JPS6230956A - Analysis for ions contained in synthetic peptide - Google Patents

Abstract

PURPOSE:To enable accurate, simultaneous analysis on a trace sample, by a separative determination of ions contained in synthetic peptides by means of a fast liquid chromatography using an ion exchange resin, a buffer liquid and an electric conduction detector. CONSTITUTION:Ions contained in synthetic peptides undergoes a separative determination by means of a fast liquid chromatography using an ion exchange resin as column, a buffer liquid of sodium hydrocarbonate, sodium carbonate or the mixture thereof or the like (elution liquid of anion), a phosphoric acid buffer liquid or a nitric acid solution (elution liquid of cations) as elution liquid and an electric conduction detector as detector. The concentration of the elution liquid shall be 0.1-100milli-mol. This enables accurate, simultaneous analysis of a trace sample.

Description

Detailed Description of the Invention The present invention relates to a method for analyzing ions contained in synthetic peptides, and more specifically to an ion exchange resin as a column, a buffer as an eluent, and an electrical conductivity as a detector. This invention relates to a method for analyzing ions contained in synthetic peptides by high performance liquid chromatography using a detector.

Generally, synthetic peptides are synthesized by solid-phase or liquid-phase methods, and in this case, synthetic peptides are produced as organic salts, often acetates, and various ions are used in the synthesis and purification process of the peptides. Therefore, synthetic peptides contain various ions as impurities. Therefore, analysis of ions as organic salts of these peptides and ions as impurities is very important for evaluating the quality of peptides.

Generally, synthetic peptides are synthesized as a salt of an organic acid such as acetic acid, and in the case of peptide synthesis by solid-phase method, for example, in the final step of synthesis, the protecting group of the amino acid is removed and the peptide is removed from the resin in the same phase. Reagents such as trifluoroacetic acid and hydrofluoric acid are used for desorption. Also,
In the peptide purification step, various methods for producing M are used depending on the properties of the peptide, and reagents such as acetic acid, trifluoroacetic acid, ammonium acetate, and sodium chloride are also used in this purification process. Also, in the liquid phase method, substantially the same operations as in the solid phase method described above are performed. Therefore, synthetic peptides are often contaminated with reagents used in the form of salts during the synthesis and purification steps.

Conventionally, the gas chromatography method has been known as a method for quantifying acetic acid present as a salt in peptides (supervised by the Examination Division of the Pharmaceutical Affairs Bureau of the Ministry of Health and Welfare, Japanese Pharmacopoeia Non-legal Drug Composition Standards 1988, (Hosha) This method requires a relatively large amount of sample, and even when applied to organic acids other than acetic acid, such as trifluoroacetic acid, there are problems with accuracy, reproducibility, and detection limits. There is.

In general, the analytical methods for inorganic ions include colorimetric method,
A method of quantitatively analyzing individual inorganic ions using atomic absorption spectrometry or the like is known. However, since the samples for synthetic peptides are extremely expensive, these analysis methods are not suitable, and there is a strong need for the development of a simultaneous analysis method that uses the same sample solution to analyze only a very small amount of sample. .

In view of these circumstances, the present inventors have conducted extensive research on methods for analyzing trace ions mixed in synthetic peptides, and have found that the analytical method of the present invention is accurate and requires only a very small amount of sample to be tested. Furthermore, the present inventors have discovered that it is also possible to simultaneously analyze anions or cations using the same sample solution to be analyzed, leading to the present invention.

That is, the present invention separates ions contained in synthetic peptides in a fixed state by high-performance liquid chromatography using an ion exchange resin as a column, a buffer as an eluent, and an electrical conductivity detector as a detector. It provides an analytical method for

The method of the present invention will be described in detail below.

In the method of the present invention, an ion exchange resin with a low ion exchange capacity for ordinary high performance liquid chromatography is used as the filler, for example, a spherical porous polymer type resin with a narrow particle size distribution of 6 to 15 μm in particle size, An ion exchange resin with an ion exchange capacity of 0.01 to 0.1 meq/g obtained by introducing an anion exchange group such as a quaternary ammonium salt or a cation exchange group such as a sulfonic acid group is used. Ru. As such an ion exchange resin, for example, Anion 5eparator is used as an exchange resin.
(Dionex III), MuX-1 (manufactured by Yokogawa Hokushin Electric)
, T8-gel lo-Anion PW (manufactured by Toyo Soda) and Cation 5e as a cation exchange resin.
parator (manufactured by Dionex), 0X-1 (manufactured by Yokogawa Hokushin Electric), T8 -gel IC-Cation (
(manufactured by Toyo Soda).

As eluents, carbonate buffers such as sodium bicarbonate solution, sodium carbonate solution, or mixtures thereof are used as eluents for anions, and phosphate buffers and nitric acid solutions are used as eluents for cations. used as.

The concentration of these eluents is between 0.1 and 100 mmol.

Although it depends on the properties of the packing material used, a sodium bicarbonate solution of around 4 mmol is usually used for the analysis of chloride ions and trifluoroacetate ions, and a solution of around 0.5 mmol is used for the analysis of fluoride ions and acetate ions. A sodium bicarbonate solution is used. Further, for analysis of sodium ions, ammonium ions, etc., a nitric acid solution of about 2 mmol is usually used.

In addition, in the method of the present invention, especially in the case of anion analysis, a column (suppressor column) is used to remove cations from the eluent in order to reduce the electrical conductivity of the eluent for the purpose of increasing sensitivity. It may also be installed in front of the electrical conductivity detector.

EXAMPLES Hereinafter, the specific content of the present invention will be explained with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Human growth hormone releasing factor (hGRF(1-44)NH! and hGR, respectively) consisting of 44 amino acids synthesized by solid phase method or 29 amino acids synthesized by liquid phase method
F(1-29) It is called NH wing. ) each about 11119
was precisely weighed and dissolved in water to prepare a 10- and 4-analyte sample solution.

Separately, add a solution of 1-2 ppm chloride ions and 5-10 ppm trifluoroacetic acid! ll&! This was used as a standard solution.

Analysis was performed by graphical methods. Table 1 shows the results of measuring the peak heights of chloride ions and trifluoroacetic acid in the sample solution and standard solution, and calculating the content of each ion using an external standard method.

Operating condition column: Dionex I (PIC-A84, 4
mm1d x 250 mlOng Eluent: 4 mmol sodium bicarbonate solution stream
Amount: 1.7 d/min Detector: Electrical conductivity detector Column temperature: Room temperature Table 1 Anion analysis results Example 2 hGRF(1-44)NH” prepared in Example 1 or hGRF(1-29 ) NHII sample solution to be analyzed and fluorine ion 1-2p prepared as a standard solution
Accurately taken 50 μt each of a solution containing 5 to 10 ppm of pm and acetic acid, and analyzed by high performance liquid chromatography under the following operating conditions.

The peak heights of fluorine ions and acetic acid in the sample solution and standard solution were measured, and the contents of each ion were calculated using an external standard method. The results are shown in Table 2.

Operating condition column: Dionek xf3 al10-A84,4
mmidX250mlOn8 Eluent: 0.5E rimole sodium carbonate solution flow rate: 1.7d/
min Detector: Electrical conductivity detector Column temperature: Room temperature Table 2 Anion analysis results Example 8 Analyte sample solution of hGRF(1-44)MHI prepared in Example 1 and ammonium prepared as a standard solution 1-2 ppm each of ion and sodium ion
Accurately aliquots of 50 .mu.t each of the solution were taken and analyzed by high performance liquid chromatography under the following operating conditions. Table 8 shows the results of measuring the peak heights of ammonium ions and sodium ions in the sample solution and standard solution, and calculating the content of each ion using an external standard method.

Operation condition column: Song Yang Soda TSK-gel lo-Cation
.

4.6 mmid XδOrr+mlong elution
Liquid: 2 mmol nitric acid effluent Amount: 1. Od/min Detector: Electrical conductivity detector Column temperature: Room temperature Table 8 Cation analysis results

[Brief explanation of the drawing]

Figures 1 and 2 are examples of chromatograms of anions separated by the method of the present invention, in which the vertical axis represents the intensity;
The horizontal axis represents retention time. In Figure 1, peaks 1 and 2 are the peaks of chloride ion and trifluoroacetate ion, respectively;
, peaks 8 and 4 are the peaks of fluorine ion and acetate ion, respectively. (min) Figure 1 (min) (Figure 2

Claims (3)

[Claims] (1) Ions contained in synthetic peptides are separated and quantified by high-performance liquid chromatography using an ion exchange resin as a column, a buffer as an eluent, and an electrical conductivity detector as a detector. Featured analysis method. (2) The ion exchange capacity of the ion exchange resin is 0.01 to 0.
．． 1. The analytical method according to claim 1, which is 1 milliequivalent/gram. (3) The analytical method according to claim 1 or 2, wherein the concentration of the buffer solution is 0.1 to 100 mmol.