Characteristic chromatogram detection method of donkey-hide gelatin blood-enriching oral liquid

Abstract

The invention discloses a specific chromatogram detection method of donkey-hide gelatin blood-enriching oral liquid. The method comprises the following steps: preparing a reference substance solution; preparing a test solution; respectively sucking the reference substance solution and the test solution, and injecting into a high performance liquid chromatograph for determination to obtain the characteristic chromatogram of the donkey-hide gelatin blood replenishing oral liquid. According to the method, each chromatographic peak in the colla corii asini blood-enriching oral liquid can be ideally separated, peak attribution is carried out on characteristic peaks, the types and the quantity of chemical components contained in the colla corii asini blood-enriching oral liquid are comprehensively reflected, and controllable product quality, safety and effectiveness are ensured. In addition, the method has important significance for effectively controlling the quality of the donkey-hide gelatin blood-enriching oral liquid.

Description

Technical Field

[0001] The present invention relates to a method for detecting the characteristic chromatogram of Ejiao Blood-tonifying Oral Liquid, belonging to the field of drug detection. Background Art

[0002] The statements here only provide the background art related to the present invention and do not necessarily constitute the prior art.

[0003] Ejiao Blood-tonifying Oral Liquid has remarkable effects. It can supplement both qi and blood, nourish yin and moisten the lungs. It is suitable for patients with anemia or deficiency of both qi and blood, presenting symptoms such as dizziness, physical fatigue, disharmony of the spleen and stomach, and physical weakness, as well as patients with low body immunity and irregular menstrual cycles.

[0004] The main components of Ejiao Blood-tonifying Oral Liquid include Ejiao, Codonopsis pilosula, Rehmannia glutinosa, Lycium barbarum, Atractylodes macrocephala, Astragalus membranaceus, etc. There is no method for detecting its characteristic chromatogram in the prior art. To ensure the quality of Ejiao Blood-tonifying Oral Liquid, a method for detecting its characteristic chromatogram is established. Summary of the Invention

[0005] Aiming at the deficiencies existing in the prior art, the purpose of the present invention is to provide a method for detecting the characteristic chromatogram of Ejiao Blood-tonifying Oral Liquid.

[0006] The present invention realizes the above purpose through the following technical solutions: A method for detecting the characteristic chromatogram of Ejiao Blood-tonifying Oral Liquid, comprising the following steps: (1) Preparation of the reference solution: Take the reference substance glycine, dissolve it with hydrochloric acid solution, add appropriate amounts of acetonitrile solutions of phenylisothiocyanate (PITC) and triethylamine, shake well, let it stand at room temperature for 1 hour, then add organic solvent to the scale and shake well to prepare the reference solution; (2) Preparation of the test solution: Take an appropriate amount of Ejiao Blood-tonifying Oral Liquid, add appropriate amounts of acetonitrile solutions of phenylisothiocyanate (PITC) and triethylamine, shake well, let it stand at room temperature for 1 hour, then add organic solvent to the scale and shake well to prepare the test solution; (3) Determination: Respectively pipette the reference solution and the test solution (usually 5 - 20 μl) and inject them into a high-performance liquid chromatograph for determination to obtain the characteristic chromatogram of Ejiao Blood-tonifying Oral Liquid. The chromatographic conditions are as follows: Chromatographic column: The filler is an octadecylsilane-bonded silica gel column; Column temperature: 20°C - 40°C; Flow rate: 0.8 - 1.2 ml / min; Detection wavelength: 220 - 260 nm; The number of theoretical plates calculated by glycine is not less than 2000; Mobile phase: Acetonitrile - 0.1% triethylamine solution (90:10) was used as mobile phase A, and methanol - 0.2% phosphoric acid solution (10:90) was used as mobile phase B; The gradient elution program was as follows: From 0 - 12 min, mobile phase A increased from 3 - 5% (v / v) to 10 - 14% (v / v); From 12 - 28 min, mobile phase A increased from 10 - 14% (v / v) to 16 - 20% (v / v); From 28 - 35 min, mobile phase A increased from 16 - 20% (v / v) to 24 - 26% (v / v); From 35 - 45 min, mobile phase A increased from 24 - 26% (v / v) to 35 - 40% (v / v); From 45 - 60 min, mobile phase A increased from 35 - 40% (v / v) to 50 - 60% (v / v); From 60 - 100 min, mobile phase A was maintained at 50 - 60% (v / v).

[0007] In a preferred embodiment, in step (1), the concentration of the glycine reference solution was 0.05 - 0.2 mg / ml.

[0008] In a preferred embodiment, in step (2), the addition amount of the organic solvent was 4 - 6 times that of Ejiao Buxue Koufuye.

[0009] In a preferred embodiment, in step (1) and step (2), the organic solvent was any one of methanol, ethanol, 50% methanol aqueous solution or 50% ethanol aqueous solution.

[0010] In a preferred embodiment, in step (1), the method for dissolving the organic solvent was any one or more of shaking dissolution, ultrasonic treatment or reflux.

[0011] In a preferred embodiment, in step (2), the method for extracting with the addition of the organic solvent was: adding the organic solvent for dissolution, shaking well, filtering, concentrating, formulating, taking the successive filtrate, and preparing the test solution.

[0012] In a preferred embodiment, in step (3), the chromatographic conditions were as follows: Column temperature: 30 °C; Flow rate: 1.0 ml / min; Detection wavelength: 254 nm; The number of theoretical plates calculated by glycine was not less than 2000; Mobile phase: Acetonitrile - 0.1% triethylamine solution (90:10) was used as mobile phase A, and methanol - 0.2% phosphoric acid solution (10:90) was used as mobile phase B; The gradient elution program is as follows: From 0 to 12 min, mobile phase A increases from 4% (v / v) to 12% (v / v); From 12 to 28 min, mobile phase A increases from 12% (v / v) to 18% (v / v); From 28 to 35 min, mobile phase A increases from 18% (v / v) to 25% (v / v); From 35 to 45 min, mobile phase A increases from 25% (v / v) to 38% (v / v); From 45 to 60 min, mobile phase A increases from 38% (v / v) to 55% (v / v); From 60 to 100 min, mobile phase A remains at 55% (v / v).

[0013] In a more preferred embodiment, the preparation method of the reference substance solution in step (1) is as follows: Take glycine reference substance, and prepare a solution containing 0.16 mg of glycine per 1 ml with 0.1 mol / L hydrochloric acid solution; Take 5 ml of the above solution, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the continued filtrate to obtain the solution.

[0014] In a more preferred embodiment, the preparation of the test sample solution in step (2) is as follows: Measure 5 ml of Ejiao Buxue Koufuye, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the continued filtrate to obtain the solution.

[0015] Beneficial effects The method of the present invention can achieve ideal separation of each chromatographic peak in Ejiao Buxue Koufuye, obtain a relatively comprehensive characteristic chromatogram of Ejiao Buxue Koufuye, and can determine the contents of Ejiao components such as L-hydroxyproline, glycine, alanine, and L-proline in Ejiao Buxue Koufuye. Moreover, the peak attribution of the characteristic peaks is carried out, comprehensively reflecting the types and quantities of chemical components contained in Ejiao Buxue Koufuye, and then conducting an overall description and evaluation of its quality to ensure the controllable quality, safety and effectiveness of the product. In addition, the operation is simple, fast, the detection cost is low, the reproducibility is good, and the information content is large, which has important significance for effectively controlling the quality of Ejiao Buxue Koufuye. Description of the drawings

[0016] Figure 1 It is the chromatogram of Experimental Example 1; Figure 2 It is the chromatogram of Experimental Example 2. Specific embodiments

[0017] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention.

[0018] Experimental Example 1 Chromatographic conditions and system suitability test: Use octadecylsilane-bonded silica gel as the filler; acetonitrile-water (4:1) as mobile phase A, and acetonitrile-0.1 mol / L sodium acetate solution (adjust the pH value to 6.5 with acetic acid) (7:93) as mobile phase B, and perform gradient elution according to the following regulations; the detection wavelength is 254 nm. The number of theoretical plates calculated based on the glycine peak should be not less than 3000.

[0019] The gradient elution program is as follows: 0 - 20 min, mobile phase A rises from 0% (v / v) to 7% (v / v); 20 - 23.9 min, mobile phase A rises from 7% (v / v) to 12% (v / v); 23.9 - 24 min, mobile phase A rises from 12% (v / v) to 15% (v / v); 24 - 39 min, mobile phase A rises from 15% (v / v) to 34% (v / v); 39 - 40 min, mobile phase A rises from 34% (v / v) to 100% (v / v); 40 - 100 min, mobile phase A remains at 100% (v / v).

[0020] Preparation of reference substance solution: Take an appropriate amount of glycine reference substance, accurately weigh it, and prepare a solution containing 0.16 mg of glycine per 1 ml with 0.1 mol / L hydrochloric acid solution. Accurately measure 5 ml of the above solution, place it in a 25-ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0021] Preparation of test solution: Accurately measure 2 ml, place it in a 10-ml volumetric flask, add 2 ml of hydrochloric acid, hydrolyze at 80 °C for 2 hours, cool, add water to the scale, and shake well. Accurately measure 5 ml of the above solution, place it in a 25-ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0022] Determination: Pipette the reference solution and the test solution (usually 5 - 20 μl) respectively, and inject them into a high performance liquid chromatograph for determination.

[0023] Experimental results: The chromatographic peaks are concentrated in the middle and good separation is not achieved.

[0024] Experimental Example 2 Chromatographic conditions and system suitability test: Use octadecylsilyl silica gel as the filler; use acetonitrile - 0.1% triethylamine solution (90:10) as mobile phase A and methanol - 0.2% phosphoric acid solution (10:90) as mobile phase B, and perform gradient elution as specified below; the detection wavelength is 254 nm. The number of theoretical plates calculated based on the glycine peak should be not less than 3000.

[0025] The gradient elution program is as follows: 0 - 12 min, mobile phase A increases from 4% (v / v) to 12% (v / v); 12 - 28 min, mobile phase A increases from 12% (v / v) to 18% (v / v); 28 - 35 min, mobile phase A increases from 18% (v / v) to 25% (v / v); 35 - 45 min, mobile phase A increases from 25% (v / v) to 38% (v / v); 45 - 60 min, mobile phase A increases from 38% (v / v) to 55% (v / v); 60 - 100 min, mobile phase A remains at 55% (v / v).

[0026] Preparation of reference solution: Take an appropriate amount of glycine reference substance, weigh accurately, and make a solution containing 0.16 mg of glycine per 1 ml with 0.1 mol / L hydrochloric acid solution. Pipette 5 ml of the above solution accurately, place it in a 25 - ml volumetric flask, add 2.5 ml of 0.1 mol / L phenylisothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the reference solution.

[0027] Preparation of test solution: Pipette 5 ml of Ejiao Buxue Koufuye accurately, place it in a 25 - ml volumetric flask, add 2.5 ml of 0.1 mol / L phenylisothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0028] Determination: Pipette the reference solution and the test solution (usually 5 - 20 μl) respectively, and inject them into a high performance liquid chromatograph for determination.

[0029] Experimental results: The chromatographic peaks are evenly distributed and can all achieve good separation.

[0030] Reference substances were used for localization. Among them, peak 2 is L-hydroxyproline, peak 5 is glycine, peak 6 is alanine, and peak 7 is L-proline, all belonging to Ejiao; medicinal materials were used for peak attribution. Peak 1 and peak 8 belong to Rehmanniae Radix Praeparata, peak 3 belongs to Lycii Fructus, peak 4 belongs to Atractylodis Macrocephalae Rhizoma, peak 9 belongs to Astragali Radix, and peak 10 belongs to Codonopsis Radix.

[0031] Experimental Example 3 The methodology of this method was investigated, and precision experiments, repeatability experiments, and sample stability experiments were carried out respectively.

[0032] (1) Precision experiment One batch of Ejiao Blood-tonifying Oral Liquid was taken, and one portion of the test sample was prepared according to the preparation method of the test sample solution in Experimental Example 2, and it was continuously injected 5 times under the chromatographic conditions in Experimental Example 2. The results are shown in Table 1 below.

[0033] Table 1 shows the results of the precision experiment The experimental results show that the precision of the characteristic chromatogram detection method for this Ejiao Blood-tonifying Oral Liquid is good.

[0034] (2) Repeatability experiment One batch of Ejiao Blood-tonifying Oral Liquid was taken, and 6 portions of the test sample were prepared according to the preparation method of the test sample solution in Experimental Example 2, and each sample was injected once under the chromatographic conditions in Experimental Example 2. The results are shown in Table 2 below.

[0035] Table 2 shows the results of the repeatability experiment The experimental results show that the repeatability of the characteristic chromatogram detection method for this Ejiao Blood-tonifying Oral Liquid is good.

[0036] (3) Stability experiment One batch of Ejiao Blood-tonifying Oral Liquid was taken, and one portion of the test sample was prepared according to the preparation method of the test sample solution in Experimental Example 2, and it was injected at 0 h, 4 h, 8 h, 12 h, and 24 h respectively under the chromatographic conditions in Experimental Example 2. The results are shown in Table 3 below.

[0037] Table 3 shows the results of the stability experiment The experimental results show that the sample stability of the characteristic chromatogram detection method for this Ejiao Blood-tonifying Oral Liquid is good within 24 hours.

[0038] Experimental Example 4 The method in Experimental Example 2 was used to detect 4 amino acid components in Ejiao in Ejiao Blood-tonifying Oral Liquid, and the content determination results are shown in Table 4 below.

[0039] Table 4 shows the test results of the four amino acid components of donkey-hide gelatin. The experimental results show that the characteristic chromatogram detection method for this donkey-hide gelatin blood-tonifying oral liquid can detect the four amino acids in donkey-hide gelatin.

[0040] Example 1 Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile - 0.1% triethylamine solution (90:10) as mobile phase A, and methanol - 0.2% phosphoric acid solution (10:90) as mobile phase B, and perform gradient elution according to the following regulations; the detection wavelength is 254 nm. The number of theoretical plates calculated by the glycine peak should be not less than 3000.

[0041] The gradient elution conditions are as follows: 0 - 12 min, mobile phase A rises from 3% (v / v) to 14% (v / v); 12 - 28 min, mobile phase A rises from 14% (v / v) to 18% (v / v); 28 - 35 min, mobile phase A rises from 18% (v / v) to 24% (v / v); 35 - 45 min, mobile phase A rises from 24% (v / v) to 40% (v / v); 45 - 60 min, mobile phase A rises from 40% (v / v) to 60% (v / v); 60 - 100 min, mobile phase A remains at 60% (v / v).

[0042] Preparation of reference substance solution: Take an appropriate amount of glycine reference substance, accurately weigh it, and make a solution containing 0.16 mg of glycine per 1 ml with 0.1 mol / L hydrochloric acid solution. Accurately measure 5 ml of the above solution, place it in a 25 ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let it stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain.

[0043] Preparation of test solution: Accurately measure 5 ml of donkey-hide gelatin blood-tonifying oral liquid (batch number: 240304), place it in a 25 ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let it stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain.

[0044] Determination: Pipette appropriate amounts of the reference solution and the test solution (usually 5 - 20 μl) respectively, and inject them into a high performance liquid chromatograph for determination.

[0045] Experimental results: All characteristic peaks were well separated, and the contents of L-hydroxyproline, glycine, alanine, and L-proline were 78.5 μg / ml, 43.5 μg / ml, 90.4 μg / ml, and 55.5 μg / ml respectively.

[0046] Example 2 Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile - 0.1% triethylamine solution (90:10) as mobile phase A, and methanol - 0.2% phosphoric acid solution (10:90) as mobile phase B, and perform gradient elution according to the following regulations; the detection wavelength is 254 nm. The number of theoretical plates calculated based on the glycine peak should be not less than 3000.

[0047] Gradient elution conditions are as follows: 0 - 12 min, mobile phase A increases from 5% (v / v) to 10% (v / v); 12 - 28 min, mobile phase A increases from 10% (v / v) to 16% (v / v); 28 - 35 min, mobile phase A increases from 16% (v / v) to 26% (v / v); 35 - 45 min, mobile phase A increases from 26% (v / v) to 38% (v / v); 45 - 60 min, mobile phase A increases from 38% (v / v) to 55% (v / v); 60 - 100 min, mobile phase A remains at 55% (v / v).

[0048] Preparation of the reference solution: Take an appropriate amount of glycine reference substance, weigh it accurately, and prepare a solution containing 0.16 mg of glycine per 1 ml with 0.1 mol / L hydrochloric acid solution. Pipette 5 ml of the above solution accurately, place it in a 25 - ml volumetric flask, add 2.5 ml of 0.1 mol / L phenylisothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let it stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the reference solution.

[0049] Preparation of the test solution: Pipette 5 ml of Ejiao Buxue Koufuye (batch number: 240304) accurately, place it in a 25 - ml volumetric flask, add 2.5 ml of 0.1 mol / L phenylisothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let it stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0050] Determination: Pipette the reference solution and the test solution (usually 5 - 20 μl) separately, and inject them into a high performance liquid chromatograph for determination.

[0051] Experimental results: All characteristic peaks were well separated, and the contents of L-hydroxyproline, glycine, alanine, and L-proline were 78.6 μg / ml, 43.4 μg / ml, 90.4 μg / ml, and 55.4 μg / ml respectively.

[0052] Example 3 Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile - 0.1% triethylamine solution (90:10) as mobile phase A, and methanol - 0.2% phosphoric acid solution (10:90) as mobile phase B, and perform gradient elution as specified below; the detection wavelength is 254 nm. The number of theoretical plates calculated based on the glycine peak should be not less than 3000.

[0053] The gradient elution conditions are as follows: 0 - 12 min, mobile phase A rises from 4% (v / v) to 12% (v / v); 12 - 28 min, mobile phase A rises from 12% (v / v) to 20% (v / v); 28 - 35 min, mobile phase A rises from 20% (v / v) to 24% (v / v); 35 - 45 min, mobile phase A rises from 24% (v / v) to 36% (v / v); 45 - 60 min, mobile phase A rises from 36% (v / v) to 55% (v / v); 60 - 100 min, mobile phase A remains at 55% (v / v).

[0054] Preparation of reference solution: Weigh an appropriate amount of glycine reference substance accurately, and prepare a solution containing 0.16 mg of glycine per 1 ml with 0.1 mol / L hydrochloric acid solution. Pipette 5 ml of the above solution accurately, place it in a 25 - ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let it stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the reference solution.

[0055] Preparation of test solution: Pipette 5 ml of Ejiao Buxue Koufuye (batch number: 240304) accurately, place it in a 25 - ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let it stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0056] Determination: Pipette the reference solution and the test solution (usually 5 - 20 μl) separately, and inject them into a high performance liquid chromatograph for determination.

[0057] Experimental results: All characteristic peaks were well separated, and the contents of L-hydroxyproline, glycine, alanine, and L-proline were 78.6 μg / ml, 43.5 μg / ml, 90.3 μg / ml, and 55.2 μg / ml, respectively.

[0058] Example 4 Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile - 0.1% triethylamine solution (90:10) as mobile phase A, and methanol - 0.2% phosphoric acid solution (10:90) as mobile phase B, and perform gradient elution according to the following regulations; the detection wavelength is 254 nm. The number of theoretical plates calculated based on the glycine peak should not be less than 3000.

[0059] The gradient elution conditions are as follows: 0 - 12 min, mobile phase A increases from 5% (v / v) to 14% (v / v); 12 - 28 min, mobile phase A increases from 14% (v / v) to 20% (v / v); 28 - 35 min, mobile phase A increases from 20% (v / v) to 25% (v / v); 35 - 45 min, mobile phase A increases from 25% (v / v) to 40% (v / v); 45 - 60 min, mobile phase A increases from 40% (v / v) to 50% (v / v); 60 - 100 min, mobile phase A remains at 50% (v / v).

[0060] Preparation of reference solution: Take an appropriate amount of glycine reference substance, accurately weigh it, and prepare a solution containing 0.16 mg of glycine per 1 ml with 0.1 mol / L hydrochloric acid solution. Accurately measure 5 ml of the above solution, place it in a 25 - ml volumetric flask, add 2.5 ml of 0.1 mol / L phenylisothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the reference solution.

[0061] Preparation of test solution: Accurately measure 5 ml of Ejiao Buxue Koufuye (batch number: 240304), place it in a 25 - ml volumetric flask, add 2.5 ml of 0.1 mol / L phenylisothiocyanate (PITC) acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0062] Determination: Pipette the reference solution and the test solution (usually 5 - 20 μl) separately, and inject them into a high performance liquid chromatograph for determination.

[0063] Experimental results: Good separation of each characteristic peak was achieved, and the contents of L-hydroxyproline, glycine, alanine, and L-proline were 78.4 μg / ml, 43.2 μg / ml, 90.4 μg / ml, and 55.1 μg / ml, respectively.

[0064] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A method for detecting the characteristic spectrum of Ejiao Blood Tonifying Oral Liquid, characterized in that, It includes the following steps: (1) Preparation of reference solution: Take glycine, dissolve it with hydrochloric acid solution, add phenyl isothiocyanate acetonitrile solution and triethylamine acetonitrile solution, shake well, let stand at room temperature, add organic solvent, shake well to prepare the reference solution; (2) Preparation of test solution: Take Ejiao Buxue Oral Liquid, add phenyl isothiocyanate acetonitrile solution and triethylamine acetonitrile solution, shake well, let stand at room temperature, add organic solvent to the scale, shake well to prepare the test solution; (3) Determination: Respectively pipette the reference solution and the test solution, inject them into a high performance liquid chromatograph for determination to obtain the characteristic chromatogram of Ejiao Buxue Oral Liquid.

2. The characteristic chromatogram detection method of Ejiao Blood-nourishing Oral Liquid according to claim 1, wherein In step (1), the concentration of the glycine reference solution is 0.05 - 0.2 mg / ml.

3. The characteristic spectrum detection method of Ejiao Blood Tonifying Oral Liquid according to claim 1, wherein, In step (1) and step (2), the organic solvent is any one of methanol, ethanol, 50% methanol aqueous solution or 50% ethanol aqueous solution.

4. The characteristic spectrum detection method of Ejiao Blood-tonifying Oral Liquid according to claim 1, characterized in that, In step (1), the method of dissolving the organic solvent is any one or more of shaking dissolution, ultrasonic treatment or reflux.

5. The characteristic spectrum detection method of Ejiao Blood-tonifying Oral Liquid according to any one of claims 1-4, characterized in that, The preparation method of the reference solution in step (1) is: Take glycine reference substance, add 0.1 mol / L hydrochloric acid solution to make a solution containing 0.16 mg of glycine per 1 ml; Take 5 ml of the above solution, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the continued filtrate to obtain.

6. The characteristic spectrum detection method of Ejiao Blood-tonifying Oral Liquid according to claim 1, wherein, In step (2), the addition amount of the organic solvent is 4 - 6 times that of Ejiao Buxue Oral Liquid.

7. The characteristic spectrum detection method of Ejiao Blood-tonifying Oral Liquid according to claim 1, characterized in that, In step (2), the method of adding organic solvent for extraction is: Add organic solvent to dissolve, shake well, filter, concentrate, prepare, and take the continued filtrate to prepare the test solution.

8. The characteristic spectrum detection method of Ejiao Blood-tonifying Oral Liquid according to claim 6 or 7, characterized in that The preparation of the test solution in step (2): Measure 5 ml of Ejiao Buxue Oral Liquid, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, let stand at room temperature for 1 hour, then add 50% acetonitrile to the scale, shake well, filter, and take the continued filtrate to obtain.

9. The characteristic spectrum detection method of Ejiao Blood Tonifying Oral Liquid according to claim 1, wherein, The chromatographic conditions in step (3) are: Chromatographic column: The filler is an octadecylsilane bonded silica gel column; Column temperature: 20°C - 40°C; Flow rate: 0.8 - 1.2 ml / min; Detection wavelength: 220 - 260 nm; The number of theoretical plates calculated by glycine is not less than 2000; Mobile phase: Use acetonitrile - 0.1% triethylamine solution (90:10) as mobile phase A, and methanol - 0.2% phosphoric acid solution (10:90) as mobile phase B; The gradient elution program is: 0 - 12 min, mobile phase A rises from 3 - 5% (v / v) to 10 - 14% (v / v); 12 - 28 min, mobile phase A rises from 10 - 14% (v / v) to 16 - 20% (v / v); 28 - 35 min, mobile phase A rises from 16 - 20% (v / v) to 24 - 26% (v / v); 35 - 45 min, mobile phase A rises from 24 - 26% (v / v) to 35 - 40% (v / v); 45 - 60 min, mobile phase A increases from 35 - 40% (v / v) to 50 - 60% (v / v); 60 - 100 min, mobile phase A is maintained at 50 - 60% (v / v).

10. The characteristic spectrum detection method of a Ejiao blood-tonifying oral liquid according to claim 9, characterized in that, In step (3), the chromatographic conditions are as follows: Column temperature: 30 °C; Flow rate: 1.0 ml / min; Detection wavelength: 254 nm; The number of theoretical plates calculated by glycine is not less than 2000; Mobile phase: Acetonitrile - 0.1% triethylamine solution (90:10) is used as mobile phase A, and methanol - 0.2% phosphoric acid solution (10:90) is used as mobile phase B; The gradient elution program is as follows: 0 - 12 min, mobile phase A increases from 4% (v / v) to 12% (v / v); 12 - 28 min, mobile phase A increases from 12% (v / v) to 18% (v / v); 28 - 35 min, mobile phase A increases from 18% (v / v) to 25% (v / v); 35 - 45 min, mobile phase A increases from 25% (v / v) to 38% (v / v); 45 - 60 min, mobile phase A increases from 38% (v / v) to 55% (v / v); 60 - 100 min, mobile phase A is maintained at 55% (v / v).

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