Preparation method of a triptolide sesquiterpene alkaloid purity standard sample

Abstract

The application discloses a preparation method of a triptolide sesquiterpene alkaloid purity standard sample, and belongs to the technical field of natural product separation and purification and standard substance preparation. The method comprises the following steps: obtaining total alkaloid crude products through ethanol extraction and acid-dissolution alkalinization extraction, enriching the total alkaloid crude products through methanol grading, pre-separating the total alkaloid crude products through a specific two-phase system of petroleum ether (60-90 DEG C)-ethyl acetate-isopropyl alcohol-water HSCCC, and finally obtaining monomer products with purity greater than or equal to 99.0% through reverse-phase preparation HPLC refining and low-temperature multiple recrystallization. The obtained standard sample has water content less than or equal to 0.5%, residual solvents meet the requirements of ICH Q3C and the Chinese Pharmacopoeia, and the impurity spectrum fingerprint similarity is greater than or equal to 0.98 under different raw material batches and amplification conditions, so that the batch consistency, traceability and storage stability are significantly improved, and the strict requirements of standard substances in quality control and analytical detection are met.

Description

Technical Field

[0001] This invention belongs to the field of natural product separation and purification and standard substance preparation technology, and particularly relates to a method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpene alkaloids. Background Technology

[0002] Sesquiterpenoid alkaloids derived from Tripterygium wilfordii (such as triptolide, triptolide, triptolide B, and triptolide II) have important applications in the quality control, pharmacokinetic and metabolic studies, clinical testing, proficiency testing, and methodological evaluation of traditional Chinese medicine and its preparations. These applications often require high-purity, well-defined, and traceable purity standard samples to support qualitative and quantitative analysis, impurity profile comparison, and cross-laboratory consistency. Because these alkaloids are complex natural products with similar structures, polarities, and multiple functional groups, and are prone to co-elution, problems such as purity fluctuations, impurity profile drift, and unstable yields can easily occur during the separation and purification process.

[0003] In existing technologies, the preparation of Tripterygium wilfordii sesquiterpenoid alkaloids is mostly aimed at obtaining research-grade monomers, often using acid-base extraction followed by a single HSCCC or semi-preparative HPLC separation. Although CN100422188C discloses methanol treatment before HSCCC to obtain monomers with a purity of approximately 95%, and CN101974005A also discloses obtaining several monomers with a content >99% through recrystallization and semi-preparative chromatography, the above methods generally do not establish quantifiable key quality attributes and closed-loop control (such as batch-to-batch similarity of impurity fingerprints, residual solvent / water content, dispensing, sealing, and storage conditions) for the purpose of "purity standard samples." Furthermore, there is insufficient constraint on the retention value of the stationary phase, slicing rules, and process fluctuation range under different raw material sources or scale-up preparation conditions, resulting in easy fluctuations in purity thresholds and impurity profiles.

[0004] In summary, although existing technologies can obtain monomers with high purity, they still have the following shortcomings: ① Lack of closed-loop control of "process-quality attributes" for purity standard samples; ② Under conditions of batch-to-batch variation / scale-up, the retention values ​​and peak resolution of the countercurrent chromatography stationary phase are prone to fluctuation, leading to segmentation errors and fluctuations in unknown impurities; ③ No technical solution was clearly proposed to improve the consistency of impurity profiles by means of key solvent system selection (such as isopropanol replacing ethanol) and methanol fractionation enrichment.

[0005] Therefore, there is an urgent need to provide a set of purification process systems for the preparation of purity standard samples. This system can simultaneously cover the acquisition of four target monomers under scalable conditions, and can improve process reproducibility and batch-to-batch consistency by defining the pH window for acid-base extraction, solvent system ratio, chromatographic separation and segmentation rules, recrystallization times and final temperature, as well as dispensing, sealing and quality index control, thereby improving the feasibility of subsequent practical applications. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a method for preparing purity standard samples of Tripterygium wilfordii sesquiterpenoid alkaloids. Specifically, this invention employs a combined route of acid-base extraction to obtain total alkaloids, methanol fractionation and enrichment, HSCCC pre-separation, preparative HPLC purification, and multiple recrystallization for finalization / dispensing, achieving controllable purity and impurity profiles, as well as batch-to-batch consistency.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids includes the following steps: (1) Extraction and preparation of total alkaloids: The root and / or stem powder of Tripterygium wilfordii was extracted by reflux with ethanol, the ethanol was recovered and cooled to obtain an extract; the extract was dissolved in an acidic solution and filtered, the pH was adjusted to 8.5-10.0 and then extracted with an organic solvent, concentrated under reduced pressure and dried to obtain crude total Tripterygium wilfordii alkaloids; (2) Methanol fractionation: The crude total alkaloids were added to methanol, stirred to dissolve, allowed to stand and filtered, and the methanol-insoluble matter was collected. (3) HSCCC pre-separation: The methanol-insoluble matter was separated by high-speed countercurrent chromatography in a two-phase solvent system of petroleum ether (60-90℃)-ethyl acetate-isopropanol-water and collected in fractions to obtain crude fractions of each component containing the target alkaloid; (4) HPLC purification: The crude fractions of each component are purified by reverse-phase C18 chromatographic column, isocratic elution is performed, the target peak is collected, and the purified solution of the corresponding monomer is obtained; (5) Resizing: After evaporating the solvent from the refined liquid, recrystallize it with hot methanol and then vacuum dry it to constant weight; (6) Separate packaging and sealing: Separate packaging under light-proof and low-humidity conditions and then seal after replacement with inert gas.

[0008] Beneficial Effects: This invention provides a method for preparing purity standard samples of Tripterygium wilfordii sesquiterpenoid alkaloids, used to obtain high-purity monomeric compounds of Tripterygium wilfordii alkaloids, Tripterygium wilfordii alkaloids, Tripterygium wilfordii alkaloid ethyl alkaloid, and / or Tripterygium wilfordii alkaloid, while meeting the requirements of controllable impurity profiles and batch-to-batch consistency for purity standard samples. The key lies in achieving simultaneous purity improvement and impurity fingerprint stability under scale-up conditions through methanol fractional enrichment + HSCCC pre-separation in a specific two-phase system + HPLC purification using a formic acid-containing reverse-phase preparation + low-temperature multiple recrystallization. The method includes: ethanol reflux extraction to obtain an extract; dissolving and filtering with an acidic solution, adjusting the pH to 8.5–10.0 with concentrated ammonia, and extracting with an organic solvent (such as dichloromethane) to obtain crude total alkaloids; fractionating the total alkaloids with methanol to obtain a methanol-insoluble enriched fraction; performing HSCCC pre-separation and segmenting according to the target peak in a petroleum ether (60–90 °C)–ethyl acetate–isopropanol–water = 6:4:5:8 two-phase system; subsequently, isocratic purification using ODS column preparative liquid chromatography with acetonitrile / water = 60:40, and recrystallizing with hot methanol 2–4 times to obtain a monomer product with a purity ≥99.0%, and a water content ≤0.5%, with residual solvents meeting the limits of ICH Q3C (or the Chinese Pharmacopoeia / related guidelines); finally, dispensing in the dark and sealing with inert gas.

[0009] In summary, this invention provides evaluation principles for impurity spectrum fingerprint similarity and process fluctuation ranges for scale-up preparation, which are used to ensure batch-to-batch consistency and traceability of purity standard samples.

[0010] Optionally, in step (1), the ethanol reflux extraction process is repeated 1 to 2 times.

[0011] Optionally, in step (1), the acidic solution is citric acid or formic acid, preferably citric acid, and is filtered using a filter membrane after dissolution.

[0012] Optionally, in step (1), the organic solvent is dichloromethane or a mixture of dichloromethane and isopropanol, and the extraction is performed 2 to 4 times. Dichloromethane is preferred.

[0013] Optionally, in step (2), the ratio of the crude total alkaloids to methanol is 1:(100-200) (g:mL).

[0014] Beneficial effects: This invention sets up "methanol fractionation" before countercurrent chromatography and uses the methanol-insoluble enriched portion as the HSCCC sample. This setting can reduce easily co-elutable polar impurities and resin / pigment interference in the sample, reduce the HSCCC load, and avoid peak tailing and segment drift. Compared with Comparative Example 2 (methanol fractionation omitted), the HSCCC stationary phase retention value increased from 50-70% to 70-90%, peak shape improved, segment error reduced, unknown impurities and total impurities significantly reduced, and the similarity of impurity spectral fingerprints improved (Table 4).

[0015] Optionally, in step (3), the volume ratio of petroleum ether (60-90°C)-ethyl acetate-isopropanol-water is 6:4:5:8.

[0016] Beneficial Effects: This invention limits the "petroleum ether (60~90℃)-ethyl acetate-isopropanol-water" system in the HSCCC two-phase system. This maintains sufficient separation and stationary phase retention values ​​under batch-to-batch variations and scale-up conditions, reducing fluctuations in co-eluted impurities. Furthermore, compared to Comparative Example 3 (isopropanol replaced by ethanol), the stationary phase retention values ​​defined by this invention are higher (70~90% vs 55~75%), the impurity fingerprint similarity is higher (≥0.98 vs 0.91~0.96), and both single unknown impurities and total impurities are lower (Table 4). This effect is not only reflected in improved purity but also in the improvement of the key quality attribute of "impurity profile consistency" in standard samples.

[0017] Furthermore, the conditions for the high-speed countercurrent chromatography separation are: rotation speed of 450–550 rpm (stationary phase is used), flow rate of 4–6 mL / min, and column temperature of 23–27℃.

[0018] Optionally, in step (4), isocratic elution is performed by acetonitrile / water, wherein the water contains 0.05% to 0.2% formic acid.

[0019] Beneficial effects: The HSCCC crude fraction is further purified by HPLC using acetonitrile / water (containing 0.05%–0.2% formic acid), combined with low-temperature multiple methanol recrystallization for sizing and low-humidity inert gas dispensing and sealing. This process allows for the acquisition of high-purity monomeric compounds while suppressing trace impurities from co-elution and stabilizing the impurity spectrum, meeting the requirements of purity standard samples for batch-to-batch consistency, residual solvent / water content, and storage stability. Compared to Comparative Example 1 (where the extraction acid / extraction solvent was replaced with acetic acid / chloroform), this invention demonstrates superior performance in terms of main peak purity, unknown impurities, and fingerprint similarity (Table 4), indicating that the above process combination is not an arbitrary combination but rather a synergistic control system formed around the intended use of the standard samples.

[0020] Optionally, in step (5), hot methanol is recrystallized 2 to 4 times, and the final temperature is controlled at 0 to 10°C to improve purity and stability.

[0021] A purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids obtained by the above preparation method contains at least one monomeric component selected from Tripterygium wilfordii aureine, Tripterygium wilfordii alkaloid, Tripterygium wilfordii alkaloid and / or Tripterygium wilfordii alkaloid, and meets the requirements of purity ≥99.0%, moisture ≤0.5%, and residual solvent meeting the requirements of ICH Q3C and the relevant guidelines of the Chinese Pharmacopoeia.

[0022] Beneficial effects: This invention utilizes a combined process window for standard samples, namely "methanol fractionation and enrichment + isopropanol two-phase system HSCCC + formic acid-containing preparation HPLC + low-temperature multiple recrystallization + low-humidity inert gas dispensing". It can achieve an impurity fingerprint similarity of ≥0.98 among three batches of samples and control a single unknown impurity to ≤0.10%.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects: This invention establishes quantifiable key quality attributes (purity, impurity fingerprint similarity, moisture content, residual solvent, dispensing, sealing, and storage conditions) around the intended use of standard samples, and provides reproducible process windows, thereby improving batch-to-batch consistency and traceability. Specifically: (1) Improve the reproducibility of total alkaloid recovery by acid-solubilization-alkalization pH window and multiple extractions with dichloromethane; (2) Methanol fractionation is used to obtain the insoluble and enriched portion of methanol, which reduces the burden of subsequent HSCCC and stabilizes the cutting process; (3) HSCCC is used for pre-separation, and HPLC is used for purification and supplemented with 2 to 4 recrystallizations (final temperature 0 to 10℃) to obtain monomers with a purity ≥99.0% and meet the purity standard sample requirements; (4) The process parameters and allowable fluctuation range are clearly defined, which facilitates the scale-up of ≥500 g raw materials and maintains batch-to-batch consistency.

[0024] (5) By recrystallization and low-humidity inert gas packaging and sealing, the levels of moisture and residual solvents can be significantly reduced and the impurity fingerprint can be stabilized, thereby improving the batch-to-batch consistency and storage stability of purity standard samples.

[0025] In summary, this invention achieves unexpected technical results through a combination of methanol fractionation and enrichment, HSCCC pre-separation in a specific two-phase system, HPLC purification with formic acid reverse-phase preparation, and low-temperature multiple recrystallization and sizing. While increasing the purity to ≥99.0%, the impurity fingerprint still maintains high similarity (≥0.98) under different raw material batches and scale-up conditions, which is significantly better than comparative processes that lack fractionation / solvent system substitution. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The molecular structure and C position diagram of triptolide; Figure 2 The infrared spectrum of triptolide; Figure 3 High-resolution mass spectrum of triptolide; Figure 4 The molecular structure and C-position diagram of Tripterygium wilfordii alkaloids; Figure 5 Infrared spectrum for determining the alkaloids of Tripterygium wilfordii; Figure 6 High-resolution mass spectrum for determining the alkaloids in Tripterygium wilfordii; Figure 7 The molecular structure formula and C position diagram of triptolide B; Figure 8 The infrared spectrum of triptolide B; Figure 9 The high-resolution mass spectrum of triptolide B; Figure 10 The molecular structure and C position diagram of Tripterygium wilfordii alkaloids; Figure 11 The infrared spectrum of triptolide; Figure 12 This is a high-resolution mass spectrum of Tripterygium wilfordii alkaloids. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] This invention discloses a method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids, used to prepare at least one high-purity monomeric compound selected from Tripterygium wilfordii aureine, Tripterygium wilfordii alkaloid, Tripterygium wilfordii alkaloid B, and Tripterygium wilfordii alkaloid C. The method includes the following steps: (1) Extraction and preparation of total alkaloids: After drying, crushing and sieving the roots and / or stems of Tripterygium wilfordii, extract them by reflux with 95 vol% ethanol at a solid-liquid ratio of 1:(8-12) (g:mL) for 2-3 hours to obtain the extract; recover the ethanol and cool to obtain the extract; dissolve the extract with 3%-8% acidic solution and filter to obtain the acidic filtrate; add concentrated ammonia to the acidic filtrate to adjust the pH to 8.5-10.0; extract with organic solvent and combine the organic phases, concentrate and dry under reduced pressure to obtain crude total Tripterygium wilfordii alkaloids; (2) Methanol fractionation: The crude total Tripterygium alkaloids were added to methanol, stirred and dissolved, allowed to stand and filtered, and the methanol-insoluble matter was collected as HSCCC sample. (3) HSCCC pre-separation: The sample was separated by high-speed countercurrent chromatography in a two-phase solvent system of petroleum ether (60-90℃)-ethyl acetate-isopropanol-water in a volume ratio of 6:4:5:8 and collected in fractions to obtain crude fractions of each component containing the target alkaloid. (4) HPLC purification: The crude fractions of each component were purified using a preparative reversed-phase C18 column, eluted isocratically with acetonitrile / water = 60:40 (v / v), and the target peaks were collected to obtain the purified solution of the corresponding monomers; (5) Fixation: After evaporating the solvent from the purified liquid, recrystallize it 2 to 4 times with hot methanol. The final recrystallization temperature is controlled at 0 to 10°C. Then, vacuum dry it to constant weight. (6) Separate packaging and sealing: Separate packaging under light-proof and low-humidity conditions and then seal after replacement with inert gas.

[0033] In some alternative embodiments, the ethanol reflux extraction in step (1) is repeated 1 to 2 times, and the extracts are combined and concentrated under reduced pressure at ≤50°C.

[0034] In some optional embodiments, the acidic dissolving solution in step (1) is a citric acid aqueous solution with a mass fraction of 3% to 8% or a formic acid aqueous solution with a volume fraction of 0.5% to 2.0%; preferably a 5% citric acid aqueous solution or a formic acid aqueous solution with a volume fraction of 0.5% to 2.0%; after dissolution, it is filtered through a 0.22 to 0.45 μm filter membrane.

[0035] In some optional embodiments, the organic solvent used for extraction after alkalization in step (1) is dichloromethane, or a mixed solvent of dichloromethane and isopropanol (volume ratio 95:5 to 85:15); the number of extractions is 2 to 4, and the extraction amount per extraction is 1.5 to 2.5 mL / g based on the mass of the raw material.

[0036] In some alternative embodiments, the volume ratio of crude total alkaloids to methanol in step (2) is 1:(100-200) (g:mL) to obtain a methanol-insoluble alkaloid enrichment.

[0037] In some alternative embodiments, the settling conditions in step (2) are: settling at 0 to 10°C for 0.5 to 2 hours and then filtering, in order to improve the batch-to-batch consistency of the methanol-poorly enriched fraction.

[0038] In some optional embodiments, the rotation speed of HSCCC in step (3) is 450-550 rpm (the stationary phase is the upper phase), the flow rate is 4-6 mL / min, the column temperature is 23-27℃, and the stationary phase retention value is 70%-90%.

[0039] In some optional embodiments, in addition to the target peak start and end points, the segmented collection in step (3) can also be assisted by the following typical elution time windows under HSCCC conditions: Tripterygium wilfordii 110-120 min, Tripterygium wilfordii 135-165 min, Tripterygium wilfordii ethyl 155-195 min, Tripterygium wilfordii methyl 220-260 min, and allow process fluctuations of ±(3-8) min.

[0040] In some optional embodiments, the alcohol in the HSCCC two-phase solvent system in step (3) is isopropanol, and the two-phase system is prepared by petroleum ether (60-90℃)-ethyl acetate-isopropanol-water in a volume ratio of 6:4:5:8, or a volume ratio fluctuation of ±10% is allowed based on this ratio.

[0041] In some alternative embodiments, in step (4), purification is performed using an ODS (reverse-phase C18) column (150 mm × 20 mm, 5 μm) under isocratic elution conditions, and the following typical retention time windows can be used to assist collection: Tripterygium wilfordii 5–7 min, Tripterygium wilfordii 8–10 min, Tripterygium wilfordii ethyl 12–14 min, Tripterygium wilfordii sine 14–16 min.

[0042] In some alternative embodiments, the HPLC collection time window in step (4) is allowed to fluctuate by ±(0.3 to 1.0) min, and the peaks are merged after the purity of the target peaks is confirmed by analytical HPLC or LC-MS.

[0043] In some alternative embodiments, the aqueous phase of the HPLC prepared in step (4) contains 0.05wt% to 0.2wt% formic acid and / or 5 to 20 mM ammonium formate to improve peak shape and reduce co-elution impurities.

[0044] In some alternative embodiments, during the dispensing and sealing process in step (6), the relative humidity of the dispensing environment is controlled at ≤30%, and the container is stored at 2-8°C in the dark.

[0045] In some alternative embodiments, the method is applicable to scale-up preparations with raw material feed amounts ≥500 g, and the amounts of ethanol, citric acid solution and dichloromethane are scaled up linearly according to the solid-liquid ratio and extraction ratio.

[0046] In some alternative embodiments, during scale-up preparation, multiple loading or parallel HSCCC columns are used to maintain the ratio of single loading volume to column volume at 0.5–2.0 mg / mL.

[0047] In some optional embodiments, the analytical HPLC conditions used for evaluating the similarity of the impurity fingerprint include at least the following: a C18 column (4.6 mm × 250 mm, 5 μm) or an equivalent column, a column temperature of 30℃ ± 2℃, a flow rate of 1.0 mL / min, and a detection wavelength of 230 nm; mobile phase A is water (containing 0.1% formic acid), and mobile phase B is acetonitrile; the gradient program is 0-5 min 45%B, 5-20 min 45%-65%B, and 20-30 min 65%-80%B; and the correlation coefficient is calculated as the similarity based on the peak area vector of the main peak and at least 5 major impurity peaks.

[0048] In some alternative embodiments, the resulting monomer product meets the quality requirements of the purity standard sample: purity ≥99.0% as determined by analytical HPLC area normalization; total impurities ≤1.0%, single unknown impurity ≤0.10%; impurity fingerprint similarity ≥0.98 among at least 3 batches of samples; moisture ≤0.5%; residual solvents meet the more stringent limits of ICH Q3C and the Chinese Pharmacopoeia / related guidelines.

[0049] A standard sample comprising at least one monomeric component selected from triptolide, triptolide, triptolide B, and / or triptolide C, and prepared by the method described above.

[0050] In some alternative embodiments, the standard sample meets at least one of the following quality requirements: the purity of the main peak as determined by the analytical HPLC area normalization method is ≥99.0%; the total impurities are ≤1.0%; the single unknown impurity is ≤0.10%; and the impurity fingerprint similarity among at least 3 batches of samples is ≥0.98.

[0051] In some alternative embodiments, the standard sample has a moisture content of ≤0.5% and the residual solvent meets the more stringent limits of the relevant guidelines of ICH Q3C and the Chinese Pharmacopoeia.

[0052] In some alternative embodiments, the standard samples are aliquoted in an environment with a relative humidity of ≤30%, sealed after being replaced with an inert gas, and stored at 2–8°C in the dark.

[0053] All raw materials used in this invention are commercially available. In this specification, the term "purity standard sample" refers to a high-purity monomeric standard substance used for analytical testing, quality control, or methodological evaluation. It has a defined chemical structure, known purity, and traceable quality attributes, and can be used as a reference substance for qualitative or quantitative analysis.

[0054] The technical solution of the present invention will be further illustrated by the following embodiments.

[0055] Example 1: Preparation and purification of 50 g raw material (1) Sample preparation: After drying the roots and stems of Tripterygium wilfordii, chop and crush them, and pass them through a 40-mesh sieve for later use.

[0056] (2) Extraction and preparation of total alkaloids: Weigh 50 g of powder, add 500 mL of 95% ethanol and reflux for 2-3 hours; recover the ethanol and cool to obtain an extract; dissolve the extract with 5 wt.% citric acid and filter; add concentrated ammonia to the filtrate to adjust the pH to 8.5-10.0; add 100 mL of dichloromethane for extraction and repeat 3 times; combine the dichloromethane layers, concentrate under reduced pressure and dry to obtain about 1.5 g of crude total alkaloids.

[0057] (3) Methanol fractionation: Take 700 mg of crude total alkaloids, add 100 mL of methanol, stir to dissolve, filter, and keep the methanol-insoluble matter for later use.

[0058] (4) HSCCC pre-separation: using a system of petroleum ether (60~90℃)-ethyl acetate-isopropanol-water = 6∶4∶5∶8, with the upper phase as the stationary phase and the lower phase as the mobile phase, the column temperature was 25℃, the rotation speed was 500 rpm (the stationary phase was the upper phase), the flow rate was 5 mL / min, and the UV 254 nm was used for monitoring; each component was collected by cutting according to the start and end points of the target peak.

[0059] (5) Preparation HPLC purification: After each component is evaporated to dryness, it is dissolved in acetonitrile and prepared using an ODS (reversed-phase C18) (150×20 mm, 5 μm) or equivalent C18 column with acetonitrile / water (containing 0.1% formic acid) = 60:40 and column temperature 25℃; collected according to the target peak start and end points; after evaporating the solvent, recrystallized three times with hot methanol at a final temperature of 5℃ to obtain white crystals.

[0060] (6) Separate packaging and sealing: Separate packaging under light-proof and low-humidity conditions and then seal after replacement with inert gas.

[0061] Table 1 Key Separation Time Window in Example 1 Table 2. Example of material balance for 50 g scale Example 2: Scale-up preparation of ≥500 g raw materials and process fluctuation range A scale-up experiment was conducted based on Example 1: the amount of raw material was 500 g, and the amounts of ethanol, citric acid solution and dichloromethane were linearly scaled up according to the solid-liquid ratio and extraction ratio; the volume ratio of the HSCCC solvent system was kept constant at 6:4:5:8, and multiple loading or parallel column connection was used to maintain the loading amount and column volume ratio.

[0062] Table 3 Key parameters and allowable fluctuation range for scale-up preparation in Example 2 Comparative Example 1: Acetic acid dissolution-ammonia treatment-dichloromethane extraction (chloroform is not within the range of extraction solvents limited by this invention). The difference from Example 1 is that the 5% citric acid solution in step (2) is replaced with a 5% acetic acid solution; the rest of the preparation steps are the same as in Example 1.

[0063] The results showed that the analytical HPLC purity and impurity profile consistency of the obtained target monomer were inferior to those of Example 1 of the present invention.

[0064] Comparative Example 2: Step (2) Methanol fractionation (direct loading of crude total alkaloids onto HSCCC) is missing. The difference from Example 1 is that step (3) methanol fractionation is omitted, and the crude total alkaloids are directly dissolved in methanol and then loaded onto HSCCC.

[0065] The results showed that the stationary phase retention value decreased and the peak shape deteriorated, leading to increased slicing error and more co-eluting impurities.

[0066] Comparative Example 3: The HSCCC solvent system with isopropanol replaced by ethanol The difference from Example 1 is that isopropanol in the HSCCC two-phase system is replaced with ethanol, while keeping the volume ratio of other solvents unchanged.

[0067] The results showed that the target peak separation was reduced and the impurity spectrum fingerprint similarity decreased.

[0068] Comparative Example 4: Acid dissolution with hydrochloric acid, alkalization with ammonia, and extraction with dichloromethane were used (the acidic solution was replaced with hydrochloric acid to verify the range of possible acid solutions). The difference from Example 1 is that the citric acid solution in step (2) is replaced with a hydrochloric acid aqueous solution with a volume fraction of 0.1% to 2.0%, while the other steps remain the same.

[0069] Table 4 Comparison of results between comparative examples and embodiments of the present invention Table 4 shows that the "main peak purity / total impurities / single unknown impurities" are all calculated based on area normalization using the same analytical HPLC method. The "impurity fingerprint similarity" is evaluated using chromatographic fingerprints from the same analytical method (with the main peak and at least five major impurity peaks as evaluation objects, and correlation coefficients calculated using peak area vectors) to characterize batch-to-batch consistency. As shown in Table 4, this invention not only improves single-sample purity but also significantly improves the consistency between stationary phase retention values ​​and impurity spectra, thereby meeting the key quality attribute requirements of purity standard samples.

[0070] Example 3: Characterization data of the target monomer (for product verification in the example) The monomer product obtained in Example 1 was subjected to ultraviolet, infrared, high-resolution mass spectrometry (HRMS), optical rotation, and other measurements. 1 H NMR, 13 Characterization using C NMR and other methods was used to confirm the structural consistency between batches. For ease of verification, key NMR data (measured values ​​compared with literature values) are summarized in Tables 5-12, and UV, IR, high-resolution mass spectrometry (HRMS), and optical rotation test data are summarized in Table 13. 1H NMR and... 13 C NMR measurement conditions: A 400 MHz superconducting nuclear magnetic resonance spectrometer was used at 25 °C, with TMS as the internal standard and deuterated chloroform (CDCl3) as the solvent.

[0071] The characterization data above are representative results; those skilled in the art should understand that different instruments, temperatures, sample concentrations, and data processing methods may cause reasonable differences in chemical shifts and coupling constants (e.g., δ is allowed ±0.05 ppm, J is allowed ±0.5 Hz). If there is any discrepancy with the original spectrum, the original spectrum / original data shall prevail.

[0072] Table 5. Tripterygium wilfordii alkaloids 1 H NMR test data The solvent used in the references is deuterated chloroform.

[0073] Table 6. Tripterygium wilfordii alkaloids 13 C NMR test data Table 7. The alkaloid content of Tripterygium wilfordii. 1 H NMR test data The solvent used in the references may be deuterated chloroform.

[0074] Table 8. The alkaloid content of Tripterygium wilfordii. 13 C NMR test data Table 9. Tripterygium alkaloid B 1 H NMR test data The solvent used in the references may be deuterated chloroform.

[0075] Table 10 Tripterygium wilfordii alkaloid B 13 C NMR test data Table 11 Tripterygium alkaloids 1 H NMR test data The solvent used in the references is deuterated chloroform.

[0076] Table 12. ¹³C NMR data of triptolide Note: The literature values ​​in Tables 5-12 are from Xu Lihong, Miao Kangli, Huang Liying, Isolation and Identification of Tripterygium wilfordii Alkaloids, China Pharmacy, 1995, 6(4): 12-13; “ / ” indicates that no relevant data was obtained.

[0077] Table 13 Test data for ultraviolet, infrared, high-resolution mass spectrometry (HRMS), and optical rotation Note: The above characterization data are typical batch results, used to demonstrate the structure and purity of the products obtained in the examples.

[0078] Figure 1 The molecular structure and C position diagram of triptolide; Figure 2 The infrared spectrum of triptolide; Figure 3 High-resolution mass spectrum of triptolide; Figure 4 The molecular structure and C-position diagram of Tripterygium wilfordii alkaloids; Figure 5 Infrared spectrum for determining the alkaloids of Tripterygium wilfordii; Figure 6 High-resolution mass spectrum for determining the alkaloids in Tripterygium wilfordii; Figure 7 The molecular structure formula and C position diagram of triptolide B; Figure 8 The infrared spectrum of triptolide B; Figure 9 The high-resolution mass spectrum of triptolide B; Figure 10 The molecular structure and C position diagram of Tripterygium wilfordii alkaloids; Figure 11 The infrared spectrum of triptolide; Figure 12 This is a high-resolution mass spectrum of Tripterygium wilfordii alkaloids.

[0079] Recommended quality indicators: The purity of the final monomer product should be confirmed by HPLC (area normalized) ≥99.0%; impurity fingerprints should be established using analytical HPLC and / or LC-MS for batch-to-batch consistency evaluation (similarity ≥0.98 for at least 3 batches); moisture content should be ≤0.5% determined by Karl Fischer chromatography; residual solvents should be evaluated by headspace-gas chromatography for at least ethanol, acetonitrile, and dichloromethane, and the following limits should be met: ethanol ≤5000 ppm, acetonitrile ≤410 ppm, dichloromethane ≤600 ppm, and the more stringent limits specified in ICH Q3C and / or the Chinese Pharmacopoeia / related guidelines should be met; after repackaging, sealing and appearance checks should be performed to support the stability verification of the purity standard samples.

[0080] Analytical methods: A C18 column (e.g., 4.6 mm × 250 mm, 5 μm) can be used, with a column temperature of approximately 30 ℃, a flow rate of 1.0 mL / min, and a detection wavelength of 230 nm; the injection volume is 10 μL; mobile phase A is water (containing 0.1% formic acid), and mobile phase B is acetonitrile; gradient program: 0-5 min 45%B, 5-20 min 45%-65%B, 20-30 min 65%-80%B; the RRT and peak area vectors of the main peak and major impurity peaks are used for similarity calculation. LC-MS: Target peak and major impurity peak information can be acquired in ESI positive ion mode for identification and fingerprint comparison. Fingerprint similarity: The original chromatographic data and processing parameters are calculated and recorded using algorithms such as correlation coefficient / cosine similarity. Residual solvents: Ethanol, acetonitrile, and dichloromethane are quantitatively analyzed using headspace GC (e.g., DB-624 or equivalent capillary column). Moisture content: Determined by Karl Fischer method (volume method or coulometric method) and test conditions are recorded; if necessary, supplement with loss on drying / thermogravimetric analysis as supporting evidence.

[0081] Impurity fingerprint similarity evaluation method: Select one batch of samples (e.g., batch A in Example 4) as the reference spectrum, and collect chromatographic data of each batch under the same analytical HPLC conditions; perform baseline correction and time alignment on the retention time range containing the main impurity peaks (which can be corrected according to the relative retention time RRT of the main peak); calculate the Pearson correlation coefficient (or cosine of the included angle) using the normalized peak area / peak height vector as the similarity index, and a similarity ≥ 0.98 is judged as batch-to-batch consistency; and record the original chromatographic data, processing software version and parameters to ensure traceability of the evaluation.

[0082] Example 4: Consistency verification of impurity profiles across multiple batches To further verify the controllability and batch-to-batch consistency of the key quality attributes of the Tripterygium wilfordii sesquiterpene alkaloid purity standard samples prepared by this invention, three batches of Tripterygium wilfordii raw materials from different sources / batch numbers (denoted as batch A, batch B, and batch C) were selected, and the purity standard samples of the target alkaloids were independently prepared according to the method in Example 1. The consistency of impurity spectra, moisture content, residual solvent, and short-term stability were verified. The samples obtained from batches A, B, and C were detected by analytical HPLC area normalization and / or LC-MS. The retention time of the main peak, relative retention time (RRT), content of the single largest impurity, and total impurity content were recorded, and the batch-to-batch consistency was evaluated by fingerprint similarity (see Table 14). The results showed that the impurity peak distribution of the three batches of samples was consistent, the fingerprint similarity was ≥0.98, and the total impurities were ≤1.0% and the single unknown impurities were ≤0.10%, indicating that the method of this invention can stably control the impurity spectra and achieve batch-to-batch consistency.

[0083] Table 14 Key quality attributes of the three batches of samples, designated A, B, and C. Example 5: Verification of Moisture and Residual Solvent Control The moisture content of the three batches of samples was determined using the Karl Fischer method, and the results were all ≤0.5%. Simultaneously, residual solvents such as ethanol and acetonitrile were tested according to ICH Q3C (or the Chinese Pharmacopoeia / related guidelines), and the contents of all residual solvents were below the corresponding limits. This indicates that the recrystallization and shaping, and the low-humidity inert gas dispensing and sealing steps of this invention can effectively control moisture and residual solvents.

[0084] Example 6: Short-term stability verification Samples from batches A, B, and C were stored at 2–8°C in the dark for one month. The purity of the main peak and the impurity spectrum were then analyzed at 0 and 4 weeks. The results showed no significant decrease in the purity of the main peak, and no new impurity peaks or significant changes were observed in the impurity spectrum. This indicates that the obtained purity standard samples have good short-term stability and meet the requirements for analytical standard samples.

[0085] The above results demonstrate that this invention can not only obtain high-purity monomeric compounds, but also achieve stable purity standard samples of Tripterygium wilfordii sesquiterpene alkaloids through specific process combinations, with stable impurity profiles, batch-to-batch consistency, controllable moisture and residual solvents.

[0086] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids, characterized in that, Includes the following steps: (1) Extract the powder of Tripterygium wilfordii root and / or stem by ethanol reflux, recover the ethanol and cool to obtain an extract; The extract was dissolved in an acidic solution and filtered. After adjusting the pH to 8.5–10.0, it was extracted with an organic solvent, concentrated under reduced pressure, and dried to obtain crude total alkaloids. (2) Add the crude total alkaloids to methanol, stir to dissolve, let stand and filter, and collect the methanol-insoluble matter; (3) The methanol-insoluble matter was separated by high-speed countercurrent chromatography in a two-phase solvent system of petroleum ether-ethyl acetate-isopropanol-water and collected in fractions to obtain crude fractions of each component containing the target alkaloid; (4) The crude fractions of each component are purified by reverse-phase C18 chromatographic column, isocratic elution is performed, the target peak is collected, and the purified solution of the corresponding monomer is obtained; (5) After removing the solvent from the purified liquid, recrystallize it with hot methanol and dry it under vacuum to constant weight to obtain the purity standard sample of the Tripterygium wilfordii sesquiterpene alkaloids.

2. The method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids according to claim 1, characterized in that, The acidic solution is citric acid.

3. The method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpene alkaloids according to claim 1, characterized in that, In step (1), the organic solvent is dichloromethane or a mixture of dichloromethane and isopropanol.

4. The method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids according to claim 3, characterized in that, The organic solvent is dichloromethane.

5. The method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids according to claim 1, characterized in that, In step (2), the ratio of crude total alkaloids to methanol is 1g:(100-200)mL.

6. The method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids according to claim 1, characterized in that, In step (3), the volume ratio of petroleum ether-ethyl acetate-isopropanol-water is 6:4:5:

8.

7. The method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids according to claim 1, characterized in that, In step (3), the conditions for high-speed countercurrent chromatography separation are: rotation speed of 450-550 rpm, stationary phase of upper phase, flow rate of 4-6 mL / min, and column temperature of 23-27℃.

8. The method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpenoid alkaloids according to claim 1, characterized in that, In step (4), isocratic elution is performed by acetonitrile / water, wherein the water contains 0.05wt% to 0.2wt% formic acid.

9. The method for preparing a purity standard sample of Tripterygium wilfordii sesquiterpene alkaloids according to claim 1, characterized in that, In step (5), hot methanol is recrystallized 2 to 4 times, and the final temperature is controlled at 0 to 10°C.

10. A purity standard sample of Tripterygium wilfordii sesquiterpene alkaloids obtained by the preparation method according to any one of claims 1-9, characterized in that, It contains at least one monomeric component selected from triptolide, triptolide, triptolide B, and triptolide C, and meets the requirements of purity ≥ 99.0% and moisture content ≤ 0.5%.

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