JPS6031055A - Sample pretreatment method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a sample pretreatment method for separating and analyzing anionic or cationic species in biological fluids using ion chromatography. Furthermore, when removing proteins from biological fluids, biological fluids can be directly injected into a small-capacity column packed with resin without using organic solvents (ethanol, acetonitrile, etc.) or strong acids (perchloric acid, trichloroacetic acid, etc.), and can be removed on the same day. It is characterized by a sample pretreatment method that allows rapid measurement of ions using a liquid.

Traditionally, colorimetric methods have been used to measure ions in biological fluids.

There are precipitation titration methods, 1 emission spectrometry methods, atomic absorption spectrometry methods, and methods using ion-selective electrodes, but recently, ion chromatography, which can separate and analyze several types of ions at the same time, has been used. When analyzing ions in biological fluids using ion chromatography, proteins and other organic substances in the sample are adsorbed onto the separation column and interfere with normal ion exchange between the ion exchange groups of the resin substrate and the ionic species. It is necessary to remove significant proteins and organic matter. However, there are extremely important problems with conventional protein removal methods that involve adding perchloric acid, trichloroacetic acid, ethanol, or acetonitrile.

First, in the method using acids, when analyzing anions in biological fluids, excess perchlorate ions, trichloroacetate ions, etc. As with other ions, ions are exchanged on the ion exchange resin, so it may interfere with the normal ion exchange reaction of the ion species to be measured, or an extremely large peak of perchlorate ion or l-lichloroacetate ion may appear on the R1 madogram. Therefore, it is difficult to limit the target anion.

On the other hand, in the method using 41 solvents, various specific phenomena occur. That is, in the ethanol or acetonitrile deproteinization method, the recovery rate of phosphate ions and calcium ions in biological fluids is extremely low, making it impossible to accurately measure their concentrations.

The present inventors have made extensive efforts to eliminate the drawbacks caused by conventional pretreatment methods and to develop a simpler sample pretreatment method when measuring ionic species in living organisms using ion chromatography. The present invention has now been completed.

That is, the present invention particularly relates to the separation and analysis of ionic species in biological fluids using ion chromatography, in which biological fluids are passed through a small-capacity column packed with polystyrene, polyacrylate, and/or these ion exchange resins, and proteins and This is a pretreatment method for biological fluids characterized by adsorbing and removing other organic substances onto the resin.

Hereinafter, the present invention will be explained in detail with reference to the drawings.

Typical examples of devices for measuring ionic species in biological fluids according to the present invention include liquid pumps, anions, It consists of an F♂ theory reed and an electrical conductivity detector.

An example of a small capacity column used in the present invention is shown in FIG.

This small capacity column body (1) can be used as it is with a commercially available syringe (
4), and a resin (2) for removing proteins and other organic matter is filled through a filter (3). Main body and filter material F,
1. It is made of plastic, and the size of the main body is 5mm to 2mm in inner diameter.
0 mm and the length ranges from 5 mm to 70 mm. The size of the column is determined by considering the adsorption capacity of the resin for proteins and organic matter and the amount of biological fluid to be injected, but preferably the column has an inner diameter of 7 am to 15 ws and a length of 10 I.
III to 60.

Silica,
Alumina, dextran. cellulose.

Polyacrylic, polystyrene, and the like can be used, but polyacrylic and polystyrene fillers are most preferred in view of adsorption ability due to hydrophobic interactions with proteins and other organic substances. Further, especially considering the molecular size of the protein, the pore size of the filler is controlled to be in the range of 100 to 100X, and preferably 250 to 500X.

It has also been found that an ion exchanger in which ion exchange groups are introduced onto a resin base material is also effective as a pretreatment for biological fluids in order to more effectively remove ionized organic substances. The exchange capacity of this ion exchanger is α1 to 5 meq/9, and when anions are analyzed, sulfonic acid groups or carboxyl groups are analyzed, and when cations are analyzed, quaternary amine groups and 3 It is preferable to use an ion exchanger having a class amine group.

The amount of various resins packed into a small capacity column is in the range of α5 to 20 wLt depending on the column size, but is preferably Fi[13 to B-.

Furthermore, depending on the purpose of measurement, the above resins may be used alone, resins with different pore sizes may be combined, or resins that do not have ion exchange groups and resins that do have ion exchange groups may be combined.

The present invention will be further explained below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 The ion chromatograph used to measure the sample was manufactured by Toyo Soda Kogyo ■ [aLc-601J (trade name), and the column for anion analysis was manufactured by Toyo Soda Kogyo ■ [sxgelIC-=A
n1on-pwj (trade name) was added to the eluent Kti 1.
3 mM potassium gluconate and 1.5 mM borax/12 thiacetonitrile (pHa5) were used.

Figure 2 shows diluted serum as a sample and a resin ([TSK gel, 5tyrene-
2507 "Day K gel 5tyrene-60j
(Toyo Soda Kogyo ■Product name) and (-TsxgelSC!
X(H”)J (Toyo Soda Kogyo ■Product name) 0.5 each
- shows a chromatogram obtained through a three-phase system). The phosphate ion is 6.6 Iθia L.

Comparative Example 1 Under the same elution conditions as in Example 1, serum was mixed with 1:1 ethanol.
Add at a ratio of 1:1, shake, and centrifuge (3500 rpm).
, 5 minutes), the supernatant was diluted to the same dilution rate as in Example 1, and the chromatogram obtained is shown in FIG. The peak of phosphate ion decreased drastically and did not show normal value.

Comparative Example λ Serum was diluted under the same eluent conditions as in Example 1, and resin (rTS K gel oI) was packed into a small volume column.
FIG. 4 shows a chromatogram obtained using -12ON (trade name, manufactured by Toyo Soda Kogyo ■, 1d).

When treated with a silica-based resin, it is not possible to completely remove organic substances other than proteins in serum, and there is an extreme baseline drop on the chromatogram due to the influence of organic substances, and the original It took 25-50 minutes to return to baseline.

Example 2 The apparatus was the same as Example 1, and the column for cation analysis was "TSKgel 0-Oation J (trade name)" manufactured by Toyo Soda Kogyo ■, and the eluent was [15mM ethylenediamine (p
Serum was diluted using resin (TSK gel 5tyrene-25) and packed into a small volume column.
0” and “TSK gel 5tyrene-60
J Toyo Soda Kogyo ■Products each 0.5m/two-phase system)
The chromatogram obtained through this process is shown in FIG.

The free calcium concentration in the serum was 2.4 mθφ.

Comparative Example 5 Under the same elution conditions as Example 2, ethanol was added to serum at 1=
Add at a ratio of 1:1, shake, and centrifuge (3500 rpm).
, 5 minutes), the supernatant was diluted at the same dilution rate as in Example 2, and the obtained chromatogram is shown in FIG.

The peak of free calcium ions in the serum was drastically reduced to less than one-fourth and did not show normal values.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the configuration of a small-capacity column filled with a serum pretreatment resin and equipped with a syringe barrel. FIG. 2 is a chromatogram of inorganic anions obtained by treating serum with a pretreatment resin. FIG. 3 is a chromatogram of inorganic anions obtained by removing protein from serum with ethanol. FIG. 4 is a chromatogram of inorganic anions obtained by treating serum with a silica-based pretreatment resin. FIG. 5 is a chromatogram of alkaline earth metal ions obtained by treating serum with a pretreatment resin. FIG. 6 is a chromatogram of alkaline earth metal ions obtained by removing protein from serum with ethanol. 1. Small capacity column 2 Pretreatment resin 5 Filter 4. Syringe 5 Phosphate ion & sulfate ion l Magnesium ion a Calcium ion Patent applicant Toyo Soda Kogyo Co., Ltd. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

Claims (1)

[Claims] 1) When separating and analyzing anions or cations in biological fluids by ion chromatography, proteins and other organic substances are removed using a small-capacity column packed with resin. Sample pretreatment method. 2) The pretreatment method according to claim 1, wherein the resin to be filled is an ion exchanger. 3) The pretreatment method according to claims 1 and 2, wherein the resin to be filled is a polystyrene base material. 4) The pretreatment method according to claims 1 and 2, wherein the resin to be filled is a polyacrylate base material.