CN107991408B

CN107991408B - Method for measuring content of index component in four-flavor Zhenzhi eye drops

Info

Publication number: CN107991408B
Application number: CN201711210212.2A
Authority: CN
Inventors: 黄志元; 曹征宇; 饶礽; 王艳萍; 龚楚良; 胡勇勇; 朱样根
Original assignee: Jiangxi Zhenshiming Pharmaceutical Co ltd
Current assignee: Jiangxi Zhenshiming Pharmaceutical Co ltd
Priority date: 2017-11-27
Filing date: 2017-11-27
Publication date: 2020-06-30
Anticipated expiration: 2037-11-27
Also published as: CN107991408A

Abstract

The invention discloses a content determination method of characteristic components oligopeptide L-tyrosyl glycine and L-tryptophan in four-flavor Zhenzhu eye drops. The method is characterized in that the four-flavor valuable layer boron matte eye drops are subjected to separation, purification and structure identification, 7 monomer compounds including oligopeptides L-tyrosylglycine and L-tryptophan are determined to be characteristic components of the four-flavor valuable layer boron matte eye drops, the content of oligopeptides L-tyrosylglycine and L-tryptophan in the four-flavor valuable layer boron eye drops is determined by an HPLC method, the content of the characteristic components oligopeptides L-tyrosylglycine and L-tryptophan is controlled, the four-flavor valuable layer boron matte eye drops can be ensured to be stable and uniform in quality, and the quality controllability of the four-flavor valuable layer boron eye drops is improved. The method is simple, convenient and quick, has strong specificity, can accurately, stably and quickly determine the content of oligopeptide L-tyrosyl glycine and L-tryptophan in the four-pearl-layer Bingpeng eye drops, and can better control the quality of the product by applying the method.

Description

Method for measuring content of index component in four-flavor Zhenzhi eye drops

Technical Field

The invention belongs to the field of traditional Chinese medicine analysis, and particularly relates to a content determination method for characteristic components in four-flavor Zhenzhu eye drops.

Background

The traditional Chinese medicine has a long history of treating diseases, however, the complexity of the components of the traditional Chinese medicine brings difficulties to the quality control of the traditional Chinese medicine, the traditional Chinese medicine is prevented from going to the world market, and the accurate control of the content of each component is required to be well carried out for comprehensively controlling the quality of the traditional Chinese medicine.

The four-flavor Zhenzhu Bingpeng eye drops mainly comprise pearl powder; the pearl layer powder is powder prepared by processing and grinding pearl layers of shells of Hyriopsis cumingii (Lea), Cristaria cristata plicata (Leach) of the Unionidae animal or Pteria martensii (Dunker) of the Pinctada martensii of the Pinctada margaritifera, is a traditional Chinese medicine, is collected in the first part of 'Chinese pharmacopoeia' 2015 edition, and has the effects of calming the liver, suppressing yang, removing nebula and the like. The pearl layer powder is extracted to obtain the pearl layer powder extracting solution, so that the effective components in the pearl layer powder can better play a role. According to the domestic reports about the chemical components of the pearl powder, the main component of the pearl powder is calcium carbonate, and the pearl powder contains a small amount of trace elements, amino acids and the like, and the research on the chemical components is not deep. The four-pearl-layer boron matte eye drops are subjected to separation, purification and structure identification, and 7 monomer compounds including oligopeptide L-tyrosyl glycine and L-tryptophan are obtained and determined through first separation and are characteristic components of the four-pearl-layer boron matte eye drops, so that a foundation is laid for improving the quality control of the four-pearl-layer boron matte eye drops.

The identification and content determination method of four pearl layer boron eye drops is recorded in the first edition of the Chinese pharmacopoeia 2015, however, the item about the pearl layer powder as the main component in the content determination only comprises the total nitrogen determination, the total nitrogen determination has no pertinence and specificity to the components of the product, and the operations of digestion, distillation, titration and the like of the nitrogen determination method are complex, the requirements on the operation proficiency of detection personnel are high, the detection consumes long time, the pollution is large, the accuracy is poor, and the quality of the pearl layer powder is difficult to scientifically evaluate and control. Only the characteristic components and the contents thereof in the pearl layer powder extracting solution and the four-flavor Zhen-zhu eye drops are further measured, the drug effect of the four-flavor Zhen-zhu eye drops can be fundamentally ensured. According to the invention, the content of oligopeptides L-tyrosylglycine and L-tryptophan in the four-taste-pearl-layer tobermorite eye drops is determined by adopting an HPLC (high performance liquid chromatography) method, and the quality of the four-taste-pearl-layer tobermorite eye drops can be ensured to be stable and uniform by controlling the content of the oligopeptides L-tyrosylglycine and L-tryptophan which are characteristic components, so that the quality controllability of the four-taste-pearl-layer tobermorite eye drops.

Disclosure of Invention

The invention aims to provide a method for measuring the content of index components in four-flavor Zhenzhu boron eye drops.

The technical scheme of the invention is as follows:

a method for measuring the content of characteristic components in four-flavor Zhenzhen Bingpeng eye drops comprises the steps of separating, purifying and identifying the structure of the Zhenzhen eye drops, determining monomeric compounds including oligopeptides L-tyrosylglycine and L-tryptophan as the characteristic components of the Zhenzhen eye drops, and controlling the content of the characteristic components oligopeptides L-tyrosylglycine and L-tryptophan to ensure the stable and uniform quality of the four-flavor Zhenzhen Bingpeng eye drops, so that the safety and the quality controllability of medicines are improved.

Further, the method comprises the following steps:

(1) preparation of control solutions: weighing a proper amount of L-tyrosyl glycine and L-tryptophan, placing the weighed L-tyrosyl glycine and L-tryptophan into a 100mL volumetric flask, adding water for dissolving and diluting, shaking up, and fixing the volume to obtain a mixed reference stock solution;

(2) preparation of a test solution: sucking four kinds of Zhenzhi eye drops, centrifuging, and taking supernatant to obtain a test solution;

(3) the determination method comprises the following steps: and (4) respectively carrying out quantitative detection on the test solution and the reference solution by adopting a high performance liquid chromatography.

Further, step (2) further comprises any one or more of the following features:

(1) absorbing the dosage of the four-flavor Zhenzhu-Boran eye drops to be 1.0-20.0 mL;

(2) the centrifugation speed is 10,000-15,000 r/min;

(3) the centrifugation temperature is 2-30 ℃.

Further, in the step (3), Eclipse XDB-C produced by Agilent, USA, is selected₁₈(3.0 × 150mm, 3.5 μm) chromatographic column for separating Zhenshiming eye drop.

Further, in the step (3), a diode array detector is used for scanning the sample at full wavelength, chromatograms at all wavelengths are analyzed and compared, characteristic peaks and peak shapes detected at 220nm are determined, and chromatographic component information is obtained to obtain a complete picture formed by the reaction system.

Further, in step (3), the assay was performed using different gradients of different mobile phase systems, and the separation was performed by a water-line gradient of 0.085% TFA + 70% acetonitrile in water to 0.1% TFA.

Further, in the step (3), the quantitative determination method comprises the steps of: respectively measuring the test solution and the reference solution by HPLC under the same chromatographic conditions, comparing the chromatogram of the test solution with the chromatogram of the reference solution, and determining the characteristic peaks of L-tyrosylglycine and L-tryptophan in the test solution according to the relative retention time and the characteristic peaks.

Further, in the step (3), the quantitative determination method includes the steps of: respectively measuring the test solution and the reference solution by HPLC under the same chromatographic condition, and calculating the contents of the L-tyrosylglycine and the L-tryptophan in the test solution by peak area according to an external standard method.

Further, the method comprises the following steps:

(1) accurately weighing 6.16mg of L-tyrosyl glycine and 3.06mg of L-tryptophan, placing in a 100mL volumetric flask, adding water to dissolve and dilute, shaking up, and fixing the volume to be used as a mixed reference stock solution;

(2) the reference stock solution can be further diluted according to the detection requirement;

(3) sucking 10mL of the four-flavor Zhenzhu-bang eye drops, centrifuging at 12,000r/min for 15min, and taking the supernatant for later use.

(4) The HPLC column used was Agilent, Eclipse XDB-C18(3.0 × 150mm, 3.5 μm);

(5) mobile phase, phase a 0.1% trifluoroacetic acid (TFA) -water, phase B0.085% trifluoroacetic acid (TFA) + 70% acetonitrile in water;

(6) gradient elution was used:

(7) flow rate: 0.1-1.0 mL/min;

(8) detection wavelength: 200-300 nm;

(9) sample introduction amount: 5-50 mu L;

(10) column temperature: 20-40 ℃;

(11) the theoretical plate number is not less than 3000 calculated according to L-tyrosyl glycine.

(12) The product contains nacreous layer powder and L-tyrosyl glycine (C)₁₁H₁₄N₂O₄) Calculated as L-tryptophan (C) not less than 0.99 μ g₁₁H₁₂N₂O₂) It should not be less than 0.46 μ g.

Still further, comprising the steps of:

(2) precisely measuring a proper amount of reference stock solution, and adding water to dilute into a solution containing 1.150-1.300 mu g and 0.550-0.660 mu g of L-tyrosyl glycine and L-tryptophan per 1ml to obtain the product.

(4) The HPLC column used was Agilent, Eclipse XDB-C18(3.0 × 150mm, 3.5 μm);

(6) gradient elution was used:

gradient elution schedule

(7) Flow rate: 0.4 mL/min;

(8) detection wavelength: 220 nm;

(9) sample introduction amount: 20 mu L of the solution;

(10) column temperature: 30 ℃;

Compared with the prior art, the invention has the following beneficial effects:

the method adopts an HPLC method to determine the contents of oligopeptide L-tyrosylglycine and L-tryptophan in the four-taste Zhenzhu eye drops, is simple, convenient and quick, has strong specificity, and can accurately, stably and quickly determine the contents of oligopeptide L-tyrosylglycine and L-tryptophan in the four-taste Zhenzhu eye drops. The method is not only beneficial to the quality control of the four-flavor Zhenzhu-Borang eye drops, but also improves the quality controllability of the product.

The inventive analysis:

the pearl layer powder as the main component of Zhenshiming eye drops is powder prepared by processing and grinding pearl layers of shells such as Hyriopisis cumingii (Lea) and Cristaria plicata (Leach) of Unionidae animals, is a traditional Chinese medicine, is collected in the 2015 part of Chinese pharmacopoeia, and has the effects of calming the liver, suppressing yang, removing nebula and the like. The pearl layer powder is hydrolyzed to obtain pearl layer powder hydrolysate, so that the effective components in the pearl layer powder can better play a role. Therefore, in order to better utilize the nacreous resources and to elucidate the chemical basis of the pharmacological effects, it is necessary to systematically study the chemical components. The research of the experiment basically determines that the nacreous layer contains chemical components such as oligopeptides and the like.

The inventor carries out separation and purification and structure identification on the Zhenshiming eye drops to obtain 7 monomer compounds including oligopeptide L-tyrosyl glycine and L-tryptophan, and establishes a method for measuring the content of the characteristic components in the Zhenshiming eye drops for the characteristic components of the Zhenshiming eye drops and better controlling the quality of the Zhenshiming eye drops.

① the selection of liquid chromatographic column is the core part of liquid chromatographic analysis, the experiment compares different chromatographic columns under the condition that the chromatographic condition is determined, and the result shows that Eclipse XDB-C produced by Agilent in America is₁₈The chromatographic column (3.0 × 150mm, 3.5 μm) has good separation effect on Zhenshiming eye drops.

② the full-wavelength scanning of the sample is carried out by using a diode array detector, and the chromatogram under each wavelength is analyzed and compared, thus determining that the number of the detected peaks under 220nm is large, the separation of each peak is good, the characteristic peak is obvious and the peak shape is good, and the full-view of the chromatographic component information and the reaction system composition can be obtained from the chromatogram as much as possible.

③ the results of the experiments using different gradients of different mobile phase systems showed that the separation was best achieved with a linear gradient of 0.085% TFA + 70% acetonitrile in water to 0.1% TFA, with the smoothest baseline.

Drawings

FIG. 1 is a chromatogram of a control L-tyrosylglycine.

FIG. 2 is a chromatogram of an L-tryptophan control.

FIG. 3 is a chromatogram of a control of L-tyrosylglycine and L-tryptophan.

FIG. 4 is a chromatogram for measuring the content of four-ingredient Zhenzhi eye drops.

FIG. 5 is a minimum detection limit chromatogram of L-tyrosylglycine.

FIG. 6 is a minimum detection limit chromatogram of L-tryptophan.

FIG. 7 is a minimum quantitation limit chromatogram of L-tyrosylglycine.

FIG. 8 is a minimum quantitation limit chromatogram of L-tryptophan.

Detailed Description

Example 1

The content of oligopeptide L-tyrosylglycine and L-tryptophan in the four-flavor Zhenzhu eye drops is determined, and the four-flavor Zhenzhu eye drops are prepared from the following components: adjusting pH of Margarita powder extract, Borneolum Syntheticum, boric acid, and Borax to 7.4, adjusting osmotic pressure to 0.29Osmol/kg with sodium chloride, ethanol, phenoxyethanol, and water for injection.

1. Laboratory apparatus and reagent

High performance liquid chromatography (1260, agilent, usa); YC-800 chromatography experiment refrigerator (XMT608, Beijing Asteriaceae science and technology development Co., Ltd.); microtablet refrigerated centrifuge (fresh co 17, seimer feishell science); an ultrasonic apparatus (KQ-300E, ultrasonic apparatus Co., Ltd., Kunshan city); acetonitrile (chromatographically pure, Tiandi, Inc., USA); methanol (chromatographically pure, Shanghai Kangchi high purity solvent Co., Ltd.); trifluoroacetic acid (TFA) (chromatographically pure, alatin); water for injection (applicant); chromatography columns (Eclipse XDB-C18, Agilent, USA); guard column (Utimate XB-C18, Shanghai Yue Xu Co.); pipette gun (200. mu.L, 1000. mu.L, Einstend, USA).

2. Chromatographic conditions

The column was Agilent, Eclipse XDB-C18(3.0 × 150mm, 3.5 μm), the mobile phase was phase A (0.1% TFA-water), phase B (0.085% TFA + 70% acetonitrile in water), and the flow rate was 0.4mL/min, the amount of sample introduction: 20 μ L, the column temperature: 30 ℃ and the detection wavelength: 220nm, as shown in Table 2 below, using a gradient elution protocol.

TABLE 2 gradient elution schedule for four-ingredient Zhenzhi eye drops

3. Preparation of control solutions

Accurately weighing 6.16mg of L-tyrosyl glycine and 3.06mg of L-tryptophan, placing in a 100mL volumetric flask, adding water to dissolve and dilute, shaking uniformly, and fixing the volume to obtain a mixed reference stock solution with the mass concentrations of 61.6 mu g/mL and 30.6 mu g/mL respectively.

Precisely measuring a proper amount of the reference stock solution, and adding water to dilute into a solution containing 1.232 mu g and 0.612 mu g of L-tyrosyl glycine and L-tryptophan per 1ml to obtain the final product.

4. Preparation of test solution

Respectively sucking 10mL of the four-ingredient Zhenzhu bonbon eye drops prepared by the prescription in each batch, centrifuging at 12,000r/min for 15min, and taking the supernatant for later use.

5. Specificity experiments

5.1 blank adjuvant influence experiment

Weighing blank adjuvants (borax, boric acid, etc.) according to the prescription, placing in volumetric flask with certain volume, adding water to dissolve and dilute to scale, shaking, filtering, collecting filtrate as sample solution, and injecting into liquid chromatograph with 20 μ L of the sample solution; as a result, no peak appears in the blank auxiliary material under the chromatographic condition within the peak-appearing time of the main peak, which shows that the auxiliary material of the product has no interference on the content determination.

5.2 mobile phase Effect test

And injecting 20 mu L of mobile phase into a liquid chromatograph, and recording a chromatogram, wherein no peak appears in the peak emergence time of the main peak, which indicates that the measurement of the relative content of the mobile phase is not interfered.

6. Detection limit and quantification limit

Precisely weighing the reference substance, adding water to dissolve, diluting with equal times to obtain a series of solutions with gradient concentration from high to low, and injecting sample respectively. The sample concentration at the signal-to-noise ratio of 3 (S/N: 3) was set as the lowest detection concentration, and the lowest detection limit was obtained from the sample amount of 20 μ L (see fig. 5 and 6). As a result, the lowest detection limits of L-tyrosylglycine and L-tryptophan were respectively: 0.091. mu.g/mL, 0.1052. mu.g/mL.

The sample concentration at S/N of 10 was taken as the lowest quantitation limit (see fig. 7 and 8). Results the lowest limit of quantitation for L-tyrosylglycine and L-tryptophan were respectively: 0.1365 μ g/mL, 0.1925 μ g/mL.

7. Linear relation

Precisely sucking 1.0mL, 1.5mL, 2.0mL, 2.5mL and 3.0mL of mixed reference stock solution respectively, putting the mixed reference stock solution into a 100mL volumetric flask, adding water to dissolve and dilute the mixed reference stock solution, shaking the mixed reference stock solution uniformly, and performing constant volume to prepare a series of standard solutions, wherein the concentrations of the standard solutions are respectively L-tyrosyl glycine: 0.616. mu.g/mL, 0.924. mu.g/mL, 1.232. mu.g/mL, 1.540. mu.g/mL, 1.848. mu.g/mL; l-tryptophan: 0.306. mu.g/mL, 0.459. mu.g/mL, 0.612. mu.g/mL, 0.765. mu.g/mL, 0.918. mu.g/mL. The experiment was carried out under chromatographic conditions under item 2, and the chromatogram was recorded.

Taking the peak area Y as an ordinate and the sampling concentration (mu g/mL) of L-tyrosyl glycine or L-tryptophan as an abscissa to obtain a regression equation, wherein the ratio of L-tyrosyl glycine: y is₁＝63.866X₂+0.8193，r²0.9998; l-tryptophan: y is₂＝450.39X₃-1.68，r²＝0.9997。

The results show that: the mass concentrations of the L-tyrosyl glycine and the L-tryptophan are respectively 0.616-1.840 mu g/mL and 0.306-0.918 mu g/mL, and have good linear relation.

8. Precision test

Taking 6 parts of sample solution of the same batch, continuously sampling for 6 times according to the chromatographic conditions under the item 2, recording the peak areas of L-tyrosyl glycine and L-tryptophan, calculating the RSD of the peak areas, and obtaining a result: RSD of L-tyrosylglycine and L-tryptophan were 0.64% and 1.33%, respectively, indicating good precision of the instrument.

9. Repeatability test

Taking 6 parts of sample solution of the same batch, carrying out sample injection measurement according to the chromatographic condition under the item 2, recording peak area, and measuring the content of the component to be measured, wherein the RSD of the 6-time content measurement values of the L-tyrosyl glycine and the L-tryptophan is 0.80-0.93 percent, and the repeatability is good.

10. Stability test

Taking 6 parts of the same batch of test solution, injecting samples for 0h, 2h, 4h, 8h, 12h and 24h respectively according to the chromatographic conditions under the item 2, measuring the peak areas of L-tyrosyl glycine and L-tryptophan, calculating the peak area RSD value, and indicating that the test solution is stable within 24h, wherein the result RSD is 1.10-1.37%.

11. Sample application recovery test

6 portions of the same batch of four-flavor Zhenzhu Boron eye drops with known L-tyrosylglycine and L-tryptophan contents (actual contents are L-tyrosylglycine: 1.320 mu g/mL and L-tryptophan: 0.630 mu g/mL) are precisely measured, 5mL of each portion is precisely added with 5mL of the above 7. mixed reference solution (L-tyrosylglycine: 1.232 mu g/mL and L-tryptophan: 0.612 mu g/mL) under item, and the recovery rate (%) is calculated by a sample measurement method (measured amount-background amount)/added amount × 100% as a result, the sample adding recovery rate measurement item meets the requirements of pharmacopoeia.

TABLE 3 sample recovery test for L-tyrosylglycine and L-tryptophan

12. Sample assay

The four-ingredient layered Bingpeng eye drops prepared by the prescription of the embodiment are respectively taken for 3 batches, 2mL are respectively taken, the sample injection determination is carried out according to the chromatographic condition under the item 2, each batch of samples are parallelly determined for 2 times, and the determination results of the 3 batches of samples are shown in the following table 4.

TABLE 4 example 1 measurement results of four-flavor Zhenzhi eye drops

Example 2

The content of oligopeptide L-tyrosylglycine and L-tryptophan in the four-flavor Zhenzhu eye drops is determined, and the four-flavor Zhenzhu eye drops are prepared from the following components: adjusting pH of Margarita powder extract, Borneolum Syntheticum, boric acid, and Borax to 7.3, adjusting osmotic pressure to 0.29Osmol/kg with sodium chloride, ethanol, phenoxyethanol, and water for injection.

1. Experimental apparatus and reagents, chromatographic conditions, and control solution preparation the same as in example 1

2. Preparation of test solution

3. Specificity experiments

3.1 blank adjuvant influence experiment

4. The detection limit, the quantitative limit and the linear relationship are the same as those in example 1

5. Precision test

Taking 6 parts of sample solution of the same batch, continuously injecting samples for 6 times according to the chromatographic conditions, recording the peak areas of L-tyrosyl glycine and L-tryptophan, calculating the RSD of the peak areas, and obtaining the result: RSD of L-tyrosyl glycine and L-tryptophan are respectively 1.66% and 1.73%, which indicates that the precision of the instrument is good.

6. Repeatability test

Taking 6 parts of sample solution of the same batch, carrying out sample injection measurement according to the chromatographic conditions, recording peak areas, and measuring the content of the component to be measured, wherein the RSD of the measured value of the 6-th peak areas of the L-tyrosyl glycine and the L-tryptophan is 0.73-1.02 percent, and the repeatability is good.

7. Stability test

Taking 6 parts of sample solution of the same batch, injecting samples for 0h, 2h, 4h, 8h, 12h and 24h respectively according to the chromatographic conditions under the item 2, measuring the peak areas of L-tyrosyl glycine and L-tryptophan, calculating the peak area RSD value, and indicating that the sample solution is stable within 24h, wherein the result RSD is 0.097-1.16%.

8. Sample application recovery test

6 parts of four-ingredient layered boron matte eye drops of the same batch with known L-tyrosyl glycine and L-tryptophan contents (actual contents are L-tyrosyl glycine: 1.295 mu g/mL and L-tryptophan: 0.600 mu g/mL) are precisely measured, 5mL of each part is precisely added into 5mL of 7 mixed control solution (L-tyrosyl glycine: 1.232 mu g/mL and L-tryptophan: 0.612 mu g/mL) in example 1, the recovery rate (%) is calculated by a sample measuring method and is (measured amount-background amount)/added amount × 100%, and the sample adding recovery rate measuring item meets the requirements of pharmacopoeia.

TABLE 5 sample recovery test for L-tyrosylglycine and L-tryptophan

9. Sample assay

The four-ingredient layered Bingpeng eye drops prepared by the prescription of the embodiment are respectively taken for 3 batches, 2mL of each solution is respectively injected and measured according to the chromatographic conditions, each batch of samples is measured in parallel for 2 times, and the measurement results of the 3 batches of samples are shown in the following table 6.

TABLE 6 EXAMPLE 2 measurement results of four-ingredient Bingpeng eye drops

Furthermore, the chromatogram of the L-tyrosylglycine control is shown in FIG. 1. The chromatogram of the L-tryptophan control is shown in FIG. 2. The chromatogram of the control mixture of L-tyrosylglycine and L-tryptophan is shown in FIG. 3. The chromatogram for measuring the content of four-ingredient Zhenzhao eye drops is shown in FIG. 4.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, and the present invention shall fall within the protection scope of the present invention as long as the technical effects of the present invention are achieved by the same means.

Claims

1. A method for measuring the content of characteristic components in four-flavor-layer tobermorite eye drops is characterized in that the four-flavor-layer tobermorite eye drops are subjected to separation, purification and structure identification, monomeric compounds including oligopeptides L-tyrosylglycine and L-tryptophan are determined to be the characteristic components of the four-flavor-layer tobermorite eye drops, the content of oligopeptides L-tyrosylglycine and L-tryptophan in the four-flavor-layer tobermorite eye drops is measured by an HPLC (high performance liquid chromatography) method, and the quality of the four-flavor-layer tobermorite eye drops can be ensured to be stable and uniform by controlling the content of the oligopeptides L-tyrosylglycine and L-tryptophan which are characteristic components, so that the quality controllability of the four; wherein:

(1) the chromatographic columns used for HPLC were: agilent, Eclipse XDB-C₁₈，3.0×150mm，3.5μm；

(2) Mobile phase, phase a 0.1% trifluoroacetic acid (TFA) -water, phase B0.085% trifluoroacetic acid (TFA) + 70% acetonitrile in water;

(3) gradient elution was used:

2. the method of claim 1, comprising the steps of:

(1) preparation of control solutions: weighing a proper amount of L-tyrosyl glycine and L-tryptophan, placing the weighed L-tyrosyl glycine and L-tryptophan into a 100mL volumetric flask, adding water for dissolving and diluting, shaking up, and fixing the volume to obtain a reference solution;

(3) and (3) determination: and (4) respectively carrying out quantitative detection on the test solution and the reference solution by adopting a high performance liquid chromatography.

3. The method of claim 2, wherein:

(1) absorbing the four-flavor Zhenzhu-Boran eye drops with the amount of 1.0-20.0 mL;

(2) the centrifugation speed is 10,000-15,000 r/min;

(3) the temperature of centrifugation is 2-30 ℃.

4. The method of claim 2, wherein a diode array detector is used to scan the sample at full wavelength, and the chromatogram at each wavelength is analyzed and compared to determine the characteristic peak and peak shape detected at 220nm, and the information of chromatographic components is obtained to obtain the complete picture of the reaction system.

5. The method of claim 2, wherein in step (3), the determining step comprises: respectively measuring the test solution and the reference solution by HPLC under the same chromatographic conditions, comparing the chromatogram of the test solution with the chromatogram of the reference solution, and determining the characteristic peaks of L-tyrosylglycine and L-tryptophan in the test solution according to the relative retention time.

6. The method of claim 2, wherein in step (3), the determining step comprises: respectively measuring the test solution and the reference solution by HPLC under the same chromatographic condition, and calculating the contents of the L-tyrosylglycine and the L-tryptophan in the test solution by peak area according to an external standard method.

7. The method of claim 2, wherein:

(1) accurately weighing 6.16mg of L-tyrosyl glycine and 3.06mg of L-tryptophan, placing in a 100mL volumetric flask, adding water to dissolve and dilute, shaking up, and fixing the volume to be used as a reference solution;

(2) further diluting the reference solution;

(3) sucking 10mL of four-flavor Zhenzhu Bingpeng eye drops, centrifuging at 12,000r/min for 15min, and taking supernatant for later use;

(4) flow rate: 0.1-1.0 mL/min;

(5) detection wavelength: 200-300 nm;

(6) sample introduction amount: 5-50 mu L;

(7) column temperature: 20-40 ℃;

(8) the theoretical plate number is not less than 3000 calculated according to L-tyrosyl glycine;

(9) the product contains L-tyrosyl glycine (C) per ml nacreous layer powder₁₁H₁₄N₂O₄) Calculated as L-tryptophan (C) not less than 0.99 μ g₁₁H₁₂N₂O₂) It should not be less than 0.46 μ g.

8. The method of claim 7,

(1) diluting: precisely measuring a proper amount of reference solution, and adding water to dilute into a solution containing 1.150-1.300 mu g and 0.550-0.660 mu g of L-tyrosyl glycine and L-tryptophan per 1ml to obtain the final product;

(2) gradient elution was used:

gradient elution schedule

(3) Flow rate: 0.4 mL/min;

(4) detection wavelength: 220 nm;

(5) sample introduction amount: 20 mu L of the solution;

(6) column temperature: at 30 ℃.