A method for detecting the content of monooligosaccharides and fingerprint of compound sophora flavescens injection

Abstract

The application belongs to the technical field of medicine, and particularly relates to a detection method for monosaccharide and oligosaccharide content and fingerprint spectrum of compound sophora flavescens injection, which comprises high performance liquid chromatography-evaporative light scattering detection, wherein the monosaccharide and oligosaccharide are D-anhydrous glucose, D-fructose, sucrose and pinacol. The application can provide an improved detection method for monosaccharide and oligosaccharide of compound sophora flavescens injection, can simultaneously detect four kinds of saccharide components in the compound sophora flavescens injection, and can construct HPLC-ELSD fingerprint spectrum by using the method, so as to provide a fast and efficient technical method for quality control of saccharide components in the compound sophora flavescens injection.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to an improved HPLC detection method for compound sophora flavescens injection. Background Art

[0002] Compound Sophora flavescens injection is a traditional Chinese medicine injection made from Sophora flavescens and Poria cocos using modern scientific methods. It has the effects of clearing heat and dampness, cooling blood and detoxifying, dispersing nodules and relieving pain. It is used to treat cancer pain and bleeding. To date, research on compound Sophora flavescens injection has primarily focused on its alkaloids and flavonoids, with relatively little research on carbohydrates. In recent years, carbohydrates in traditional Chinese medicine, as a class of bioactive ingredients, have become a hot topic of research, possessing diverse functions such as immunomodulation, anti-tumor, antioxidant, hypoglycemic, and anti-lung cancer. Currently, there are few reports in the literature on the types and contents of monooligosaccharides in compound sophora flavescens injection: except for the HPLC-ELSD determination of pinitol content in compound sophora flavescens injection published by Li Bowen et al. (Journal of Chinese Traditional Medicine Information, Vol. 21, No. 2, February 2014, pp. 83-85), and the determination of D-anhydrous glucose in Liu Xiaoqian's doctoral thesis "Research on Control Technology and Standards of Production Process of Compound Sophora flavescens Injection & Exploratory Research on Reducing Toxicity of Vinorelbine by Liposome Technology", there are no other documents recording the types and contents of monooligosaccharides in compound sophora flavescens injection.

[0003] Currently, high performance liquid chromatography is used in traditional Chinese medicine to detect the content of monosaccharides and oligosaccharides in injections.

[0004] Patent CN103543222A discloses a method for detecting the content of carbohydrate components in Reduning injection. The method uses HPLC-ELSD to simultaneously determine the contents of fructose and D-anhydrous glucose in Reduning injection. However, the chromatogram obtained by this method shows a low degree of separation between fructose and D-anhydrous glucose, and cannot separate pinitol, which has a retention time between fructose and D-anhydrous glucose. Therefore, this method cannot be used for carbohydrate detection in Fufang Kushen Injection.

[0005] Zhang Xue et al. published an HPLC-CAD method for the simultaneous determination of monosaccharide and disaccharide content in Bletilla striata (International Journal of Pharmaceutical Research, Vol. 45, No. 2, February 2018, pp. 154-157). The method employed an Xbridge Amide column for separation, with an isocratic elution using acetonitrile-0.2% ethylamine (78:22) as the mobile phase, a flow rate of 1 ml / min, a column temperature of 30°C, and a CAD detector internal temperature of 30°C. D-fructose, mannose, D-anhydrous glucose, and sucrose were detected. However, the inventors found that this method was not suitable for the determination of sugar components in compound sophora flavescens injection. The main drawbacks were poor separation between chromatographic peaks, excessive interfering substances, inability to perform content determination, and an unstable baseline.

[0006] Huang Qinwei et al. published a study on the quantitative determination of monosaccharides and oligosaccharides in Guanxinning Injection (Chinese Patent Medicine, Vol. 34, No. 7, July 2012, pp. 1299-1303). The method described employed HPLC-ELSD, using a sugar-based filler (Prevail Carbohydrate ES column, 4.6 mm × 250 mm, 5 μm); a mobile phase of acetonitrile-water (79:21); a volume flow rate of 1.0 mL / min; an ELSD detector; a drift tube temperature of 100°C; and an N₂ flow rate of 2.8 L / min. However, the inventors discovered that this method was unsuitable for the detection of sugar components in Fufang Kushen Injection. The primary drawback was that fructose and pinitol appeared as a mixed peak in the sample chromatogram detected using this method, and they were completely unresolvable. Therefore, pinitol could not be detected under these chromatographic conditions, and fructose quantification was inaccurate.

[0007] Therefore, in order to accurately control the carbohydrate components in compound sophora flavescens injection, it is necessary to provide a detection method that can simultaneously complete the content determination and fingerprint spectrum of multiple carbohydrate components in compound sophora flavescens injection, and provide a fast and efficient detection method for the detection of carbohydrate components in compound sophora flavescens injection. Summary of the Invention

[0008] In view of the above technical status, the present invention provides a method for detecting the content and fingerprint of monooligosaccharides in compound sophora flavescens injection, which comprises high performance liquid chromatography-evaporative light scattering detection, wherein the monooligosaccharides are D-anhydrous glucose, D-fructose, sucrose and pinitol.

[0009] In the method of the present invention, as one embodiment, the chromatographic column in the high performance liquid chromatography-evaporative light scattering detection method is a Prevail Carbohydrate ES column with a specification of 4.6 mm×250 mm and 5 μm.

[0010] In the method of the present invention, as one of the embodiments, the mobile phase in the high performance liquid chromatography-evaporative light scattering detection method is a gradient solution of acetonitrile-water.

[0011] In the method of the present invention, as one embodiment, the gradient elution conditions in the high performance liquid chromatography-evaporative light scattering detection method are as follows:

[0012] Time (min) Acetonitrile (%) water(%) 0-25 85 15 25-30 85-70 15-30 30-45 70 30

[0013] In the method of the present invention, as one embodiment, the flow rate of the mobile phase in the high performance liquid chromatography-evaporative light scattering detection method in the method is 0.95 to 1.05 ml / min, preferably 1 ml / min.

[0014] In the method of the present invention, as one embodiment, the column temperature in the high performance liquid chromatography-evaporative light scattering detection method in the method is 13-20°C, preferably 15°C.

[0015] In the method of the present invention, as one embodiment, the injection volume of the low-concentration reference substance and sample in the high-performance liquid chromatography-evaporative light scattering detection method is 10 μl, and the injection volume of the high-concentration reference substance is 20 μl.

[0016] In the method of the present invention, as one embodiment, the evaporation temperature of the evaporative light detector in the high performance liquid chromatography-evaporative light scattering detection method is 59-61°C, preferably 60°C.

[0017] In the method of the present invention, as one of the embodiments, the atomization temperature of the evaporative light detector in the high performance liquid chromatography-evaporative light scattering detection method in the method is 59-61°C, preferably 60°C.

[0018] In the method of the present invention, as one of the embodiments, the carrier gas in the high performance liquid chromatography-evaporative light scattering detection method in the method is nitrogen, and the flow rate is 1.4 to 1.6 L / min, preferably 1.5 L / min.

[0019] In the method of the present invention, as one embodiment, the blank solution in the high performance liquid chromatography-evaporative light scattering detection method is prepared as follows: acetonitrile-water = 50:50 mixed solution, that is,

[0020] In the method of the present invention, as one embodiment, the reference solution in the high performance liquid chromatography-evaporative light scattering detection method is prepared as follows: accurately weigh appropriate amounts of D-fructose reference, pinitol reference, D-anhydrous glucose reference, and sucrose reference, add blank solution to make a mixed reference solution containing 1.00 mg of D-fructose, 0.19 mg of pinitol, 0.90 mg of D-anhydrous glucose, and 0.20 mg of sucrose per 1 ml, shake well, and prepare two portions in the same manner.

[0021] In the method of the present invention, as one of the embodiments, the test solution in the high performance liquid chromatography-evaporative light scattering detection method is prepared as follows: 1 ml of each batch of compound sophora flavescens injection is accurately measured and placed in a 20 ml volumetric flask, a blank solution is added to the scale, shaken, filtered, and the filtrate is taken as the test solution.

[0022] In the method of the present invention, as one embodiment, the method for detecting the oligosaccharide content of the compound sophora flavescens injection of the present invention comprises detecting by high performance liquid chromatography-evaporative light scattering detection, wherein the conditions of high performance liquid chromatography-evaporative light scattering are:

[0023]

[0024] (1) Blank solution preparation: acetonitrile-water = 50:50 mixed solution;

[0025] (2) Preparation of reference solution: Accurately weigh appropriate amounts of D-fructose reference, pinitol reference, D-anhydrous glucose reference, and sucrose reference, add blank solution to make a mixed reference solution containing 1.00 mg of D-fructose, 0.19 mg of pinitol, 0.90 mg of D-anhydrous glucose, and 0.20 mg of sucrose per 1 ml, shake well, and prepare two portions in the same manner.

[0026] (3) Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection into a 20 ml volumetric flask, add blank solution to the mark, shake well, filter, and use the filtrate as the test solution;

[0027] (4) Detection: Inject the blank solution, reference solution, and test solution in this order, and then use the external standard two-point method to calculate the content of D-fructose, pinitol, D-anhydrous glucose, and sucrose in the test solution.

[0028] In the method of the present invention, as one of the embodiments, the fingerprint detection method of the compound sophora flavescens injection of the present invention comprises: constructing a fingerprint of the compound sophora flavescens injection containing D-anhydrous glucose, D-fructose, sucrose and pinitol.

[0029] In the method of the present invention, as one embodiment, the method includes: the method includes high performance liquid chromatography-evaporative light scattering detection for detection, wherein the conditions of high performance liquid chromatography-evaporative light scattering are:

[0030]

[0031] (1) Blank solution preparation: acetonitrile-water = 50:50 mixed solution;

[0032] (2) Preparation of reference solution: Accurately weigh appropriate amounts of D-fructose reference, pinitol reference, D-anhydrous glucose reference, and sucrose reference, add blank solution to make a mixed reference solution containing 1.00 mg of D-fructose, 0.19 mg of pinitol, 0.90 mg of D-anhydrous glucose, and 0.20 mg of sucrose per 1 ml, shake well, and prepare two portions in the same manner.

[0033] (3) Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection into a 20 ml volumetric flask, add blank solution to the mark, shake well, filter, and use the filtrate as the test solution;

[0034] (4) Construction of standard fingerprint: blank solution, reference solution, and test solution were injected in order to construct a standard fingerprint of compound sophora flavescens injection containing D-anhydrous glucose, D-fructose, sucrose, and pinitol;

[0035] (5) Detection: Inject the blank solution, reference solution, and test solution in this order, and then calculate the contents of D-fructose, pinitol, D-anhydrous glucose, and sucrose in the test solution using the external standard two-point method.

[0036] In the method of the present invention, as one of the embodiments, the sample can be injected in the following manner:

[0037] order sample Number of needles 1 blank solution 1 needle 2 Reference solution 5 needles (continuous needle insertion) 3 Test solution 1 needle

[0038] In the method of the present invention, as one of the embodiments, the standard fingerprint includes three unknown peaks, a D-fructose chromatographic peak, a pinitol chromatographic peak, a D-anhydroglucose chromatographic peak and a sucrose chromatographic peak.

[0039] In the method of the present invention, as one of the embodiments, in the standard fingerprint, the relative retention times of the three unknown peaks are 0.100-0.130, preferably 0.12; 0.135-0.150, preferably 0.14; 0.170-0.190, preferably 0.18; the relative retention time of D-fructose is 0.660-0.690, preferably 0.67; the relative retention time of pinitol is 0.695-0.730, preferably 0.70; the relative retention time of D-anhydrous glucose is 1.00; and the relative retention time of sucrose is 1.130-1.153, preferably 1.14.

[0040] In the method of the present invention, as one of the implementation plans, in the standard fingerprint, the relative peak areas of the three unknown peaks are 0.32, 0.03, and 2.36, respectively; the relative peak area of ​​D-fructose is 2.32; the relative peak area of ​​pinitol is 0.10; the relative peak area of ​​D-anhydrous glucose is 1.00; and the relative peak area of ​​sucrose is 0.19.

[0041] The greatest advantage of the method of the present invention over conventional methods is that it can simultaneously detect D-fructose and pinitol. Under conventional chromatographic conditions, when D-fructose and pinitol coexist, fructose and pinitol form one chromatographic peak and are poorly separated. The method of the present invention solves this problem and simultaneously determines the contents of the four sugars and establishes a carbohydrate fingerprint.

[0042] Compared with the existing detection methods for compound sophora flavescens injection, the present invention adopts high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method, which can simultaneously determine the four sugar components in compound sophora flavescens injection, and use this method to construct a chromatographic fingerprint, providing a fast and efficient technical method for quality control of compound sophora flavescens injection, reducing the inspection workload. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the result diagram of blank and negative samples in Example 1.

[0044] Figure 2 This is a linear graph of the index components of D-fructose, pinitol, D-anhydrous glucose and sucrose in Example 1.

[0045] Figure 3 This is the fingerprint of the standard reference substance in Example 2.

[0046] Figure 4 Fingerprints of different batches of test samples in Example 2 are superimposed.

[0047] Figure 5 This is the repeatability superposition fingerprint of different batches of test samples in Example 2.

[0048] Figure 6 This is the stability fingerprint in Example 2.

[0049] Figure 7 This is the double time fingerprint in Example 2.

[0050] Figures 8-1 to 8-3 This is a graph showing the results of investigating different chromatographic columns in Example 3.

[0051] Figures 9-1 to 9-4 This is a graph showing the results of investigating different mobile phase gradients in Example 3.

[0052] Figures 10-1 to 10-5 This is a graph showing the results of investigating different column temperatures in Example 3.

[0053] Figures 11-1 to 11-3 This is a graph showing the results of investigating different flow rates in Example 3.

[0054] Figures 12-1 to 12-3 This is a graph showing the results of investigating different evaporation temperatures in Example 3.

[0055] Figures 13-1 to 13-3 This is a graph showing the results of investigating different atomization temperatures in Example 3.

[0056] Figures 14-1 to 14-3 This is a graph showing the results of investigating different carrier gas flow rates in Example 3. DETAILED DESCRIPTION

[0057] The following examples and test examples are used to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0058] instrument

[0059]

[0060] Reference substances

[0061]

[0062]

[0063] Reagents

[0064] name batch number source level Methanol 10984507849 Merck KGaA HPLC Acetonitrile SHBK9452 Merck KGaA HPLC

[0065] Test sample

[0066]

[0067]

[0068] Example 1 Method for Detecting the Content of Monooligosaccharides in Compound Sophora flavescens Injection

[0069] 1. Chromatographic conditions, sample preparation, system suitability requirements, calculation formulas, and limit requirements

[0070]

[0071]

[0072] 2Verify the specific content

[0073] 2.1 System Applicability

[0074] (1) Experimental steps

[0075] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution, filtered to obtain.

[0076] Preparation of reference solution: Accurately weigh appropriate amounts of D-fructose reference, pinitol reference, D-anhydrous glucose reference, and sucrose reference, add blank solution to make a mixed reference solution containing 1.00 mg of D-fructose, 0.19 mg of pinitol, 0.90 mg of D-anhydrous glucose, and 0.20 mg of sucrose per 1 ml, shake well, and obtain.

[0077] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place it in a 20 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0078] Injection order

[0079] order sample Number of needles 1 blank solution 1 needle 2 Reference solution 5 needles (continuous needle insertion) 3 Test solution 1 needle

[0080] (2) Results report

[0081] The RSD values ​​of the peak area and retention time of the reference solution after 5 consecutive injections.

[0082] Table 2.1-1 Peak area and retention time results of reference solution

[0083]

[0084]

[0085] Table 2.1-2 System applicability results

[0086]

[0087] (3) Conclusion

[0088] According to the results, the peak areas of the reference solution injected continuously for 5 times, the RSDs of the retention times of D-fructose, pinitol, D-anhydrous glucose and sucrose were 0.15%, 0.12%, 0.09% and 0.11% respectively, all less than 1.3%, and the RSDs of the peak areas were 1.1%, 0.8%, 0.7% and 1.7% respectively, all less than 5.0%, the theoretical plate numbers of the content determination indicators were all greater than 5000, and the tailing factors were all less than 1.3%, which met the requirements.

[0089] 2.2 Exclusivity

[0090] (1) Experimental steps

[0091] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution, filtered to obtain.

[0092] Preparation of 0.25% Tween 80 solution: weigh 0.25g Tween 80, add water to dissolve to 100ml, shake well, filter, and take the filtrate as 0.25% Tween 80 solution.

[0093] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1.

[0094] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place it in a 20 ml volumetric flask, add blank solution to the scale, shake well, and set aside.

[0095] Preparation of filter membrane interference samples: Take the test solution, centrifuge one part and filter one part, discarding different volumes (1ml, 3ml, 5ml, 7ml, 9ml).

[0096] Injection procedure requirements

[0097] Injection order

[0098] order sample Number of needles 1 blank solution 1 needle 2 0.25% Tween 80 solution 1 needle 3 Reference solution 1 needle 4 Test solution - centrifugation 1 needle 5 Test solution - filtered 1 ml 1 needle 6 Test solution - filtered 3ml 1 needle 7 Test solution - filtered 5ml 1 needle 8 Test solution - filtered 7ml 1 needle 9 Test solution - filtered 9ml 1 needle

[0099] (2) Results report

[0100] See also Figure 1 ;in Figure 1 Results for blank and negative samples

[0101] Table 2.2-1 Results of filter interference experiment (percentage of area of ​​centrifuged sample after discarding different volumes)

[0102] / D-fructose % Pineol% D-anhydrous glucose % sucrose% 1ml 103.04 90.63 101.10 99.11 3ml 105.20 84.02 101.17 99.50 5ml 104.66 93.00 100.79 98.59 7ml 104.81 95.96 102.21 99.90 9ml 106.98 97.21 101.97 102.40

[0103] (3) Conclusion

[0104] The results show that the blank solution, blank mobile phase, and 0.25% Tween 80 solution did not interfere with the sample. The relative percentage of the area of ​​the index component of the test solution after discarding different volumes and the area of ​​the index component of the directly injected test solution ranged from 84.02.0% to 106.98%, indicating that the filter membrane interfered with the test sample and no further filtration was performed. (See Figure 1 )

[0105] 2.3 Linearity and range

[0106] (1) Experimental steps

[0107] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution.

[0108] Linear stock solution: Accurately weigh appropriate amounts of D-fructose reference substance, pinitol reference substance, D-anhydrous glucose reference substance, and sucrose reference substance, add blank solution to make a mixed reference solution containing 2.02 mg of D-fructose, 0.39 mg of pinitol, 1.80 mg of D-anhydrous glucose, and 0.40 mg of sucrose per 1 ml, shake well, and obtain.

[0109] 33% reference solution: Accurately measure 1.5 ml of the mixed reference solution, place it in a 10 ml volumetric flask, dilute to the scale with blank solution, shake well, and obtain.

[0110] 40% reference solution: Accurately measure 2 ml of the mixed reference solution, place it in a 10 ml volumetric flask, dilute to the scale with blank solution, shake well, and obtain.

[0111] 60% reference solution: Accurately measure 3 ml of the mixed reference solution, place it in a 10 ml volumetric flask, dilute to the scale with blank solution, shake well, and obtain.

[0112] 80% reference solution: Accurately measure 4 ml of the mixed reference solution, place it in a 10 ml volumetric flask, dilute to the scale with blank solution, shake well, and obtain.

[0113] 100% reference solution: Accurately measure 5 ml of the mixed reference solution, place it in a 10 ml volumetric flask, dilute to the scale with blank solution, shake well, and obtain.

[0114] 140% reference solution: Accurately measure 3.5 ml of the mixed reference solution, place it in a 5 ml volumetric flask, dilute to the scale with the blank solution, shake well, and obtain.

[0115] Injection procedure requirements

[0116] Injection order

[0117]

[0118]

[0119] (2) Results report

[0120] The regression equations, correlation coefficients and linear plots of each index component are as follows (plotted with the reciprocal of mass and the reciprocal of peak area) (see Figure 2 , Figure 2 (Linear graph of D-fructose, pinitol, D-anhydrous glucose and sucrose)

[0121] (3) Conclusion

[0122] The linear correlation coefficient should be ≥0.999, which meets the standard.

[0123] 2.4 Sensitivity

[0124] (1) Experimental steps

[0125] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution.

[0126] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1.

[0127] Quantitation limit and detection limit solutions: The reference solution was diluted stepwise with the blank solution. When the pinitol signal-to-noise ratio (S / N) was 10:1, it was used as the quantitation limit solution. When the pinitol signal-to-noise ratio (S / N) was 2-3, it was used as the detection limit solution.

[0128] Injection procedure requirements

[0129] Injection order

[0130] order sample Number of needles 1 blank solution 1 needle 2 Reference substance 1 solution Pin 5 (continuous test) 3 Quantitative limit solution 6-pin 4 Detection limit solution 2 needles

[0131] (3) Result report

[0132] Table 2.4-1 Statistics of Quantitation Limit Results

[0133] 1 2 3 4 5 6 average RSD Pineol peak area 11907 11998 11542 11337 10572 11062 11403 4.29 Retention time 26.096 26.245 26.291 26.291 26.072 26.11 26.18 0.36

[0134] Table 2.4-2 Sensitivity test results

[0135]

[0136]

[0137] (3) Conclusion

[0138] From the results, it can be seen that after continuous injection of the quantitative limit solution, the RSD value of the pinitol peak retention time was less than 1.3%, and the peak area was less than 5.0%; the quantitative limit of pinitol was 404 ng, and the detection limit was 303 ng.

[0139] 2.5 Repeatability

[0140] (1) Experimental steps

[0141] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution.

[0142] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1 and prepare two portions in the same way.

[0143] Preparation of test solution (6 portions): Accurately measure 1 ml of compound sophora flavescens injection into a 20 ml volumetric flask, add blank solution to the mark, shake well, and use this as the test solution. Repeat the procedure for 6 portions.

[0144] Injection procedure requirements

[0145] Injection order

[0146] order sample Number of needles 1 blank solution 1 needle 2 Reference substance 1 solution Pin 5 (continuous test) 3 Reference substance 2 solution 2 needles 4 Reference substance 2 solution (20 μl) 2 needles 5 Test sample-1 solution 1 needle 6 Test sample-2 solution 1 needle 7 Test sample-3 solution 1 needle 8 Test sample-4 solution 1 needle 9 Test sample-5 solution 1 needle 10 Test sample-6 solution 1 needle 11 Reference substance 1 solution 1 needle

[0147] (2) Results report

[0148] Table 2.5 Repeatability test results

[0149]

[0150]

[0151] (3) Conclusion

[0152] The RSD of the results of D-fructose, pinitol, D-anhydrous glucose and sucrose content in 6 test samples was no more than 5.0%, indicating that the test samples had good repeatability.

[0153] 2.6 Solution stability

[0154] (1) Experimental steps

[0155] Blank solvent preparation: acetonitrile-water = 50:50 mixed solution.

[0156] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1 and prepare two portions in the same way.

[0157] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place it in a 20 ml volumetric flask, add blank solution to the scale, shake well, and use it as the test solution.

[0158] Injection procedure requirements

[0159] Injection order

[0160]

[0161]

[0162] (2) Results report

[0163] Table 2.6 Solution stability test results

[0164]

[0165] (3) Conclusion

[0166] The reference solution and the test solution were placed at room temperature for 24 hours. The RSDs of the D-fructose, pinitol, D-anhydrous glucose and sucrose contents in the test samples were all no more than 5.0%.

[0167] The percentage of the indicator component area at each time point to the indicator component area at 0 hour, the results of the reference and test solutions at each time point compared with the initial results, the relative content was between 95.76% and 104.284%, indicating that the solution had good stability within 24 hours.

[0168] 2.7 Accuracy

[0169] (1) Experimental steps

[0170] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution.

[0171] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1 and prepare two portions in the same way.

[0172] 50% recovery solution: Accurately measure 0.5 ml of compound sophora flavescens injection and place it in a 20 ml volumetric flask. Add 5 ml of the reference solution and then add blank solution to the mark. Shake well and filter to prepare 50% recovery solution (prepare 3 portions in the same way).

[0173] 100% recovery solution: Accurately measure 0.5 ml of compound sophora flavescens injection and place it in a 20 ml volumetric flask. Add 10 ml of the reference solution and then add blank solution to the mark. Shake well and filter to prepare 100% recovery solution (prepare 3 portions in the same way).

[0174] 150% recovery solution: Accurately measure 0.5 ml of compound sophora flavescens injection and place it in a 20 ml volumetric flask. Add 15 ml of the reference solution and then add blank solution to the mark. Shake well and filter to prepare 150% recovery solution (prepare 3 portions in the same way).

[0175] Injection procedure requirements

[0176] Injection order

[0177] order sample Number of needles 1 blank solution 1 needle 2 Reference substance 1 solution Pin 5 (continuous test) 3 Reference substance 2 solution 2 needles 4 Reference substance 2 solution (20 μl) 2 needles 5 50%-1 recovery solution 2 needles 6 50%-2 recovery solution 2 needles 7 50%-3 recovery solution 2 needles 8 100%-1 recovery solution 2 needles 9 100%-2 recovery solution 2 needles 10 100%-3 recovery solution 2 needles 11 150%-1 recovery solution 2 needles 12 150%-2 recovery solution 2 needles 13 150%-3 recovery solution 2 needles 14 Reference substance 1 solution 1 needle

[0178] (2) Results report

[0179] Recovery rate calculation formula:

[0180] Table 2.7-1 D-fructose content recovery test results

[0181]

[0182]

[0183] Table 2.7-2 Pineol content recovery test results

[0184]

[0185] Table 2.7-3 D-anhydrous glucose content recovery test results

[0186]

[0187] Table 2.7-4 Sucrose content recovery test results

[0188]

[0189] (3) Conclusion

[0190] The recovery rates of D-fructose in the test samples ranged from 111.78% to 123.47%, the recovery rates of pinitol ranged from 98.02% to 111.01%, the recovery rates of D-anhydrous glucose ranged from 105.98% to 114.83%, and the recovery rates of sucrose ranged from 105.98% to 114.83%. The RSD values ​​of the 9 recoveries were 3.16%, 4.86%, 3.08%, and 3.57%, respectively, all less than 5.0%, meeting the requirements.

[0191] 2.8 Sample determination

[0192] (1) Experimental steps

[0193] Blank solvent preparation: acetonitrile-water = 50:50 mixed solution.

[0194] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1 and prepare two portions in the same way.

[0195] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection from different batches, place in a 20 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0196] Injection procedure requirements.

[0197] Injection order

[0198]

[0199]

[0200] (2) Results report

[0201] Table 2.8 Content determination results

[0202]

[0203]

[0204] Example 2 Fingerprint Detection Method of Compound Sophora Flavescentis Injection

[0205] 1. Chromatographic conditions, elution conditions, sample preparation, etc.

[0206]

[0207]

[0208] 2. Verify content

[0209] 2.1 System Applicability

[0210] (1) Experimental steps

[0211] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution, filtered to obtain.

[0212] Preparation of reference solution: Accurately weigh appropriate amounts of D-fructose reference, pinitol reference, D-anhydrous glucose reference, and sucrose reference, add blank solution to make a mixed reference solution containing 1.00 mg of D-fructose, 0.19 mg of pinitol, 0.90 mg of D-anhydrous glucose, and 0.20 mg of sucrose per 1 ml, shake well, and obtain.

[0213] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place it in a 20 ml volumetric flask, add blank solution to the scale, shake well, and use it as the test solution.

[0214] The injection sequence and requirements are shown in the table below.

[0215] Injection order

[0216] order sample Number of needles 1 blank solution 1 needle 2 Reference solution 5 needles (continuous needle insertion) 3 Test solution 1 needle 4 Reference solution 1 needle

[0217] (2) Results report

[0218] The RSD values ​​of the peak area and retention time of the reference solution after 5 consecutive injections.

[0219] Table 2.1-1 Peak area and retention time results of reference solution

[0220]

[0221]

[0222] Table 2.1-2 System applicability results

[0223]

[0224] (3) Conclusion

[0225] According to the results, the peak areas of the reference solution injected continuously for 5 times, the RSDs of the retention times of D-fructose, pinitol, D-anhydrous glucose and sucrose were 0.15%, 0.12%, 0.09% and 0.11% respectively, all less than 1.3%, and the RSDs of the peak areas were 1.1%, 0.8%, 0.7% and 1.7% respectively, all less than 5.0%, the theoretical plate numbers of the content determination indicators were all greater than 5000, and the tailing factors were all less than 1.3%, which met the requirements.

[0226] 2.2 Fingerprint establishment

[0227] (1) Experimental steps

[0228] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution, filtered to obtain.

[0229] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1.

[0230] Each batch of test solution: Accurately measure 1 ml of each batch of compound sophora flavescens injection, place it in a 20 ml volumetric flask, add blank solution to the scale, shake well, and use it as the test solution.

[0231] The injection sequence and requirements are shown in the table below.

[0232] Injection order

[0233] order sample Number of needles 1 blank solution 1 needle 2 Reference solution Pin 5 (continuous test) 3 Test sample-1 solution 1 needle 4 Test sample-2 solution 1 needle 5 Test sample-3 solution 1 needle 6～21 Test sample 4-19 solution 1 injection per test sample 22 Test sample-20 solution 1 needle 23 Reference solution 1 needle

[0234] (2) Results report

[0235] Based on the chromatographic fingerprints of 20 batches of Fufang Kushen Injection, the data were processed using the 2012 edition of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" recommended by the Pharmacopoeia Committee. The chromatographic peak of test sample 1 (20181034) was used as the reference spectrum. The median method was used with a time window of 0.1. After multi-point correction, full peak matching was performed to generate the standard reference fingerprint and common pattern. For details, see Figures 3-4 .

[0236]

[0237] Table 2.2-2 Non-common peak results for each batch

[0238] Test sample Non-common peak area Total peak area Non-common peak area ratio 20181034 60091.77 4795683.23 1.25％ 20181138 63116.29 5147190.69 1.23％ 20181139 66488.50 5625149.45 1.18％ 20181203 57340.95 4603919.20 1.25％ 20181204 56063.72 4492491.74 1.25％ 20181209 98792.50 4928487.34 2.00％ 20181212 128336.95 5269675.50 2.44％ 20181213 60720.33 4850477.85 1.25％ 20181214 65671.81 5177579.33 1.27％ 20181215 107623.46 5063312.90 2.13％ 20190404 84625.12 8065353.75 1.05％ 20190405 85207.14 7199973.97 1.18％ 20190406 296286.55 7713478.94 3.84％ 20190407 97240.03 7714585.06 1.26％ 20190408 81956.49 7835904.60 1.05％ 20190409 319642.52 7769513.36 4.11％ 20190410 97023.32 7608901.36 1.28％ 20190412 305995.68 7557559.65 4.05％ 20190413 94951.65 7693269.20 1.23％ 20190414 282277.93 7542182.24 3.74％

[0239] (3) Conclusion

[0240] After comparison with the reference substance, it can be concluded that peaks 1, 2, and 3 are unknown peaks, peak 4 is D-fructose, peak 5 is pinitol, peak 6 is D-anhydrous glucose, and peak 7 is sucrose.

[0241] From the results, the fingerprint similarity between the 20 batches of samples and the control is greater than 0.9, and the proportion of non-common peak area is less than 5.0%.

[0242] 2.3 Repeatability

[0243] (1) Experimental steps

[0244] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution, filtered to obtain.

[0245] Preparation of reference solution: Refer to the preparation method of reference solution under 3.1.

[0246] Preparation of test solution: Accurately measure 1 ml of 6 portions of compound sophora flavescens injection of the same batch number, place them in a 20 ml volumetric flask, add blank solution to the scale, shake well, and use as the test solution.

[0247] The injection sequence and requirements are shown in the table below.

[0248] Injection order

[0249] order sample Number of needles 1 blank solution 1 needle 2 Reference solution Pin 5 (continuous test) 3 Test sample-1 solution 1 needle 4 Test sample-2 solution 1 needle 5 Test sample-3 solution 1 needle 6 Test sample-4 solution 1 needle 7 Test sample-5 solution 1 needle 8 Test sample-6 solution 1 needle 9 Reference solution 1 needle

[0250] (2) Results report

[0251] Based on the reproducible chromatograms, the same processing method as the sample was used to calculate the similarity using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines". The relative retention time and relative peak area were calculated using Peak 6 (D-anhydrous glucose) as a reference. (See Figure 5 )

[0252] Table 2.3-1 Similarity results of repetitive common peak patterns

[0253]

[0254] Table 2.3-2 Relative retention time results of reproducible common peaks

[0255]

[0256]

[0257] Table 2.3-3 Repetitive common peak relative peak area results

[0258]

[0259] (3) Conclusion

[0260] From the results, we can see that the similarity among the 6 test samples is greater than 0.99, and the RSD values ​​of the relative retention time and relative peak area of ​​each common peak are less than 5.0%, so the repeatability is good.

[0261] 2.4 Solution stability and double time spectrum

[0262] (1) Experimental steps

[0263] Preparation of blank solution: acetonitrile-water = 50:50 mixed solution, filtered to obtain.

[0264] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1.

[0265] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place it in a 20 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0266] The injection sequence and requirements are shown in the following table.

[0267] Injection order

[0268]

[0269]

[0270] (2) Results report

[0271] Based on the stability chromatogram, the same treatment method as the sample was used to calculate the similarity using the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System" and the relative retention time and relative peak area were calculated using Peak 6 (D-anhydrous glucose) as a reference. (See Figures 6-7 )

[0272] Table 2.4-1 Stability common peak pattern similarity results

[0273]

[0274] Table 2.4-2 Relative retention time results of common stability peaks

[0275]

[0276]

[0277] Table 2.4-3 Relative peak area results of common stability peaks

[0278]

[0279] (3) Conclusion

[0280] The results show that the sample similarity is greater than 0.99 within 24 hours. The relative retention time RSD values ​​of the common peaks are all less than 2.0%, and the peak area RSD values ​​are all less than 10.0%. Therefore, the sample is stable within 24 hours. No peaks appear in the chromatogram after twice the time, indicating a good result.

[0281] Example 3 Investigation of different chromatographic conditions

[0282] In order to obtain the detection method of the present invention, this experiment investigated and screened the chromatographic column, mobile phase and gradient, column temperature, flow rate, temperature, etc. in the detection method, as shown below. Other methods and conditions in this experiment refer to the operations in Examples 1 and 2.

[0283] 3.1 Investigation of different chromatographic columns

[0284] (1) Experimental steps

[0285] Chromatographic conditions:

[0286] Three different chromatographic columns were investigated: Waters Xbridge Amide (3.5 μm, 4.6 mm × 250 mm), TechMate NH2-ST (5 μm 80A 4.6 × 250 mm), and Prevail Carbo-hydrate ES column (5 μm, 4.6 mm × 250 mm);

[0287] (2) Experimental results

[0288] Chromatographic columns Chromatogram Waters Xbridge Amide(3.5μm,4.6mm×250mm) See also Figure 8-1 TechMate NH2-ST(5μm 80A 4.6*250mm) See also Figure 8-2 Prevail Carbo-hydrate ES column (5μm, 4.6mm×250mm) See also Figure 8-3

[0289] Considering the separation, baseline noise and chromatographic peak shape, the best chromatographic column is Prevail Carbo-hydrate ES column 4.6mm×250mm, 5μm Sel No.J2910088.

[0290] 3.2 Investigation of different mobile phase gradients

[0291] (1) The following four different mobile phase gradients were investigated, where mobile phase D was acetonitrile and mobile phase C was water.

[0292]

[0293] (2) Experimental results

[0294]

[0295]

[0296] The separation degree of fructose and pinitol was used as an indicator for investigation, and the mobile phase gradient 4 was finally optimized as the best mobile phase gradient condition.

[0297] 3.3 Investigation of different column temperatures

[0298] (1) Five different column temperatures were investigated: 35°C, 25°C, 20°C, 15°C, and 13°C;

[0299] (2) Experimental results

[0300]

[0301] In summary, the column temperature range of 13℃-35℃ shows that when the temperature is lower, the separation degree of fructose and pinitol is better. Considering the instrument and the surrounding environment, the column temperature is temporarily set at 15℃.

[0302] 3.4 Investigation of different flow rates

[0303] (1) Three different flow rates were investigated: 0.95 ml / min, 1 ml / ml, and 1.05 ml / min;

[0304] (2) Experimental results

[0305]

[0306]

[0307] In summary, the flow rate has no significant effect on the peak shape of the chromatogram. The RSD of the separation between D-fructose and pinitol under various conditions is 1.72%, with no significant difference. Therefore, the flow rate is set to 1 ml / min.

[0308] 3.5 Investigation of different evaporation temperatures

[0309] (1) Three different evaporation temperatures were investigated: 59°C, 60°C, and 61°C;

[0310] (2) Experimental results

[0311]

[0312] In summary, the evaporation temperature has no significant effect on the peak shape of the chromatogram. The RSD of the separation between D-fructose and pinitol under various conditions is 0.27%, with no significant difference. Therefore, the evaporation temperature is set at 60℃.

[0313] 3.6 Investigation of different atomization temperatures

[0314] (1) Three different atomization temperatures were investigated: 59°C, 60°C, and 61°C;

[0315] (2) Experimental results

[0316]

[0317]

[0318] In summary, the evaporation temperature showed that the atomization temperature had no significant effect on the peak shape of the chromatogram. The RSD of the separation between D-fructose and pinitol under various conditions was 2.69%, with no significant difference. Therefore, the atomization temperature was set at 60°C.

[0319] 3.7 Investigation of different carrier gas flow rates

[0320] (1) Three different carrier gas flow rates were investigated: 1.4 L / min, 1.5 L / ml, and 1.6 L / min;

[0321] The remaining chromatographic conditions are:

[0322] (2) Experimental results

[0323]

[0324] In summary, the carrier gas flow rate has no significant effect on the peak shape of the chromatogram. The RSD of the separation between D-fructose and pinitol under various conditions is 2.18%, with no significant difference. Therefore, the carrier gas flow rate is set to 1.5 L / min.

Claims

1. A method for detecting the content of mono-oligosaccharides and fingerprint of compound sophora flavescens injection, characterized in that: The method includes high performance liquid chromatography-evaporative light scattering detection for detection, wherein the detection objects are monooligosaccharides and pinitol, and the monooligosaccharides are D-anhydrous glucose, D-fructose, and sucrose; the chromatographic column in the high performance liquid chromatography-evaporative light scattering detection method is a Prevail Carbohydrate ES column with a specification of 4.6 mm×250 mm and 5 μm; the mobile phase in the high performance liquid chromatography-evaporative light scattering detection method is an acetonitrile-water gradient solution, wherein the gradient elution conditions are as follows: The flow rate of the mobile phase in the high performance liquid chromatography-evaporative light scattering detection method in the method is 0.95 to 1.05 ml / min; The column temperature in the high performance liquid chromatography-evaporative light scattering detection method in the method is 13°C to 20°C; The evaporation temperature of the evaporative light detector in the high performance liquid chromatography-evaporative light scattering detection method is 59-61°C; The atomization temperature of the evaporative light detector in the high performance liquid chromatography-evaporative light scattering detection method in the method is 59 to 61°C; In the method, the carrier gas in the high performance liquid chromatography-evaporative light scattering detection method is nitrogen, and the carrier gas flow rate is 1.4 to 1.6 L / min.

2. The method according to claim 1, characterized in that The flow rate of the mobile phase in the high performance liquid chromatography-evaporative light scattering detection method in the method is 1 ml / min.

3. The method according to claim 1, characterized in that The column temperature in the high performance liquid chromatography-evaporative light scattering detection method in the method is 15°C.

4. The method according to claim 1, wherein In the method, the injection volume in the high performance liquid chromatography-evaporative light scattering detection method is 10 μl or 20 μl.

5. The method according to claim 1, wherein The evaporation temperature of the evaporative light detector in the high performance liquid chromatography-evaporative light scattering detection method in the method is 60°C.

6. The method according to claim 1, characterized in that The atomization temperature of the evaporative light detector in the high performance liquid chromatography-evaporative light scattering detection method in the method is 60°C.

7. The method according to claim 1, characterized in that The carrier gas flow rate in the high performance liquid chromatography-evaporative light scattering detection method in the method is 1.5 L / min.

8. The method according to claim 1, characterized in that The blank solution in the high performance liquid chromatography-evaporative light scattering detection method is prepared by: preparing a mixed solution of acetonitrile and water in a ratio of 50:

50.

9. The method according to claim 1, characterized in that Preparation of the reference solution in the HPLC-ELSD method: Accurately weigh appropriate amounts of D-fructose reference, pinitol reference, D-anhydrous glucose reference, and sucrose reference, add blank solution to prepare a mixed reference solution containing 1.00 mg of D-fructose, 0.19 mg of pinitol, 0.90 mg of D-anhydrous glucose, and 0.20 mg of sucrose per 1 ml, and shake well to obtain the solution.

10. The method according to claim 1, characterized in that Preparation of the test solution in the HPLC-ELSD method: accurately measure 1 ml of each batch of compound sophora flavescens injection, place it in a 20 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

11. The method according to any one of claims 1 to 10, characterized in that: The method comprises detecting the contents of oligosaccharides and pinitol in the compound sophora flavescens injection by high performance liquid chromatography-evaporative light scattering detection, wherein the conditions of the high performance liquid chromatography-evaporative light scattering are: (1) Blank solution preparation: acetonitrile-water = 50:50 mixed solution; (2) Preparation of reference solution: Accurately weigh appropriate amounts of D-fructose reference, pinitol reference, D-anhydrous glucose reference, and sucrose reference, add blank solution to make a mixed reference solution containing 1.00 mg of D-fructose, 0.19 mg of pinitol, 0.90 mg of D-anhydrous glucose, and 0.20 mg of sucrose per 1 ml, and shake well. (3) Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection into a 20 ml volumetric flask, add blank solution to the mark, shake well, filter, and use the filtrate as the test solution; (4) Detection: Inject the blank solution, reference solution, and test solution in this order, and then use the external standard two-point method to calculate the content of D-fructose, pinitol, D-anhydrous glucose, and sucrose in the test solution.

12. The method according to any one of claims 1 to 10, characterized in that: The method comprises: constructing a fingerprint spectrum of the compound sophora flavescens injection containing D-anhydrous glucose, D-fructose, sucrose and pinitol.

13. The method according to claim 12, characterized in that The method includes: the method includes high performance liquid chromatography-evaporative light scattering detection method for detection, wherein the conditions of high performance liquid chromatography-evaporative light scattering are: (1) Blank solution preparation: acetonitrile-water = 50:50 mixed solution; (2) Preparation of reference solution: Accurately weigh appropriate amounts of D-fructose reference, pinitol reference, D-anhydrous glucose reference, and sucrose reference, add blank solution to make a mixed reference solution containing 1.00 mg of D-fructose, 0.19 mg of pinitol, 0.90 mg of D-anhydrous glucose, and 0.20 mg of sucrose per 1 ml, shake well, and prepare two portions in the same manner. (3) Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection into a 20 ml volumetric flask, add blank solution to the mark, shake well, filter, and use the filtrate as the test solution; (4) Construction of standard fingerprint: blank solution, reference solution, and test solution were injected in order to construct the standard fingerprint of compound sophora flavescens injection containing D-anhydrous glucose, D-fructose, sucrose, and pinitol; (5) Detection: Inject the blank solution, reference solution, and test solution in this order, and then calculate the contents of D-fructose, pinitol, D-anhydrous glucose, and sucrose in the test solution using the external standard two-point method.

14. The method according to claim 13, characterized in that The standard fingerprint spectrum includes three unknown peaks, a D-fructose chromatographic peak, a pinitol chromatographic peak, a D-anhydrous glucose chromatographic peak and a sucrose chromatographic peak.

15. The method according to claim 14, characterized in that In the standard fingerprint, the relative retention times of the three unknown peaks are 0.100-0.130, 0.135-0.150, and 0.170-0.190, respectively; the relative retention time of D-fructose is 0.660-0.690; the relative retention time of pinitol is 0.695-0.730; the relative retention time of D-anhydrous glucose is 1.00; and the relative retention time of sucrose is 1.130-1.

153.

16. The method according to claim 15, characterized in that In the standard fingerprint, the relative retention times of the three unknown peaks are 0.12, 0.14, and 0.18, respectively; the relative retention time of D-fructose is 0.67; the relative retention time of pinitol is 0.70; and the relative retention time of sucrose is 1.14.

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