JPH01229972A - Determining method of low carboxylic acid in body fluid material - Google Patents

Abstract

PURPOSE:To enhance the reaction rate between a low carboxylic acid to be measured and a labelling esterified agent and to make it possible to perform accurate analysis as a result, by treating a body fluid sample with adsorbing agent in a reverse phase type, and removing the interfering substance in the sample. CONSTITUTION:0.2ml of human plasma undergoes ultrafiltration by using a Centriflow CF 25, and liquid of 1ml is obtained. In this liquid, a 10ml of 7% NaHCO3 solution is added, and the solution having pH of about 8.0-8.5 is prepared. Said solution is loaded in a C18 cartridge. The passed liquid is picked up into a Sep-Pak common-plug test tube made of glass, and 6NHCl is added so as to obtain acidity. Thereafter, 0.5ml of tri-n-butyl phosphate is added and extracted by 3min oscillation. After centrifugal separation, 0.3ml of tri-n-butyl phosphate is picked up into another brown test tube. 0.2ml of methanol of 0.2ml and 0.1ml of ADAM solution (3mg/ml acetone solution) are added. The solution is made to react for about 2hr at room temperature. In addition, 1ml of standard oxalic acid liquid (0.2, 0.5, 1.0 and 2.0mug/ml) and 5ml of reacting liquid that is handled by the same way are injected into HPLC. The determination is performed by an absolute calibration curve method utilizing a peak height.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a method for quantifying lower carboxylic acids present in body fluid materials such as plasma.

[Prior Art] Various lower carboxylic acids exist in body fluids such as blood, and abnormal concentrations of these acids are often indicators of specific diseases, dysfunctions, or metabolic disorders. Of interest for physical or clinical examination. For example, oxalic acid is associated with kidney and urolithiasis, propionic acid is associated with propionic acidemia, and acetoacetic acid and β-hydroquinoacetic acid are indicators for understanding the pathology of diabetes and are associated with abnormal glucose metabolism. In addition, pyruvic acid and lactic acid are associated with glycolysis, α-ketoisocaproic acid, α-ketoisovaleric acid, and α-ketoglutaric acid are associated with abnormal amino acid metabolism, and malic acid, fumaric acid, and citric acid are associated with metabolic regulation. It is taken up for analysis as a component.

An enzyme method for oxalic acid is available as a method for measuring lower carboxylic acids in such body fluid materials, such as plasma. In this method, oxalate is decomposed with oxalate oxidase and the amount of oxalate is measured based on the pH change due to the generation of carbon dioxide gas. However, this method is difficult to apply due to complicated operations and poor accuracy (the concentration of oxalic acid in blood is only 1/50-100 of that in urine).
There is a drawback. In addition, as a method for measuring oxalic acid in urine rather than plasma, oxalic acid is extracted with trilobyl phosphate, esterified with 9-anthryldiazomethane (abbreviated as ADAM), and then subjected to high-performance liquid chromatography. A method of separating by chromatography (abbreviated as H'LC) and measuring fluorescence (Bulletin of Urology, Vol. 29, No. 3, pp. 287-292, 1983
). However, this method cannot be directly applied to plasma, which contains a large amount of various substances compared to urine.

[Problems to be Solved by the Invention] The inventor thought to improve the above-mentioned urinary injection using ADAM so that it could be applied to body fluid materials as a method for determining the concentration of lower carboxylic acids in body fluid materials. Ta.

[Means to solve the problem] The main reason why the urinary oxalation method cannot be directly applied to body fluid materials is that there are large amounts of substances (other than oxalic acid) that consume ADAM in body fluid materials. I found out that it is. Possible interfering substances include higher fatty acids such as stearic acid, palmitic acid, and oleic acid, prostaglandins, and phospholipids.
Since these are all acidic substances like lower carboxylic acids, there has been no suitable method to separate and remove only the interfering substances. It has also been found that the presence of other fat-soluble substances (fat, fat-soluble vitamins, etc.) in body fluid materials is also a cause of interfering with layer separation during solvent extraction and reducing extraction efficiency. While considering various methods for pretreating body fluid materials, the inventor used a reversed-phase chromatography adsorbent as a pretreatment agent, and found that the above-mentioned interfering substances could be efficiently removed by this method. I found it. This invention was made based on such knowledge.

[Structure of the Invention] That is, the present invention provides a method for quantifying the carboxylic acid by esterifying a lower carboxylic acid in a protein-depleted body fluid material with a labeled esterifying agent and measuring the label. The present invention provides a method characterized in that the material is treated with a reverse phase type absorbent.

This invention also provides a kit that combines instruments and reagents for carrying out the above method, including: (1) a filter with an ultrafiltration membrane, (2) a container containing a weak alkaline solution, (2) a reversed-phase column, (2) Combine a container containing an acid solution, ■ a container containing phosphoric acid lower alkyl ester, ■ a container containing lower alcohol, ■ a container containing 9-anthryldiazomethane solution, and ■ a small test tube or centrifuge tube. Shino provides a kit for analyzing lower carboxylic acids in body fluid materials.

Furthermore, the present invention is not limited to the above-mentioned method, but is composed of a filter, a filter holder, and a short column as an instrument convenient for pretreatment in various body fluid component analysis methods, and the filter is provided at least in the center of the filtration surface. The filter holder is a short cylinder with a receiving part that matches the shape of the filter and a through hole in the center of the receiving part, and the short column is a short column with one end connected to the filter holder. The cylinder is filled with column packing material and a funnel-shaped liquid receiving port is attached to the holder side.
A body fluid material pretreatment device is provided.

[Embodiments] (1) In the method of this invention, the body fluid material includes:
Included are materials that contain all or most of the liquid components of blood and tissue, such as plasma, serum, tissue extracts, and the like.

Protein removal can also be carried out by precipitation with trichloroacetic acid, perchloric acid, etc., or is conveniently carried out by ultrafiltration. Ultrafiltration is usually performed using a membrane filter. Such filters include those commercially available under the names of Centriflow CF-25, Ultracent-30, and the like.

Examples of lower carboxylic acids include acetic acid, propionic acid, citrate, malonic acid, succinic acid, malic acid, fumaric acid, citric acid, lactic acid, pyruvic acid, β-hydroquinebutyric acid, acetoacetic acid, α-ketocaproic acid, α-Ketoisovaleric acid, α
- includes aliphatic carboxylic acids with up to 6 carbon atoms, usually 5 or 4 ([!II or more), such as ketoglutaric acid, with simple substituents such as hydroquine or oxo groups. Most of the lower carboxylic acids are normal biological components, but they also include substances that do not normally exist in living organisms, such as substances that appear when drugs are injected.

Labels in the labeled esterification agent include fluorescent labels, ultraviolet absorption labels, and the like. In addition, moieties that perform esterification include alcoholic hydroxyl groups or their active forms (for example, halogens such as chlorine and bromine, sulfonyloquines such as methylsulfonyloquino and tolylsulfonyloquine, diazo, etc.). A specific example of the fluorescently labeled esterifying agent is 9-anthrylnoadimethane (A
DAM), 9-chloromethylanthracene, 7-acedoquine-4-bromomethylcoumarin, 4-bromomethyl-
7-Medoxycoumarin, N-(7-nidrobenzo2-
oxa-1,3-diazol-4-yl)aziridine (
NBD anolinone) etc. are included. Further, examples of the ultraviolet absorption labeled esterification agent include p-bromphenacylbromide, 9-phenylphenanlubromide, and the like.

For reversed phase type adsorbents, the carrier material contains lipophilic groups,
Examples include adsorbents having, for example, 15-30% chemically bonded aliphatic groups. As the carrier material, silica is most often used, but polystyrene, dextran, etc. can also be used. As lipophilic groups, octylnoryl group,
Higher alkylinlyl groups such as octadenlucylyl groups are preferred, and these are introduced by reaction with the support material in the form of a silyl-lylating agent, for example a silyl halide.

Such adsorbents and columns packed with them are known, and it is convenient to use commercially available ones. Suitable ones include, for example, Sep-Pak C
I8 Cartridge (Japan Waters Limited)
, Chemco Sesob 0DS-B (Kemco Tsuji), and Pond Elut Cl8 (Analyte Chem International).

This method is carried out, for example, as follows.

First, body fluid material, such as plasma, is deproteinized, preferably by ultrafiltration. The protein removal solution is adjusted to near neutrality or weakly alkaline, for example pH 6-9, with a weak alkaline solution that does not contain carboxylic acid, such as an aqueous sodium bicarbonate solution, and is preferably treated with a reversed phase type adsorbent packed in a column. do. The effluent or filtrate is acidified, for example by treatment with an acid such as hydrochloric acid or sulfuric acid, and an organic solvent for extraction, preferably trilower esters of phosphoric acid (lower esters generally have 1 to 1 carbon atoms), is used for extraction.
6, usually 1-5 saturated or unsaturated aliphatic hydrocarbons),
For example, extraction is performed with tributyl phosphate, tripropyl phosphate, triamyl phosphate, dibutylmethyl phosphate, diethylvinyl phosphate, etc., or a mixture thereof. At this time, the amino acids remain without being extracted. preferably adding a lower alcohol, such as methanol or ethanol, to the extract;
Labeled esterification agent, e.g. h″ of excess A D A M
Add a solvent solution, preferably an acetone solution. After reacting at room temperature with slight heating, the reaction mixture is subjected to high-performance liquid chromatography (IPLC), and the amount of frucnoyone containing the target acid ester is quantified using a label. HP
The L C is preferably carried out using a silica- or alumina-based reversed-phase packing, such as a column packed with octane resin. Such columns are known and are commercially available, for example, under the names Nucleonol 5C8, Licrosolve 5C8, Nmupac Brev C8, etc. For detection, a fluorescence detector, an ultraviolet detector, etc. are used depending on the label.

In this way, carboxylic acids of known concentrations are measured, a calibration curve is created, and analytes of unknown concentrations are quantified. Since the lower carboxylic acid content in blood samples is extremely high, such as several micrograms/Fl&, it is
The reagents and solvents used must be of extremely high purity.

In the method below, Cebu Pack CI8 was used as an adsorbent.
When using Cartrits, the pH is suitably around 8-9. Since the reaction rate of esterification with ADAM is slow in a phosphoric acid ester, it is preferable to add methanol, and the amount added is preferably around 30-40%. ADA
The concentration of M is suitably 0.02% or more in the case of 1μ9/1Q oxalic acid, and preferably about 0.05-0.1%.

The reaction time reached approximately 85% in 30 minutes at room temperature and was complete in 15 hours. In HPLC, when using the medium polarity packing column Nucleonol 5C8, an acetonitrile/water system is suitable as the mobile phase, and the ratio is 7.
3 is preferred.

(2) In the kit of the present invention, the containers and instruments are made of plastic or glass. As the reagent, solvent, and adsorbent, those explained in Section 1-(1) are used. It is desirable that these be sealed by a stopper or by melt sealing.

(3) The device of the invention is suitably made of corrosion-resistant materials, such as plastic or glass.

As the filter, an ultrafiltration membrane, a semipermeable membrane, etc. are used. Ultrafiltration membranes are used that leave substances with a molecular weight above a certain value of 20,000 to 50,000 on the membrane, and pass substances with a molecular weight below that, such as polystyrene, vinyl chloride, acrylic copolymers, and polysaccharide-based membranes. It is made by gluing a membrane onto paper. The filter is concave, preferably conical, at least in its central portion.

The short tube in the filter holder preferably has a circular cross section, and suitably has an inner diameter that does not vary significantly. The receiving portion preferably has a shape that allows it to come into close contact with the filter.

The connecting portion between the short column and the boulder is preferably removable, and can be connected, for example, by grinding.

In addition to reversed phase type adsorbents, column packing materials include
It can be filled with various fillers depending on the purpose, such as silica gel, alumina, ion exchange resin, molecular nitrogen, porous adsorption resin, etc. The exposed surfaces of the filler layer are preferably covered with a porous or escape-preventing material such as glass wool. The funnel-shaped liquid receiver [2] is constituted by the inner surface of the short wall.

Furthermore, a collection container such as a centrifuge tube or a test tube can be connected to the other end of the holder.

[Effects] The quantitative method of the present invention removes various interfering substances existing in the blood sample in advance by treating the body fluid sample with a reversed-phase adsorbent. The reaction rate with the esterifying agent increases, and as a result, highly accurate analysis becomes possible. Further, the amount of labeled esterifying agent used is small, and the reaction system is easy to handle.

However, since it only requires a simple operation of treatment with a reversible type adsorbent, it has the advantage of being easy to implement and can be implemented in a short time.

The kit of the present invention contains all the reagents and equipment necessary to carry out the above-mentioned measurement method, so it can be carried out using, for example, several grams or more! This eliminates the waste of purchasing reagents and solvents that require only a small cliff of about 1a in sales units such as 5009 or Shoulder Q. In addition, there is no need to prepare extra equipment in case it is not possible to carry out the test in a timely manner due to lack of equipment, etc., and appropriate dimensions of the equipment can be selected in advance, which is convenient for aligning the implementation conditions.

The device of this invention is used for the following purposes. When analyzing drugs or biological components in body fluids 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 a pretreatment. For this purpose, protein removal and separation and purification means are required in accordance with each purpose, but it is unavoidable that there is a disadvantage that sample loss is involved in this process. However, in order to obtain as accurate a value as possible, it is desirable to purify the target substance by a means that can efficiently remove protein and purify the substance in as short a time as possible, with little loss of sample.

The device of this invention was created to achieve the above-mentioned purpose, and since it is equipped with a filter and a filler, body fluid materials can be pretreated with just this device. Furthermore, since the filtration membrane and the adsorbent are attached to separate cylinders and the cylinders are detachable, refilling and cleaning are extremely convenient.

In addition, the filter holder is provided with a receiving part that matches the shape of the filter, and a through hole is provided in the center of the receiving part, so the filter does not tear and the filtrate falls in an orderly manner. Moreover, since a funnel-shaped liquid receiving port is attached to one end of the Noyote column, the fallen filtrate is guided to the column in an orderly manner.

[Examples] Hereinafter, the present invention will be described in more detail based on Examples.

Example 1 (Quantitative Method) (Method) Human plasma 2 mρ was ultrafiltered (toooc, 30 minutes) using Centriflow CF25 (Amicon), and the filtrate L+ (7%-N a I-I CO3 solution 10 μ The p[I was adjusted to about 8.0 to 8.5 by adding diluted water. This solution was loaded into a SEBUPAC C18 cartridge, and the passed liquid was transferred to a glass stoppered test tube (inner diameter 8 ++ on, length 8 cm,
After adding 6N-HC and 20 μC to acidify (pI-I l, O ~ 2.0), add 0.5 mQ of tri-n-butyl phosphate, and shake the system for 3 minutes with a shaker. I finally extracted it. Centrifugation (3000 rpm,
After I 0 min), tri-n-butyl phosphate layer 0,3
Transfer txQ to another brown test tube and add 0.2 m of methanol.
Q,, A D A M solution C3H/mQ acetone solution:
(Prepared before use) O, Iu& were added and allowed to react at room temperature for about 2 hours. Separate oxalic acid standard solution (0,2,05, l O12,
0μ9/zQ)lx(was operated in the same manner. Reaction solution 5μa
was injected into I-I PLC and quantified using the absolute detection line method based on peak height.

(HPLC measurement conditions) The column was a reversed-phase Nucleonol 5C8 (inner diameter 4°6 n
+ m, length 25 cm), acetonitrile-water mixture (7:3) was used as the mobile phase, and the flow rate was 1. OmQ was set to 1 minute.

Fluorescence detection uses an excitation wavelength of 254 nm and a measurement wavelength of 412 nm.
I went there.

(Identification of oxalate diester in blood) Oxalate in plasma was identified by treating human plasma with oxalate oxidase.

That is, after protein removal from human plasma by ultrafiltration, it was divided into two test tubes, and one was injected with 50 μm2 of oxalate oxidase (Boehringer Mannheim) (0°56 U/shoulder Q).
) was added, and the reaction was carried out at 37°C for 130 minutes. 50 μQ of water was added to the other, and the same operation and analysis were conducted. the result,
Regarding the oxalate okinguse-treated sample, the peak assumed to be that of oxalate noester disappeared because the oxalate was decomposed by the enzyme. Furthermore, oxalic acid standard solution 1.0μ910. As a result of analysis with the addition of IyQ, the peak assumed to be derived from oxalate diester in plasma overlapped with the peak of the oxalate standard solution, and the peak height increased. From the data of the above two tests, the peak with a retention time of about 14 minutes was confirmed to be an ester compound of oxalic acid in plasma.

(Additional recovery rate) Add 0.4 to 2.0μ9 of oxalic acid to plasma 2xC,
The recovery rate was determined under two-legged measurement conditions. As shown in Table 1, good results of 98.34 to 100.0% were obtained.

(Calibration curve) Oxalic acid bleaching solution 0.2 to 2.0 μg/m (1 m degree and peak height showed good correlation (r=0.9994) (Figure 1). Detection limit was 0. 1 μg/M (l plasma), and the coefficient of variation of this quantitative method was good at 0.59 to 2.43% in the concentration range of 0.2 to 1.07 gr/+πQ.

(Correlation between illegality and enzymatic method) The enzymatic method is widely used for the analysis of oxalic acid contained in urine and food [Method of Enzymatic Analysis (Method of Enzymatic Analysis).

) p. 1542] and illegality.

The enzymatic method has a high limit of quantification, so 20 and 40μi/mQ
Using the oxalic acid concentration, this solution was illegally diluted 20 times and measured. As a result, r = 0.9 for illegal and enzymatic methods.
A good correlation with 930 was obtained (Fig. 2).

Table 1 (Note) * indicates the average Hanato standard deviation.

Example 2 (Kit) ■A filter with an ultrafiltration membrane (the membrane should filter out substances with molecular m below a certain value of 20,000 to 50,000.The shape is as shown in Figure 3) ■7% Naf ( Ampoule containing CO3 aqueous solution ■ Reverse-phase tanto ram (filling material is μ Bonda Pack 018 with low polarity) ■ Ampoule containing 6 N -1-1C & ■ Ampoule containing tri-n-butyl phosphate ■ Adding methanol Ampoule ■Acetone solution of 9-anthrylnoazomethane (3m9
/ Ampoule containing n(2) ■Inner diameter 8 mm, length 8
cm stoppered glass test tube Example 3 (apparatus) In FIG. 3, the apparatus of the present invention includes a filter 1,
It consists of a filter holder 2 and a short column 3. The filter 1 is preferably a conical ultrafiltration membrane. The boulder 2 is provided with a receiving part 22 in a short cylinder 21 that can be brought into close contact with the filter 1. A through hole 23 is bored 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 thereof is filled with a filler 32. Filter plates 33 are in contact with both the upper and lower sides of the filler. A liquid receiving port 34 is provided on the side connected to the holder 2. A test tube 4 is connected to the other side of the column. The holder 2, column 3 and test tube 4 are made of a corrosion-resistant material such as glass or polypropylene or acrylic resin.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the test m line 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, 3, t...liquid receiving port. Figure 1 0 0.5 Yu, 0 1
5 2.0 Figure 2

Claims (3)

[Claims] (1) In the method of quantifying the carboxylic acid by esterifying the lower carboxylic acid in the deproteinized body fluid material with a labeled esterifying agent and measuring the label, the sample is esterified with a reversed-phase absorbent before esterification. A method characterized by processing. (2) [1] Filter with ultrafiltration membrane, [2] Container containing weak alkaline solution, [3] Reverse phase short column, [4] Container containing acid solution, [5] Lower phosphoric acid Container containing alkyl ester, [6] Container containing lower alcohol, [7] Container containing 9-anthryldiazomethane solution,
and [8] A kit for analyzing lower carboxylic acids in body fluid materials, which comprises a combination of small test tubes or centrifuge tubes. (3) Consisting of a filter, a filter holder, and a short column, the filter has a recess at least in the center of its filtration surface, and the filter holder has a receiving part that matches the shape of the filter inside the short cylinder. A short column is a short column with one end connected to a filter holder, filled with column packing material, and a funnel-shaped liquid receiving port attached to the holder side. A body fluid material pretreatment device.