UPLC characteristic spectrum detection method for immature bitter orange and Chinese herbaceous peony powder

Abstract

The invention belongs to the field of pharmaceutical analysis, and particularly discloses a UPLC (Ultra Performance Liquid Chromatography) specific chromatogram detection method of immature bitter orange and Chinese herbaceous peony powder, which comprises the following steps: taking octadecylsilane chemically bonded silica as a filling agent for a chromatographic column; acetonitrile is used as a mobile phase A, and a 0.1-0.3% phosphoric acid solution is used as a mobile phase B; the gradient elution procedure lasts for 0-23 minutes, and 5%-47% A is obtained. The characteristic chromatogram established through specific chromatographic conditions can be used for calibrating characteristic components of different categories and different polarities including alkaloids, organic acids, flavones, flavonoid glycosides and the like. The calibration of the components realizes the quality control of the immature bitter orange and paeonia lactiflora powder, ensures the clinical curative effect of the immature bitter orange and paeonia lactiflora powder, and has practical popularization and application values.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical analysis, in particular to a HPLC characteristic chromatogram detection method of Zhishai Shaoyao Powder. BACKGROUND

[0002] Ancient classic prescription Zhishai Shaoyao Powder is from Zhang Zhongjing's <Synopsis of Golden Chamber>·<Treatment of the Twenty-First Pulse and Syndrome of Postpartum Disease in Women>, which has the functions of breaking qi and blood, relieving restlessness and relieving pain. It is widely spread in later generations because of its excellent curative effect on treating abdominal pain in women after childbirth. The prescription is made into a powder by combining two Chinese medicines, Zhishi and Shaoyao. Li Chenyu et al. analyzed and researched <Ancient Literature Analysis and Research of Classic Prescription Zhishai Shaoyao Powder> and determined that Shaoyao is Baishao and Zhishi is Zhishi charcoal, and each is equally divided. It is used to treat postpartum abdominal pain, acute pelvic inflammatory disease, acute cholecystitis, intestinal diseases, and sequelae of stroke.

[0003] The quality control of ancient classic prescription can ensure its curative effect in modern application to be consistent with the ancient records. Especially through liquid chromatography fingerprint, the authenticity of ancient classic prescription can be identified to prevent the circulation of fake and inferior products. In addition, different batches of ancient classic prescription preparations can be analyzed to ensure the consistency of their chemical components, which is crucial to ensure their clinical efficacy and safety. However, there are few quality researches on Zhishai Shaoyao Powder preparations at present, especially the research on comprehensively reflecting the characteristics of complex components in Zhishai Shaoyao Powder through liquid chromatography fingerprint is still a blank, which limits the research and development and clinical popularization and application of modern Zhishai Shaoyao Powder preparations. SUMMARY

[0004] To solve the above problems, the present application provides a UPLC characteristic chromatogram detection method of Zhishai Shaoyao Powder, which comprises the following steps: 1) Preparation of reference solution of reference substance: take alkaloids, organic acids, flavonoids and / or flavonoid glycosides reference substance, and dissolve in methanol to obtain the reference solution of reference substance; 2) Preparation of test solution: take the Zhishai Shaoyao Powder to be tested, add methanol solution for extraction, filter the extract, and take the filtrate to obtain the test solution; 3) Inject the reference solution of reference substance and the test solution into the UPLC instrument respectively, and record the chromatogram; the chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel as the filler; mobile phase: acetonitrile as the mobile phase A, and 0.1-0.3% phosphoric acid solution as the mobile phase B; gradient elution program 0-23 min, 5%→47% A.

[0005] Further, the reference solution of step 1) contains 5-15 μg of each reference substance per 1 ml.

[0006] Further, the alkaloids in the control are synephrine, the organic acids are gallic acid, the flavones are catechin, naringenin, nobiletin, tangeritin and / or hesperidin, and the flavonoid glycosides are paeoniflorin, albiflorin, benzoylpaeoniflorin, naringin, narirutin, hesperidin, neohesperidin and / or neonorbeckin.

[0007] Further, the mass-volume ratio of the to-be-tested Zhishirenyangpian and the methanol solution in step 2) is 0.4-0.8 g:25-50 ml; the methanol solution is 70% methanol; and the methanol solution is 70-100% methanol, preferably 70% methanol.

[0008] Further, the to-be-tested Zhishirenyangpian is a powder mixture of Zhishiren carbon and Baishao. The mass ratio of Zhishiren carbon and Baishao is 1:1.

[0009] Further, the extraction in step 2) is ultrasonic extraction, with a power of 600 W, a frequency of 40 kHz, and a time of 20-40 min.

[0010] Further, in the chromatographic conditions in step 3), the chromatographic column is: ACQUITY UPLC® BEH C18, 1.7 μm, 2.1 mm x 100 mm; the column temperature is 25-35°C, the injection volume is 1-3 μL, the flow rate is 0.25-0.35 mL·min -1 , the detection wavelength is 220-230 nm, preferably the column temperature is 30°C, the injection volume is 2 μL, the flow rate is 0.3 mL·min -1 , and the detection wavelength is 230 nm.

[0011] Further, the chromatogram of the test solution should exhibit 17 characteristic peaks, wherein the peak corresponding to the hesperidin reference is the S peak, and the relative retention time of each characteristic peak to the S peak should be within ±10% of the specified value, with the specified value being peak 1:0.083, peak 2:0.146, peak 3:0.421, peak 4:0.603, peak 5:0.665, peak 6:0.835, peak 7:0.896, peak 8:0.917, peak 9:0.962, peak 11:1.045, peak 12:1.302, peak 13:1.427, peak 14:1.454, peak 15:1.508, peak 16:1.914, and peak 17:2.104.

[0012] Further, the peak 1 corresponds to alkaloids, peak 2 corresponds to organic acids, peaks 3, 13, 15, 16 and 17 correspond to flavones, and peaks 4-6, 8 and 9-11 correspond to flavonoid glycosides.

[0013] Further, the component corresponding to the peak 1 is synephrine, the component corresponding to the peak 2 is gallic acid, the component corresponding to the peak 3 is catechin, the component corresponding to the peak 4 is paeonol glycoside, the component corresponding to the peak 5 is paeoniflorin, the component corresponding to the peak 6 is neohesperidin, the component corresponding to the peak 8 is naringin, the component corresponding to the peak 9 is naringin, the component corresponding to the peak 10 is hesperidin, the component corresponding to the peak 11 is neohesperidin, the component corresponding to the peak 13 is naringenin, the component corresponding to the peak 14 is benzoylpaeoniflorin, the component corresponding to the peak 15 is hesperetin, and the component corresponding to the peak 16 is nobiletin, and the component corresponding to the peak 17 is tangeretin.

[0014] The UPLC characteristic spectrum detection method of the Citri Fructus and Paeoniae Dispersion can determine 17 characteristic peaks by the characteristic spectrum established by specific chromatographic conditions, and further identify 15 characteristic components including alkaloids, organic acids, flavones, flavone glycosides and other different categories and different polarities by using reference substances. These components are mainly active components of the carbon of Citri Fructus, and benzoylpaeoniflorin with significant pharmacological activity in Paeoniae is identified from these components. The identification of these more characteristic chemical components realizes the quality control of the Citri Fructus and Paeoniae Dispersion, guarantees the clinical efficacy of the Citri Fructus and Paeoniae Dispersion, and has practical popularization and application value.

[0015] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present application.

[0016] The above content of the present application will be further explained in detail by the following embodiment. However, it should not be understood that the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Different mobile phase type chromatograms; Figure 2 Different phosphoric acid concentration chromatograms; Figure 3 Different gradient chromatograms; Figure 4 Different wavelength chromatograms; Figure 5 Different column temperature investigation; Figure 6 Different flow rate investigation; Figure 7 Different sample amount investigation; Figure 8 Delay investigation; Figure 9Extraction solvent investigation; Figure 10 Extraction time investigation; Figure 11 Solvent addition amount investigation; Figure 12 Comparison of characteristic chromatograms of single herb pieces and Zhishisheyang powder samples; Figure 13 Chromatographic peak identification; Figure 14 Specificity; Figure 15 Verification figures of characteristic chromatograms of 5 batches of Zhishisheyang powder samples; Figure 16 Control characteristic chromatogram. DETAILED DESCRIPTION

[0018] The reagents, reagents, and equipment used in the embodiments of the present application are known products, which are obtained by purchasing commercially available products. The Zhishisheyang powder is prepared as follows: The Zhishisheyang powder is prepared as follows:

[0019] Example 1: Detection of characteristic chromatogram of Zhishisheyang powder of the present application 1) Preparation of reference solution of control Take syringid, gallic acid, catechin, paeoniflorin, albiflorin, benzoylpaeoniflorin, naringin, aurantiamarin, hesperidin, neohesperidin, neoreinerin, nobiletin, tangeretin, naringenin, and hesperetin control, accurately weigh, add methanol to prepare a mixture solution containing 10 μg of each control per 1 mL as a reference solution of control; 2) Preparation of test solution Take 0.5 g of Zhishisheyang powder, place it in a conical flask with a plug, add 50 ml of 70% methanol, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake, filter, take the filtrate, and obtain; 3) Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, and record the chromatogram. The chromatographic conditions are as follows: Chromatographic column: ACQUITY UPLC® BEH C18, 1.7 μm, 2.1 mm × 100 mm; detection wavelength: 230 nm; column temperature: 30℃, flow rate: 0.3 mL·min -1 ; injection amount: 2 μL; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; gradient elution program: 4) Analysis of chromatogram The chromatogram of the test solution should present 17 characteristic peaks, of which 15 peaks correspond to the retention time of the reference peak of the corresponding control substance respectively. The peak corresponding to the reference peak of hesperidin is the S peak, and the relative retention time of the other two peaks, peak 7 and peak 12, to the S peak is calculated, which should be within ±10% of the specified value: 0.896 (peak 7), 1.302 (peak 12).

[0020] Example 2 Detection of the characteristic spectrum of Zhishai Shaoyao Powder of the application 1) Preparation of the control substance reference solution Take the hesperidin control substance, accurately weigh and add methanol to prepare a solution containing 10 μg per 1 mL as the control substance reference solution; 2) Preparation of the test solution Take 0.5 g of Zhishai Shaoyao Powder, place it in a conical flask with a plug, add 50 ml of 70% methanol, tightly plug, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate, which is obtained; 3) Accurately take 10 μl of the reference solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, and record the chromatogram. The chromatographic conditions are as follows: Chromatographic column: ACQUITY UPLC® BEH C18, 1.7 μm, 2.1 mm × 100 mm; detection wavelength: 230 nm; column temperature: 30 ℃, flow rate: 0.3 mL·min -1 ; injection volume: 2 μL; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; gradient elution program: 4) Analysis of the chromatogram The chromatogram of the test solution should present 17 characteristic peaks, of which the peak corresponding to the reference of hesperidin is the S peak, and the relative retention time of each characteristic peak to the S peak is calculated, which should be within ±10% of the specified value: 0.083 (peak 1), 0.146 (peak 2), 0.421 (peak 3), 0.603 (peak 4), 0.665 (peak 5), 0.835 (peak 6), 0.896 (peak 7), 0.917 (peak 8), 0.962 (peak 9), 1.045 (peak 11), 1.302 (peak 12), 1.427 (peak 13), 1.454 (peak 14), 1.508 (peak 15), 1.914 (peak 16), 2.104 (peak 17); Wherein, peak 1 corresponds to the component of synephrine, peak 2 corresponds to the component of gallic acid, peak 3 corresponds to the component of catechin, peak 4 corresponds to the component of paeonol glycoside, peak 5 corresponds to the component of paeoniflorin, peak 6 corresponds to the component of neorein, peak 8 corresponds to the component of naringin, peak 9 corresponds to the component of naringin, peak 10 corresponds to the component of hesperidin, peak 11 corresponds to the component of neohesperidin, peak 13 corresponds to the component of naringenin, peak 14 corresponds to the component of benzoylpaeoniflorin, peak 15 corresponds to the component of hesperetin, peak 16 corresponds to the component of nobiletin, and peak 17 corresponds to the component of tangeretin.

[0021] The beneficial effects of the present application are illustrated by the following experimental examples: Experimental Example 1 Fingerprint spectrum research of Citri Fructus and Paeoniae Radix powder 1. Chromatographic conditions In the previous test process, the existing fingerprint spectrum of Citri Fructus and the fingerprint spectrum of Paeoniae Radix cannot effectively separate the characteristic components of the two traditional Chinese medicines in one chromatogram, which leads to the inability to control the quality of Citri Fructus and Paeoniae Radix powder. Through the exploration and attempt of chromatographic conditions, the characteristic spectrum conditions of Citri Fructus and Paeoniae Radix powder are preliminarily determined as follows, and the detection is carried out according to the conditions.

[0022] Chromatographic conditions: octadecylsilane bonded silica gel as filler (ACQUITY UPLC® BEH C18, 1.7 μm, 2.1 mm × 100 mm); gradient elution with acetonitrile as mobile phase A and acid solution as mobile phase B; flow rate is 0.3 ml per minute; column temperature is 30°C; detection wavelength is 230 nm; gradient elution program: 1.1 Inorganic phase screening in mobile phase On the basis of the proposed chromatographic conditions, three different types of mobile phases, acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% formic acid, and acetonitrile-0.1% glacial acetic acid, were used to investigate the peak shape and separation of the chromatogram of the test solution of Citri Fructus and Paeoniae Radix powder extracted by 70% methanol, in order to determine the best type of mobile phase. The results are shown in Figure 1 .

[0023] From Figure 1 It can be seen that under the conditions of the three mobile phases, the retention time and separation of peaks 3-17 have no obvious difference; but under the conditions of acetonitrile-0.1% phosphoric acid, the peak shape and separation of peak 1 (synephrine) and peak 2 (gallic acid) are better, and under the conditions of acetonitrile-0.1% formic acid and acetonitrile-0.1% glacial acetic acid, peak 1 and peak 2 are all superimposed with the inverted peak part of the solvent peak, affecting the identification and integration of the chromatographic peaks. Therefore, the composition of the mobile phase is selected as acetonitrile-0.1% phosphoric acid.

[0024] 1.2 Inorganic phase phosphoric acid concentration screening in mobile phase On the basis of the inorganic phase acid species determined in the mobile phase, the acid concentration was further changed, and four different types of mobile phase were used respectively, acetonitrile-0.3% phosphoric acid, acetonitrile-0.5% phosphoric acid, acetonitrile-1.0% phosphoric acid, acetonitrile-3.0% phosphoric acid, to investigate the peak shape and separation of the chromatogram, in order to determine the best phosphoric acid concentration. The results are shown in Figure 2 .

[0025] From Figure 2 It can be seen that with the increase of phosphoric acid concentration, peak 4 (albiflorin glycoside) gradually collapses, and when the phosphoric acid concentration reaches 3.0%, peak 4 almost disappears. The reason is that the key component albiflorin glycoside has an unsaturated lactone ring structure, which makes the component not stable enough. If the acidity of the mobile phase is too strong, the lactone ring chemical bond will be broken and opened, and albiflorin glycoside and its degradation products cannot be separated. In order to ensure the stability of the component and the normal peak shape of the chromatographic peak, the concentration of phosphoric acid solution is determined to be 0.1%~0.3%.

[0026] 1.3 Mobile phase gradient screening On the basis of the determination of the mobile phase, the mobile phase gradient was adjusted in the same time range (0~23 min) to determine the best chromatographic conditions. The results are shown in Figure 3 , Table 1.

[0027] Table 1 Separation under different gradients From the results, it can be seen that different gradient conditions have a greater impact on the separation of peaks 7 and 13 (naringenin). Specifically, when the acetonitrile termination concentration is large (greater than 47%), peak 7 is shifted or coincides with peaks 8~10, which affects the effective separation and detection of the main flavonoid components naringin, naringin, and hesperidin, and peaks 13 and 14 also coincide, which cannot effectively separate the main active components naringenin and benzoyl peony glycoside. When the acetonitrile termination concentration is small (less than 47%), peaks 12~15 are shifted as a whole, and when the acetonitrile termination concentration is 40%, peak 15 coincides with the adjacent interfering peak, which affects the effective separation and detection of the main active component naringenin, and because the acetonitrile termination ratio is too low, peak 17 cannot be eluted within 23 minutes. It shows that the gradient change range of acetonitrile in the gradient elution process can only be in the range of 5%~47%, so that the 17 characteristic components in the Fructus Aurantii and Paeoniae Powder can be presented in the chromatogram within 23 min.

[0028] 1.4 Detection wavelength investigation On the basis of the determination of the mobile phase and elution program, the diode array detector was used to scan the full wave band of the test solution, and the chromatograms of the test solution at 230 nm, 260 nm, 300 nm, and 330 nm were extracted respectively. See Figure 4The results show that the chromatographic peaks are more obvious at a detection wavelength of 230 nm for 0-23 min, and therefore the detection wavelength is determined to be 230 nm.

[0029] 1.5 Column temperature investigation On the basis of the determined chromatographic conditions, the column temperature was investigated at 25 °C, 30 °C and 35 °C, respectively. As shown in Table 1.5, the results show that the chromatograms are not much different when the column temperature is 25 °C, 30 °C and 35 °C, and effective separation can be achieved, and therefore the column temperature is temporarily determined to be 30 °C for subsequent investigation. Figure 5 1.6 Flow rate investigation On the basis of the determined chromatographic conditions, the flow rate was investigated at 0.25 mL / min, 0.30 mL / min and 0.35 mL / min, respectively. As shown in Table 1.6, the results show that the chromatograms are not much different when the flow rate is 0.25 mL / min, 0.30 mL / min and 0.35 mL / min, and effective separation can be achieved, and therefore the flow rate is temporarily determined to be 0.30 mL / min for subsequent investigation. Figure 6

[0030] 1.7 Injection volume investigation On the basis of the determined chromatographic conditions, the injection volume was investigated at 1 μl, 2 μl and 3 μl, respectively. As shown in Table 1.7, the results show that the chromatograms are not much different when the injection volume is 1 μl, 2 μl and 3 μl, and therefore the injection volume is temporarily determined to be 2 μl for subsequent investigation. Figure 7

[0031] 1.8 Delay test On the basis of the determined chromatographic conditions, the chromatogram acquisition time was extended to 46 min. As shown in Table 1.8, the results show that there is basically no chromatographic peak after 23 min, and therefore the sample detection time is determined to be 23 min. Figure 8

[0032] 1.9 Determination of chromatographic conditions The chromatographic conditions and system suitability test were as follows: octadecylsilane-bonded silica gel (ACQUITY UPLC® BEH C18, 1.7 μm, 2.1 mm x 100 mm) was used as the filler; acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B for gradient elution; the flow rate was 0.3 ml per minute; the column temperature was 30 °C; the detection wavelength was 230 nm; and the injection volume was 2 μl. The theoretical plate number should not be less than 5000 according to the peak of hesperidin.

[0033] 2. Preparation method of test sample 2.1 Investigation of extraction solvent ​​​​Take Dried Fructus Aurantii and Radix Paeoniae Alba 0.5 g, put it in a conical flask with a plug, add water, 30% methanol, 70% methanol and methanol 50 ml respectively, seal tightly, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the filtrate, that is, as shown in Figure 9 .

[0034] From Figure 9 It can be seen that when 70% methanol and methanol are used as extraction solvents, the amount of chromatographic peak information is large, so the extraction solvent of the test sample is temporarily determined as 70% methanol for subsequent investigation.

[0035] 2.2 Extraction time investigation Take Dried Fructus Aurantii and Radix Paeoniae Alba 0.5 g, put it in a conical flask with a plug, add 70% methanol 50 ml, seal tightly, ultrasonic treatment (power 600 W, frequency 40 kHz), respectively for 20 minutes, 30 minutes and 40 minutes, cool, shake well, filter, take the filtrate, that is, as shown in Figure 10 .

[0036] From Figure 10 It can be seen that when the extraction time is 20 minutes, 30 minutes and 40 minutes, the chromatograms are not much different, and they can all achieve sufficient extraction, so the extraction time is temporarily determined as 30 minutes for subsequent investigation.

[0037] 2.3 Solvent addition amount investigation Take Dried Fructus Aurantii and Radix Paeoniae Alba 0.5 g, put it in a conical flask with a plug, add 80% methanol 25 ml, 50 ml and 75 ml respectively, seal tightly, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, take the filtrate, that is, as shown in Figure 11 .

[0038] From Figure 11 It can be seen that when the solvent addition amount is 25 ml and 50 ml, the characteristic spectrum chromatographic peak area is moderate, so the solvent addition amount is temporarily determined as 50 ml.

[0039] 2.4 Test sample preparation method determination Take Dried Fructus Aurantii and Radix Paeoniae Alba 0.5 g, put it in a conical flask with a plug, add 70% methanol 50 ml, seal tightly, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the filtrate, that is, as shown in

[0040] 3, Methodology investigation 3.1 Chromatographic peak attribution and identification Preparation of test sample solution: prepare Dried Fructus Aurantii and Radix Paeoniae Alba sample test sample solution according to the above proposed experimental conditions.

[0041] Preparation of reference solution: accurately weigh ephedrine, gallic acid, catechin, paeoniflorin, albiflorin, benzoylpaeoniflorin, naringin, narirutin, hesperidin, neohesperidin, neoremeritrin, nobiletin, tangeretin, naringenin, hesperetin reference substance, and add methanol to prepare a solution containing 100 μg of each reference substance per 1 mL.

[0042] Preparation of negative control solution: prepare the sample solutions of single medicine Fructus Aurantii Carbonisatus and Radix Paeoniae Alba according to the experimental conditions.

[0043] Identify and assign the peaks of the sample characteristic chromatogram of Zhishishaoyao Powder. As shown in Table 2. Figure 12 、 13 ; Table 2.

[0044] Table 2 Identification and assignment of characteristic peaks 3.2 Specificity Preparation of blank solution: use 70% methanol solution as blank.

[0045] Preparation of mixed reference solution: accurately weigh ephedrine, gallic acid, catechin, paeoniflorin, albiflorin, benzoylpaeoniflorin, naringin, narirutin, hesperidin, neohesperidin, neoremeritrin, nobiletin, tangeretin, naringenin, and hesperetin reference substance, and add methanol to prepare a mixed solution containing 10 μg per 1 mL.

[0046] Preparation of test solution: prepare the test solution of Zhishishaoyao Powder sample according to the experimental conditions.

[0047] Inject the above three solutions respectively, and record the chromatogram. See Figure 14 . The results show that the blank solution has no interference at the peak position of the characteristic peak, and the test sample chromatogram presents 17 characteristic peaks, which correspond to the retention time of 15 characteristic peaks in the mixed reference solution chromatogram. The method has good specificity.

[0048] 3.3 Precision examination Take the test solution of Zhishishaoyao Powder sample (batch number: ZSSYS-01), and inject 6 times continuously according to the experimental method, 2 μl each time, and record the retention time and peak area of each characteristic peak. The results are shown in Tables 3 and 4.

[0049] Table 3 Precision examination-retention time Table 4 Precision examination-peak area The results showed that the RSD of the retention time of each characteristic peak was 0.01%-0.09%, and the RSD of the peak area was 0.08%-2.39%. The method had good precision.

[0050] 3.4 Reproducibility Investigation Precisely weigh 6 portions of Zhishiyaojiansan sample (batch number: ZSSYS-01), prepare and determine according to the proposed experimental method. The results are shown in Tables 5 and 6.

[0051] Table 5 Reproducibility Investigation-Relative Retention Time Table 6 Reproducibility Investigation-Relative Peak Area The results showed that the RSD of the relative retention time of each characteristic peak was 0.01%-0.08%, and the RSD of the relative peak area was 0.26%-9.66%. The method had good reproducibility.

[0052] 3.5 Stability Investigation Under the above experimental conditions, the same test solution was taken and determined at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h. The results are shown in Tables 7 and 8.

[0053] Table 7 Stability Investigation-Retention Time Table 8 Stability Investigation-Peak Area The results showed that the RSD of the relative retention time of the corresponding characteristic peak was 0.04%-0.48%, and the RSD of the peak area was 0.19%-12.09%. The sample solution was stable within 24 hours.

[0054] From the above results, it can be seen that the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good.

[0055] 2.3 Method Validation The detection method validated by the method validation was used to determine the characteristic chromatogram of 5 batches of Zhishiyaojiansan samples, and the relative retention time and relative peak area were calculated. The results are shown in Tables 9 and 10. Figure 15

[0056] Table 9 Relative Retention Time of 5 Batches of Zhishiyaojiansan Samples Table 10 Relative Peak Area of 5 Batches of Zhishiyaojiansan Samples ​According to the principle of relative retention time stability and the detection of each batch sample, 17 peaks with good repeatability were selected as characteristic peaks. Figure 16 The results showed that when peak 7 was used as the S peak, the relative retention time RSD of the characteristic peaks of 5 batches of Jishai Shayaosan samples was less than 1%.

[0057] Finally, it was stipulated that the RSD of the relative retention time of each characteristic peak met the requirements in the above investigations, and the method was good. The test sample chromatogram should show 17 characteristic peaks, of which the peak corresponding to the hesperidin reference was the S peak. The relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within ±10% of the specified value, which was 0.083 (peak 1, synephrine), 0.146 (peak 2, gallic acid), 0.421 (peak 3, catechin), 0.603 (peak 4, tectoridin), 0.665 (peak 5, paeoniflorin), 0.835 (peak 6, neohesperidin), 0.896 (peak 7), 0.917 (peak 8, narirutin), 0.962 (peak 9, naringin), 1.045 (peak 11, neohesperidin), 1.302 (peak 12), 1.427 (peak 13, naringenin), 1.454 (peak 14, benzoylpaeoniflorin), 1.508 (peak 15, hesperetin), 1.914 (peak 16, citropten), and 2.104 (peak 17, tangeretin).

[0058] In summary, the characteristic spectrum established by the specific chromatographic conditions can identify 17 characteristic peaks, covering different large categories of alkaloids, organic acids, flavonoids, flavonoid glycosides, and other characteristic components of different polarities. These components are mainly attributed to Jishai carbon, which is the main active ingredient of Jishai carbon. At the same time, benzoylpaeoniflorin, which has significant pharmacological activity in Baishao, was identified from these components. The identification of these more characteristic chemical components realizes the quality control of Jishai Shayaosan, guarantees the clinical efficacy of Jishai Shayaosan, and has practical application value.

Claims

1. A method for detecting the UPLC feature map of Zhishi Shaoyao San, characterized in that: It includes the following steps: 1) Preparation of reference solution: Take alkaloids, organic acids, flavonoids and / or flavonoid glycosides as reference standards, dissolve them in methanol to obtain the reference solution; 2) Preparation of test solution: Take the Zhishi Shaoyao powder to be tested, add methanol solution to extract, filter the extract, and take the filtrate to obtain the test solution; 3) Inject the reference solution and the test solution into the UPLC instrument separately and record the chromatograms; the chromatographic conditions are as follows: Chromatographic column: octadecylsilane-bonded silica gel as the packing material; mobile phase: acetonitrile as mobile phase A, and 0.1-0.3% phosphoric acid solution as mobile phase B; gradient elution program: 0-23 min, 5%→47%A.

2. The UPLC feature map detection method according to claim 1, characterized in that: Step 1) Each 1 ml of the reference solution contains 5-15 μg of each reference standard.

3. The UPLC feature map detection method according to claim 2, characterized in that: The reference standard contains synephrine as an alkaloid, gallic acid as an organic acid, catechin, naringenin, norihesperidin, tangeretin and / or hesperidin as flavonoids, and paeoniflorin, paeoniflorin lactone, benzoylpaeoniflorin, naringenin, rutin-naringenin, hesperidin, neohesperidin and / or neohesperidin.

4. The UPLC feature map detection method according to claim 1, characterized in that: Step 2) The mass-to-volume ratio of the tested Zhishi Shaoyao powder to the methanol solution is 0.4~0.8g:25~50ml; the methanol solution is 70~100% methanol.

5. The UPLC feature map detection method according to claim 1 or 4, characterized in that: The Zhishi Shaoyao Powder to be tested is a powder made from a mixture of charred Zhishi (Fructus Aurantii Immaturus) and white peony root. The mass ratio of the charred immature bitter orange and the white peony root is 1:

1.

6. The UPLC feature map detection method according to claim 1, characterized in that: Step 2) The extraction is ultrasonic extraction, with a power of 600W, a frequency of 40kHz, and a time of 20~40min.

7. The UPLC feature map detection method according to claim 1, characterized in that: Step 3) The chromatographic conditions described are as follows: Column: ACQUITY UPLC® BEH C18, 1.7 μm, 2.1 mm × 100 mm; Column temperature: 25–35 °C; Injection volume: 1–3 μL; Flow rate: 0.25–0.35 mL∙min -1 The detection wavelength is 230~330nm.

8. The UPLC feature map detection method according to claim 1, characterized in that: The chromatogram of the test solution should show 17 characteristic peaks, among which the peak corresponding to the hesperidin reference is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: Peak 1: 0.083, Peak 2: 0.146, Peak 3: 0.421, Peak 4: 0.603, Peak 5: 0.665, Peak 6: 0.835, Peak 7: 0.896, Peak 8: 0.917, Peak 9: 0.962, Peak 11: 1.045, Peak 12: 1.302, Peak 13: 1.427, Peak 14: 1.454, Peak 15: 1.508, Peak 16: 1.914, Peak 17: 2.

104.

9. The UPLC feature map detection method according to claim 8, characterized in that: The component corresponding to peak 1 is an alkaloid, the component corresponding to peak 2 is an organic acid, the components corresponding to peaks 3, 13, 15, 16 and 17 are flavonoids, and the components corresponding to peaks 4-6, 8 and 9-11 are flavonoid glycosides.

10. The UPLC feature map detection method according to claim 9, characterized in that: Peak 1 corresponds to synephrine, peak 2 corresponds to gallic acid, peak 3 corresponds to catechin, peak 4 corresponds to paeoniflorin, peak 5 corresponds to paeoniflorin, peak 6 corresponds to neohesperidin, peak 8 corresponds to rutin, peak 9 corresponds to naringin, peak 10 corresponds to hesperidin, peak 11 corresponds to neohesperidin, peak 13 corresponds to naringenin, peak 14 corresponds to benzoylpaeoniflorin, peak 15 corresponds to hesperidin, peak 16 corresponds to norihesperidin, and peak 17 corresponds to tangeretin.

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