JP2003315320A - Aluminum concentration measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an aluminum concentration measuring method for measuring aluminum concentration with high accuracy. <P>SOLUTION: The aluminum measuring method brings a sample including aluminum and a complexing agent into reaction in a buffer solution so as to form an aluminum complex, and measures aluminum concentration in the sample based on the concentration of the aluminum complex in the reacted mixed solution. As a reagent for preparing the buffer solution, one of MES, MOPS, PIPES, HEPES, HEPPSO, EPPS, ACES, ADA or CHES is used. When one of the reagents is used for preparing the buffer solution, aluminum concentrations in the complexing agent and the buffer solution become small, that is, a smaller blank value, thereby measuring the aluminum concentration in the sample with high accuracy. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an aluminum concentration measuring method for measuring the aluminum concentration in a sample,
In particular, it relates to a method of complexing aluminum to measure the aluminum concentration.

[0002]

2. Description of the Related Art In the United States and Japan, the regulation of the aluminum content in medicines will be enforced, so that the measurement of the aluminum concentration in medicines has become important. Until now, the trace aluminum concentration has been measured mainly by the flameless atomic absorption method or ICP-AES (inductively coupled plasma-auger).
electron spectroscopy), ICP-MS (inductively couple
d plasma-mass spectroscopy). In the United States, attempts have been made to measure the aluminum concentration in injectables for over 10 years, most of which are based on the flameless atomic absorption method.

Flameless atomic absorption method, ICP-AES, ICP-M
The S method is originally highly sensitive to pure samples (ppb to ppb
It is possible to measure aluminum concentration at (t level). However, it is difficult to measure a trace amount of aluminum concentration in a complex matrix such as an injection, a pretreatment operation is required, and sensitivity and reproducibility are remarkably lowered. (S.No
moto et al., Biomed. Res. Trace Elements, Vol.8, 2
45 (1997))

In addition, flameless atomic absorption method and ICP-AE
In addition to the S and ICP-MS methods, as a method for measuring aluminum concentration, aluminum in a sample is complexed, and the concentration of the aluminum complex formed is measured by a wet analysis method such as a titration method or an analysis method such as a fluorescence analysis method. The method is known. When aluminum is complexed and analyzed, since only aluminum or only aluminum and some elements form a complex, the influence of the matrix can be reduced, so a method for measuring trace aluminum concentrations in a complex matrix. Is being watched as.

[0005]

When complexing aluminum, a sample containing aluminum is reacted with a complexing agent, but a buffer solution is added in order to carry out the complexing reaction at a constant pH. There are many.

[0006] Here, various reagents such as potassium phosphate and monopotassium phthalate are known as the buffer solution reagent. However, whichever buffer solution reagent is used, the buffer solution reagent contains impurities as impurities. The less aluminum that is included, the better.

However, some of the conventionally used buffer solution reagents do not always contain a small amount of aluminum contained as impurities. If aluminum is contained in the reagent for the buffer solution, there is a problem that the measurement accuracy of the aluminum concentration is reduced.

The present invention has been made in view of the above circumstances, and an object thereof is to provide an aluminum concentration measuring method capable of measuring the aluminum concentration with high accuracy.

[0009]

[Means for Solving the Problems] Good buffer agents, which have been extensively studied to achieve the above-mentioned objects, are known in the field of biochemistry but are rarely used in high performance liquid chromatography (HPLC) measurement. As a result of focusing on a group of reagents called "Good", we found that among Good's buffers, there are reagents suitable as buffer adjustment reagents for preparing the buffer added during the complexation reaction of aluminum. It was

That is, according to the present invention for achieving the above object, a sample containing aluminum and a complexing agent are reacted in a buffer solution to form an aluminum complex, and the aluminum complex is added to a reaction mixture solution containing the aluminum complex. An aluminum measuring method for measuring the aluminum concentration in the sample from the concentration of an aluminum complex, wherein as a reagent for preparing a buffer solution for preparing the buffer solution, 2-morpholinoethanesulfonic acid (hereinafter referred to as "2-Morpholinoethanesulfonic acid") is used. , MES
, 3-morpholinopropanesulfonic acid (3-Morp
holinopropsnesulfonic acid (hereinafter referred to as MOPS), piperazine-1,4-bis (2-ethanesulfonic acid) (Pi
perazine-1,4-bis (2-ethanesulfonic acid), below, PIP
ES), 2- [4- (2-hydroxyethyl) -1
-Piperazinyl] ethanesulfonic acid (2- [4- (2-Hydroxy
ethyl) -1-piperazinyl] ethanesulfonic acid,
HEPES), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl)] propanesulfonic acid (2-Hydroxy-3- [4- (2-hydroxyethyl) -1-piperaz
inyl] propanesulfonic acid, monohydrate, below, HEP
PSO), 3- [4- (2-hydroxyethyl)-
1-piperazinyl] propanesulfonic acid (3- [4- (2-hyd
roxyethyl) -1-piperazinyl] propanesulfonic aicid,
Hereinafter referred to as EPPS), N- (2-acetamido) -2-
Aminoethanesulfonic acid (N- (2-Acetamido) -2-aminoeth
anesulfonic acid, hereinafter referred to as ACES), N- (2-acetamido) iminodiacetic acid (N- (2-Acetamido) iminodiace
tic acid, hereinafter referred to as ADA), N-cyclohexyl-
2-Aminoethanesulfonic acid (N-Cyclohexyl-2-aminoet
hanesulfonic acid (hereinafter, referred to as CHES)).

[0011]

EFFECTS OF THE INVENTION MES, MOPS, P
When any of IPES, HEPES, HEPPSO, EPPS, ACES, ADA, and CHES is used, the aluminum concentration measurement value in the solution containing the complexing agent and the buffer solution and containing no sample for concentration measurement, that is, blank Since the value becomes small, the aluminum concentration in the sample can be measured with high accuracy.

[0012]

Other Embodiments Here, preferably, the aluminum concentration measuring method is a method of separating an aluminum complex from the reaction mixture using liquid chromatography and detecting the separated aluminum complex with a photometer. . In this way, by separating the aluminum complex from the reaction mixture using liquid chromatography and detecting it with a photometer, the influence of other components other than the aluminum complex can be reduced, so that the aluminum concentration can be more accurately measured. Can be measured.

A drug can be used as the sample.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. As the complexing agent, both a chelating agent that forms a chelate with aluminum and a complexing agent that does not form a chelate can be used. Examples of chelating agents include lumogallion, 8-quinolinol (oxine), and DH.
AB (2,2'-dihydroxyazobenzene), EDTA (ethylenediaminetetraacetic acid), aluminone, eriochrome, cyanine R,
Alizarin S, Stilbazo, Alizarin Garnet R,
Hongtachrome Blue Black R, Bontachrome VSW,
Morin or the like can be used.

The sample for measuring the aluminum concentration is not particularly limited and includes, for example, raw materials for pharmaceuticals, pharmaceuticals, plant tissues, animal tissues, raw materials for health foods, health foods, raw materials for drinking water, drinking water, raw materials for cosmetics, Cosmetics, tea, alcoholic beverages, tap water, environmental water, seawater, lake water, river water, industrial wastewater, industrial water, research reagents, industrial raw materials, antibodies, vaccines consisting of each antigen, serum, urine, plasma, blood, humans and Animal body fluids and the like can be used. The drugs include injections, infusions,
Includes dialysate, antibiotics, blood products, etc.

The sample is preferably in the form of a liquid, but when it is not a liquid, it is made into a liquid sample by solubilization treatment or extraction treatment.

As the buffer solution preparation reagent for preparing the buffer solution, MES, MOPS, PIPES, HEPES, HEPPSO, EPPS, A
Use CES, ADA, CHES. All of these reagents are included in the reagent for preparing a buffer solution widely known in the field of biochemistry as Good's buffer solution.

As a method for measuring the concentration of the aluminum complex in the reaction mixture of the sample, the complexing agent and the buffer solution, a gravimetric method, a volumetric method such as a titration method, an optical analysis method and the like can be used. Here, the optical analysis method includes an absorptiometry method and a fluorescence analysis method. In the case of absorptiometry, as the buffer solution preparation reagent, aluminone, eriochrome, cyanine R,
Alizarin S, 8-quinolinol, stilbazo, etc. are used, and in the case of the fluorescence analysis method, as a reagent for preparing a buffer solution,
Alizarin Garnet R, Hongtachrome Blue Black R, Bontachrome VSW, 8-quinolinol, Morin,
Lumo gallion etc. are used.

When the concentration of the aluminum complex is measured by an optical analysis method, a batch type measurement or a flow injection method is used without separating the reaction mixture of the sample, the complexing agent and the buffer solution. Although it is possible to detect aluminum complex with an absorptiometer or a fluorometer,
Depending on the measurement sample, in addition to the aluminum complex, it may contain components that can be detected by an absorptiometer or a fluorometer.So, separate the aluminum complex from the reaction mixture by liquid chromatography, and separate the separated aluminum complex with an absorptiometer. Alternatively, detection by a fluorometer is preferable because the aluminum concentration in the sample can be measured more accurately.

FIG. 1 shows a high performance liquid chromatograph for separating an aluminum complex from a reaction mixture (hereinafter, referred to as
It is a figure explaining the general structure of 10 called HPLC.

In FIG. 1, the mobile phase solvent (eluent) stored in advance in the solvent tank 12 is introduced into the column 16 by the pump 14 at a predetermined speed, for example, about 1.0 mL / min. The mobile phase solvent may be a solvent suitable for holding and eluting the aluminum complex, such as a buffer solution or a mixed solution of a buffer solution and an organic solvent.

In the injection section 18, a reaction mixture prepared by reacting the sample with the complexing agent in a buffer solution is injected into the column 16 by a syringe (not shown). Column 1
6 is, for example, a stainless tube, for example, an inner diameter of 4.6 m
It is said to be m x 150 mm long. The reaction mixture injected from the injection unit 18 is separated by the column 16 and the detector 2
Introduced to zero. The result detected by the detector 20 is recorded in the recording device 22.

[0023]

EXAMPLE Next, as a complexing agent, lumogallion or 8-
A quinolinol is used to separate an aluminum complex from a mixed solution of a complexing agent and various buffer solutions described later (hereinafter, this mixed solution is referred to as a blank solution) by the HPLC apparatus 10 shown in FIG. An example detected in 1. will be described.
In this example, a fluorescence photometer (fluorescence detector) was used as the detector 20.

First, a calibration curve was prepared for each of the case of using lumogallion and the case of using 8-quinolinol. [Preparation of calibration curve when using lumogalion as complexing agent] Preparation of reagents and samples Reagents were prepared as follows. Preparation of chelating reagent (reagent 1) Lumogalion (chelating reagent for analysis) [Dojindo Laboratories], hydrochloric acid (ultra high purity) [Kanto Chemical Co., Inc.], and ultrapure water (ultra high purity) [Kanto] Chemical Co., Ltd.] was used to prepare a solution of lumogallion 1 mmol / L and hydrochloric acid 1 mol / L. Here, the ultra-high purity reagent is a reagent whose level is guaranteed to be ppt or less with respect to aluminum and other trace elements. The hydrochloric acid is added because the aluminum contained in the measurement sample is once ionized by making the mixed solution of the chelating reagent and the sample acidic. Therefore, other acids such as nitric acid can be used instead of hydrochloric acid, and a neutral chelating reagent can be used when the acid is added to the measurement sample in advance. An organic solvent may be added to the chelating reagent. Preparation of buffer solution (reagent 2) MES (2-Morpholinoethanesulfonic acid, monohydrate)
(Analytical reagent) [Dojindo Laboratories Inc.], sodium hydroxide (ultra high purity) [Kanto Chemical Co., Inc.], ultrapure water (ultra high purity) [Kanto Chemical Co., Ltd.] 0.5 mol / L MES buffer
(pH 7.0) was prepared. Preparation of eluent (reagent 3) 2-propanol (HPLC grade) [Kanto Chemical Co., Inc.],
Using sodium acetate (special grade reagent) [Wako Pure Chemical Industries, Ltd.], hydrochloric acid (ultra high purity) [Kanto Chemical Co., Inc.], and ultrapure water (ultra high purity) [Kanto Chemical Co., Ltd.] 2-propanol
A 25 W / V%, 0.1 mol / L acetate buffer solution (pH 4.6) was prepared. Preparation of samples for calibration curve Preparation of aluminum standard solution (for analysis) for atomic absorption using ultra high purity nitric acid (ultra high purity) [Kanto Chemical Co., Ltd.] and ultra pure water (ultra high purity) [Kanto Chemical Co., Ltd.] ] Diluted with a 0.1 mol / L nitric acid solution prepared from
5, 20, 25, 30, 35, 40, 45, 50 μg / L
The aluminum concentration was adjusted. Preparation of Injection Solution (Reaction Mixture) 50 μL of the chelating reagent (Reagent 1) was placed in a 1.5 mL sample vial, 300 μL of the calibration curve preparation sample was added thereto, and the mixture was rapidly stirred and mixed. Next, 600 μL of the buffer solution (reagent 2) was added, and again rapidly stirred and mixed. Since the reaction mixture becomes neutral by adding the reagent 2, aluminum ions and lumogallion rapidly react and aluminum chelate is quickly formed. Then, the solution was left standing at room temperature (25 ° C.) for 5 minutes to prepare an injection solution (reaction mixture solution). Measurement of aluminum concentration 20 μL of the reaction mixture prepared above was sampled
It was injected into the injection part 18 of the PLC 10 and measured. The measurement conditions of HPLC10 are shown below. By this method, each calibration curve preparation sample was measured twice. Also, the aluminum concentration is 0,
Each sample of 5, 10 μg / L was measured 5 times to confirm reproducibility. HPLC measurement conditions Flow rate 1.0 mL / min Column Develosil LAL 4.6 mm id × 150 mm (Nomura Chemical Co., Ltd.) Column temperature 40 ° C Wavelength Ex. (Excitation wavelength) = 505 nm, Em. (Detection wavelength) = 574n
m measurement result As shown in FIG. 2, the quantitative property was confirmed at least between 0 and 50 μg / L. Moreover, as shown in FIG. 3, the reproducibility was also good. In addition, in FIG. 3, the CV value (= coeffici
ent of variation) is a value calculated by dividing the standard deviation σ by the average value.

[Preparation of calibration curve when 8-quinolinol is used as complexing agent] The reagent for preparing the injection liquid (reaction mixture) is an aluminum measurement kit (Dojindo Co., Ltd.) using 8-quinolinol as a chelating reagent. Laboratory: Kumamoto) was used according to the recommended method. That is, 50 μL of a chelating reagent solution containing 8-quinolinol and hydrochloric acid was placed in a 1.5 mL sample vial, and 150 μL of the same sample for preparing a calibration curve as in the case of lumogarion was added thereto and rapidly stirred and mixed. Next, add 400 μL of the buffer solution adjusted to pH 7.5, and again,
The mixture was rapidly stirred and mixed, and allowed to stand at room temperature (25 ° C.) for 10 minutes to prepare an injection liquid (reaction mixture liquid). Measurement of Aluminum Concentration 200 μL of the reaction mixture prepared as described above was sampled and injected into the injection part 18 of the HPLC 10 for measurement. HPLC 1
The measurement conditions of 0 are shown below. The eluent is a solution containing a surfactant and having a pH of 7.0. By this method, each calibration curve preparation sample was measured twice. HPLC measurement conditions Flow rate 1.0 mL / min Column Aluminum analysis column 4.6 mm id × 50 m
m (Shinotest Co., Ltd.) Column temperature 25 ° C Wavelength Ex. = 370 nm, Em. = 504 nm Measurement results Similar to the case of Lumogarion, the calibration curve showed good linearity.

[Measurement of Aluminum Concentration in Blank Solution]
Next, the aluminum concentration in the blank solution was measured based on the prepared calibration curve. Preparation of Reagents Chelating reagents and eluents include lumogallion and 8-
Both quinolinol was prepared in the same manner as in the case of preparing the calibration curve. Preparation of buffer solution The following buffer solution preparation reagents, sodium hydroxide (ultra high purity) [Kanto Chemical Co., Inc.], ultrapure water (ultra high purity) [Kanto Chemical Co., Ltd.] Was used to prepare a pH 7.0 buffer solution in which the concentration of each buffer solution preparation reagent was 0.5 mol / L. Buffer solution reagents MES, MOPS, BES, PIPES, HEPES, TES, HEPPSO, EPPS, M
OPSO, Bis-Tris, ACES, ADA, CHES, potassium phosphate Here, BES is N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (N, N-Bis (2-hydroxy
ethyl) -2-aminoethanesulfonic acid), and TES means N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid.
aminoethanesulfonic acid), and MOPSO means 2-
Hydroxy 3-morpholinopropanesulfonic acid (2-Hydro
xy-3-morpholinopropanesulfonic acid), Bis-
Tris is bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis (2-hydroxyethyl) imin
It means otris (hydroxymethyl) methane). The buffer solution preparation reagents are all analysis reagents, potassium phosphate is manufactured by Wako Pure Chemical Industries, Ltd., and the others are manufactured by Dojindo Laboratories, Inc. Preparation of Blank Solution and Measurement of Aluminum Concentration For both lumogalion and 8-quinolinol, a blank solution (injection solution) was prepared in the same manner as in the case of preparing the calibration curve, except that the measurement sample was not added. The method for measuring the aluminum concentration is also the same as in the case of creating the calibration curve. Measurement results Table 1 shows the measurement results. As shown in Table 1, MES, MOP
When S, PIPES, HEPES, HEPPSO, EPPS, ACES, ADA, CHES is used as a reagent for preparing a buffer solution, the measured value, that is, the blank value becomes a small value even if any complexing agent is used. It can be seen that the use of the buffer solution of 1) enables reliable measurement of the aluminum concentration. (Table 1) ────────────────────────────── Measured value of buffer solution reagent (ppb) Lumogalion 8-quinolinol ── ──────────────────────────── MES 1.5 4.0 MOPS 3.2 11.6 BES 89.2 178.6 PIPES 1.4 6.6 HEPES 3.7 16.1 TES 10.3 39.1 HEPPSO 4.2 9.2 EPPS 2.4 5.2 MOPSO 9.4 33.5 Bis-Tris 127 522.6 ACES 4.9 7.0 ADA 4.8 5.3 CHES 2.1 7.3 Potassium phosphate 121 538.3 ─────────────────────────── ────

Although the present invention has been described above based on the embodiments, the present invention is not limited to the embodiments, and the present invention includes various modifications and improvements based on the knowledge of those skilled in the art. Can be implemented in.

[Brief description of drawings]

FIG. 1 is a diagram illustrating a general configuration of a high performance liquid chromatograph used to carry out the present invention.

FIG. 2 is a diagram showing a calibration curve created in Examples.

FIG. 3 is a diagram showing the reproducibility of a calibration curve.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/00 G01N 33/00 D // G01N 30/04 30/04 P 30/26 30/26 A 30 / 48 30/48 K 30/74 30/74 EF 31/22 122 31/22 122 F term (reference) 2G042 AA01 BC12 CA02 CA10 CB03 DA06 DA08 EA01 FA01 FA06 FA14 FA20 FB02 GA05 HA07 2G052 AA00 AB01 AD26 AD46 GA27 HB07 2G054 AA10 AB07 BA04 BB04 CA10 CE01 EA06 GA04 GB01

Claims (3)

[Claims] 1. A sample containing aluminum and a complexing agent,
An aluminum measuring method of reacting in a buffer solution to form an aluminum complex, and measuring an aluminum concentration in the sample from a concentration of the aluminum complex in a reaction mixture containing the aluminum complex, wherein the buffer solution is prepared. As a buffer preparation reagent for
2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), 2- [4- (2
-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl)] propanesulfonic acid (HEPPSO), 3- [4- ( 2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS),
N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N- (2-acetamido) iminodiacetic acid (AD
A), N-cyclohexyl-2-aminoethanesulfonic acid
(CHES) is included, The aluminum concentration measuring method characterized by the above-mentioned. 2. The method for measuring aluminum concentration according to claim 1, wherein the aluminum complex is separated from the reaction mixture using liquid chromatography, and the separated aluminum complex is detected with a photometer. 3. The aluminum concentration measuring method according to claim 1, wherein the sample is a drug.