阅读说明：本技术 一种测定氨基酸原料药中痕量糖类杂质的方法 (Method for determining trace carbohydrate impurities in amino acid bulk drug ) 是由 惠人杰 姜峰 薛灵杰 杨荻 于 2020-06-16 设计创作，主要内容包括：本发明公开了一种测定氨基酸原料药中痕量糖类杂质的方法,属于药物分析检测技术领域。本发明采用离子交换树脂处理待测氨基酸原料药样品,吸附氨基酸原料药中糖分,再采用高效液相色谱-蒸发光散射检测(HPLC-ELSD)测定氨基酸原料药中5种糖类杂质,采用极性有机相-水为流动相进行梯度洗脱,极性键合相色谱柱进行分离,以氮气作为辅气,直接进样。经验证,该方法可以使各成分实现基线分离,采用外标法测定氨基酸原料药中5种糖类杂质的含量。本发明方法灵敏度高,操作简便,准确度高,适合同时测定氨基酸原料药中5种糖类杂质的含量。(The invention discloses a method for determining trace carbohydrate impurities in an amino acid raw material medicine, and belongs to the technical field of medicine analysis and detection. The method comprises the steps of treating an amino acid raw material medicine sample to be detected by using ion exchange resin, adsorbing sugar in the amino acid raw material medicine, determining 5 kinds of sugar impurities in the amino acid raw material medicine by using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD), performing gradient elution by using a polar organic phase-water as a mobile phase, separating by using a polar bonded phase chromatographic column, and directly injecting samples by using nitrogen as auxiliary gas. The verification proves that the method can realize baseline separation of all components, and the content of 5 kinds of carbohydrate impurities in the amino acid bulk drug is determined by adopting an external standard method. The method has the advantages of high sensitivity, simple and convenient operation and high accuracy, and is suitable for simultaneously determining the content of 5 kinds of carbohydrate impurities in the amino acid raw material medicine.)

1. A pretreatment method for enriching sugar in amino acid raw material medicines is characterized in that the pretreatment method comprises the steps of adsorbing amino acid in the amino acid raw material medicines by adopting ion exchange resin, washing the resin by adopting ultrapure water, and collecting and concentrating the sugar in the amino acid raw material medicines; the rinsing volume of the ultrapure water is 100-250 mL.

2. The pretreatment method of claim 1, wherein the amino acid drug substance comprises one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

3. The pretreatment method according to claim 1 or 2, wherein the ion exchange resin is a cation exchange resin or an anion exchange resin, and comprises a D113 type resin, a D001 resin, or a 732 strong acid styrene cation exchange resin.

4. The pretreatment method according to any one of claims 1 to 3, wherein the adsorption time is 3 to 5 hours.

5. The pretreatment method according to any one of claims 1 to 4, wherein the method comprises the steps of:

(1) alternately treating the resin with pure water, an acidic solution and an alkaline solution; the acid solution is inorganic acid water solution, and the alkaline solution is inorganic alkaline water solution;

(2) treating the amino acid sample solution with resin;

(3) filtering, rinsing the resin with ultrapure water, and collecting filtrate.

6. The pretreatment method of any one of claims 1 to 5, wherein the sugar comprises one or more of fructose, glucose, sucrose, maltose and lactose.

7. A method for detecting sugar in amino acid bulk drug is characterized by comprising the following steps: (1) pretreating an amino acid drug substance by using the pretreatment method of any one of claims 1 to 6; (2) and detecting sugar in the amino acid bulk drug by adopting high performance liquid chromatography-evaporative light scattering.

8. The method of claim 7, wherein the high performance liquid chromatography conditions are: the conditions of the high performance liquid chromatography are as follows: the chromatographic column is a Lichropher amino column with the specification of 4.6mm multiplied by 100mm and 5 mu m.

9. The method according to claim 7 or 8, wherein the high performance liquid chromatography uses acetonitrile and water as eluent, gradient elution mode; the gradient elution conditions were: 0-3min, 10-15% of water and 85-90% of acetonitrile; 3-23min, 12-25% of water and 75-88% of acetonitrile; 23-35min, 10-15% of water and 85-90% of acetonitrile.

10. Use of the method of any one of claims 7-9 for quality monitoring of a drug substance.

Technical Field

The invention relates to a method for determining trace carbohydrate impurities in an amino acid raw material medicine, and belongs to the technical field of medicine analysis and detection.

Background

Amino acids (Amino acids) are the basic units of proteins (proteins) which are the first nutritional elements in the human body, and play an important role in the growth, reproduction, immunity and the like of human beings. The production method of amino acid mainly includes extraction method, fermentation method, chemical synthesis and enzyme method. The traditional extraction method, chemical synthesis method and enzyme method are difficult to achieve the purpose of industrial production due to higher cost of precursors and complex process, and the fermentation method is generally adopted in the industry at present. Sugar is introduced into the process flow as a sugar source of microorganisms in the fermentation process and may become trace impurities in the amino acid bulk drug, but because a high-sensitivity detection means is lacked, the impurity analysis of residual sugar in the amino acid bulk drug is not reported at present.

The most commonly used methods of carbohydrate analysis are mainly high performance liquid chromatography, with common detectors being ultraviolet, differential refractive and evaporative light scattering detectors. The saccharides have no characteristic ultraviolet absorption characteristic, and can be detected by an ultraviolet detector for determination after being subjected to derivatization to carry out chromophoric groups, and the operation steps are complicated when the ultraviolet detector is used for determination. Although the differential refractometer can directly measure saccharide substances, the sensitivity of the refractometer is low, the influence of temperature is large, and the differential refractometer cannot be compatible with gradient elution. However, Evaporative Light Scattering Detectors (ELSDs) do not suffer from these limitations, and the detection principle is based on the general ability of particles to cause light scattering, so that the response of ELSDs is not affected by the specific properties of the sample, making up for the deficiencies of conventional detectors for high performance liquid chromatography.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention adopts high performance liquid chromatography-evaporative light scattering detection to simultaneously detect the contents of fructose, glucose, sucrose, maltose and lactose, and has high sensitivity, good repeatability and high precision; the invention is suitable for detecting trace fructose, glucose, sucrose, maltose, lactose and sugar impurities in the amino acid bulk drug.

The invention provides a pretreatment method for enriching sugar in amino acid raw material medicines, which comprises the steps of firstly adopting ion exchange resin to adsorb amino acid in the amino acid raw material medicines, then adopting ultrapure water to flush the resin, and collecting and concentrating the sugar in the amino acid raw material medicines; the rinsing volume of the ultrapure water is 100-250 mL.

In one embodiment of the present invention, the amino acid drug substance includes, but is not limited to, twenty basic amino acids: glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

In one embodiment of the present invention, the ion exchange resin is cation exchange resin and anion exchange resin, including but not limited to D113 type resin, D001 resin or 732 strong acid styrene cation exchange resin.

In one embodiment of the invention, the adsorption time is 3 to 5 hours.

In one embodiment of the present invention, the ion exchange resin is treated with pure water, acidic solution and alkaline solution alternately; the acidic solution is inorganic acid aqueous solution, and the alkaline solution is inorganic alkaline aqueous solution.

In one embodiment of the invention, the method comprises the steps of:

(1) alternately treating the resin with pure water, an acidic solution and an alkaline solution;

(2) treating the amino acid sample solution with resin;

(3) filtering, rinsing the resin with ultrapure water, and collecting filtrate.

In one embodiment of the invention, the rinsing volume of the ultrapure water is 100-.

The second purpose of the invention is to provide a method for detecting sugar in amino acid bulk drug, which comprises the following steps: (1) the amino acid raw material medicine is pretreated by the pretreatment method; (2) and detecting sugar in the amino acid bulk drug by adopting high performance liquid chromatography-evaporative light scattering.

In one embodiment of the invention, the sugar comprises one or more of fructose, glucose, sucrose, maltose and lactose.

In one embodiment of the present invention, the high performance liquid chromatography conditions are: the chromatographic column is a Lichropher amino column with the specification of 4.6mm multiplied by 100mm and 5 mu m; acetonitrile and water are used as eluent, and a gradient elution mode is adopted.

In one embodiment of the present invention, the gradient elution conditions are: 0-3min, 10-15% of water and 85-90% of acetonitrile; 3-23min, 12-25% of water and 75-88% of acetonitrile; 23-35min, 10-15% of water and 85-90% of acetonitrile.

In one embodiment of the invention, a polar bonded phase column is selected, sample injection is performed in a direct sample injection mode, and sample measurement is performed in an evaporative light scattering detector monitoring and gradient elution mode.

In one embodiment of the invention, an inert gas is used as a carrier gas; the temperature of the drift tube is 40-50 ℃; the gain value is 1-8; sample introduction volume: 20 mu L of the solution; and (3) sample introduction mode: and directly injecting a sample.

In one embodiment of the invention, the column temperature is 20-40 ℃; the temperature of the drift tube is 30-35 ℃; the gain value is 2-6; the carrier gas is nitrogen.

In one embodiment of the invention, the gradient elution mode: the gradient range is 55-95%, the time is 20-30 min, the initial gradient is maintained for 3min, and the end gradient is maintained for 3 min.

The third purpose of the invention is to provide an application of the detection method in quality monitoring of the bulk drug.

The invention has the beneficial effects that:

the invention adopts high performance liquid chromatography-evaporative light scattering detection to simultaneously detect the contents of fructose, glucose, sucrose, maltose and lactose, the recovery rate is 87.6-107%, the detection limits of the fructose, glucose, sucrose, maltose and lactose are respectively 75.0, 66.6, 20.8, 25.0 and 34.6mg/kg (S/N is 3), and the quantification limits are respectively 238.8, 212.3, 96.2, 123.0 and 125.0mg/kg (S/N is 10); RSD of peak areas of 5 kinds of sugars such as fructose, glucose, sucrose, maltose, lactose and the like is respectively 0.70%, 0.76%, 0.62%, 0.56% and 0.43%; the method has the advantages of high sensitivity, good repeatability and high precision, and is suitable for detecting trace fructose, glucose, sucrose, maltose and lactose impurities in the amino acid bulk drug.

Drawings

FIG. 1 shows the adsorption capacity of three resins for amino acids and the recovery rate of glucose in example 2; wherein A is the average time required for amino acid adsorption, and B is the recovery rate of sugar.

FIG. 2 is a resin rinse option of example 2.

FIG. 3 is a liquid chromatogram, wherein A is a blank solution liquid chromatogram, and B is a mixed standard solution chromatogram of fructose, glucose, sucrose, maltose and lactose measured in example 3; 1: fructose, 2: glucose, 3: sucrose, 4: maltose, 5: lactose.

FIG. 4 is a liquid chromatogram under different elution conditions in example 4; wherein, A: isocratic condition 1; b: isocratic condition 2; c: gradient condition 1; d: gradient condition 2; e: gradient condition 3; f: gradient condition 4; 1: fructose; 2: glucose; 3: galactose; 4: sucrose; 5: maltose; 6: lactose.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

1. Test materials: resin D113, resin D001, and 732 strong acid styrene cation exchange resins (abbreviated as 732 cation exchange resins) were purchased from Shanghai Aladdin Biotech Ltd.

2. Determination of amino acids by the Indantrione method:

1mL of sample solution is added with 1mL of acetic acid buffer solution with pH of 5.4 and 2mol/L and 1mL of ninhydrin color development solution, mixed uniformly, heated in a boiling water bath at 100 ℃ for 15min, and cooled by tap water. After standing for 5min, 3mL of 60% ethanol was added for dilution, and after shaking up, the absorbance at 570nm was determined (the resulting color was stable within 60 min).