Construction method of characteristic chromatogram of tripterygium wilfordii or derivative products thereof and determination method of content of triptolide

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a specific chromatogram of tripterygium wilfordii or a derivative product thereof and a determination method of the content of triptolide. The construction method of the specific chromatogram of the tripterygium wilfordii or the derivative product of the tripterygium wilfordii comprises the step that ultra-high performance liquid chromatography is adopted for detection, and chromatographic conditions include that octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, a phosphoric acid-containing aqueous solution is used as a mobile phase B, and gradient elution is carried out. The construction method of the UPLC characteristic chromatogram of the tripterygium wilfordii or the derivative product of the tripterygium wilfordii is higher in efficiency, more stable in base line, more uniform in distribution of characteristic peaks, better in separation degree, easier to position and qualitatively analyze and more in characteristic peaks, and provides a basis for quality detection and medication safety of the tripterygium wilfordii or the derivative product of the tripterygium wilfordii.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for constructing a characteristic spectrum of tripterygium wilfordii or its derivatives and a method for determining the content of tripterygium wilfordii. Background Art

[0002] Tripterygium wilfordii Hook.f. is a dried and peeled root of the Celastraceae plant. It is bitter and cool in nature, and has the effects of clearing away heat and detoxifying, dispelling wind and activating blood circulation, and relieving swelling and pain. In traditional Chinese medicine, it is mostly used to treat rheumatoid arthritis, immune diseases, kidney disease, tumors and other diseases, such as rheumatoid arthritis, low back pain, peripheral neuritis, leprosy, osteomyelitis, systemic lupus erythematosus, etc. Tripterygium wilfordii is commonly known as heartbreak grass and mountain arsenic. The whole plant has different degrees of toxicity. In clinical practice, the root wood is often peeled off the second layer of bark to reduce toxic side effects and ensure safe use.

[0003] The chemical components of Tripterygium wilfordii are complex and diverse, and the active ingredients mainly include alkaloids, glycosides, terpenes, lignin, etc. The Chinese Pharmacopoeia has not included Tripterygium wilfordii since its 1963 edition; the Ministry of Health's Drug Standards for Chinese Patent Medicines and Prescriptions only include the index components, content and detection methods of Tripterygium wilfordii ethanol extracts, and lack standards and quality control methods for Tripterygium wilfordii water solvent preparations.

[0004] The fingerprint established for Tripterygium wilfordii in the prior art has 18 common characteristic peaks and identified 6 of them, but the response values ​​of some characteristic peaks are too small and close to the baseline, and the characteristic is not obvious. Especially for the preparation made from the water extract of Tripterygium wilfordii, after the process treatment, its effective ingredients are obviously different from the medicinal material itself. The characteristic spectrum established for the standard decoction of Tripterygium wilfordii in the prior art only identifies 4 common characteristic peaks, and some characteristic peaks are obviously not separated from the baseline, which is easily affected by the chromatographic column or laboratory and difficult to reproduce; there are also problems such as interference from miscellaneous peaks and long running time; the prior art cannot control the quality of triptolide in Tripterygium wilfordii; therefore, how to improve the detection standard of Tripterygium wilfordii or its derivatives and comprehensively control the quality of the finished product is a technical problem to be solved by the present invention. Summary of the invention

[0005] Therefore, the first object of the present invention is to provide a method for constructing a characteristic spectrum of Tripterygium wilfordii or its derivatives. According to the characteristics of Tripterygium wilfordii or its derivatives, this method strengthens specific identification and multi-component and holistic quality control, and establishes a characteristic spectrum of this variety. The method has a large number of characteristic peaks with high separation and obvious characteristic, and can perform comprehensive quality control on Tripterygium wilfordii or its derivatives.

[0006] The second object of the present invention is to provide a method for determining the content of triptolide in Tripterygium wilfordii or its derivatives, which method can effectively and accurately determine the content of triptolide so as to better monitor the quality of Tripterygium wilfordii preparations.

[0007] The present invention provides a method for constructing a characteristic spectrum of Tripterygium wilfordii or its derivatives, comprising detecting by ultra-high performance liquid chromatography, wherein the chromatographic conditions include:

[0008] Octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, and aqueous solution containing phosphoric acid was used as mobile phase B. The gradient elution procedure included:

[0009] 0→6min, the volume percentage of mobile phase A is: 22%→25%, the volume percentage of mobile phase B is: 78%→75%;

[0010] 6min→10min, the volume percentage of mobile phase A is: 25%→31%, the volume percentage of mobile phase B is: 75%→69%;

[0011] 10min→15min, the volume percentage of mobile phase A is: 31%→33%, the volume percentage of mobile phase B is: 69%→67%;

[0012] 15min→24min, the volume percentage of mobile phase A is: 33%→40%, the volume percentage of mobile phase B is: 67%→60%;

[0013] 24min→30min, the volume percentage of mobile phase A is: 40%→42%, the volume percentage of mobile phase B is: 60%→58%;

[0014] 30min→36min, the volume percentage of mobile phase A is: 42%→90%, and the volume percentage of mobile phase B is: 58%→10%.

[0015] Optionally, the chromatographic conditions include at least one of the following:

[0016] (1) The detection wavelength is 210-270nm; optionally, 220nm;

[0017] (2) The flow rate is 0.2-0.4 ml / min; optionally, the flow rate is 0.3 ml / min;

[0018] (3) Column temperature 20-30°C; optionally, column temperature 25°C;

[0019] (4) The injection volume is 0.5-2 μL; optionally, the injection volume is 1 μL;

[0020] (5) The phosphoric acid concentration in the aqueous solution containing phosphoric acid in mobile phase B is 0.015-0.1% v / v; optionally, the phosphoric acid concentration is 0.05% v / v.

[0021] Optionally, the preparation of the test solution is also included, including:

[0022] 1) Weigh the Tripterygium wilfordii sample, add extraction solvent to extract, and obtain an extract;

[0023] 2) separating the extract from solid and liquid, taking the supernatant and eluting it with an elution solvent to obtain an eluate;

[0024] 3) The eluate is spin-dried, and the re-dissolving solvent is added to re-dissolve, the solid and liquid are separated, and the liquid is taken to obtain the test sample solution.

[0025] Optionally, the preparation of the test solution satisfies at least one of the following:

[0026] A. The ratio of the mass of the Tripterygium wilfordii test sample to the volume of the solvent is (0.5-2):(10-50); the mass to volume ratio is g / mL;

[0027] B. The extraction method is ultrasonic extraction or reflux extraction;

[0028] C. Extraction time is ≥30 min, preferably 30-60 min;

[0029] D. The solid-liquid separation is independently selected from centrifugation or filtration;

[0030] E. The solvent is selected from one or more of ethyl acetate and methanol; preferably, the extraction solvent is ethyl acetate; preferably, the elution solvent is ethyl acetate containing 0.2% v / v formic acid; preferably, the re-dissolution solvent is methanol;

[0031] F. The elution is performed by column chromatography; preferably, the column chromatography is neutral alumina column chromatography; more preferably, the neutral alumina column is pretreated with ethyl acetate.

[0032] Optionally, the construction method further comprises the steps of preparing a reference solution using at least one of triptolide and triptolide as a solubilizing agent, and obtaining a reference spectrum of the reference solution by detecting the reference solution using ultra-high performance liquid chromatography in the construction method;

[0033] Preferably, each 1 mL of the triptolide reference solution contains 5-20 μg of triptolide; preferably, 10 μg;

[0034] Preferably, each 1 mL of triptolide reference solution contains 5-20 μg of triptolide; preferably, 10 μg;

[0035] Preferably, the solvent used in the preparation of the reference solution is selected from methanol or a methanol aqueous solution with a volume fraction of not less than 70%;

[0036] Optionally, the Tripterygium wilfordii or its derivative products include one or more of Tripterygium wilfordii medicinal materials, Tripterygium wilfordii decoction pieces or Tripterygium wilfordii preparations.

[0037] Optionally, the Tripterygium wilfordii preparation includes a standard decoction of Tripterygium wilfordii slices and a Tripterygium wilfordii formula granule.

[0038] The present invention also provides a quality detection method of Tripterygium wilfordii or its derivative products, comprising the steps of comparing a characteristic spectrum of the product to be detected with a control characteristic spectrum of Tripterygium wilfordii or its derivative products;

[0039] The characteristic spectrum of the product to be tested is obtained according to the construction method;

[0040] The reference characteristic spectrum of Tripterygium wilfordii or its derivative product is selected from any one of the following (1)-(4):

[0041] (1) It has 9 common characteristic peaks, the peak corresponding to the reference peak of the triptolide reference substance is the S1 peak, the relative retention times of peaks 2-3 and S1 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 2-3 are 1.15 and 1.68, respectively; the peak corresponding to the reference peak of the triptolide reference substance is the S2 peak, the relative retention times of peaks 4-6 and peaks 8-9 and S2 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 4-6 and peaks 8-9 are 0.63, 0.66, 0.78, 1.11 and 1.20, respectively;

[0042] (2) It has 9 common characteristic peaks, of which 2 peaks correspond to the retention times of reference peaks of triptolide and triptolide reference substances, respectively; the peak corresponding to the reference peak of triptolide reference substance is S1 peak; the relative retention times of peaks 2-3 and S1 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 2-3 are 1.15 and 1.68, respectively; peak 7 is S2 peak; the relative retention times of peaks 4-6 and 8-9 and S2 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 4-6 and 8-9 are 0.63, 0.66, 0.78, 1.11 and 1.20, respectively;

[0043] (3) Characteristic spectra of Tripterygium wilfordii and / or its preparations obtained by the construction method using a single batch or multiple batches of Tripterygium wilfordii and / or its derivatives as test samples;

[0044] (4) Using multiple batches of Tripterygium wilfordii and / or its derivatives as test samples, the characteristic spectra obtained according to the construction method are averaged or median to prepare a control characteristic spectrum.

[0045] Optionally, the peak 1 corresponds to triptolide; the peak 2 corresponds to syringaresinol; the peak 6 corresponds to peritassine A; the peak 7 corresponds to triptolide; the peak 8 corresponds to euphorbia pine; and the peak 9 corresponds to triptolide.

[0046] The present invention also provides a method for determining the content of triptolide in Tripterygium wilfordii or its derivative products, comprising obtaining a characteristic spectrum of the Tripterygium wilfordii product according to the construction method;

[0047] Reference substances included triptolide and triptolide.

[0048] The technical solution of the present invention has the following advantages:

[0049] 1. The method for constructing the characteristic spectrum of Tripterygium wilfordii or its derivatives provided by the present invention comprises the following steps: using octadecylsilane bonded silica gel as a filler, the mobile phase comprises an aqueous solution containing phosphoric acid and acetonitrile, and obtaining 9 common characteristic peaks through a specific gradient elution program. Compared with the existing HPLC characteristic spectrum of Tripterygium wilfordii or its derivatives, the method for constructing the UPLC characteristic spectrum of Tripterygium wilfordii or its derivatives of the present invention is more efficient, about 30 minutes, the baseline is more stable, the characteristic peaks are more evenly distributed, the separation is better, and they can all be separated from the baseline, which is easier to locate and qualitatively analyze; compared with the prior art, there are more characteristic peaks, and 6 characteristic components of Tripterygium wilfordii, Eugenol, Peritassine A, Tripterygium wilfordii, Eucommia ulmoides, and Tripterygium wilfordii are identified, providing a basis for the quality detection and drug safety of Tripterygium wilfordii or its derivatives.

[0050] 2. The quality detection method of Tripterygium wilfordii or its derivatives provided by the present invention compares the characteristic spectrum of the Tripterygium wilfordii product to be tested with the control characteristic spectrum of Tripterygium wilfordii or its derivatives, so as to detect the quality of the Tripterygium wilfordii or its derivatives more comprehensively and rapidly, thereby proving that the quality of the Tripterygium wilfordii or its derivatives is effective and reliable.

[0051] 3. The method for determining the content of triptolide in Tripterygium wilfordii or its derivatives provided by the present invention can quickly identify the content of triptolide in Tripterygium wilfordii or its derivatives and perform quality control by establishing a characteristic spectrum of Tripterygium wilfordii or its derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0053] Figure 1 is the UPLC chromatogram using pure methanol in Experimental Example 1 of the present invention;

[0054] Figure 2 is the UPLC chromatogram using 95% v / v ethanol in Experimental Example 1 of the present invention;

[0055] Figure 3 is the UPLC chromatogram using pure ethyl acetate (5 mL of pure ethyl acetate reconstituted) in Experimental Example 1 of the present invention;

[0056] Figure 4 is the UPLC chromatogram using chloroform in Experimental Example 1 of the present invention;

[0057] Figure 5 is the UPLC chromatogram using 50% v / v methanol in Experimental Example 1 of the present invention;

[0058] Figure 6 is the UPLC chromatogram using 80% v / v methanol in Experimental Example 1 of the present invention;

[0059] Figure 7 is a UPLC chromatogram using ethyl acetate-chloroform (1:1, v / v) in Experimental Example 1 of the present invention;

[0060] Figure 8 is a UPLC chromatogram using dichloromethane-methanol (49:1, v / v) in Experimental Example 1 of the present invention;

[0061] Fig. 9 This is the UPLC chromatogram of pure ethyl acetate (1 mL of pure ethyl acetate reconstituted) used in Experimental Example 1 of the present invention;

[0062] Fig.10 is a UPLC chromatogram using ethyl acetate-methanol (8:2, v / v) in Experimental Example 1 of the present invention;

[0063] Fig.11 is a UPLC chromatogram using ethyl acetate-methanol (7:3, v / v) in Experimental Example 1 of the present invention;

[0064] Fig.12 is the UPLC chromatogram using ethyl acetate-methanol (1:1, v / v) in Experimental Example 1 of the present invention;

[0065] Fig.13 is the UPLC chromatogram without column chromatography in Experimental Example 1 of the present invention;

[0066] Fig.14 is a UPLC chromatogram using a Waters Sep-Pak neutral alumina solid phase extraction column in Experimental Example 1 of the present invention;

[0067] Fig.15 is the UPLC chromatogram using AR neutral alumina in Experimental Example 1 of the present invention;

[0068] Fig.16 is the UPLC chromatogram using AR acidic alumina in Experimental Example 1 of the present invention;

[0069] Fig.17 is the UPLC chromatogram using AR basic alumina in Experimental Example 1 of the present invention;

[0070] Fig.18 is a UPLC chromatogram using a polyamide column in Experimental Example 1 of the present invention;

[0071] Fig.19 is a UPLC chromatogram of the aqueous solution discarded under water extraction in Experimental Example 1 of the present invention;

[0072] Fig. 20 is the UPLC chromatogram of the test solution under water extraction in Experimental Example 1 of the present invention;

[0073] Fig.21 is a UPLC chromatogram using a SPE C18 solid phase extraction column (20% v / v methanol) in Experimental Example 1 of the present invention;

[0074] Fig. 22 is a UPLC chromatogram using a SPE C18 solid phase extraction column (40% v / v methanol) in Experimental Example 1 of the present invention;

[0075] Fig.23 is a UPLC chromatogram using a SPE C18 solid phase extraction column (20% v / v acetonitrile) in Experimental Example 1 of the present invention;

[0076] Fig.24 It is the UPLC chromatogram using a silica gel column (petroleum ether-ethyl acetate (8:2, v / v)) in 3.4.3.1 of Experimental Example 1 of the present invention;

[0077] Fig.25 It is the UPLC chromatogram using a silica gel column (petroleum ether-ethyl acetate (1:1, v / v)) in 3.4.3.1 of Experimental Example 1 of the present invention;

[0078] Fig.26 It is the UPLC chromatogram using a silica gel column (petroleum ether-ethyl acetate (2:8, v / v)) in 3.4.3.1 of Experimental Example 1 of the present invention;

[0079] Fig. 27 is the UPLC chromatogram using a silica gel column (ethyl acetate) in 3.4.3.1 of Experimental Example 1 of the present invention;

[0080] Fig.28is the UPLC chromatogram using a silica gel column (ethyl acetate) in 3.4.3.2 of Experimental Example 1 of the present invention;

[0081] Fig.29 is the UPLC chromatogram using a silica gel column (ethyl acetate-methanol (8:2, v / v)) in 3.4.3.2 of Experimental Example 1 of the present invention;

[0082] Fig.30 is the UPLC chromatogram using a silica gel column (ethyl acetate-methanol (1:1, v / v)) in 3.4.3.2 of Experimental Example 1 of the present invention;

[0083] Fig.31 is the UPLC chromatogram using a silica gel column (ethyl acetate-methanol (2:8, v / v)) in 3.4.3.2 of Experimental Example 1 of the present invention;

[0084] Fig.32 is the UPLC chromatogram using a silica gel column (methanol) in 3.4.3.2 of Experimental Example 1 of the present invention;

[0085] Fig.33 is a UPLC chromatogram using ethyl acetate (containing 0.2% v / v formic acid) in Experimental Example 1 of the present invention;

[0086] Fig.34 is a UPLC chromatogram using ethyl acetate (containing 0.05% v / v formic acid) in Experimental Example 1 of the present invention;

[0087] Fig.35 This is the UPLC chromatogram of using methanol as the extraction solvent and ethyl acetate as the reconstitution in Experimental Example 1 of the present invention;

[0088] Fig.36 is a UPLC chromatogram using ethyl acetate as the extraction solvent in Experimental Example 1 of the present invention;

[0089] Fig.37 is the UPLC chromatogram of the blank solution in 4.1 in Experimental Example 1 of the present invention;

[0090] Fig.38 is the UPLC chromatogram of the test solution in 4.1 in Experimental Example 1 of the present invention;

[0091] Fig.39 It is the UPLC chromatogram with a wavelength of 220 nm in Experimental Example 1 of the present invention;

[0092] Fig.40 It is the UPLC chromatogram with a wavelength of 230 nm in Experimental Example 1 of the present invention;

[0093] Fig.41 It is the UPLC chromatogram with a wavelength of 254 nm in Experimental Example 1 of the present invention;

[0094] Fig.42 It is the UPLC chromatogram with a wavelength of 268 nm in Experimental Example 1 of the present invention;

[0095] Fig.43 It is the UPLC chromatogram with a wavelength of 280 nm in Experimental Example 1 of the present invention;

[0096] Fig.44 is the UPLC chromatogram of chromatographic column 1 in Experimental Example 1 of the present invention;

[0097] Fig.45 is the UPLC chromatogram of chromatographic column 2 in Experimental Example 1 of the present invention;

[0098] Fig.46 is the UPLC chromatogram of chromatographic column 3 in Experimental Example 1 of the present invention;

[0099] Fig.47 is the UPLC chromatogram of chromatographic column 4 in Experimental Example 1 of the present invention;

[0100] Fig.48 is the UPLC chromatogram at a column temperature of 25° C. in Experimental Example 1 of the present invention;

[0101] Fig.49 is the UPLC chromatogram at a column temperature of 30° C. in Experimental Example 1 of the present invention;

[0102] Fig.50 is the UPLC chromatogram at a column temperature of 35° C. in Experimental Example 1 of the present invention;

[0103] Fig.51 is the UPLC chromatogram using gradient condition 1 in Experimental Example 1 of the present invention;

[0104] Fig.52 is the UPLC chromatogram using gradient condition 2 in Experimental Example 1 of the present invention;

[0105] Fig.53 is the UPLC chromatogram using gradient condition 3 in Experimental Example 1 of the present invention;

[0106] Fig.54 is the UPLC chromatogram using gradient condition 4 in Experimental Example 1 of the present invention;

[0107] Fig.55 is the UPLC chromatogram using acetonitrile-0.05% v / v formic acid in Experimental Example 1 of the present invention;

[0108] Fig.56 is the UPLC chromatogram using acetonitrile-0.05% v / v glacial acetic acid in Experimental Example 1 of the present invention;

[0109] Fig.57 is the UPLC chromatogram using acetonitrile-0.1% v / v phosphoric acid in Experimental Example 1 of the present invention;

[0110] Fig.58 is the UPLC chromatogram using acetonitrile-0.04% v / v phosphoric acid in Experimental Example 1 of the present invention;

[0111] Fig.59 is the UPLC chromatogram using acetonitrile-0.03% v / v phosphoric acid in Experimental Example 1 of the present invention;

[0112] Fig.60 is the UPLC chromatogram using acetonitrile-0.025% v / v phosphoric acid in Experimental Example 1 of the present invention;

[0113] Fig.61 is the UPLC chromatogram using acetonitrile-0.015% v / v phosphoric acid in Experimental Example 1 of the present invention;

[0114] Fig.62 is the UPLC chromatogram using acetonitrile-0.05% v / v phosphoric acid in Experimental Example 1 of the present invention;

[0115] Fig.63 It is a specific localization map of the LC / MS / MS analysis structure in Experimental Example 1 of the present invention; (wherein, from top to bottom, they are respectively Tripterygium wilfordii, Syringoresinol, Peritassine A, Tripterygium wilfordii alkaloid, Eucommia ulmoides, and Tripterygium wilfordii alkaloid);

[0116] Fig.64 is the UPLC chromatogram of the standard decoction of Tripterygium wilfordii slices in Experimental Example 1 of the present invention;

[0117] Fig.65 It is the chromatogram of the test product obtained in the specific property investigation in Experimental Example 1 of the present invention;

[0118] Fig.66 It is the negative control chromatogram obtained in the specific property investigation in Experimental Example 1 of the present invention;

[0119] Fig.67 is the chromatogram at a flow rate of 0.25 mL / min in Experimental Example 1 of the present invention;

[0120] Fig.68 is the chromatogram at a flow rate of 0.30 mL / min in Experimental Example 1 of the present invention;

[0121] Fig.69 is the chromatogram at a flow rate of 0.35 mL / min in Experimental Example 1 of the present invention;

[0122] Fig.70 This is the chromatogram at a column temperature of 20° C. in Experimental Example 1 of the present invention;

[0123] Fig.71 This is the chromatogram at a column temperature of 25° C. in Experimental Example 1 of the present invention;

[0124] Fig.72 This is the chromatogram at a column temperature of 30° C. in Experimental Example 1 of the present invention;

[0125] Fig.73 It is the reference characteristic spectrum of the standard decoction of Tripterygium wilfordii slices in Experimental Example 2 of the present invention;

[0126] Fig.74 It is the common peak pattern of UPLC characteristic spectra of 19 batches of Tripterygium wilfordii medicinal materials in Experimental Example 3 of the present invention (S1-S19: 20231207, 2023121101, 2023121102, 20231226, 20240129, 20240202, 2024022701, 2024022702, 2024022703, 2024022704, 2024022705, 2024022706, 2024022707, 2024022708, 2024022709, 2024022710, 2024041201, 2024041202, 2024041203);

[0127] Fig.75 It is the common pattern of the comparison atlas of Chinese medicinal materials, decoction pieces and standard decoctions in Experimental Example 4 of the present invention (wherein S1 is the comparison atlas of Tripterygium wilfordii medicinal materials, S2 is the comparison atlas of Tripterygium wilfordii decoction pieces, and S3 is the comparison atlas of the standard decoction of Tripterygium wilfordii decoction pieces);

[0128] Fig.76 It is the common peak pattern of the UPLC characteristic spectra of three batches of Tripterygium wilfordii formula granules in Experimental Example 5 of the present invention (S1-S3: 2406001Y, 2406002Y, 2406003Y); DETAILED DESCRIPTION

[0129] The following experimental examples are provided for a better understanding of the present invention, but are not limited to the best implementation mode described, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the present invention with the features of other prior arts shall fall within the protection scope of the present invention.

[0130] If no specific experimental steps or conditions are specified in the experimental examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0131] In the following experimental examples of the present invention, the instruments, reagents and reagents involved include:

[0132] Chromatograph 1: Thermo Fisher Vanquish UPLC chromatography system, including quaternary pump, autosampler, chromatography column oven, diode array UV detector, and Chromeleon chromatography management system.

[0133] Chromatograph 2: Waters H-CLASS UPLC chromatography system, including quaternary solvent manager (ACQ-QSM), automatic sample injector (ACQ-FTN), original imported chromatography column oven (ACQ-CM), diode array UV detector (ACQ-PDA), and Empower chromatography management system.

[0134] Electronic analytical balance: METTLER TOLEDO (Swiss Mettler) ME36S, XS204, XS205, XSE205 one hundred thousandth balance.

[0135] Ultrasonic cleaner: SK5200H Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0136] Column: Waters CORTECS UPLCShield RP (2.1×100mm, 1.6μm); Waters ACQUITYUPLC HSS T3 (2.1×100nm, 1.8μm); Waters CORTECS UPLC T3 (2.1×100mm, 1.6μm); WatersCORTECS C18 (2.1×100mm, 1.6μm).

[0137] Test drug: The freeze-dried powder of the standard decoction of Tripterygium wilfordii slices can be prepared by conventional methods in the art, for example, in the present invention, it is prepared according to the following steps: take about 100 g of the Tripterygium wilfordii slices and put them in a casserole, add 900 mL of deionized water and soak them for 30 minutes, first boil them over high heat (500W), then simmer them over low heat (300W) for 120 minutes, and filter them through a 200-mesh filter cloth while hot; add 700 mL of deionized water to the residue, boil them over high heat (500W), then simmer them over low heat (300W) for 60 minutes, filter them through a 200-mesh filter cloth while hot, combine the two filtrates, quickly cool the filtrate to room temperature, concentrate them under reduced pressure and low temperature (65°C) to a concentrated extract with a relative density of about 1.04, place them in a freeze dryer, freeze-dry them (-40°C, 110kpa) until dry, and grind them into powder to obtain the freeze-dried powder of the standard decoction of Tripterygium wilfordii slices.

[0138] The Tripterygium wilfordii formula granules can be prepared by conventional methods in the art. For example, in the present invention, it is prepared according to the following steps: take 10,000 g of Tripterygium wilfordii slices, add 9 times of water and boil for 2 hours, filter, then add 7 times of water and boil for 1 hour, filter, combine the filtrate, concentrate the filtrate into a clear paste, dry (or dry and crush), then add 110 g of maltodextrin, mix well, granulate, and make 1000 g to obtain the Tripterygium wilfordii formula granules.

[0139] Triptolide reference substance (batch number: CFN99196, purchased from ChemFaces, purity ≥ 98%); Triptolide reference substance (batch number: WP23101608, purchased from Vicogene, purity ≥ 98%);

[0140] A total of 19 batches of samples were collected from five locations across the country, including Taining County, Sanming City, Fujian Province, Yangxin County, Huangshi City, Hubei Province, Chengbu County, Shaoyang City, Hunan Province, Shuangbai County, Chuxiong Prefecture, Yunnan Province, and Jianhe County, Qiandongnan Prefecture, Guizhou Province. The sources and numbers are shown in Table 1.

[0141] Table 1

[0142]

[0143]

[0144] Acetonitrile and methanol were of chromatographic grade, and water was ultrapure water; other reagents were of analytical grade.

[0145] Example 1 Construction method and methodological verification of UPLC characteristic spectrum of standard decoction of Tripterygium wilfordii slices

[0146] 1. Solution Preparation

[0147] 1.1 Preparation of reference solution

[0148] Preparation of reference solution:

[0149] Take the triptolide reference substance and add methanol to prepare a triptolide reference substance solution containing 10 μg of triptolide per 1 ml.

[0150] Take the triptolide reference substance and add methanol to prepare a triptolide reference substance solution containing 10 μg of triptolide per 1 ml.

[0151] 1.2 Preparation of test solution:

[0152] Take 0.5g of the freeze-dried powder of the standard decoction of Tripterygium wilfordii slices, put it in a stoppered conical flask, add 10ml of pure ethyl acetate, weigh it, and perform ultrasonic extraction (power 500W, frequency 40kHz) for 30 minutes, make up the missing weight with ethyl acetate, centrifuge for 10 minutes (8000r / min), take the supernatant, pass it through a pretreated neutral alumina column (100-200 mesh; 2g; inner diameter 10mm; the pretreatment is elution with 30ml of ethyl acetate), elute with 30ml of ethyl acetate (containing 0.2% v / v formic acid), take the eluate, spin dry it, add 1ml of methanol to dissolve it, filter it, take the filtrate to obtain the test solution.

[0153] 2. Determination method

[0154] Accurately pipette 1 μL of the test solution and the reference solution respectively, inject into the liquid chromatograph, measure, and record the chromatogram; the preliminary chromatographic conditions are as follows: octadecylsilane bonded silica gel as the filler, chromatographic column: Waters CORTECS UPLC Shield RP (2.1 mm × 100 mm, particle size 1.6 μm), acetonitrile as mobile phase A, 0.05% v / v phosphoric acid as mobile phase B, gradient elution according to Table 2, flow rate of 0.3 ml / min, column temperature of 25 ° C, detection wavelength of 220 nm; the number of theoretical plates calculated based on the triptolide peak should be no less than 5000.

[0155] Table 2 Gradient elution program

[0156]

[0157] 3. Optimization of the test solution preparation method

[0158] 3.1 Investigation of extraction solvent

[0159] Take 0.5 g of freeze-dried powder of standard decoction of Tripterygium wilfordii slices (20231226Y) and put it in a stoppered conical flask. Accurately add 10 ml each of pure methanol, 95% v / v ethanol, pure ethyl acetate, chloroform, 50% v / v methanol, 80% v / v methanol, ethyl acetate-chloroform (1:1, v / v), and dichloromethane-methanol (49:1, v / v), weigh the weight, and perform ultrasonic extraction (power 500 W, frequency 40 kHz) for 30 minutes. The missing weight should be supplemented with solvent, centrifuged for 10 minutes (8000r / min), the supernatant was taken, passed through a pretreated neutral alumina column (100-200 mesh; 2g; inner diameter of 10mm; the pretreatment was eluted with 30ml ethyl acetate), eluted with 30ml ethyl acetate (containing 0.2% formic acid), the eluate was taken, spin-dried, added 5ml methanol to re-dissolve, filtered, accurately aspirated 1μL of the filtrate, injected into the ultra-high performance liquid chromatograph, and measured according to the chromatographic conditions described under 2. The results are shown in Figure 1-8.

[0160] Depend on Figure 1-8 It can be seen that the peak response of the extraction is larger when different concentrations of methanol are used as the extraction solvent, but when ethyl acetate is used as the extraction solvent, the 29min peak response in the spectrum is larger. Therefore, the effect of mixed solutions of ethyl acetate and methanol in different proportions as the extraction solvent on the extraction of Tripterygium wilfordii freeze-dried powder was further investigated.

[0161] 3.2 Investigation of mixed solutions of ethyl acetate and methanol in different proportions

[0162] Take 0.5g of freeze-dried powder of the standard decoction of Tripterygium wilfordii slices (20231226Y), place it in a stoppered conical flask, accurately add 10ml of pure ethyl acetate, ethyl acetate-methanol (8:2, v / v), ethyl acetate-methanol (7:3, v / v), and ethyl acetate-methanol (1:1, v / v), weigh the weight, and perform ultrasonic extraction (power 500W, frequency 40kHz) for 30 minutes. Make up the missing weight with the corresponding solvent, centrifuge for 10 minutes (8000r / min), take the supernatant, pass it through a pretreated neutral alumina column (100-200 mesh; 2g; inner diameter 10mm; the pretreatment is elution with 30ml ethyl acetate), elute with 30ml ethyl acetate (containing 0.2% v / v formic acid), take the eluate, spin dry, add 1ml methanol to re-dissolve, filter, accurately aspirate 1μL of the filtrate, inject it into the ultra-high performance liquid chromatograph, and measure according to the chromatographic conditions described under 2. Results are shown in Figure 9-12 .

[0163] Depend on Figure 9-12 It can be seen that the peak response is larger when ethyl acetate-methanol (1:1, v / v) is used as the extraction solvent, so ethyl acetate-methanol (1:1, v / v) is preferably used as the extraction solvent in this embodiment.

[0164] 3.3 Column chromatography filler investigation

[0165] Take 0.5g of freeze-dried powder of standard decoction of Tripterygium wilfordii slices (20231226Y), place it in a stoppered conical flask, add 10ml of ethyl acetate-methanol (1:1, v / v), weigh it, and perform ultrasonic extraction (power 500W, frequency 40kHz) for 30 minutes. Make up the missing weight with the corresponding solvent, centrifuge for 10 minutes (8000r / min), take the supernatant, spin dry, add 1ml of methanol to dissolve, filter, accurately draw 1μL of the filtrate, inject it into the ultra-high performance liquid chromatograph, and measure it according to the chromatographic conditions described under 2. The results are shown in Fig.13 .

[0166] Take 0.5g of freeze-dried powder of the standard decoction of Tripterygium wilfordii slices (20231226Y), place it in a stoppered conical flask, add 10ml of ethyl acetate-methanol (1:1, v / v), weigh it, perform ultrasonic extraction (power 500W, frequency 40kHz) for 30 minutes, make up the missing weight with the corresponding solvent, centrifuge for 10 minutes (8000r / min), take the supernatant, and elute it through a pretreated Waters Sep-Pak neutral alumina solid phase extraction column, AR neutral alumina, AR acidic alumina, AR basic alumina, polyamide column (the pretreatment is 30ml ethyl acetate elution), and elute with 30ml ethyl acetate (containing 0.2% v / v formic acid), take the eluate, spin dry, add 1ml methanol to dissolve it, filter, accurately aspirate 1μL of the filtrate, inject it into the ultra-high performance liquid chromatograph, and measure it according to the chromatographic conditions described under 2. Results are shown in Figure 14-18 .

[0167] Depend on Figure 13-18 It can be seen that after elution through Waters Sep-Pak neutral alumina solid phase extraction column, neutral alumina and acidic alumina columns, two peaks were lost; after elution through alkaline alumina column, more peaks were lost; after elution through polyamide column, there was no obvious difference; therefore, it was further investigated whether the two lost peaks could be eluted.

[0168] 3.4 Collecting missing peaks

[0169] 3.4.1 Using water extraction, see if the two missing peaks can be eluted.

[0170] Preparation method of the test sample: Take 0.2g of freeze-dried powder of the standard decoction of Tripterygium wilfordii slices (20231226Y), accurately add 20ml of methanol, perform ultrasonic extraction (power 500W, frequency 37kHz) for 30 minutes, centrifuge for 5 minutes (4000r / min), accurately transfer 10ml of supernatant, spin dry, add 5ml of water to dissolve, transfer to a centrifuge tube (10ml), centrifuge for 10 minutes (4000r / min), separate the solid and liquid, discard the aqueous solution, add 2ml of methanol to dissolve the precipitate, filter, and take the filtrate to obtain the test solution. Accurately aspirate 1μL of the test solution and the discarded aqueous solution, respectively inject them into the ultra-high performance liquid chromatograph, and measure according to the chromatographic conditions described under 2. Results are shown in Figure 19-20 .

[0171] Depend on Fig.13 and Figure 19-20 It can be seen that the chromatogram after water extraction collected the two missing peaks, but did not collect triptolide, so this method is not applicable.

[0172] 3.4.2 Use SPE C18 solid phase extraction column and elute with different solvents to see if the two missing peaks can be eluted.

[0173] Preparation method of test sample (20% v / v methanol): Take 0.1g of freeze-dried powder of standard decoction of Tripterygium wilfordii slices (20231226Y), accurately add 10ml of methanol, ultrasonic extraction (power 500W, frequency 37kHz) for 30 minutes, centrifuge for 10 minutes (4000r / min), take the supernatant, spin dry, add 3ml of water to dissolve, pass through a pretreated SPE C18 solid phase extraction column (the pretreatment is activated with methanol and water), first rinse with 2ml20%v / v methanol, discard, and then elute with 2ml20%v / v methanol, take the eluate, take the subsequent filtrate, and obtain the test sample solution.

[0174] Preparation method of test sample (40% v / v methanol): Take 0.1g of freeze-dried powder of standard decoction of Tripterygium wilfordii slices (20231226Y), accurately add 10ml of methanol, ultrasonic extraction (power 500W, frequency 37kHz) for 30 minutes, centrifuge for 10 minutes (4000r / min), take the supernatant, spin dry, add 3ml of water to dissolve, pass through a pretreated SPE C18 solid phase extraction column (the pretreatment is activated with methanol and water), first rinse with 2ml40%v / v methanol, discard, and then elute with 2ml40%v / v methanol, take the eluate, take the subsequent filtrate, and obtain the test sample solution.

[0175] Preparation method of test sample (20% v / v acetonitrile): Take 0.1g of freeze-dried powder of standard decoction of Tripterygium wilfordii slices (20231226Y), accurately add 10ml of methanol, ultrasonic extraction (power 500W, frequency 37kHz) for 30 minutes, centrifuge for 10 minutes (4000r / min), take the supernatant, spin dry, add 3ml of water to dissolve, pass through a pretreated SPE C18 solid phase extraction column (the pretreatment is activated with acetonitrile and water), first rinse with 2ml20%v / v acetonitrile, discard, and then elute with 2ml20%v / v acetonitrile (containing 0.2%v / v formic acid), take the eluate, take the subsequent filtrate, and obtain the test sample solution.

[0176] Accurately pipette 1 μL of each of the above test solutions and inject them into the ultra-high performance liquid chromatograph, and measure them according to the chromatographic conditions described under 2. The results are shown in Figure 21-23 .

[0177] Depend on Figure 21-23 It can be seen that when using the SPE C18 solid phase extraction column and eluting with different solvents, there is no missing peak, but the baseline is not flat, so this method is not applicable.

[0178] 3.4.3 Using silica gel column

[0179] 3.4.3.1 Use a silica gel column and perform gradient elution with petroleum ether-ethyl acetate (8:2, v / v), petroleum ether-ethyl acetate (1:1, v / v), petroleum ether-ethyl acetate (2:8, v / v), and ethyl acetate to see if the two missing peaks can be collected.

[0180] Prepare the test solution according to the preparation method of the test solution in 1.2, and determine it according to the chromatographic conditions described under 2. The results are shown in Figure 24-27 .

[0181] Depend on Fig.13 and Figure 24-27 It can be seen that there are basically no missing peaks in the ethyl acetate chromatogram; therefore, elution with ethyl acetate was further investigated.

[0182] 3.4.3.2 Use a silica gel column and perform gradient elution with ethyl acetate, ethyl acetate-methanol (8:2, v / v), ethyl acetate-methanol (1:1, v / v), ethyl acetate-methanol (2:8, v / v) and pure methanol to see if the two missing peaks can be collected.

[0183] Prepare the test solution according to the preparation method of the test solution in 1.2, and measure it according to the chromatographic conditions described under 2. The results are shown in Figure 28-32 .

[0184] Depend on Figure 28-32 It can be seen that after elution with ethyl acetate, the baseline is relatively flat, the response is small, and there is no peak loss; but the peak shape of triptolide is poor; therefore, further consideration is given to optimizing the eluent for adjustment.

[0185] 3.4.4 Use Waters Sep-Pak neutral alumina solid phase extraction column and add 0.2% v / v formic acid and 0.05% v / v formic acid in ethyl acetate for elution.

[0186] Prepare the test solution according to the preparation method of the test solution in 1.2, and measure it according to the chromatographic conditions described under 2. The results are shown in Figure 33-34 .

[0187] Depend on Figure 33-34 It can be seen that ethyl acetate (containing 0.2% v / v formic acid) can collect two loss peaks, but the baseline in the first 6 minutes is not flat; ethyl acetate (containing 0.05% v / v formic acid) did not collect two loss peaks; therefore, ethyl acetate (containing 0.2% v / v formic acid) was used as the eluent, and further consideration was given to improving the baseline in the first 6 minutes.

[0188] 3.4.5 Improving the Baseline

[0189] Pure methanol was used as the extraction solvent. After extraction, the extract was dried by rotation, re-dissolved in ethyl acetate, and passed through a Waters Sep-Pak neutral alumina solid phase extraction column. The results are shown in Fig.35 ;like Fig.35 As shown, the peak response is extremely small and this method is not applicable.

[0190] Pure ethyl acetate was used as the extraction solvent, and the extraction was carried out through a Waters Sep-Pak neutral alumina solid phase extraction column. The results are shown in Fig.36 ;like Fig.36 As shown, the peak response becomes smaller, but the baseline is flat and the characteristic peaks are obvious, so the present invention is best to select pure ethyl acetate as the extraction solvent.

[0191] In summary, the present invention determines the following method for preparing the test solution: 0.5 g of the freeze-dried powder of the standard decoction of Tripterygium wilfordii slices is taken and placed in a stoppered conical flask, 10 ml of pure ethyl acetate is added, the weight is weighed, ultrasonic extraction (power 500 W, frequency 40 kHz) is performed for 30 minutes, the missing weight is supplemented with ethyl acetate, centrifuged for 10 minutes (8000 r / min), the supernatant is taken, and passed through a pretreated Waters Sep-Pak neutral alumina solid phase extraction column (2 g; the pretreatment is eluted with 30 ml of ethyl acetate), eluted with 30 ml of ethyl acetate (containing 0.2% v / v formic acid), the eluate is taken, spin-dried, 1 ml of methanol is added to dissolve, filtered, and the filtrate is taken to obtain the test solution.

[0192] 4. Optimization of Chromatographic Conditions

[0193] The freeze-dried powder of the standard decoction of Tripterygium wilfordii slices was finely ground (20231226Y), 0.5 g was taken, placed in a stoppered conical flask, 10 ml of pure ethyl acetate was added, the weight was weighed, ultrasonic extraction (power 500 W, frequency 40 kHz) was performed for 30 minutes, the missing weight was supplemented with ethyl acetate, centrifuged for 10 minutes (8000 r / min), the supernatant was taken, passed through a pretreated neutral alumina column (100-200 mesh; 2 g; inner diameter 10 mm; the pretreatment was eluted with 30 ml of ethyl acetate), eluted with 30 ml of ethyl acetate (containing 0.2% v / v formic acid), the eluate was taken, dried, 1 ml of methanol was added for re-dissolution, filtered, and the filtrate was taken to obtain the test solution. The test solution was investigated as follows:

[0194] 4.1 Blank Survey

[0195] Accurately pipette 1 μL of the test solution and blank solution (pure methanol) respectively, inject into the liquid chromatograph, and measure according to the chromatographic conditions described under 2. The results are shown in Figure 37-38 .

[0196] Depend on Figure 37-38 It can be seen that there is a large peak at 29 minutes in both the test solution and the blank solution, so this peak is not selected as the characteristic peak.

[0197] 4.2 Investigation of detection wavelength

[0198] The test solution was taken separately and measured according to the chromatographic conditions described under 2, the only difference being that the detection wavelengths were 220nm, 230nm, 254nm, 268nm, and 280nm, respectively. Figure 39-43 .

[0199] Depend on Figure 39-43 It can be seen that compared with other wavelengths, at the detection wavelength of 220nm, there are more chromatographic peaks, the chromatographic peak response is large and the distribution is uniform. Therefore, the present invention selects 220nm as the best detection wavelength for characteristic spectrum determination.

[0200] 4.3 Investigation of different chromatographic columns

[0201] The test solution was taken separately and measured according to the chromatographic conditions described under item 2, the only difference being that different chromatographic columns (chromatographic column 1: Waters ACQUITY UPLC HSS T3 (2.1×100nm, 1.8μm); chromatographic column 2: Waters CORTECSUPLC T3 (2.1×100mm, 1.6μm); chromatographic column 3: Waters CORTECS UPLCShield RP (2.1×100mm, 1.6μm); chromatographic column 4: Waters CORTECS C18 (2.1×100mm, 1.6μm)) were used for measurement. The results are shown in Figures 44-47 .

[0202] Depend on Figures 44-47 It can be seen that the overall peak shape of the chromatogram measured by using Waters CORTECS UPLCShield RP (2.1×100 mm, 1.6 μm) as the chromatographic column is good and evenly distributed. Therefore, the present invention selects Waters CORTECS UPLCShield RP as the best.

[0203] 4.4 Investigation of different column temperatures

[0204] Take the test solution and measure it according to the chromatographic conditions described in item 2. The only difference is that the column temperature is 25℃, 30℃ and 35℃ respectively. The results are shown in Figures 48-50 .

[0205] Depend on Figures 48-50 It can be seen that increasing the column temperature can help separate the two small peaks around 25 minutes, but when the column temperature is 25°C, the separation effect of each characteristic peak is best and the peak shape is better. Therefore, the present invention selects a column temperature of 25°C as the best.

[0206] 4.5 Optimization of elution conditions

[0207] The test solution was taken separately and measured according to the chromatographic conditions described under 2. The only difference was that the gradient elution procedure was different (gradient conditions 1-4, see Table 4-7). The results are shown in Figure 51-54 .

[0208] Depend on Figure 51-54 It can be seen that gradient condition 4 has the best separation effect and the peaks are distributed more evenly. Therefore, gradient condition 4 is temporarily selected as the gradient elution program for further investigation.

[0209] Table 4 Gradient conditions 1

[0210]

[0211] Table 5 Gradient conditions 2

[0212]

[0213] Table 6 Gradient Condition 3

[0214]

[0215] Table 7 Gradient Condition 4

[0216]

[0217] 4.6 Investigation of mobile phase

[0218] The test solution was taken separately and determined according to the chromatographic conditions described under item 2, except that the mobile phase B was 0.05% v / v formic acid, 0.05% v / v glacial acetic acid, 0.1% v / v phosphoric acid, 0.04% v / v phosphoric acid, 0.03% v / v phosphoric acid, 0.025% v / v phosphoric acid, 0.015% v / v phosphoric acid, and 0.05% v / v phosphoric acid, respectively. The results are shown in the table below. Figure 55-62 .

[0219] Depend on Figure 55-62 It can be seen that the peak separation effect is best when the mobile phase B is 0.05% v / v phosphoric acid, so the present invention uses acetonitrile-0.05% v / v phosphoric acid as the best mobile phase.

[0220] In summary, the present invention determines the following chromatographic conditions: octadecylsilane bonded silica gel as filler chromatographic column: Waters CORTECS UPLCShield RP (2.1mm×100mm, particle size 1.6μm), acetonitrile as mobile phase A, 0.05% v / v phosphoric acid as mobile phase B, gradient elution according to Table 8, flow rate of 0.3ml / min, column temperature of 25°C, detection wavelength of 220nm; theoretical plate number calculated based on triptolide peak should be not less than 5000. Thus, the following is obtained: Fig.64The UPLC chromatogram of the standard decoction of Tripterygium wilfordii slices was obtained, and the main chromatographic peaks in the chromatogram were identified. The results are shown in Table 9.

[0221] Table 8 Gradient elution program

[0222]

[0223] Table 9 System suitability results

[0224]

[0225] 4.7 Identification of characteristic peaks

[0226] An in-depth study was conducted on the identification of characteristic peaks. The ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS) method was used to analyze the Tripterygium wilfordii samples. According to the multi-level mass spectrometry information of the samples and combined with relevant literature, the target peaks were identified and located using reference substances. The identification results of the target components are shown in Table 10, and it was confirmed that peak 2 was syringaresinol, peak 7 was Peritassine A, peak 9 was triptolide, peak 10 was dasyphylline, peak 11 was triptolide, and peak 12 was triptolide A.

[0227] Table 10 Identification results of target components of Tripterygium wilfordii

[0228]

[0229] Based on the characteristic peaks determined above, the chromatograms of Tripterygium wilfordii and each reference substance are shown in Fig.63 shown.

[0230] 5. Chromatographic conditions and system suitability test

[0231] 5.1 Chromatographic conditions

[0232] Octadecylsilane bonded silica was used as the filler, the chromatographic column was Waters CORTECS UPLCShield RP (2.1 mm × 100 mm, particle size 1.6 μm), acetonitrile was used as the mobile phase A, 0.05% v / v phosphoric acid was used as the mobile phase B, gradient elution was performed according to Table 11, the flow rate was 0.3 ml / min, the column temperature was 25°C, and the detection wavelength was 220 nm; the theoretical plate number calculated based on the triptolide peak should be no less than 5000.

[0233] Table 11 Gradient elution program

[0234]

[0235] 5.2 Solution preparation

[0236] 5.2.1 Preparation of reference solution

[0237] Preparation of reference solution:

[0238] Take the triptolide reference substance and add methanol to prepare a triptolide reference substance solution containing 10 μg of triptolide per 1 ml.

[0239] Take the triptolide reference substance and add methanol to prepare a triptolide reference substance solution containing 10 μg of triptolide per 1 ml.

[0240] 5.2.2 Preparation of test solution:

[0241] Take 0.5g of the freeze-dried powder of the standard decoction of Tripterygium wilfordii slices, put it in a stoppered conical flask, add 10ml of pure ethyl acetate, weigh it, and perform ultrasonic extraction (power 500W, frequency 40kHz) for 30 minutes, make up the missing weight with ethyl acetate, centrifuge for 10 minutes (8000r / min), take the supernatant, pass it through a pretreated neutral alumina column (100-200 mesh; 2g; inner diameter 10mm; the pretreatment is elution with 30ml of ethyl acetate), elute with 30ml of ethyl acetate (containing 0.2% v / v formic acid), take the eluate, spin dry it, add 1ml of methanol to dissolve it, filter it, take the filtrate to obtain the test solution.

[0242] 5.3 Determination method

[0243] Accurately pipette 1 μL of the test solution and reference solution respectively, inject into the liquid chromatograph, measure according to the chromatographic conditions under 5.1, and record the chromatogram, such as Fig.64 The chromatograms shown in FIG. 1 and the system suitability parameters shown in Table 12 were obtained.

[0244] Table 12 System suitability results

[0245]

[0246] 6. Validation of analytical methods

[0247] 6.1 Precision

[0248] 6.1.1 Instrument precision test

[0249] Take the same portion of the test solution of standard decoction of Tripterygium wilfordii slices, prepare the test sample according to the method of “Preparation of test sample solution” in 5. Chromatographic conditions and system suitability conditions, inject the sample 6 times continuously, determine the relative retention time and relative peak area of ​​each characteristic peak according to the chromatographic conditions in 5. Chromatographic conditions and system suitability conditions, and calculate the RSD results of the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 13-14.

[0250] Table 13 Instrument precision relative retention time results

[0251]

[0252] Table 14 Instrument precision relative peak area results

[0253]

[0254]

[0255] It can be seen from Tables 13-14 that the relative retention time RSD of each characteristic peak is less than 2%, and the relative peak area RSD is less than 5.2%, indicating that the precision of the instrument is good.

[0256] 6.1.2 Method repeatability test

[0257] Take the same portion of standard decoction of Tripterygium wilfordii slices and prepare 6 portions repeatedly according to the method of "Preparation of test solution" under 5. Chromatographic conditions and system suitability conditions. Sample injection and analysis are performed. The relative retention time and relative peak area of ​​each characteristic peak are determined according to the chromatographic conditions under 5. Chromatographic conditions and system suitability conditions. The results are shown in Tables 15-16.

[0258] Table 15 Method repeatability relative retention time results

[0259]

[0260] Table 16 Method repeatability relative peak area results

[0261]

[0262]

[0263] It can be seen from Tables 15-16 that the relative retention time RSD of each characteristic peak is less than 2%, indicating that the method has good repeatability.

[0264] 6.1.3 Intermediate precision (different operators)

[0265] Three inspectors took two portions of the same standard decoction of Tripterygium wilfordii slices, prepared the test solution according to the "Preparation of test solution" in 5. Chromatographic conditions and system suitability conditions, injected and analyzed the samples according to the chromatographic conditions in 5. Chromatographic conditions and system suitability conditions, and used the same equipment to determine the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 17-18.

[0266] Table 17 Relative retention time results for different personnel

[0267]

[0268] Table 18 Relative peak area results of different personnel

[0269]

[0270]

[0271] It can be seen from Tables 17-18 that the RSD of the relative retention time of each characteristic peak is less than 2%, indicating that the intermediate precision of this method among different personnel is good.

[0272] 6.2 Exclusivity

[0273] The test sample was reconstituted with methanol as the solvent. 1 μL of the test sample solution and negative control solution (pure methanol) were accurately pipetted and injected into the ultra-high performance liquid chromatograph respectively. The test was performed according to the chromatographic conditions under 5.1. The results were as follows: Figure 65-66 The results showed negative without interference.

[0274] 6.3 Stability test

[0275] Take the same sample solution of standard decoction of Tripterygium wilfordii slices and inject it at 0, 4, 8, 12, 16 and 24 h according to the chromatographic conditions in 5.1. Determine the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 19-20.

[0276] Table 19 Relative retention time results of stability test

[0277]

[0278]

[0279] Table 20 Relative peak area results of stability test

[0280]

[0281] It can be seen from Tables 19-20 that the relative retention time RSD of each characteristic peak is less than 2%, indicating that the test solution is stable within 24 hours and meets the measurement requirements.

[0282] 6.4 Durability test

[0283] 6.4.1 Investigation of different flow rates

[0284] The same sample solution of the standard decoction of Tripterygium wilfordii slices was taken and measured according to the chromatographic conditions under 5.1. The only difference was that different flow rates (0.25 mL / min, 0.3 mL / min, and 0.35 mL / min) were used for the measurements. The results are shown in Tables 21-22 and Figures 67-69 .

[0285] Table 21 Relative retention time results at different flow rates

[0286]

[0287]

[0288] Table 22 Relative peak area results at different flow rates

[0289]

[0290] From Table 21-22 and Figures 67-69 It can be seen that different flow rates have a certain influence on the relative retention time and relative peak area of ​​each characteristic peak, so it is recommended to select 0.3mL / min as the flow rate for the characteristic spectrum determination method of the standard decoction of Tripterygium wilfordii slices.

[0291] 6.4.2 Investigation of different column temperatures

[0292] Take the same sample solution of the standard decoction of Tripterygium wilfordii slices and measure it at 20℃, 25℃ and 30℃ respectively according to the chromatographic conditions in 5.1. The results are shown in Tables 23-24 and Figure 70-72 .

[0293] Table 1 Relative retention time results at different column temperatures

[0294]

[0295] Table 24 Relative retention time results at different column temperatures

[0296]

[0297]

[0298] From Table 23-24 and Figure 70-72 It can be seen that different column temperatures have a certain influence on the relative peak area of ​​the characteristic peak, so it is recommended to fix the column temperature at 25℃.

[0299] Among the 9 common peaks of the characteristic spectrum of the standard decoction of Tripterygium wilfordii slices established based on the above methodological investigation results, triptolide is the S1 peak and triptolide is the S2 peak. The relative retention time is calculated. Each chromatographic peak is affected to a certain extent by column temperature and flow rate factors. In order to adapt to its durability, it is recommended to control the specified value range within ±10%; and it is recommended to fix the chromatographic column Waters CORTECS UPLC Shield RP (2.1x100mm, 1.6μm).

[0300] The characteristic spectrum of the test sample of the standard decoction of Tripterygium wilfordii slices (lyophilized powder) should show 9 characteristic peaks, among which the retention time of peak 1 should correspond to the retention time of the reference peak of the reference substance of triptolide, and the retention time of peak 7 should correspond to the retention time of the reference peak of the reference substance of triptolide. The peak corresponding to triptolide is the S1 peak, and the relative retention time of peaks 2 to 3 and S1 is calculated. The peak corresponding to triptolide is the S2 peak, and the relative retention time of peaks 4 to 9 and S2 is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified values ​​are: 1.15 (peak 2), 1.68 (peak 3), 0.63 (peak 4), 0.66 (peak 5), 0.78 (peak 6), 1.11 (peak 8), 1.20 (peak 9).

[0301] Verification based on the above analytical method showed that the method was simple to operate, had accurate results and good reproducibility.

[0302] Example 2 Construction of characteristic spectra of multiple batches of standard decoctions of Tripterygium wilfordii slices

[0303] According to the determination method of the characteristic spectrum determined in Example 1, 19 batches of standard decoctions of Tripterygium wilfordii slices were determined by this method. The results are shown in Tables 25-26.

[0304] Table 2519 batches of Tripterygium wilfordii slices standard decoction relative retention time results

[0305]

[0306] Table 26 Relative peak area results of 19 batches of standard decoctions of Tripterygium wilfordii slices

[0307]

[0308]

[0309] The results showed that the relative retention time of each characteristic peak of 19 batches of standard decoctions of Tripterygium wilfordii slices were all within ±10% of the specified value, meeting the requirements.

[0310] Example 3 Establishment of the control characteristic spectrum

[0311] 19 batches of standard decoction of Tripterygium wilfordii slices (20231207Y, 2023121101Y, 2023121102Y, 20231226Y, 20240129Y, 20240202Y, 2024022701Y, 2024022702Y, 2024022703Y, 2024022704Y, 2024022705Y, 2024022706Y, 2024022707Y, 2024022708Y, 2024022709Y, 2024022710Y, 2024041201Y, 2024041202Y, 2024041203Y), detected according to the characteristic spectrum determination method of the standard decoction of Tripterygium wilfordii slices provided in Example 1, and the results are as shown in Example 2.

[0312] Results: The characteristic spectra of 19 batches of standard decoctions of Tripterygium wilfordii slices were fitted with the fingerprint similarity evaluation software "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee to generate reference characteristic spectra. Through the identification of characteristic peaks by UPLC, 9 common characteristic peaks with more appropriate response values, better separation and higher purity were obtained. The chromatographic peak numbers were rearranged as peak 1-peak 9 according to the order of chromatographic peaks. Fig.73 Peak 1 was determined to be triptolide, peak 2 was syringaresinol, peak 6 was Peritasine A, peak 7 was triptolide, peak 8 was euphorbia pine, and peak 9 was triptolide.

[0313] Tripterygium wilfordii includes alkaloids, diterpene lactones and other substances. Among them, alkaloids represented by triptolide have the highest content and certain biological activity; diterpene lactones are the most active chemical components and are prone to cause poisoning, while triptolide is the most active epoxy diterpene lactone compound in triptolide and is the main component causing toxic side effects. The same method can determine the main active ingredients and main substances in the standard decoction of triptolide slices. Therefore, triptolide was selected as the S1 peak and triptolide as the S2 peak, and the relative retention time of each characteristic peak was calculated.

[0314] Depend on Fig.73It can be seen that there should be 9 characteristic peaks in the chromatogram of the test sample, among which the retention time of peak 1 and peak 7 should be the same as the retention time of the peak in the chromatogram of the corresponding reference substance; Peak 1 corresponding to the peak of the reference substance of triptolide is taken as the S1 peak, and the relative retention time of peaks 2-3 and S1 is calculated; Peak 7 corresponding to the peak of the reference substance of triptolide is taken as the S2 peak, and the relative retention time of peaks 4-9 and S1 is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified value is 1.15 (peak 2), 1.68 (peak 3), 0.63 (peak 4), 0.66 (peak 5), 0.78 (peak 6), 1.11 (peak 8), 1.20 (peak 9). Peak 1 is triptolide, peak 2 is syringinol, peak 6 is Per itass i ne A, peak 7 is triptolide, peak 8 is eucomycin, and peak 9 is triptolide.

[0315] Example 4 Construction of UPLC characteristic spectrum of Tripterygium wilfordii medicinal material

[0316] 19 batches of Tripterygium wilfordii medicinal materials (batch numbers: 20231207, 2023121101, 2023121102, 20231226, 20240129, 20240202, 2024022701, 2024022702, 2024022703, 2024022704, 2024022705, 2024022706, 2024022707, 2024022708, 2024022709, 2024022710, 2024041201, 2024041202, 2024041203) were detected by the characteristic spectrum determination method of the standard decoction of Tripterygium wilfordii slices of Example 1. The results are shown in Tables 27-28 and Fig.74 From Table 27-28 and Fig.74 It can be seen that the relative retention times of the characteristic peaks of the characteristic spectra of 19 batches of Tripterygium wilfordii medicinal materials are all within the specified value range.

[0317] Table 27 Relative retention time results of characteristic spectra of 19 batches of Tripterygium wilfordii

[0318]

[0319]

[0320] Table 18 Results of relative peak area determination of characteristic spectra of 19 batches of Tripterygium wilfordii medicinal materials

[0321]

[0322]

[0323] Example 5 Study on the characteristic spectrum transfer relationship of Tripterygium wilfordii medicinal materials, Tripterygium wilfordii slices and standard decoction of Tripterygium wilfordii slices

[0324] According to the 19 batches of Tripterygium wilfordii medicinal materials, 19 batches of Tripterygium wilfordii decoction pieces, and 19 batches of Tripterygium wilfordii decoction pieces standard decoction freeze-dried powder selected for the study, the determination was carried out according to their respective characteristic spectrum methods. The results are shown in Table 29 and Fig.75 As shown. The number of characteristic peaks obtained is consistent, all of which are 9 characteristic peaks, indicating that the main chemical components of Tripterygium wilfordii have not changed during the processing of medicinal materials into Tripterygium wilfordii slices and during the water decoction process, and the material basis is consistent; the relative retention time specified for each characteristic peak is within the range of ±10% of the specified value of the reference spectrum of the standard decoction of Tripterygium wilfordii slices, and the relative retention time of each characteristic peak is corresponding, indicating that the material basis of Tripterygium wilfordii slices has not changed after the water decoction process, and the quantity transfer relationship is good. It is recommended to stipulate that the relative retention time of the overall characteristic peak information should be used as a distinguishing evaluation indicator for the characteristic quantity transfer of the standard decoction of Tripterygium wilfordii slices.

[0325] Table 29 Comparison of relative retention time of medicinal materials, decoction pieces and standard decoctions

[0326]

[0327] Example 6 Construction of UPLC characteristic spectra of multiple batches of Tripterygium wilfordii granules

[0328] Three batches of Tripterygium wilfordii granules (batch numbers: 2406001Y, 2406002Y, 2406003Y) were taken and tested according to the characteristic spectrum determination method of the standard decoction of Tripterygium wilfordii slices in Example 1. The results are shown in Tables 30-31 and Fig.76 From Table 30-31 and Fig.76 It can be seen that the relative retention time specified for each characteristic peak of the characteristic spectra of the three batches of Tripterygium wilfordii formula granules is within the specified value range.

[0329] Table 30 Relative retention time results of characteristic spectra of three batches of Tripterygium wilfordii granules

[0330]

[0331] Table 31 Relative peak area results of characteristic spectra of three batches of Tripterygium wilfordii granules

[0332]

[0333] Example 7 Method for Detecting the Content of Triptolide in Batches of Tripterygium Wilfordii Granules

[0334] Three batches of Tripterygium wilfordii formula granules (batch numbers: 2406001Y, 2406002Y, 2406003Y) were taken and tested according to the characteristic spectrum determination method of the standard decoction of Tripterygium wilfordii slices in Example 1, and the content of triptolide was calculated. The results are shown in Table 32. As can be seen from Table 32, this method is simple and easy to operate, and has the advantages of good separation effect, high sensitivity, good repeatability, etc., and therefore can be used for the determination of triptolide in Tripterygium wilfordii formula granules.

[0335] Table 32 Determination results of triptolide content in 3 batches of Tripterygium wilfordii formula granules

[0336]

[0337] Obviously, the above experimental examples are only examples for clear explanation, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A method for constructing a characteristic spectrum of Tripterygium wilfordii or its derivatives, characterized in that: The method includes ultra-high performance liquid chromatography detection, and the chromatographic conditions include: Octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, and aqueous solution containing phosphoric acid was used as mobile phase B. The gradient elution procedure included: 0→6min, the volume percentage of mobile phase A is: 22%→25%, the volume percentage of mobile phase B is: 78%→75%; 6min→10min, the volume percentage of mobile phase A is: 25%→31%, the volume percentage of mobile phase B is: 75%→69%; 10min→15min, the volume percentage of mobile phase A is: 31%→33%, the volume percentage of mobile phase B is: 69%→67%; 15min→24min, the volume percentage of mobile phase A is: 33%→40%, the volume percentage of mobile phase B is: 67%→60%; 24min→30min, the volume percentage of mobile phase A is: 40%→42%, the volume percentage of mobile phase B is: 60%→58%; 30min→36min, the volume percentage of mobile phase A is: 42%→90%, and the volume percentage of mobile phase B is: 58%→10%.

2. The construction method according to claim 1, characterized in that: The chromatographic conditions include at least one of the following: (1) The detection wavelength is 210-270nm; optionally, 220nm; (2) The flow rate is 0.2-0.4 ml / min; optionally, the flow rate is 0.3 ml / min; (3) Column temperature 20-30°C; optionally, column temperature 25°C; (4) The injection volume is 0.5-2 μL; optionally, the injection volume is 1 μL; (5) The phosphoric acid concentration in the aqueous solution containing phosphoric acid in mobile phase B is 0.015-0.1% v / v; optionally, the phosphoric acid concentration is 0.05% v / v.

3. The construction method according to claim 1 or 2, characterized in that: It also includes the preparation of test solution, including: 1) Weigh the Tripterygium wilfordii sample, add extraction solvent to extract, and obtain an extract; 2) separating the extract from solid and liquid, taking the supernatant and eluting it with an elution solvent to obtain an eluate; 3) The eluate is spin-dried, and a redissolving solvent is added to dissolve the eluate, and the solid-liquid separation is performed. The liquid is taken to obtain the test solution.

4. The construction method according to claim 3, characterized in that: The preparation of the test solution satisfies at least one of the following: A. The ratio of the mass of the Tripterygium wilfordii test sample to the volume of the solvent is (0.5-2):(10-50); the mass to volume ratio is g / mL; B. The extraction method is ultrasonic extraction or reflux extraction; C. Extraction time is ≥30 min, preferably 30-60 min; D. The solid-liquid separation is independently selected from centrifugation or filtration; E. The solvent is selected from one or more of ethyl acetate and methanol; preferably, the extraction solvent is ethyl acetate; preferably, the elution solvent is ethyl acetate containing 0.2% v / v formic acid; preferably, the re-dissolution solvent is methanol; F. The elution is performed by column chromatography; preferably, the column chromatography is neutral alumina column chromatography; more preferably, the neutral alumina column is pretreated with ethyl acetate.

5. The construction method according to any one of claims 1 to 4, characterized in that: The construction method further comprises the steps of preparing a reference solution by using at least one solubilizing agent selected from triptolide and triptolide, and obtaining a reference spectrum of the reference solution by detecting the reference solution using ultra-high performance liquid chromatography in the construction method; Preferably, each 1 mL of the triptolide reference solution contains 5-20 μg of triptolide; preferably, 10 μg; Preferably, each 1 mL of triptolide reference solution contains 5-20 μg of triptolide; preferably, 10 μg; Preferably, the solvent used in the preparation of the reference solution is selected from methanol or a methanol aqueous solution with a volume fraction of not less than 70%.

6. The construction method according to any one of claims 1 to 5, characterized in that: The tripterygium wilfordii or its derivative products include one or more of tripterygium wilfordii medicinal materials, tripterygium wilfordii decoction pieces or tripterygium wilfordii preparations.

7. The construction method according to claim 6, characterized in that: The tripterygium wilfordii preparations include standard decoction of tripterygium wilfordii slices and tripterygium wilfordii formula granules.

8. A method for detecting the quality of Tripterygium wilfordii or its derivative products, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the product to be tested with the control characteristic spectrum of Tripterygium wilfordii or its derivative product; The characteristic spectrum of the product to be tested is obtained according to the construction method described in any one of claims 1 to 7; The reference characteristic spectrum of Tripterygium wilfordii or its derivative product is selected from any one of the following (1)-(4): (1) It has 9 common characteristic peaks, the peak corresponding to the reference peak of the triptolide reference substance is the S1 peak, the relative retention times of peaks 2-3 and S1 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 2-3 are 1.15 and 1.68, respectively; the peak corresponding to the reference peak of the triptolide reference substance is the S2 peak, the relative retention times of peaks 4-6 and peaks 8-9 and S2 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 4-6 and peaks 8-9 are 0.63, 0.66, 0.78, 1.11 and 1.20, respectively; (2) It has 9 common characteristic peaks, of which 2 peaks correspond to the retention times of reference peaks of triptolide and triptolide reference substances, respectively; the peak corresponding to the reference peak of triptolide reference substance is S1 peak; the relative retention times of peaks 2-3 and S1 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 2-3 are 1.15 and 1.68, respectively; peak 7 is S2 peak; the relative retention times of peaks 4-6 and 8-9 and S2 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 4-6 and 8-9 are 0.63, 0.66, 0.78, 1.11 and 1.20, respectively; (3) a characteristic spectrum of Tripterygium wilfordii and / or its preparation obtained by the construction method according to any one of claims 1 to 7 using a single batch or multiple batches of Tripterygium wilfordii and / or its derivatives as test samples; (4) Using multiple batches of Tripterygium wilfordii and / or its derivatives as test samples, the characteristic spectra obtained according to the construction method of any one of claims 1 to 7 are used to prepare a control characteristic spectrum by averaging or median.

9. The quality detection method of Tripterygium wilfordii or its derivative products according to claim 8, characterized in that: The peak 1 corresponds to triptolide; the peak 2 corresponds to syringaresinol; the peak 6 corresponds to peritassine A; the peak 7 corresponds to triptolide; the peak 8 corresponds to euonymus serrata; and the peak 9 corresponds to triptolide.

10. A method for determining the content of triptolide in Tripterygium wilfordii or its derivative products, characterized in that: The method comprises obtaining a characteristic spectrum of the tested Tripterygium wilfordii product according to the construction method of any one of claims 1 to 7; Reference substances included triptolide and triptolide.

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