpoint of suppressing the interference of contaminants and / or shortening the analysis time, it is preferable to use a single column. It is preferable to switch and use a plurality of columns by a column switching method. Here, "column switching" is a liquid chromatography using a plurality of columns, and depending on the adsorption or elution of the component to be measured in one column (A), the other column (B) A method of changing at least one of the flow path of the mobile phase and the connection between the column (A) and the column (B) (for details of this column switching, see, for example, Takeo Yamabe “Ultra High Performance Liquid Chromatography”). Graph ", 101-120
Page, industry book, 1989).

The column switching method that can be used in the present invention is not particularly limited. For example, of the fractions preliminarily separated in the first column (pre-column), only the necessary fraction is separated in the second column (main column). A column switching method of precisely separating the column can be preferably used. In such an embodiment, unnecessary fractions can be eluted from the analysis system at high speed by bypassing the second column, thereby shortening the overall analysis time. In this case, it is preferable to use a plurality of mobile phases having different mobilities from the viewpoint that it is easy to precisely analyze the fraction containing dehydromevalonolactone while measuring the rapid discharge of the contaminants from the analysis system. preferable. Furthermore, if necessary, three or more columns including a plurality of main columns having different separation characteristics may be used, and a plurality of switching valves may be used to facilitate rapid change in the solvent composition.

In the present invention, when measuring mevalonic acid in urine, for example, the usual Isocratic method (single column,
In the case of analysis with a single mobile phase composition), the analysis time becomes slightly longer (for example, about 30 to 60 minutes per analysis of one sample) due to the presence of contaminants in the sample. Further, in the continuous analysis of a plurality of samples, there is a possibility that the peak derived from the previously injected sample overlaps with the peak of the next injected sample.

On the other hand, when the gradient method (changing the composition of the mobile phase) is used to shorten the analysis time, the time for column washing and column conditioning after elution of dehydromevalonolactone is rather long. Therefore, the analysis time per sample is, for example, about 40 minutes or more.

On the other hand, when the column switching method described above is used and a plurality of types of mobile phase compositions are used in correspondence with a plurality of columns, dehydromevalonolactone can be separated while rapidly separating contaminants. Since it is easy to perform precise analysis, for example, the analysis time per sample is 16
It takes about 20 minutes. Particularly in the continuous analysis of a plurality of samples, the influence of the unidentified component of the previous sample can be remarkably reduced in the analysis of the next sample, so that stable routine analysis can be performed in a short analysis time.

In the present invention, from the viewpoint of suppressing the interference of contaminants and shortening the analysis time, for example, the following column switching may be performed using two types of columns and two types of mobile phases. preferable.

With reference to the block diagram of FIG. 2, the switching valve 2 is set to the valve position A before the sample is injected from the autosampler 1. In this case, the first
The mobile phase of (1) is sent from the first pump 3 to the drain through the autosampler 1, the precolumn (first column) 4, and the switching valve 2. in this case,
The second mobile phase is sent from the second pump 5, passes through the main column (second column) 6 and the detector 7, and is sent directly to the drain.

After the sample is injected into the autosampler 1, the switching valve 2 remains at the valve position A until a predetermined time (the time when the elution of the fraction containing dehydromevalonolactone from the precolumn 4 starts). Is. That is, the sample is roughly separated by the pre-column 4, and the fraction eluted from the pre-column 4 before the fraction containing dehydromevalonolactone is sent to the drain after passing through the switching valve 2.

On the other hand, after the sample is injected into the autosampler 1, the switching valve 2 is set to the valve position B at a predetermined time (when the elution of the fraction containing dehydromevalonolactone starts from the precolumn 4). 3), the fraction containing dehydromevalonolactone eluted from the pre-column 4 passes through the switching valve 2, is further precisely separated in the main column 6, is detected by the detector 7, and is then sent to the drain.

After the fraction containing dehydromevalonolactone has been transferred to the main column 6, the switching valve 2 is returned to the valve position A (FIG. 2) and eluted from the precolumn 4 after the fraction containing dehydromevalonolactone. The fraction to be discharged is sent to the drain after passing through the switching valve 2.

(Detector) In the present invention, known detectors for liquid chromatography can be used without particular limitation. More specifically, for example,
It is possible to use an absorptiometric detector (ultraviolet and / or visible), a fluorescence detector (FL), a differential refraction detector (RI), an infrared absorptiometry detector (IR), a mass spectrometry (MS) detector, etc. it can.

From the viewpoint of balance between simplicity and sensitivity,
In the present invention, it is preferable to use an absorption photometric detector. The measurement wavelength in the absorption photometric detection is not particularly limited, but from the viewpoint of detection selectivity, it is preferable to use a wavelength of 215 nm or more, more preferably 220 to 240 nm (particularly around 230 nm).

Hereinafter, the present invention will be described more specifically with reference to Examples.

[0057]

Example 1 (Confirmation of the structure of dehydromevalonolactone) Commercially available mevalonic acid standard product (Chemical formula 1) was treated in the same manner as the sample described below to give dehydromevalonolactone (Chemical formula 3). Was confirmed to be generated.

That is, 0.4 ml of a 10% aqueous solution of perchloric acid was added to 1.0 ml of a commercially available mevalonic acid standard product (reagent special grade, manufactured by Sigma), and the mixture was reacted by stirring at room temperature (22 to 25 ° C.) for 10 minutes. It was To the reaction product, 5 ml of 1M phosphate buffer (pH = 2.0) was added, and about 3 g of anhydrous sodium sulfate was added, followed by extraction with 10 ml of ethyl acetate. The organic phase thus obtained was dried at 50 ° C. under a nitrogen stream.

The obtained residue was dissolved in 0.3 ml of pre-column mobile phase and then purified by HPLC under the following conditions.

HPLC device: Brand name Intelligent Pump L
-6200, Hitachi column: column for reverse phase chromatography (trade name: In
tersil-80A, manufactured by GL Sciences Inc., C ₁₈ -OD
S-silica gel-containing chemically bonded column), diameter 4.6 mm, length 250 mm Column temperature: 25 ° C. Mobile phase: 8 mM phosphate buffer solution (containing 1.3% tetrahydrofuran) Flow rate: 0.8 ml / min Detector : Ultraviolet / visible absorption detector (SPD-10A,
Shimadzu Corporation), measurement wavelength: 230 nm Injection volume: 50 μl The sample purified as described above was deuterated chloroform (CDC).
l ₃ ) and then dissolved in proton NMR (trade name: XL-
300, manufactured by Varian). The obtained NMR spectrum is shown in FIG.

In the spectrum shown in FIG. 4, peaks corresponding to methyl (a), methylene (b and c) and methine (d) are recognized. The chemical shifts of these peaks are reported in the literature (Ajit Sanghivi et al., Biochem. Bioph.
ys. Acta., 444 , 727-733, 1976) by-product Δ ² -3-CH ₃ -Mevalonic acid lactone.
It is in good agreement with (dehydromevalonolactone; Chemical formula 3).

As shown in FIG. 4, the integrated value of each peak is 45.0: 30.7: 31.5: 15.
It was 0. The structure (a: b: c: d =) shown in (Chemical Formula 3)
3: 2: 2: 1) is also supported by these integrated values.

Example 2 (Analysis of Mevalonic Acid Standard and Sample) Mevalonic acid in a sample (plasma or urine) was introduced into dehydromevalonolactone and then analyzed by HPLC.

That is, 10% in 1.0 ml of a sample (plasma or urine) (or a predetermined amount of the above mevalonic acid standard product)
Add 0.4 ml of perchloric acid aqueous solution, shaker (trade name: Mi
croTube Mixer MT-360, manufactured by Tommy Seiko Co., Ltd.)
After reacting by shaking at room temperature for 10 minutes using a centrifuge (trade name: Micro-cooling centrifuge 1700, manufactured by Kubota, rotor radius: 7.3 cm), 4 ° C., 117
It was centrifuged for 15 minutes under the condition of 50 G (12000 rpm).

To 1.0 ml of the supernatant after the above centrifugation treatment, 1M
After adding 5 ml of phosphate buffer (pH = 2.0) and 3 g of anhydrous sodium sulfate, the mixture was shaken with 10 ml of ethyl acetate using a shaker (custom-made product, manufactured by Ikeda Rika Co., Ltd.) Mevalonolactone was extracted with ethyl acetate. Then, using a centrifuge (trade name: H-107S, domestic centrifuge, rotor radius: 18 cm), 450 G (15
The organic phase was recovered by centrifugation under the conditions of (00 rpm).
9 ml of this organic phase was dried at 50 ° C. under a nitrogen stream. At the time of this dry solidification operation, the inner wall of the test tube used for dry solidification was washed with ethyl acetate containing 0.1% perchloric acid about 2 to 3 times to dehydrate and lactonize unreacted mevalonic acid. Completed.

The residue thus obtained is 0.25 m
1 of the following mobile phase 1 (8 m containing 2.5% CH ₃ CN
After being dissolved in M phosphate buffer, pH = 2.2), it was analyzed by HPLC under the following conditions.

HPLC device: Brand name Intelligent Pump L
-6200, Hitachi pre-column: column for reverse phase chromatography (trade name: LQ-D Type, manufactured by Labo Co-Tech, C ₁₈ -OD
Chemically bonded column containing S silica gel) Diameter 4.6 mm, length 30 mm Main column: column for reverse phase chromatography (trade name: Intersil-80A, manufactured by GL Sciences Inc., C ₁₈₎
-ODS silica gel), diameter 4.6 mm, length 250 m
m Column temperature: 25 ° C. First mobile phase: 2.5% acetonitrile (CH ₃ CN)
8 mM phosphate buffer (pH = 2.2) containing 2nd mobile phase: 8 mM phosphate buffer (pH = 2.2) containing 1.3% tetrahydrofuran Flow rate: 0.8 ml / min Detector : UV / visible absorption detector (measurement wavelength: 230
nm) Injection amount: 50 μl In the above HPLC analysis, the following column switching analysis was performed using the above-mentioned two types of columns and two types of mobile phases.

Referring to FIG. 2, before the sample is injected into the autosampler 1 and less than 3.5 minutes after the sample is injected, the switching valve 2 is set to the valve position A and the sample is pre-columned. The fraction roughly separated by 4 and eluted before the fraction containing dehydromevalonolactone was passed through the switching valve 2 and then sent to the drain.

On the other hand, at the time of 3.5 minutes after the sample was injected into the autosampler 1, the switching valve 2 was set to the valve position B (FIG. 3), and the portion containing dehydromevalonolactone eluted from the precolumn 4 was added. The image was passed through the switching valve 2, precisely separated by the main column 6, detected by the detector 7, and then sent to the drain.

After the fraction containing dehydromevalonolactone has been transferred to the main column 6 (autosampler 1
4.5 minutes after the sample was injected into the column, the switching valve 2 was returned to the valve position A (FIG. 2), and the fraction eluted from the precolumn 4 after the fraction containing dehydromevalonolactone was switched. After passing through the valve 2, the liquid was sent to the drain.

The HPLC chromatogram (peak of dehydromevalonolactone) obtained when 100 ng / ml and 1 ng / ml of mevalonic acid (standard product) were used as the above samples is shown in FIG. In Fig. 5, "In
te. ATT ”is the detector sensitivity attenuation (atte
nute) (the smaller Inte. ATT, the higher the sensitivity of the detector).

Further, the HPLC chromatograms obtained when two types of plasma (A and B) and two types of urine (A and B) as samples were used as the above samples are shown in FIGS. 6 and 7, respectively. . In these figures, the peak of dehydromevalonolactone is D-
It is indicated by the symbol MVL.

Example 3 (linearity of calibration curve) As the concentration of the above mevalonic acid (standard product), 5 ng / ml, 10 ng / ml, 50 ng / m
1, 100 ng / ml, 500 ng / ml, 1000 n
g / ml, 2000 ng / ml, and 4000 ng /
HPLC analysis was performed in the same manner as in Example 1 except that eight kinds of 8 ml samples were used.

The relationship between the HPLC peak height (vertical axis; μV) of dehydromevalonolactone thus obtained and the mevalonic acid standard product concentration (horizontal axis; ng / ml) is shown below (Table 1). And it shows in the graph (calibration curve) of FIG.

[0075]

[Table 1]

As shown in Table 1 and FIG. 8 above, the measurement method of the present invention provided a good linear relationship between the mevalonic acid standard concentration and the HPLC peak height.

Example 4 (Reproducibility of measured value of standard product) As a concentration of mevalonic acid (standard product) of 50 ng / ml, the same measurement was repeated 8 times (N = 8), except that Analysis was carried out by HPLC in the same manner as in Example 1.

The measured values of the HPLC peak height (μV) thus obtained are shown below (Table 2).

[0079]

[Table 2]

From the above eight measurements, X (average value)
= 1394.6 μV, SD (standard deviation) = 40.4 μ
Good reproducibility was obtained with V and CV = SD / X (average value) = 2.90%.

Example 5 (Simultaneous reproducibility of plasma measured values) Plasma samples C and D were used as the above samples, and the same measurement was repeated 8 times (N = 8), as in Example 1. Analysis was performed by HPLC.

The measured value of the HPLC peak height (μV) obtained by such a measurement was converted into the mevalonic acid concentration using the calibration curve obtained in Example 3. The mevalonic acid concentration values thus obtained are shown below (Table 3).

[0083]

[Table 3]

As shown in Table 3 above, good reproducibility was obtained for both plasmas C and D by the eight measurements.

<Plasma C> X (average value) = 6.2 ng / ml SD (standard deviation) = 0.20 ng / ml CV = SD / X (average value) = 3.2% <Plasma D> X (average value) Value) = 15.9 ng / ml SD (standard deviation) = 0.49 ng / ml CV = SD / X (mean value) = 3.1% Example 6 (simultaneous reproducibility of urine measurement values) The same measurement was repeated 8 times using the samples C and D (N =
The analysis was performed by HPLC in the same manner as in Example 1 except for 8).

The measured value of the HPLC peak height (V) obtained by such a measurement was converted into the mevalonic acid concentration using the calibration curve obtained in Example 3. The mevalonic acid concentration values thus obtained are shown below (Table 4).

[0087]

[Table 4]

As shown in Table 4 above, good reproducibility was obtained for both of the measured urine C and D by the measurement of 8 times.

<Urine C> X (mean) = 101.3 ng / ml SD (standard deviation) = 2.79 ng / ml CV = SD / X (mean) = 2.75% <Urine D> X (mean) Value) = 455.5 ng / ml SD (standard deviation) = 12.01 ng / ml CV = SD / X (mean) = 2.64% Example 7 (addition recovery test using plasma sample) As the above sample, Using plasma samples E and F, 1 volume of mevalonic acid aqueous solution was added to 9 volumes (volume part) of each sample, and the concentration of added mevalonic acid was 0 ng / ml and 5 n, respectively.
g / ml, 10 ng / ml, and 50 ng / ml, and then HPLC analysis was performed in the same manner as in Example 1 except that the same measurement as in Example 1 was performed, and the quantitative value of each sample I asked.

In these measurements, the HPLC peak height (μV) of the obtained dehydromevalonolactone was calculated as
The mevalonic acid concentration was converted using the calibration curve obtained in Example 3. The quantitative values of mevalonic acid thus obtained are shown below (Table 5) and in the graph of FIG.

[0091]

[Table 5]

"Recovery rate" shown in Table 5 above
Is a numerical value defined as follows.

Recovery rate (%) = 100 × {(quantitative value)-(quantitative value when the added amount is 0 ng / ml)} / added amount As shown in Table 5 and FIG. 9, the measured plasma E and Good recovery rates were obtained for all of F.

Example 8 (Additional recovery test using urine sample) Urine specimens E and F were used as the above samples, and 1 volume of mevalonic acid aqueous solution was added to 9 volumes (volume part) of each sample and added. Mevalonic acid concentrations of 0 ng / ml and 50 ng, respectively
/ Ml, 100 ng / ml, and 500 ng / ml, and then, except that the same measurement as in Example 1 was performed, HPLC analysis was performed in the same manner as in Example 1, and the quantitative value of each sample was determined. I asked.

In these measurements, the HPLC peak height (μV) of the obtained dehydromevalonolactone was calculated as
The mevalonic acid concentration was converted using the calibration curve obtained in Example 3. The quantitative values of mevalonic acid thus obtained are shown below (Table 6) and in the graph of FIG.

[0096]

[Table 6]

As shown in Table 6 and FIG. 10, a good recovery rate was obtained for each of the measured urine E and F.

Example 9 (Comparison between HPLC method and GC-MS method) The following (Table 7)
Mevalonic acid was measured by the HPLC method of the present invention in the same manner as in Example 1 except that 10 types of samples (6 types of plasma and 4 types of urine) shown in 1 were used.

On the other hand, the conventional GC-MS method (measurement conditions are described in the above-mentioned literature:
Mevalonic acid was measured according to "Medical and Pharmacy, 27 (4) , 939-945, 1992", and the above-mentioned HPL
It was compared with the data of Method C. The measured values obtained by the above measurement are shown below (Table 7) and in the graph of FIG.

In Table 7 below, the description such as "+5 ng / ml" means that the concentration of mevalonic acid added was "9% by volume of plasma or urine, and 1% by volume of standard aqueous mevalonic acid solution was added. After being adjusted to the “concentration described in the table”, it is used as a sample for measurement.

[0101]

[Table 7]

As shown in the graph of FIG.
Regarding the measured value (x) by the LC method and the measured value (y) by the GC-MS, the following good correlation (correlation coefficient γ = 0.997) was obtained.

Y = 0.864x-1.27

[0104]

As described above, according to the present invention, after converting mevalonic acid in a sample to dehydromevalonolactone,
Provided is a method for measuring mevalonic acid, which comprises measuring the dehydromevalonolactone by liquid chromatography.

In the present invention, not only can mevalonic acid in a sample be efficiently converted to dehydromevalonolactone by a simple operation, but the dehydromevalonolactone can be treated with a special pretreatment (for example, GC-MS treatment). Liquid chromatography (eg, U chromatography) without the trimethylsilylation and silica gel column purification required for
It can be easily measured by using a V detector). Therefore, according to the present invention, mevalonic acid can be measured with good sensitivity and with good measurement accuracy and stability.

Furthermore, according to the present invention, purification by silica column after extraction with an organic solvent (indispensable in the conventional GC-MS method) is not essential, and the pretreatment of chromatography is simplified as a whole. As compared with the GC-MS method, mevalonic acid can be measured very easily.

Even if high performance liquid chromatography (HPLC) is used as the liquid chromatography in the present invention, the equipment cost is about several million at most, and unlike the above-mentioned GC-MS, the maintenance is simple and easy. No dedicated operator is required for the actual measurement operation. Therefore, according to the present invention, there is provided a method for measuring mevalonic acid, which has low device cost and running cost.

Furthermore, the ultraviolet absorption of dehydromevalonolactone to be detected by liquid chromatography in the present invention is very strong (molar extinction coefficient is several tens to several hundreds of times that of mevalonic acid), and during the above-mentioned UV detection. (Eg wavelength 2
Since it is possible to use the longer wavelength side (for example, 215 nm or more) with less interfering substances (compared to 10 nm), it is possible to measure mevalonic acid with higher sensitivity and accuracy.

[Brief description of drawings]

FIG. 1 is an embodiment of the present invention (HPLC method) and conventional GC.
FIG. 6 is a flowchart showing the operating procedure of mevalonic acid analysis with the MS method.

FIG. 2 is a schematic diagram for explaining a column switching method that can be preferably used in the present invention, and shows a state in which mobile phases are separately fed to two types of columns.

FIG. 3 is a schematic diagram for explaining a column switching method that can be preferably used in the present invention, and shows a state in which two types of columns are connected in series.

FIG. 4 is a chart showing a proton NMR spectrum of dehydromevalonolactone obtained under the same treatment conditions as in the present invention.

FIG. 5 is a chart showing a chromatogram obtained by subjecting a standard mevalonic acid product to pretreatment by the method of the present invention and then performing HPLC analysis.

FIG. 6 is a chart showing a chromatogram obtained by subjecting plasma (two types) as samples to pretreatment by the method of the present invention and performing HPLC analysis.

FIG. 7 is a chart showing a chromatogram obtained by subjecting urine (two types) as samples to pretreatment by the method of the present invention and then performing HPLC analysis.

FIG. 8: Calibration curve showing the relationship between the mevalonic acid concentration and the HPLC peak height (μV) of dehydromevalonolactone obtained by pretreatment and HPLC analysis of aqueous solutions of standard mevalonic acid with various concentrations. Is a graph of.

FIG. 9 is a graph showing the results of a mevalonate-added recovery test when plasma was used as a measurement sample.

FIG. 10 is a graph showing the results of a mevalonic acid addition recovery test when urine was used as a measurement sample.

FIG. 11 is a graph showing the correlation between the mevalonic acid concentration measured value (ng / ml) by the HPLC method of the present invention and the mevalonic acid concentration measured value (ng / ml) by the conventional GC-MS method.

[Explanation of symbols]

1 ... Autosampler, 2 ... Switching valve, 3 ... First
Pump, 4 ... pre-column (first column), 5 ... second
Pump, 6 ... Main column (second column), 7 ... Detector.

Claims (13)

[Claims] 1. A method for measuring mevalonic acid, which comprises converting mevalonic acid in a sample to dehydromevalonolactone and then measuring the dehydromevalonolactone by liquid chromatography. 2. The method for measuring mevalonic acid according to claim 1, wherein the dehydromevalonolactone is derived by lactonizing and dehydrating the mevalonic acid in the presence of an acid. 3. The method for measuring mevalonic acid according to claim 2, wherein the acid is Bronsted acid. 4. The method for measuring mevalonic acid according to claim 3, wherein the acid is a strong acid. 5. The method for measuring mevalonic acid according to claim 4, wherein the acid is perchloric acid. 6. The mevalon according to claim 1, wherein the mevalonlactone in the sample is extracted and then dehydromevalonolactone is derived by extracting the mevalonolactone in the sample by subjecting the sample containing the mevalonic acid-converted mevalonolactone to extraction with a solvent. Method of measuring acid. 7. The method for measuring mevalonic acid according to claim 6, wherein the sample containing mevalonolactone converted from mevalonic acid is subjected to a centrifugal separation treatment, and then an extraction operation with a solvent is performed. 8. In the liquid chromatography,
The method for measuring mevalonic acid according to claim 1, wherein mevalonic acid is detected by measuring absorbance in the ultraviolet. 9. The method for measuring mevalonic acid according to claim 8, wherein a wavelength of 215 nm or more is used as a measurement wavelength in the absorbance measurement in the ultraviolet region. 10. A plurality of columns including a first column and a second column are used in the liquid chromatography, and the first column is prepared in response to the elution of the fraction containing dehydromevalonolactone from the first column. Second on the downstream side of the column
The method for measuring mevalonic acid according to claim 1, wherein the columns are connected in series. 11. After the transfer of the fraction containing dehydromevalonolactone from the first column to the second column is completed, the second column is cut off from the first column, and the first and second columns are cut off. The method for measuring mevalonic acid according to claim 10, wherein the mobile phase is separately flowed to each of the columns. 12. In the liquid chromatography, a plurality of mobile phases containing a first and a second mobile phase are used, the first mobile phase is passed through the first column, and the second mobile phase is passed through the second column. The method for measuring mevalonic acid according to claim 10, wherein a mobile phase is flown. 13. The first mobile phase is flowed through the first and second columns connected in series only when the fraction containing dehydromevalonolactone is eluted from the first column. 12. The method for measuring mevalonic acid according to item 12.