JPS61128166A - Simultaneous analysis of creatine and creatinine - Google Patents

Abstract

PURPOSE:To perform the simultaneous analysis of creatine and creatinine with high accuracy, by applying the eluate, which was obtained by applying a specimen after protein removal treatment to reversed-phase partition chromatography, to reversed-phase partition chromatography of an acidic moving phase to measure the ultraviolet absorbancy of the eluate. CONSTITUTION:A specimen such as serum, to which a perchloric acid solution was added under mixing, is centrifugally separated to obtain a protein removed supernatant solution which is, in turn, flowed through a reversed-phase chromatographic column, wherein an octadecyl group or a trimethyl group was chemically bonded to a silica gel, by using a buffer solution with pH of 2.0-7.0 as a moving phase to obtain a creatin and creatinine-containing solution from which impurities in the specimen were removed. This eluate is applied to reversed-phase ion-pair chromatography by using a buffer solution, of which the pH is adjusted to 2.0-3.0 by phosphoric acid, as a moving phase to separate and elute creatin and creatinine. The ultraviolet absorbancy of this eluate is measured at a wavelength of 120nm. By this method, simultaneous analysis is enabled with high sensitivity and good reproducibility.

Description

Detailed description of the invention (a) Industrial application field This invention utilizes reverse phase column chromatography to
This invention relates to a simultaneous analysis method for separating and analyzing creatine and creatinine in a sample.

(B) Conventional technology Creatine and creatinine-containing samples, for example, creatine in serum increases in primary muscle diseases, myositis, muscle atrophy, hyperthyroidism, etc., and creatinine in serum is an accurate indicator of renal dysfunction. known as. Conventionally, both components have been measured by a colorimetric method using Jaffe reaction, but there are some problems in reproducibility and sensitivity. Furthermore, as a method for analyzing both components using high-performance liquid chromatography (HPLC), it is known to separate the two components using gradient elution, perform a post-column reaction using ninhydrin as a reaction reagent, and perform fluorescence detection. (Hiraga, Y.
,.

Kinoshita, T.; J, Chromato
gr, 226.43 (1981)), this method is somewhat complicated as a means of measuring only both components. Regarding the measurement of only serum creatinine, a method has been reported in which creatinine is separated by HPLC using a mobile phase of a fixed composition and measured using an ultraviolet absorbance detector (Ambrose, R.T., Ketc
hum, D., F., Sm1th, J.

W, ; C11n, Chem, 29.256
(1983) ), there is no similar report regarding creatine. In addition, creatine in the above serum was f(PLC)
When performing separation using an ultraviolet absorbance detector,
Due to sensitivity requirements, it was necessary to set the wavelength to a short wavelength around 210 nm, and measurement was difficult due to the influence of impurities in serum.

As mentioned above, there is no known method for simultaneously analyzing both creatine and the components of a creatinine-containing sample with high sensitivity, good reproducibility, and a simple method.

(c) Purpose This invention was made in the above situation, and
The purpose of this invention is to provide a method for separating creatine and the components of a creatinine-containing sample and easily analyzing both components with high sensitivity and good reproducibility.

(D) Structure This invention provides creatine and creatinine-containing samples obtained either as they are or by previously subjecting the samples to deproteinization treatment and then subjecting them to reverse phase column chromatography to remove impurities in the samples. The creatinine-containing eluate is subjected to reverse-phase ion pair column chromatography with an acidic mobile phase to separate and elute creatine and creatinine, and the ultraviolet absorbance of the obtained eluate is measured to analyze creatine and creatinine. This provides a simultaneous analysis method for creatine and creatinine.

Creatine and creatinine-containing samples to be analyzed by the method of the present invention include not only aqueous solutions containing both components, but also biological serum and urine.

In the method of this invention, conventional methods can be used to remove protein from the sample, such as adding perchloric acid to the sample, shaking, and centrifuging to remove proteins.

Further, the reversed phase column chromatography performed after the above-mentioned protein removal treatment can be performed by the following method. In other words, as a separation column, a chemically bonded reverse phase column is used, which has a stationary phase in which silica gel is used as a carrier and octadecyl groups, octyl groups, trimethyl groups, etc. are chemically bonded to this.
For example, BondElut C18 and BondElut C8 (Anal
(manufactured by YTICHEM FTERNATIONA 1), etc. As a mobile phase, p++ is 2.0 to 7.0
Buffers such as sodium phosphate and potassium phosphate are used.

Further, the reversed phase ion pair chromatography in this invention can be performed by the following method.

In other words, as a separation column, a chemically bonded reverse phase column having a silica gel carrier and a 1C1@$ stationary phase for octadecyl, octyl, trimethyl groups, etc. is used, such as Shim-pack manufactured by Kinsei Seisakusho.
CL C-OD S, Zorbax OD S
Examples of mobile phases include buffers such as sodium phosphate and potassium phosphate, organic solvents such as acetonitrile for adjusting retention time, and ion pairing reagents such as sodium alkyl sulfate and alkanesulfonic acid. Sodium, salts of strong acids and strong bases, etc. are added as appropriate, and the pH of the resulting mixture is adjusted to 2.0-2.0 with phosphoric acid, etc.
The one adjusted to 3.0 is used.

Detection of both components is performed by measuring the absorbance of ultraviolet light having a wavelength of approximately 210 nm.

Furthermore, if the sample does not contain any contaminants, refractive index can be used instead of ultraviolet absorbance.

(e) Examples Next, the method of this invention will be explained by way of examples.

A sample of a standard solution containing the pure creatine and creatinine shown below on the test plate was analyzed under the following analysis conditions. The obtained chromatogram is shown in Figure 1, and it can be seen that creatine and creatinine were analyzed with high sensitivity. This analysis was repeated with good reproducibility.

i) 50 μl of an aqueous solution containing 1 μ/d1 each of sample creatine and creatinine (use of special grade reagent manufactured by Wako Pure Chemical Industries) ii) Analysis conditions a Separation column Shim-pack CL C-〇 D S
(5, Q++n φ
Sodium phosphate buffer (pH 2°1) and acetonitrile are mixed at a volume ratio of 3:1, and the pH of this mixture is adjusted to 2.1 with phosphoric acid.

C Separation conditions Column temperature 55°C Mobile phase delivery rate 2ml/ml 1nd Ultraviolet absorbance measurement equipment Quality Ultraviolet absorbance detector 5PD-2A (210nm absorbance) Practical basis 1 Human serum (200μβ) was analyzed in ii) of Example 1 above. The chromatogram obtained by analysis under the same conditions as above is shown in FIG.

According to FIG. 2, it can be seen that creatine and creatinine are separated with high sensitivity, and then a large amount of impurities are separated. This analysis was repeated and creatine and creatinine could be analyzed with high sensitivity and good reproducibility.

Mifilo I scolded Human serum (200 μl) was analyzed under the following conditions.

i) Protein removal treatment of sample Add 100 μg of 1N perchloric acid solution to the above sample, shake, centrifuge at 12000 r, p, m for 2 minutes, and collect the supernatant.

ii) Elution of both creatine and creatinine components 100μ# of the above supernatant was injected into Bond Elut C18 (Ana
lytichem Internationala1)
pass. Furthermore, 400 μl of 0.4 M sodium phosphate buffer (pH 2,0) is injected into the column to elute creatine and creatinine.

iii) Analysis of both components 50μβ of the above eluate was analyzed according to the analysis conditions ii) of Example 1, and the obtained chromatogram is shown in FIG.

As is clear from FIG. 3, the large peak of impurities after 10 minutes in the chromatogram of Example 2 (FIG. 2) has disappeared. This analysis was repeated with good reproducibility.

In view of the results of Examples 2 and 3 above, when the sample contains a large amount of creatine and impurities other than creatinine, such as serum or urine, the sample is pre-deproteinized as in Example 3 and then subjected to reverse phase treatment. It is possible to reduce the time required for analysis by subjecting the eluate to column chromatography to remove impurities and extracting t volumes of creatine and creatinine, and then subjecting the eluate to reversed-phase ion pair chromatography. I understand.

(C) Effects According to the present invention, both components of creatine and a creatinine-containing sample can be easily analyzed with high sensitivity and good reproducibility.

[Brief explanation of the drawing]

Figure 1 shows a chromatogram obtained in an example in which creatine and a standard sample containing creatinine were analyzed using the method of this invention.
Figures 2 and 3 are chromatograms obtained in an example in which human serum was analyzed by the method of the present invention.
4. −”−. Agent: Patent Attorney Shinobu Nogawa / Inj. (Now) Figure 2 (Now) (Order)

Claims (1)

[Claims] 1. A creatine and creatinine containing sample as it is, or a creatine and creatinine containing eluate obtained by previously subjecting the sample to protein removal treatment and then subjecting it to reverse phase column chromatography to remove impurities in the sample, Creatine and creatinine are analyzed by subjecting them to reverse phase ion pair column chromatography in an acidic mobile phase to separate and elute creatine and creatinine, and measuring the ultraviolet absorbance of the obtained eluate to analyze creatine and creatinine. Simultaneous analysis method.