CN112415115A - Detection method of blood-replenishing and milk-producing preparation - Google Patents
Detection method of blood-replenishing and milk-producing preparation Download PDFInfo
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Abstract
The invention discloses a detection method of a blood-enriching and milk-producing preparation, which comprises the following steps: (1) preparing a reference substance solution; (2) preparing a test solution; (3) and (5) detecting by HPLC-ELSD. The method has the advantages of simple operation, high accuracy, good specificity, high precision and good reproducibility, and is favorable for overall control of the preparation.
Description
Technical Field
The invention belongs to a medicine detection technology, and particularly relates to a detection method of a blood-enriching and milk-producing preparation.
Background
How to effectively evaluate the quality of the traditional Chinese medicine is one of the key problems faced by the modernization research of the traditional Chinese medicine. The traditional Chinese medicine components are complex, and the existing mode of evaluating the quality of the traditional Chinese medicine by using 1-2 chemical components cannot represent the integrity and complexity of the chemical components of the traditional Chinese medicine and effectively evaluate the quality of the traditional Chinese medicine.
The blood-enriching and milk-producing particle is formed by adding and reducing a formula of Yulu Yin from a secret formula of traditional Chinese medicine efficacy test at ancient and modern times and treasury [1], consists of nine traditional Chinese medicines of astragalus, angelica, white paeony root, tuckahoe, liquorice, cowherb seed, szechuan lovage rhizome, bitter orange and platycodon root, belongs to three new traditional Chinese medicines (Chinese medicine character Z0000082) in the original country, and is mainly used for postpartum hypogalactia caused by deficiency of qi and blood.
In recent years, pharmacological, toxicological and clinical applications of the blood-enriching and milk-producing granules are reported, but no relevant quality standard research is found. At present, the content of astragaloside IV is determined by thin layer chromatography in quality standard (WS3-575(Z-082) -2003(Z)) executed by blood-enriching raw milk particles. From the aspect of quality control, the method is simple, only quantitative control is carried out on the monarch drug astragalus, and no quality control index is established for other medicinal materials in the other side.
Disclosure of Invention
The invention aims to provide an HPLC-ELSD method for effectively controlling the internal quality of the blood-enriching raw milk preparation, shortening the detection time and saving the detection cost aiming at the current detection situation of the blood-enriching raw milk preparation, simultaneously determining the contents of astragaloside, ferulic acid, glycyrrhizic acid, cowherb seed flavonoid glycoside, naringin, neohesperidin and platycodin D in the blood-enriching raw milk granules and being beneficial to comprehensively controlling the quality of the product.
A detection method of a blood-enriching and milk-producing preparation comprises the following steps:
(1) preparation of control solutions:
weighing semen Vaccariae flavonoid glycoside, paeoniflorin, ferulic acid, naringin, neohesperidin, platycodin D, astragaloside IV and glycyrrhizic acid reference substances respectively, placing in a volumetric flask, adding methanol, ultrasonically dissolving, fixing volume to scale, shaking up, and making into reference substance solution;
(2) preparation of a test solution:
taking a quantitative blood-enriching raw milk preparation, placing the blood-enriching raw milk preparation in a conical flask or a volumetric flask, adding methanol, carrying out ultrasonic or reflux extraction for 10-20 min, cooling, taking supernatant, repeatedly extracting for 2-3 times, combining the supernatant, concentrating under reduced pressure to dryness, adding methanol solution to constant volume, filtering with a microporous membrane, and taking a subsequent filtrate to obtain a sample solution of the blood-enriching raw milk preparation;
(3) HPLC-ELSD detection:
respectively sucking the reference solution and the test solution obtained in the step (1) and the step (2), and respectively injecting the reference solution and the test solution into an HPLC-ELSD chromatograph for determination;
wherein, the chromatographic conditions are as follows: the chromatographic column is C18A reverse phase chromatography column; the detector is ELSD (drift tube temperature: 40 ℃); the column temperature is 30-35 ℃; the mobile phase A is 0.1-0.2% formic acid water solution, the mobile phase B is acetonitrile, and the flow rate is 0.8-1.2 ml/min; analysis time 52 minutes; gradient elution, elution order is as follows in table 1:
TABLE 1 gradient elution procedure
The step (1) of preparing the reference substance solution is preferably carried out by the following method:
accurately weighing appropriate amount of each reference substance, placing in a volumetric flask, adding methanol, ultrasonic dissolving and fixing volume to scale, and making into solution containing cowherb seed flavonoid glycoside 2500 μ g/ml, paeoniflorin 2500 μ g/ml, ferulic acid 2500 μ g/ml, naringin 2500 μ g/ml, neohesperidin 2500 μ g/ml, platycodin D2500 μ g/ml, astragaloside 2500 μ g/ml and glycyrrhizic acid 2500 μ g/ml.
The preparation of the test solution in the step (2) is preferably carried out by the following method:
preparing a blood-enriching and milk-producing preparation test solution:
a. the blood-enriching and milk-producing preparation is tablets, capsules and granules: taking the content of the blood-replenishing raw emulsion preparation, grinding, precisely weighing 1-2g, placing into a conical flask or a volumetric flask, adding 30-40mL of methanol, performing ultrasonic treatment for 10-15min, taking the supernatant, repeatedly extracting for 2-3 times, combining the supernatants, concentrating under reduced pressure to dryness, adding 1mL of methanol to dissolve and fix the volume, filtering with a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the blood-replenishing raw emulsion sample solution;
b. the blood-enriching and milk-generating preparation is a mixture and an oral liquid: and (3) uniformly mixing the blood-replenishing and milk-producing preparation, precisely measuring 5-10 ml, placing the mixture into a 25-100 ml volumetric flask, adding methanol, carrying out ultrasonic or reflux extraction for 10-20 min, cooling, diluting to a scale with methanol, shaking up, filtering with a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain a sample solution of the blood-replenishing and milk-producing preparation.
The ultrasonic treatment time in the step (2) is preferably 10 minutes.
The chromatographic column in the step (3) is preferably a Diamonsil C18 chromatographic column, and the specification of the chromatographic column is as follows: 250X4.6mm, 5 μm.
The mobile phase A in the step (3) is 0.1% formic acid aqueous solution.
The flow rate in the step (3) is 1.0 ml/min.
In the step (3), the sample injection amount is 10 μ L.
The invention has the beneficial effects that: the detection method can simultaneously determine the contents of astragaloside IV, ferulic acid, glycyrrhizic acid, cowherb seed flavonoid glycoside, naringin, neohesperidin and platycodin D in the blood-enriching and milk-producing preparation, and the method is simple, convenient, quick and accurate and is beneficial to comprehensively controlling the preparation.
The technical scheme of the invention is that the methodology investigation process is as follows:
1 Material
1.1 instruments
Shimadzu high performance liquid chromatograph (Shimadzu 20A) with ELSD (LT II), Shimadzu corporation, Japan; KQ5200B ultrasonic cleaner, ultrasonic instruments ltd, kunshan; an MS205DU model electronic balance, mettler-toledo instruments (shanghai) ltd, precision 0.01 mg; an ATX224 model electronic balance, Shimadzu, Japan, with an accuracy of 0.1 mg.
1.2 reagent
Vaccaria flavonoid glycoside (batch No. 111853-. Methanol is analytically pure (national pharmaceutical group); formic acid is chromatographically pure (national pharmaceutical group); acetonitrile is chromatographically pure (national drug group); the water is ultrapure water. The blood-replenishing and milk-producing granules are provided by Jiuzhi Zhitang GmbH, under the batch numbers 20180401, 20180402 and 20180507.
2 methods and results
2.1 chromatographic conditions, sample pretreatment mode, selection of chromatographic column
2.1.1 examination of chromatographic conditions
Due to the complex types of components in the blood-enriching and milk-producing granules, wherein astragaloside IV and platycodin D are absorbed at the tail end in an ultraviolet region, and the chromatographic behaviors of the vaccaria flavonol glycoside and the paeoniflorin are similar, so that the separation is difficult. Repeated after repeated groping, so the experiment finally adopts 0.1 percent formic acid water solution-acetonitrile as a mobile phase, gradient elution is carried out on a C18 chromatographic column, an evaporation light detector is used for detection, the peak shapes of 8 target chromatographic peaks are good, and the tailing factor, the sensitivity and the separation degree all meet the quantitative technical requirements.
2.1.2 examination of sample pretreatment
In the optimization of the pretreatment of the sample, acetonitrile, methanol and 50% methanol solution are respectively adopted to extract and compare the blood-enriching raw milk particle sample.
50% methanol extraction
Taking 0.5g of sample, adding 10mL of 50% methanol, carrying out ultrasonic extraction for 10min, taking supernatant, repeating for three times, combining the supernatants, concentrating under reduced pressure to dryness, adding methanol solution to constant volume, filtering with microporous membrane, taking subsequent filtrate, and obtaining chromatogram thereof as shown in figure 1.
Extraction of acetonitrile
Taking 0.5g of sample, adding 10mL of acetonitrile, carrying out ultrasonic extraction for 10min, taking supernatant, repeating for three times, combining the supernatants, carrying out reduced pressure concentration to dryness, adding methanol solution to constant volume, filtering with a microporous membrane, taking subsequent filtrate, and obtaining a chromatogram of the subsequent filtrate, wherein the chromatogram is shown in figure 2.
③ extraction of methanol
Taking 1g of sample, adding 10mL of methanol, performing ultrasonic extraction for 10min, taking supernatant, repeating for three times, combining the supernatants, concentrating under reduced pressure to dryness, adding methanol solution to constant volume, filtering with microporous membrane, taking subsequent filtrate, and obtaining chromatogram thereof as shown in FIG. 3.
The results show that when acetonitrile is used as an extraction solvent, the extraction efficiency of 8 components is very low, and some components cannot be detected even under chromatographic conditions, possibly due to poor solubility of each component. When the 50% methanol solution is used for extraction, the chromatogram is more complex than that of methanol extraction, and more interference peaks influencing the target chromatographic peak exist, which is not beneficial to quantitative analysis. Therefore, methanol was chosen as the final extraction solvent.
2.1.3 investigation of the column
This experiment investigated two different brands of chromatography columns [ Diamonsil C18(250X4.6mm, 5 μm) and Hypersil C18 ODS，(250x4.6mm，5μm)]As a result, the chromatographic peak separation of the flavonoid glycoside and paeoniflorin of cowherb seed was poor when the latter was used, while the separation was good when the former was used.
2.1.4 examination of gradient elution sequence
The experiment investigated different elution gradients,
sample treatment mode: taking 1g of sample, adding 30mL of methanol, performing ultrasonic extraction for 10min, taking supernatant, repeatedly extracting for 3 times, combining supernatants, concentrating under reduced pressure to dryness, adding 1mL of methanol to dissolve to constant volume, filtering with 0.45 μm microporous membrane, and taking subsequent filtrate; a chromatographic column: hypersil C18 ODS, 5 μm (250X4.6 mm); mobile phase: a0.1% aqueous formic acid solution, B acetonitrile; flow rate: 1 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: 30 ℃; a detector: ELSD; temperature of the drift tube: 40 deg.C
Gradient elution order 1:
0.01min(10％B)---30min(40％B)---45min(95％B)---55min(95％B)---55.01min (10％B)---62min(stop)
the resulting chromatogram is shown in FIG. 4.
Gradient elution order 2:
0.01min(10％B)---5min(12％B)---10min(15％B)---15min(20％B)---20min(25％ B)---35min(40％B)---50min(95％B)---52min(95％B)---52.01min(10％B)---55min(stop)
the resulting chromatogram is shown in FIG. 5.
Gradient elution order 3:
0.01min(10％B)---30min(40％B)---45min(95％B)---48min(95％B)---48.01min (10％B)---52min(stop)
the resulting chromatogram is shown in FIG. 6.
The results show that chromatographic peak separation degree of the cowherb seed flavonoid glycoside and paeoniflorin in the chromatograms obtained by the gradient elution sequences 1 and 2 is poor, so that the gradient elution sequence 3 is selected.
2.2 chromatographic conditions
The optimal chromatographic conditions of the blood-enriching and milk-producing particles are obtained by observing conditions such as a mobile phase system, a sample pretreatment mode, drift tube temperature, a chromatographic column, elution conditions and the like and repeatedly optimizing the conditions, wherein the optimal chromatographic conditions are as follows: diamonsil C18(250X4.6mm, 5 μm); mobile phase 0.1% aqueous formic acid-acetonitrile, gradient elution (procedure see table 2); the volume flow is 1 mL/min; column temperature: 30 ℃; the detector is ELSD (drift tube temperature: 40 ℃); the amount of the sample was 10. mu.L.
TABLE 2 gradient elution procedure
2.3 grinding the prepared particles of the test solution, precisely weighing about 1g, adding 30mL of methanol, performing ultrasonic treatment for 10min, taking the supernatant, repeatedly extracting for 3 times, combining the supernatants, concentrating under reduced pressure to dryness, adding 1mL of methanol to dissolve and fix the volume, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate.
2.4 preparation of control solution A proper amount of each control is precisely weighed, and added with methanol to prepare solutions respectively containing 2500 μ g/ml of cowherb seed flavonoid glycoside, 2500 μ g/ml of paeoniflorin, 2500 μ g/ml of ferulic acid, 2500 μ g/ml of naringin, 2500 μ g/ml of neohesperidin, 2500 μ g/ml of platycodin D, 2500 μ g/ml of astragaloside and 2500 μ g/ml of glycyrrhizic acid, thus obtaining the product.
2.5 methodological considerations
2.5.1 Linear relationship examination of the precision absorption of the mixed reference stock solution prepared by the method under the '2.4' item,
respectively placing 0.25 ml, 2ml, 2.5 ml, 4 ml and 5ml into a 10ml measuring flask, fixing the volume with methanol, and shaking up to obtain a series of mixed reference substance solutions with mass concentration. Respectively sucking 10 μ l of each of the above series of mixed reference solutions, injecting into liquid chromatograph, and recording chromatogram. The peak area natural logarithm was plotted against the natural logarithm of the concentration to obtain the calibration curve equation for each component, as shown in Table 3.
TABLE 3 Linear relationship of the components
2.5.2 precision test the same test sample (batch No. 20180401) was prepared into test sample solution according to the method under item "2.3", and 6 times of sample injection was carried out under the chromatographic condition of item "2.2", and the peak areas RSD of vaccaria flavonoid glycoside, paeoniflorin, ferulic acid, naringin, neohesperidin, platycodin D, astragaloside A and glycyrrhizic acid were respectively 0.56%, 1.34%, 1.85%, 1.02%, 0.89%, 1.21%, 1.53% and 2.01%, which indicated that the instrument had good precision.
2.5.3 repeatability test the same test sample (lot No. 20180401), preparing 6 test sample solutions in parallel according to the method under item "2.3", measuring under the chromatographic condition of item "2.2", and measuring the contents of vaccaria flavonoid glycoside, paeoniflorin, ferulic acid, naringin, neohesperidin, platycodin D, astragaloside IV and glycyrrhizic acid RSD to be 1.06%, 2.11%, 0.94%, 1.38%, 1.45%, 2.33%, 0.95% and 1.74%, respectively, which shows that the method has good repeatability.
2.5.4 stability test the sample (batch No. 20180401) prepared by the method of item "2.3" was analyzed according to the liquid chromatography conditions under item "2.2", and the sample was injected at different time points of 0, 2, 6, 12, 18, 24h and analyzed, the sample injection amount was 10 μ l, and the peak area RSD values of vaccaria flavonoid glycoside, paeoniflorin, ferulic acid, naringin, neohesperidin, platycodin D, astragaloside IV and glycyrrhizic acid were 1.32%, 2.01%, 1.42%, 2.11%, 2.04%, 3.01%, 2.14% and 3.27%, respectively, and the results showed that the sample solution had little change in the chromatographic peak area within 24h and good stability.
2.5.5 sample application recovery test about 1g of uniform sample (batch No. 20180401) was precisely weighed, 6 portions were placed in a 50ml measuring flask, appropriate amounts of each control were added, sample solutions were prepared according to the method of item "2.3", and the recovery was calculated by performing the measurement under the liquid chromatography condition of item "2.2". As a result, the average sample recovery rates of vaccaria flavonoid glycoside, paeoniflorin, ferulic acid, naringin, neohesperidin, platycodin D, astragaloside IV and glycyrrhizic acid are 97.72%, 97.67%, 95.87%, 96.70%, 99.47%, 96.31%, 95.51% and 96.06%, respectively, and the RSD is 3.69%, 2.44%, 0.31%, 3.44%, 1.63%, 0.82%, 3.70% and 2.89%, respectively.
TABLE 4 sample recovery test results
2.5.6 content determination of sample 3 batches of granules were prepared, and the test solution was prepared according to the method of item "2.3", and the content was calculated under the conditions of liquid chromatography under the item "2.2", and the results are shown in table 5.
Table 5 results of content measurement of sample (n ═ 3)
Drawings
FIG. 1: a chromatogram obtained by extracting a sample with 50% methanol;
FIG. 2: extracting a chromatogram of a sample from acetonitrile;
FIG. 3: a chromatogram obtained by extracting a sample with methanol;
FIG. 4: elution of the chromatogram obtained for gradient sequence 1;
FIG. 5: elution of the chromatogram obtained for gradient sequence 2;
FIG. 6: elution of the chromatogram obtained for gradient sequence 2;
fig. 7 is a chromatogram of the mixed standard of example 1, wherein the peaks are 1: cowherb seed flavonoid glycoside, 2: paeoniflorin, 3: ferulic acid, 4: naringin, 5: neohesperidin, 6: platycodin D, 7: astragaloside IV, 8: glycyrrhizic acid;
fig. 8 is a sample chromatogram of the blood-enriching raw milk particles of example 1, wherein each peak is 1: cowherb seed flavonoid glycoside, 2: paeoniflorin, 3: ferulic acid, 4: naringin, 5: neohesperidin, 6: platycodin D, 7: astragaloside IV, 8: glycyrrhizic acid.
Detailed Description
Example 1 detection method of blood-enriching lactogenesis granules
1.1 drugs and reagents
Vaccaria flavonoid glycoside (batch No. 111853-. Methanol is analytically pure (national pharmaceutical group); formic acid is chromatographically pure (national pharmaceutical group); acetonitrile is chromatographically pure (national drug group); the water is ultrapure water. The blood-replenishing and milk-producing granule is supplied by Jiuzhitong corporation, lot number 20180301.
1.2 instruments
Shimadzu high performance liquid chromatograph (Shimadzu 20A) with ELSD (LT II), Shimadzu corporation, Japan; KQ5200B ultrasonic cleaner, ultrasonic instruments ltd, kunshan; an MS205DU model electronic balance, mettler-toledo instruments (shanghai) ltd, precision 0.01 mg; an ATX224 model electronic balance, Shimadzu, Japan, with an accuracy of 0.1 mg.
2 detection method and results
2.1 preparation of control solutions
Accurately weighing appropriate amount of control substances (semen Vaccariae flavonoid glycoside, penoniflorin, ferulic acid, naringin, neohesperidin, platycodin D, astragaloside IV, glycyrrhizic acid), placing in volumetric flask, adding methanol, ultrasonic dissolving and fixing volume to scale to obtain solutions containing 2500 μ g/ml of semen Vaccariae flavonoid glycoside, 2500 μ g/ml of paeoniflorin, 2500 μ g/ml of ferulic acid, 2500 μ g/ml of naringin, 2500 μ g/ml of neohesperidin, 2500 μ g/ml of platycodin D, 2500 μ g/ml of astragaloside IV, and 2500 μ g/ml of glycyrrhizic acid, to obtain control solution.
2.2 preparation of test solutions
Taking the content of the blood-replenishing raw milk particles, grinding, precisely weighing 1g, placing in a conical flask or a volumetric flask, adding 30mL of methanol, carrying out ultrasound for 10min, taking the supernatant, repeatedly extracting for 3 times, combining the supernatants, concentrating under reduced pressure to dryness, adding 1mL of methanol to dissolve to a constant volume, filtering with a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the sample solution of the blood-replenishing raw milk.
2.3 chromatographic conditions
A chromatographic column: diamonsil C18(250X4.6mm, 5 μm); mobile phase 0.1% aqueous formic acid-acetonitrile, gradient elution (procedure see table 6); the volume flow is 1 mL/min; column temperature: 30 ℃; the detector is ELSD (drift tube temperature: 40 ℃); the amount of the sample was 10. mu.L.
TABLE 6 gradient elution procedure
2.4 measurement of content
Respectively sucking the reference solution and the test solution obtained by 2.1 and 2.2, respectively injecting the reference solution and the test solution into an HPLC-ELSD combined instrument for determination, wherein the reference chromatogram and the test chromatogram are respectively shown in the figure 7 and the figure 8, and the content determination results are shown in the following table 7:
table 7 measurement results of contents of respective components (mg/g, n ═ 3)
Claims (8)
1. A detection method of a blood-enriching and milk-producing preparation comprises the following steps:
(1) preparation of control solutions:
weighing semen Vaccariae flavonoid glycoside, paeoniflorin, ferulic acid, naringin, neohesperidin, platycodin D, astragaloside IV and glycyrrhizic acid reference substances respectively, placing in a volumetric flask, adding methanol, ultrasonically dissolving, fixing volume to scale, shaking up, and making into reference substance solution;
(2) preparation of a test solution:
taking a quantitative blood-enriching raw milk preparation, placing the blood-enriching raw milk preparation in a conical flask or a volumetric flask, adding methanol, carrying out ultrasonic or reflux extraction for 10-20 min, cooling, taking supernatant, repeatedly extracting for 2-3 times, combining the supernatant, concentrating under reduced pressure to dryness, adding methanol solution to constant volume, filtering with a microporous membrane, and taking a subsequent filtrate to obtain a sample solution of the blood-enriching raw milk preparation;
(3) HPLC-ELSD detection:
respectively sucking the reference solution and the test solution obtained in the step (1) and the step (2), and respectively injecting the reference solution and the test solution into an HPLC-ELSD chromatograph for determination;
wherein, the chromatographic conditions are as follows: the chromatographic column is C18A reverse phase chromatography column; the detector is ELSD (drift tube temperature: 40 ℃); the column temperature is 30-35 ℃; the mobile phase A is 0.1-0.2% formic acid water solution, the mobile phase B is acetonitrile, and the flow rate is 0.8-1.2 ml/min; analysis time 52 minutes; gradient elution, elution order is as follows in table 1:
TABLE 1 gradient elution procedure
2. The method of claim 1, wherein: the step (1) of preparing the reference substance solution is carried out according to the following method:
accurately weighing appropriate amount of each reference substance, placing in a volumetric flask, adding methanol, ultrasonic dissolving and fixing volume to scale, and making into solution containing cowherb seed flavonoid glycoside 2500 μ g/ml, paeoniflorin 2500 μ g/ml, ferulic acid 2500 μ g/ml, naringin 2500 μ g/ml, neohesperidin 2500 μ g/ml, platycodin D2500 μ g/ml, astragaloside 2500 μ g/ml and glycyrrhizic acid 2500 μ g/ml.
3. The method of claim 1, wherein: the preparation of the test solution in the step (2) is carried out according to the following method:
preparing a blood-enriching and milk-producing preparation test solution:
a. the blood enriching preparation is tablets, capsules and granules: taking the content of the blood-replenishing raw emulsion preparation, grinding, precisely weighing 1-2g, placing into a conical flask or a volumetric flask, adding 30-40mL of methanol, performing ultrasonic treatment for 10-15min, taking the supernatant, repeatedly extracting for 2-3 times, combining the supernatants, concentrating under reduced pressure to dryness, adding 1mL of methanol to dissolve and fix the volume, filtering with a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the blood-replenishing raw emulsion sample solution;
b. the blood-enriching and milk-generating preparation is a mixture and an oral liquid: and (3) uniformly mixing the blood-replenishing and milk-producing preparation, precisely measuring 5-10 ml, placing the mixture into a 25-100 ml volumetric flask, adding methanol, carrying out ultrasonic or reflux extraction for 10-20 min, cooling, diluting to a scale with methanol, shaking up, filtering with a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain a sample solution of the blood-replenishing and milk-producing preparation.
4. The method of claim 1, wherein: in the step (3), the sample injection amount is 10 μ L.
5. A method according to claim 1 or 3, characterized in that: the ultrasonic treatment time in the step (2) is 10 minutes.
6. The method of claim 1, wherein: the chromatographic column in the step (3) is a Diamonsil C18 chromatographic column, and the specification of the chromatographic column is as follows: 250x4.6mm, 5 μm.
7. The method of claim 1, wherein: the mobile phase A in the step (3) is 0.1% formic acid aqueous solution.
8. The method of claim 1, wherein: the flow rate in the step (3) is 1.0 ml/min.
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