CN111638291B

CN111638291B - Method for determining trace carbohydrate impurities in amino acid bulk drug

Info

Publication number: CN111638291B
Application number: CN202010545666.0A
Authority: CN
Inventors: 惠人杰; 姜峰; 薛灵杰; 杨荻
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2021-11-16
Anticipated expiration: 2040-06-16
Also published as: CN111638291A

Abstract

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.

Description

Method for determining trace carbohydrate impurities in amino acid bulk drug

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).

Example 1: pretreatment method for enriching sugar in amino acid raw material medicine

(1) Resin pretreatment: precisely weighing 732 parts of cation exchange resin 10.0g, placing the cation exchange resin in a 250mL conical flask, washing the flask with water until the effluent is colorless, treating the flask with 70mL of 5% (v/v) HCl overnight, and washing the flask with water until the effluent is neutral; then treated with 5 percent (m/v) NaOH with the same volume overnight, and washed with water to be neutral; finally, the mixture was treated with 70mL of 5% (v/v) HCl overnight and washed with water to neutrality for further use.

(2) Amino acid sample pretreatment: taking 1 part of the pretreated resin, placing in a 250mL conical flask, precisely weighing 0.20g of the amino acid sample, dissolving in 100mL of water, adding the solution into the conical flask, and placing in a magnetic stirrer at room temperature for static adsorption at low speed until the amino acid is completely adsorbed. The solution was filtered, the filtrate was rotary evaporated to about 30mL, then lyophilized at-80 ℃ and the lyophilized material reconstituted with 1mL of water for use.

Example 2: selection of pretreatment conditions

(1) Selection of resin species:

a mixed solution of arginine and glucose (arginine concentration 2g/mL and glucose concentration 1mg/mL in the mixed solution) was treated in the same manner as in example 1 except that 732 cation exchange resins were replaced with a macroporous adsorption resin, a D113 weak acid type cation exchange resin and a D001 type cation exchange resin, and the adsorption of amino acids was examined by the ninhydrin method under the same conditions as in example 1, and the results are shown in FIG. 1.

The adsorption of amino acid and saccharide impurities by adopting macroporous adsorption resin is similar, and the sugar recovery rate is too low. As can be seen from FIG. 1, the adsorption of the amino acid by the D113 type resin was completed within 4.5 hours, and the adsorption ability was poor. The D001 type resin can complete the adsorption of amino acid within 3h, the adsorption capacity is good, but a large amount of glucose is attached to the resin, and the recovery rate is only 57.81%. 732 resin can complete the adsorption of amino acid within 4h, and the recovery rate of glucose is higher and reaches 77.59%. Therefore, preferably, 732 cation exchange resins are selected to treat the amino acid sample.

(2) Selection of resin flush volume:

glucose standard solution having a concentration of 1mg/mL was treated in the same manner as in example 1 except that the volumes of ultrapure water were changed to 50, 100, 150, 200 and 250mL, and the other conditions were the same as in example 1, and the results are shown in FIG. 2.

As can be seen from fig. 2, the recovery rate of glucose tends to increase and decrease with the increase of the flush volume, and the recovery rate of glucose peaks when the flush volume is 150mL, and therefore, it is preferable that the resin flush volume is 150 mL.

Example 3: method for detecting sugar in amino acid bulk drug

The pretreatment method of example 1 is applied to extract and adsorb the pigment in the eggs, and then the HPLC-ELSD and external standard method are combined to carry out quantitative analysis on the pigment content in the eggs, and the method comprises the following specific steps:

1. establishing a standard curve:

(1) preparing a mixed reference solution: accurately weighing fructose 2.0mg, glucose 3.0mg, sucrose 2.3mg, maltose 3.0mg and lactose 3.0mg respectively, placing in a 10mL volumetric flask, dissolving with water and fixing volume to scale mark to obtain a mixed reference solution;

(2) HPLC-ELSD determination:

20 mu L of mixed reference substance solution and blank solvent water are taken for measurement under the following chromatographic conditions:

a chromatographic column: lichropher NH2 column (4.6mm × 250mm, 5 μm) is selected; the column temperature is 30 ℃; the mobile phase was acetonitrile-water 75:25(v/v), the elution conditions are shown in table 1; the flow rate is 1.0 mL/min; the drift tube temperature is 40 ℃; the carrier gas was nitrogen (pressure 350 kPa); the gain value is 2; the sample injection amount is 20 mu L, and the sample injection mode is as follows: and directly injecting a sample.

The chromatogram obtained by the method is shown in figure 3, and the figure shows that the solvent has no influence on the determination of each component and has good specificity; and each component can be well separated, the theoretical plate number of each peak is more than 50000, each adjacent peak is completely separated, and the peak emergence sequence is as follows in sequence: fructose, glucose, sucrose, maltose, lactose.

TABLE 1 gradient elution conditions

(3) Linear test

Preparing a series of mixed reference substance solutions with concentration, precisely sucking 20 μ L, measuring under the chromatographic conditions, and drawing a standard curve by taking the peak area of each component as the ordinate (y) and the corresponding mass concentration as the abscissa (x). The regression equation and correlation coefficient results are shown in table 2.

Table 2 linear regression equations and associated coefficients for the five saccharides (n ═ 5)

(4) Quantitative and detection limit determination

And diluting the reference stock solution by adopting a stepwise dilution method, injecting 20 mu L of the reference stock solution, calculating the detection limit by using the signal-to-noise ratio S/N which is 3, and calculating the quantification limit by using the signal-to-noise ratio S/N which is 10. The detection limits of fructose, glucose, sucrose, maltose and lactose were 75.0, 66.6, 20.8, 25.0 and 34.6mg/kg (S/N ═ 3), respectively, and the quantitation limits were 238.8, 212.3, 96.2, 123.0 and 125.0mg/kg (S/N ═ 10), respectively.

(5) Precision test

20. mu.L of the mixed control solution was aspirated, and the measurement was performed under the above-mentioned chromatographic conditions, and the RSD value of the peak area of each component was calculated by measuring 6 times in succession. The results show that the RSD of the peak areas of 5 sugars such as fructose, glucose, sucrose, maltose, lactose and the like are respectively 0.70%, 0.76%, 0.62%, 0.56% and 0.43%, and the instrument precision is good.

2. Method for detecting sugar in amino acid bulk drug

(1) preparing a test solution: weighing 0.20g of amino acid sample into a 250mL conical flask, adding 100mL of water for dissolving, adding 732 cation exchange resin, and statically adsorbing for 4h at room temperature. The solution was filtered, the resin was rinsed with 150mL of ultrapure water, and the filtrates combined. The filtrate was rotary evaporated to 25% of the original volume and lyophilized at-80 ℃. Redissolving the residue after freeze-drying with 1mL of water to obtain a test solution;

(2) HPLC-ELSD determination: taking 20 mu L of test solution and blank solvent water, and measuring according to the following chromatographic conditions: a chromatographic column: lichropher NH2 column (4.6mm × 250mm, 5 μm) is selected; the column temperature is 30 ℃; the mobile phase was acetonitrile-water 75:25(v/v), the elution conditions are shown in table 1; the flow rate is 1.0 mL/min; the drift tube temperature is 40 ℃; the carrier gas was nitrogen (pressure 350 kPa); the gain value is 2; the sample injection amount is 20 mu L, and the sample injection mode is as follows: and directly injecting a sample.

And obtaining the content of the 5 kinds of carbohydrate impurities in the amino acid sample to be detected by referring to the standard curve.

Example 4: selection of elution conditions

Referring to the method of example 3, except that the elution conditions were changed, the assay was optimized according to several elution conditions shown in Table 3, and the other conditions were the same as example 1, and the separation of each elution condition is shown in FIG. 4.

TABLE 3 elution conditions

The liquid chromatogram is shown in FIG. 4, when eluted isocratically under isocratic conditions 1, i.e., 25% aqueous phase isocratic, the 5 carbohydrates did not achieve baseline separation. When the separation of 5 saccharides was improved under isocratic condition 2, i.e., 20% aqueous isocratic elution, the chromatographic peak shape and signal-to-noise ratio were poor, and the response of the disaccharide component (

compounds

4, 5, 6) was low.

Based on the above results, when the separation was performed by gradient elution instead, the monosaccharide components (compounds 1, 2) did not achieve effective separation and the disaccharide component response was low when the elution was performed under gradient condition 1, i.e., the gradient elution was performed in such a manner that 25% to 15% of the aqueous phase was decreased. When elution was performed under

gradient conditions

2, 3, and 4, respectively, 5 saccharides achieved baseline separation, better peak shape, and higher disaccharide component response. Among the three gradient conditions, the background noise under the gradient condition 2 is the smallest, the total peak area is the largest, and the number of theoretical plates is more than 10000.

Example 5: accuracy and reproducibility of the method

(1) Recovery rate of added standard

And (3) adding 9 parts of arginine which is a raw material drug with known residual sugar impurity content into low, medium and high quality mixed standard solutions, wherein each concentration is parallel to 3 times, feeding 20 mu L of sample, recording the peak area of a chromatographic peak, and calculating the sample recovery rate of various saccharide compounds according to the standard curve. The results are shown in Table 4.

TABLE 4 recovery of residual sugar from amino acids 5 (n ═ 9)

(2) Repeatability test

6 parts of the mixed control solution was prepared, 20. mu.L of each solution was precisely aspirated, measurement was performed under the above-mentioned chromatographic conditions, the peak area was recorded, and the RSD value of the peak area of each component was calculated. The results show that the RSD of the peak areas of fructose, glucose, sucrose, maltose and lactose are respectively 1.1%, 1.4%, 2.1%, 2.0% and 1.3%, and the method has good reproducibility.

Example 6: the method is applied to detecting the amino acid bulk drug

Commercially available arginine samples and lysine acetate samples were taken and measured according to the detection method and conditions of example 3, respectively, and the content of fructose, glucose, sucrose, maltose and lactose in the samples was calculated by an external standard method. The results show that 5 kinds of sugar impurities such as fructose, glucose, sucrose, maltose, lactose and the like are not detected in the two amino acid samples.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for detecting sugar in amino acid bulk drug is characterized by comprising the following steps: (1) pretreating amino acid raw material medicines: firstly, adsorbing amino acid in the amino acid raw material medicine by using ion exchange resin, filtering, then flushing the resin by using ultrapure water, and collecting and concentrating sugar in the amino acid raw material medicine; the rinsing volume of the ultrapure water is 100-250 mL; (2) detecting sugar in the amino acid raw material medicine by adopting high performance liquid chromatography-evaporative light scattering;

wherein the ion exchange resin is 732 strong acid styrene cation exchange resin;

the high performance liquid chromatography adopts acetonitrile and water as eluent and adopts a gradient elution mode; the gradient elution conditions were: 0min, 12% water and 88% acetonitrile; 3min, 12% water and 88% acetonitrile; 12 min, 25% water and 75% acetonitrile; 15min, 25% water and 75% acetonitrile; 23min, 12% water and 88% acetonitrile; 30min, 12% water and 88% acetonitrile;

the chromatographic column is a Lichropher amino column with the specification of 4.6mm multiplied by 100mm and 5 mu m;

the sugar is one or more of fructose, glucose, sucrose, maltose and lactose.

2. The 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 process according to claim 1, characterized in that the adsorption time is 3-5 h.

4. The method according to claim 1, characterized in that it comprises the following steps:

(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.

5. Use of the method of any one of claims 1 to 4 for quality monitoring of amino acid drug substances.