JP2766869B2 - Determination of lower carboxylic acids in body fluid materials - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for quantifying a lower carboxylic acid present in a body fluid material such as plasma.

[Prior Art] Various lower carboxylic acids are present in body fluids such as blood. However, abnormalities in these concentrations often indicate specific diseases, dysfunctions or metabolic disorders. Interesting in laboratory or laboratory tests. For example, oxalic acid is associated with renal and urolithiasis, propionic acid is associated with propionic acidemia, acetoacetic acid and β-hydroxybutyric acid are both indicators of diabetes pathology and are associated with abnormal glucose metabolism. Pyruvic acid and lactic acid are related to glycolysis, α-ketoisocaproic acid, α-ketoisovaleric acid and α-ketoglutaric acid are related to amino acid metabolism disorders, and malic acid, fumaric acid and citric acid are metabolic regulators It is picked up as a component for analysis.

As a method for measuring a lower carboxylic acid in such a body fluid material, for example, plasma, there is an enzymatic method for oxalic acid. In this method, oxalic acid is decomposed with oxalate oxidase, and the amount of oxalic acid is measured based on a pH change caused by generation of carbon dioxide gas. However, this method has the drawback that the operation is complicated and that it is difficult to apply due to poor accuracy (the concentration of oxalic acid in blood is only 1/100 to 100 in urine). As a method for measuring oxalic acid in urine but not in plasma, oxalic acid is extracted with tri-n-butyl phosphate, oxalic acid is esterified with 9-anthryldiazomethane (abbreviated as ADAM), and high-performance liquid chromatography is performed. Graphy (HP
Separation by LC) and measurement of fluorescence (Urology Bulletin 29
Vol. 3, pages 287-292, 1983). However, this method cannot be directly applied to plasma containing a large amount of various substances incomparably with urine.

[Problems to be Solved by the Invention] As a method of quantifying lower carboxylic acid in a body fluid material, the present inventor has sought to improve the urinary oxalic acid determination method using ADAM so as to be applicable to body fluid material. .

[Means for solving the problem] A major reason that the urinary oxalic acid determination method cannot be directly applied to body fluid material is that a large amount of substances (other than oxalic acid) that consume ADAM are present in the body fluid material. I found that. Examples of such interfering substances include stearic acid, palmitic acid, higher fatty acids such as oleic acid, prostaglandins, phospholipids, etc., all of which are acidic substances like lower carboxylic acids.
Heretofore, there has been no suitable method for separating and removing only interfering substances. It was also found that the presence of other fat-soluble substances (fats, fat-soluble vitamins, etc.) in the body fluid material hindered layer separation during solvent extraction and reduced the extraction rate. The present inventors have been studying the pretreatment of body fluid materials by various methods, and have found that when the reverse phase type adsorbent for chromatography is used as a pretreatment agent, the interfering substance can be efficiently removed by this. . The present invention has been made based on such findings.

[Constitution of the Invention] That is, the present invention relates to a method of quantifying a carboxylic acid by esterifying a lower carboxylic acid in a deproteinized liquid material with a labeled esterifying agent and measuring the label, wherein the sample is prepared before esterification. Is treated with a reverse-phase-type absorbent.

In addition, the present invention provides a kit combining an instrument and a reagent for performing the above method, a filter having an ultrafiltration membrane, a container containing a weak alkaline solution, a reverse-phase short column, and an acid solution. A container containing a lower alkyl phosphate ester, a container containing a lower alcohol, a container containing a 9-anthryldiazomethane solution, and a small test tube or a centrifuge tube; a lower carboxylic acid in a body fluid material; This is to lower the kit for analysis.

Further, the present invention is not limited to the above-described method, and includes a filter, a filter holder, and a short column as a convenient instrument for pretreatment in various body fluid component analysis methods, and the filter is provided at least at a central portion of a filtration surface. The filter holder is provided with a receiving portion corresponding to the shape of the filter in a short cylinder, and a through hole is provided at the center of the receiving portion.The short column has a short end connected to the filter holder. An object of the present invention is to provide a body fluid material pretreatment device in which a column is filled with a column filler and a funnel-shaped liquid receiving port is provided on the holder side.

[Embodiments] (1) In the method of the present invention, the body fluid material includes a material containing all or most of liquid components of blood and tissue, such as plasma, serum, and tissue extract.

Deproteinization can be carried out by precipitation with trichloroacetic acid, perchloric acid or the like, but it is convenient to carry out ultrafiltration. Ultrafiltration is usually performed using a membrane filter. Examples of such a filter include those commercially available under the names of Centriflo-CF-25 and Ultracent-30.

Lower carboxylic acids include acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, malic acid, fumaric acid, citric acid, lactic acid, pyruvic acid, β-hydroxybutyric acid, acetoacetic acid, α-ketocaproic acid, α-ketoiso Valeric acid, α
-Includes aliphatic carboxylic acids having 6 or less carbon atoms, usually 5 or 4 carbon atoms, such as ketoglutaric acid, which have a simple substituent such as a hydroxy group or an oxo group. The majority of lower carboxylic acids are normal biological components, but also include substances that are not normally present in the body, such as those that appear when a drug is administered.

As the label in the labeled esterifying agent, a fluorescent label,
UV-absorbing labels and the like are included. Further, the portion that performs the esterification action includes an alcoholic hydroxyl group or an active form thereof (for example, halogen such as chlorine and bromine, sulfonyloxy such as methylsulfonyloxy and tolylsulfonyloxy, and diazo.). Specific examples of the esterifying agent include 9-anthryldiazomethane (ADAM), 9-chloromethylanthracene, 7-acetoxy-4-bromomethylcoumarin, and 4-bromomethyl-
7-methoxycoumarin, N- (7-nitrobenz-2-
Oxa-1,3-diazol-4-yl) aziridine (NBD
Aziridine) and the like. Examples of the ultraviolet-absorbing labeled esterifying agent include p-bromophenacyl bromide, 9-
Phenylphenacyl bromide and the like.

Reverse phase type adsorbents include adsorbents in which a lipophilic group, for example, a higher aliphatic group, for example, 15-30% is chemically bonded to a carrier substance. As the carrier material, silica is most frequently used, but polystyrene, dextran and the like can also be used. As the lipophilic group, an octylsilyl group,
Preferred are higher alkylsilyl groups such as octadecylsilyl groups, which are introduced by reacting the carrier material in the form of a silylating agent, for example a silyl halide.
Such an adsorbent and a column filled with the adsorbent are known, and it is convenient to use a commercially available one. Suitable products are commercially available, for example, under the names of Sep-Pak C18 cartridge (Nippon Waters Limited), Chemcosep ODS-B (Chemco), and BondElute C18 (Analytechem International).

This method is implemented, for example, as follows. First, the body fluid material, for example, plasma, is deproteinized, preferably by ultrafiltration. The deproteinized solution is adjusted to near neutral or weakly alkaline, for example, pH 6-9 with a weak alkali solution containing no carboxylic acid, for example, an aqueous solution of sodium hydrogen carbonate, and treated with a reverse phase type adsorbent, preferably packed in a column. I do. The effluent or filtrate is acidified by treatment with, for example, an acid such as hydrochloric acid or sulfuric acid, and the organic solvent for extraction is preferably a tri-lower ester of phosphoric acid (lower esters are generally saturated with 1 to 6 carbon atoms, usually 1 to 5 carbon atoms). Or unsaturated aliphatic hydrocarbons), for example, tributyl phosphate, tripropyl phosphate, triamyl phosphate, dibutylmethyl phosphate, diethyl vinyl phosphate and the like, or a mixture thereof. At this time, the amino acid remains without being extracted. Preferably, a lower alcohol such as methanol or ethanol is added to the extract, and a solution of a labeled esterifying agent such as an excess of ADAM in an organic solvent, preferably acetone is added. Usually, the reaction is carried out at room temperature with slight heating, followed by high-performance liquid chromatography (HPLC) treatment, and the fraction containing the ester of the desired acid is quantified by labeling. The HPLC is preferably carried out on a column packed with a silica or alumina based reversed phase filler, for example an octylsilane resin. Such columns are known and include, for example, nucleosyl 5C8, Lichrosolve 5C8,
It is commercially available under the name of Shim-Pak Prep C8. For the detection, for example, a fluorescence detector, an ultraviolet detector, or the like is used according to the label. In this way, measurement is performed for a carboxylic acid having a known concentration, a calibration curve is prepared, and quantification is performed for a sample having an unknown concentration. Since the lower carboxylic acid content in blood samples is extremely small, such as several micrograms / ml,
Reagents and solvents used for quantification must be of extremely high purity.

In the above method, Seppak C18 is used as an adsorbent.
When a cartridge is used, the pH is preferably around 8-9. Since esterification by ADAM has a slow reaction rate in a phosphate ester, it is preferable to add methanol, and the addition amount is preferably around 30-40%. The concentration of ADAM is
When oxalic acid is 1 μm / ml, 0.02% or more is appropriate.
About -0.1% is preferable. The reaction time is approximately 30 minutes at room temperature.
It reaches 85% and is complete in 1.5 hours. In the HPLC, when the medium-polar packing column nucleosyl 5C8 is used, an acetonitrile / water system is suitable as the mobile phase, and the ratio is preferably 7.3.

(2) In the kit of the present invention, the container and the instrument are made of plastic or glass. As the reagent, solvent and adsorbent, those described in the above section (1) are used. These are desirably sealed by stoppering or sealing.

(3) The device of the present invention is suitably made of a corrosion resistant material such as plastic or glass.

As the filter, an ultrafiltration membrane, a semipermeable membrane, or the like is used. As the ultrafiltration membrane, a membrane that leaves a substance having a molecular weight of a certain value of 20,000 to 50,000 or more on the membrane and allows a substance having a molecular weight lower than that to pass is used. For example, a polystyrene, vinyl chloride / acryl copolymer / polysaccharide membrane is used. Is made of paper glued to paper. The filter is at least centrally recessed and is preferably conical.

The short cylinder of the filter holder preferably has a circular cross section and does not have a large change in inner diameter. It is preferable that the receiving portion has a shape that can be in close contact with the filter. The connection between the short column and the holder is preferably detachable, and can be connected by, for example, rubbing.

As the column filler, various fillers such as silica gel, alumina, ion exchange resin, molecular sieve, porous adsorption resin, and the like can be filled according to the purpose, in addition to the reversed-phase type adsorbent. The exposed surface of the filler layer is desirably covered with an escape-preventing material such as porous or glass wool. The funnel-shaped liquid receiving port may be constituted by the inner surface of the short cylinder wall. In addition, a collection container such as a centrifuge tube or a test tube can be connected to the other end of the holder.

[Effect] The quantification method of the present invention removes a variety of interfering substances present in a blood sample in advance by treating a body fluid sample with a reversed-phase type adsorbent. The reaction rate with the esterifying agent increases,
As a result, accurate analysis becomes possible. In addition, the amount of the labeled esterifying agent used is small, and the handling of the reaction system is facilitated. In addition, there is an advantage that the operation is easy and the operation can be performed in a short time because only the simple operation of the treatment with the reverse phase type adsorbent is performed.

Since the kit of the present invention contains all the reagents and instruments necessary to carry out the above-described assay,
For example, it is possible to avoid waste such as purchasing reagents and solvents that require only a small amount of several grams or ml in sales units such as 500 g or ml. In addition, there is no need to provide extra equipment in case of inability to implement in a timely manner due to lack of equipment, etc.
Appropriate equipment dimensions can be selected in advance, which is convenient for adjusting the working conditions.

The device of the present invention is used for the following purposes. When analyzing a drug or a biological component in a body fluid such as serum, plasma, or tissue fluid, it is necessary to purify the substance by removing impurities other than the substance to be analyzed or substances that make analysis difficult as pretreatment. For this purpose, protein removal and separation / purification means are required for each purpose, but it is unavoidable that there is a drawback that the sample is lost at this time. However, in order to obtain as accurate a value as possible, it is desirable to purify the target substance by means capable of efficiently removing proteins and purifying the substance in the shortest possible time and with little loss of the sample.

The device of the present invention has been created in order to achieve the above-described object, and since the filter and the filler are set, the body fluid material can be pretreated by itself.
In addition, since the filtration membrane and the adsorbent are attached to separate cylinders and the cylinders are detachable, it is very convenient for refilling and washing. In addition, the filter holder is provided with a receiving portion corresponding to the shape of the filter, and a through hole is provided at the center thereof, so that the filter is not broken and the filtrate falls down in an orderly manner. In addition, since a funnel-shaped liquid receiving port is provided at the upper end of the short column, the dropped filtrate is guided to the column in an orderly manner.

EXAMPLES Hereinafter, the present invention will be described more specifically based on examples.

Example 1 (Quantitative method) (Method) 7% -Na was added to 1 ml of a filtrate obtained by ultrafiltration (1000 G, 30 minutes) of 2 ml of human plasma using Centriflow CF25 (Amicon).
The pH was adjusted to about 8.0 to 8.5 by adding 10 μl of HCO ₃ solution. This solution was loaded onto a Sepupak C18 cartridge, and the passing solution was passed through a glass stoppered test tube (inner diameter 8 mm, length 8 cm, 4 ml capacity).
The mixture was acidified (pH 1.0 to 2.0) by adding 20 μl of 6N-HCl, 0.5 ml of tri-n-butyl phosphate was added, and the mixture was shake-extracted with a shaker for 3 minutes. Centrifugation (3000 rpm, 10
Minutes), take 0.3 ml of the tri-n-butyl phosphate layer into another brown test tube, add 0.2 ml of methanol and 0.1 ml of ADAM solution (3 mg / ml acetone solution: prepared at the time of use), and allow to stand at room temperature for about 2 hours. Reacted. Separately, oxalic acid standard solution (0.2, 0.5, 1.0, 2.0μ
g / ml) 1 ml was handled similarly. 5 μl of the reaction solution was injected into the HPLC and quantified using the absolute calibration curve method based on the peak height.

(HPLC measurement conditions) The column is reverse-phase nucleosyl 5C8 (inner diameter 4.6 mm, length
25cm), and an acetonitrile-water mixture (7: 3) was used as a mobile phase, and the flow rate was 1.0 ml / min. Excitation wavelength 254n for fluorescence detection
m, at a measurement wavelength of 412 nm.

(Identification of oxalic acid diester in blood) Identification of oxalic acid in plasma was performed by treating human plasma with oxalate oxidase. That is, after deproteinizing human plasma by ultrafiltration, it is divided into two test tubes,
On the other hand, 50 μl (0.56 U / ml) of oxalate oxidase (Boehringer Mannheim) was added and reacted at 37 ° C. for 30 minutes. To the other, 50 μl of water was added, and the same operation and analysis were performed. As a result, in the oxalate oxidase-treated sample, the oxalic acid was decomposed by the enzyme, and the peak assumed to be that of oxalic acid diester disappeared. Oxalic acid standard solution 1.0 μg / 0.1 for human plasma
As a result of analysis by adding ml, the peak of the oxalic acid standard solution overlapped the peak assumed to be oxalic acid diester in plasma, and the peak height increased. From the data of the above two tests, the peak at a retention time of about 14 minutes was confirmed to be a diester compound of oxalic acid in plasma.

(Addition recovery rate) 0.4 to 2.0 μg of oxalic acid was added to 2 ml of plasma, and the recovery rate under the above measurement conditions was determined. As shown in Table 1,
Good results of 98.34 to 100.0% were obtained.

(Calibration curve) The oxalic acid standard solution had a good correlation between the concentration of 0.2 to 2.0 µg / ml and the peak height (r = 0.9994) (Fig. 1). The detection limit is 0.1 μg / ml plasma, and the coefficient of variation of this assay is 0.2 to 1.
It was as good as 0.59 to 2.43% in the concentration range of 0 μg / ml.

(Correlation between this method and enzymatic method) An enzymatic method commonly used for the analysis of oxalic acid in urine and food [Method of Enzymatic Method]
Analysis (Method of Enz. Anal.) P. 1542] and the present method were examined.

Since the enzymatic method has a high limit of quantification, oxalic acid concentrations of 20 and 40 μg / ml were used. In this method, the solution was diluted 20-fold and measured. As a result, good correlation was obtained with r = 0.9930 between the present method and the enzymatic method (FIG. 2).

Example 2 (Kit) A filter having an ultrafiltration membrane (the membrane filters substances having a molecular weight of 20,000 to 50,000 or less. The shape is as shown in FIG. ₃ ) A 7% aqueous NaHCO ₃ solution was charged. Ampoule reversed-phase short column (filler is low-polarity μ Bonder Pack C1
8) Ampoule containing 6N-HCl Ampoule containing tri-n-butyl phosphate Ampoule containing methanol Acetone solution of 9-anthryldiazomethane (3 mg / n
l) An ampoule containing a glass stoppered glass test tube having an inner diameter of 8 mm and a length of 8 cm Example 3 (Equipment) In FIG.
, A filter holder 2, and a short column 3. As the filter 1, a conical ultrafiltration membrane is preferable. Holder 2 contains filter 1 in short cylinder 21
And a receiving portion 22 that can be in close contact with. A through hole 23 is formed in the center of the receiving portion 22. In the short column 3, one end of a short tube 31 is connected to the holder 2, and the inside is filled with a filler 32. Filter plates 33 are in contact with the upper and lower sides of the filler. At the connection side with the holder 2, the liquid receiving port 34
Is attached. A test tube 4 is connected to the other side of the column. The holder 2, the column 3, and the test tube 4 are made of glass or a corrosion-resistant material such as a polypropylene-based or acrylic-based resin.

[Brief description of the drawings]

FIG. 1 is a graph showing a calibration curve in Example 1. FIG. 2 is a graph showing the correlation between the method of the present invention and the enzyme method in Example 1. FIG. 3 is a sectional view showing an example of the device of the present invention. 1 ... Filter, 2 ... Filter holder, 3 ...
Short column, 4 ... Test tube, 22 ... Receiving part, 32 ...
Filler, 33 ... Filter plate, 34 ... Liquid receiving port.

Continued on the front page (72) Inventor Hiromasa Araki 2-3-3 Morinomiya, Joto-ku, Osaka-shi, Osaka Fuso Pharmaceutical Co., Ltd. Research and Development Center (56) References JP-A-58-196455 (JP, A)

Claims (3)

(57) [Claims] 1. A method of quantifying a carboxylic acid by esterifying a lower carboxylic acid in a deproteinized body fluid material with a labeled esterifying agent and measuring a label, wherein the sample is subjected to reverse phase absorption before esterification. A method characterized by treating with an agent. 2. A filter having an ultrafiltration membrane, a vessel containing a weak alkaline solution, a reversed-phase short column, a vessel containing an acid solution, a vessel containing a lower alkyl phosphate ester, and a vessel containing a lower alcohol. A kit for analyzing lower carboxylic acids in body fluid materials, comprising a combination of a container containing a 9-anthryldiazomethane solution and a small test tube or a centrifuge tube. 3. A filter comprising a filter, a filter holder, and a short column, wherein the filter has a hollow at least in the center of the filter cotton, and the filter holder has a receiving portion in the short cylinder which matches the shape of the filter. The short column is provided with a through-hole in the center of the receiving part, and the short column is filled with a reversed-phase type column filler in a short cylinder having one end connected to a filter holder, and a funnel-shaped liquid receiving liquid is provided on the holder side. A pretreatment device provided with a mouth for removing a substance that interferes with the determination of a lower carboxylic acid in a body fluid material from the body fluid.