A method for establishing a fingerprint spectrum of a shiyu jiutai pill and a method for determining multiple component contents

Abstract

The application provides a method for establishing a fingerprint spectrum of Jiyu Baotai pills and a method for determining multiple component contents, relates to the technical field of traditional Chinese medicine quality analysis, and comprises the following steps: preparing a mixed control solution and a test solution, then determining the test solution and the control solution by UHPLC, recording chromatograms and comparing, identifying common peaks, and establishing a fingerprint spectrum of Jiyu Baotai pills. The method has the advantages of simplicity, stability, high precision, good reproducibility and the like; the method can also comprehensively reflect the types and quantities of chemical components contained in Jiyu Baotai pills, and then the quality of Jiyu Baotai pills is described and evaluated as a whole. The chemical components of the fingerprint spectrum of Jiyu Baotai pills detected by the method are relatively more, the height proportion of each characteristic peak is moderate, the baseline is relatively stable, the separation degree and peak shape are good, and the column efficiency is high.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality analysis technology, and in particular to a method for establishing a fingerprint spectrum of Siyu Baotai Pill and a method for determining the content of multiple components. Background Technology

[0002] Siyu Baotai Pills originate from the "Baochan Wuyou San" formula recorded on page 5 of the volume of "Taichan Xinfa" (Heart Method of Pregnancy and Childbirth) written by Yan Chunxi (Chengzhai) in the Qing Dynasty, and have been improved with added ingredients. Its formula includes 19 Chinese herbs: Astragalus membranaceus, Codonopsis pilosula, Poria cocos, Atractylodes macrocephala (stir-fried with wheat bran), Glycyrrhiza uralensis, Angelica sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Rehmannia glutinosa, Colla corii asini, Taxillus chinensis, Cuscuta chinensis, Artemisia argyi (charred), Schizonepeta tenuifolia, Magnolia officinalis (processed with ginger), Citrus aurantium (stir-fried with wheat bran), Fritillaria cirrhosa, Notopterygium incisum, and deer antler powder. This formula primarily uses Astragalus membranaceus, Angelica sinensis, Ligusticum chuanxiong, and Paeonia lactiflora to replenish qi, nourish blood, and promote blood circulation. It is supplemented with Cuscuta chinensis to tonify the liver and kidneys, benefit essence, and stabilize the fetus. Donkey-hide gelatin, Artemisia argyi, and Taxillus chinensis promote menstruation, nourish blood, warm the uterus, and stabilize the fetus. Deer antler powder prevents uterine coldness, Fritillaria cirrhosa facilitates delivery, Rehmannia glutinosa nourishes yin, replenishes blood, benefits essence, and fills marrow. Magnolia officinalis is added to soothe the chest and regulate qi, while Schizonepeta tenuifolia lifts the fetal essence and is also an essential medicine for postpartum dizziness. Notopterygium incisum promotes blood circulation and relieves limb congestion. Licorice root is used as a guiding herb to unblock meridians, benefit qi and blood, and detoxify various medicines. The combined effects of these herbs are nourishing and menstrual-regulating, gentle yet effective. The herbs are sweet and warm in nature, stabilizing pregnancy before delivery and promoting delivery during labor. This formula has the effects of replenishing qi and blood, protecting the fetus, and stabilizing the fetus.

[0003] Due to the influence of factors such as the origin, harvesting time, and processing techniques of Chinese medicinal materials, the content of medicinal components varies greatly. Furthermore, the efficacy of Chinese herbal medicine (TCM) compound preparations is the result of the synergistic effect of multiple components. These universal issues pose significant challenges to the quality evaluation system of TCM compound preparations, making industry standard upgrades extremely difficult. Therefore, exploring a high-throughput multi-component detection method that can provide comprehensive information on the overall characteristics of TCM compound preparations can offer a reference for the construction of a TCM compound preparation quality control system. The existing standard for Siyu Baotai Pill only specifies the inspection items for its appearance and pill form, with a single control indicator. In addition, current technology only uses traditional high-performance liquid chromatography (HPLC) to determine a single active ingredient (paeoniflorin) in Siyu Baotai Pill, resulting in a one-sided quality evaluation of Siyu Baotai Pill. Its intrinsic quality is difficult to fully control, and its overall quality cannot be adequately reflected.

[0005] Existing technologies mostly focus on the quantitative analysis of single components, lacking a comprehensive evaluation of the overall quality of drugs. Traditional Chinese medicine (TCM) fingerprinting technology can be applied to research in the field of TCM quality control, comprehensively reflecting the intrinsic overall quality of TCM preparations. This provides an effective technical means for the characteristic and holistic analysis of TCM compound prescriptions. However, existing methods still have shortcomings in the establishment and application of fingerprinting, resulting in insufficient coverage of drug components, neglecting the effective correlation of principal, assistant, adjuvant, and guide herbs in prescription formulation, and limiting the quality control model. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a method for establishing a fingerprint spectrum of Siyu Baotai Pill and a method for determining the content of multiple components. This method has the advantages of being simple, stable, highly precise, and reproducible. Furthermore, this method can comprehensively reflect the types and quantities of chemical components contained in Siyu Baotai Pill, thereby providing an overall description and evaluation of its quality. Moreover, the fingerprint spectrum of Siyu Baotai Pill detected by this method shows a relatively large number of chemical components, a moderate proportion of characteristic peak heights, a relatively stable baseline, good resolution and peak shape, and high column efficiency.

[0007] In a first aspect, the present invention provides a method for establishing a fingerprint spectrum of a pregnancy-preserving pill, comprising the following steps: (1) Preparation of mixed reference solution: Prepare a mixed reference solution containing chlorogenic acid, paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin, magnolol and atractylodes lactone I with methanol. (2) Preparation of test solution: Weigh the Dingsiyu Baotai Pill, add methanol, and extract by ultrasonication to obtain the test solution; (3) Establishment of fingerprint spectrum: The mixed reference solution and the test solution were determined by UHPLC, the chromatograms were recorded and compared, the common peaks were identified, and the fingerprint spectrum for fertility and pregnancy maintenance was established. The chromatographic conditions for the UHPLC are as follows: Chromatographic column: Waters ACQUITY UPLC BEH C8, 2.1 mm × 100 mm, 1.7 mm; Mobile phase: Acetonitrile as phase A, and 0.1% (v / v) trifluoroacetic acid (TFA) aqueous solution as phase B, for gradient elution; Detection wavelength: 230 nm; Flow rate: 0.4 mL / min; Column temperature: 30℃; Injection volume: 2 μL.

[0008] Preferably, in step (1), each 1 mL of the mixed reference solution contains 5 µg of chlorogenic acid, 30 µg of paeoniflorin, 10 µg of hyperoside, 5 µg of ferulic acid, 5 µg of glycyrrhizin, 100 µg of naringin, 50 µg of neohesperidin, 3.5 µg of magnolol, and 23 µg of atractylodes lactone I.

[0009] As a preferred embodiment, in step (2), the specific conditions for preparing the test solution are as follows: accurately weigh the Siyu Baotai pill, accurately add methanol, weigh the mass, extract by ultrasound, cool and make up the mass, shake well, take the supernatant, filter through a filter membrane, and the test solution is obtained.

[0010] Preferably, the power of the ultrasound is 130~150W, more preferably 135~145W; the frequency of the ultrasound is 35~45KHz.

[0011] In this technical solution, the power of the ultrasound is controlled at 135~145W; the frequency of the ultrasound is 35~45 KHz, which has a good extraction effect on the sample and is conducive to obtaining better chromatographic peaks and peak shapes.

[0012] Preferably, the ultrasound duration is 27-32 minutes.

[0013] Preferably, the filter membrane has a pore size of 0.22 µm.

[0014] Preferably, the methanol in steps (1) and (2) is independently a methanol solution with a volume percentage concentration of 70% to 75%.

[0015] In this technical solution, a methanol solution with a volume percentage concentration of 70%~75% is used to extract the sample, which helps to improve the extraction efficiency and obtain chromatographic peaks with high peak height and good peak shape.

[0016] Preferably, the methanol in step (2) is a methanol aqueous solution with a volume percentage concentration of 75%, the ultrasonic power is 135~145W, and the ultrasonic frequency is 35~45 KHz.

[0017] This technical solution improves sample pretreatment by employing a novel sample pretreatment method. It utilizes an ultrasonic-assisted extraction with a 75% (v / v) methanol-water solution at an optimal material-to-liquid ratio of 1:50. The optimized extraction technology makes sample processing simpler and more efficient, improving the extraction recovery rate and purity of the target components.

[0018] Preferably, the mass ratio of the fertility-preserving pill to the volume ratio of the methanol solution is 1 g: (45~55) mL.

[0019] Preferably, in step (3), the gradient elution procedure is as follows: 0~2 min, A 5%→14%; 2~5 min, A 14%→16%; 5~8 min, A 16%→30%; 8~10 min, A 30%→40%; 10~12 min, A 40%→70%; 12~13 min, A 70%→85%; 13~15 min, A 85%.

[0020] Preferably, the number of common peaks is 24, among which... .

[0021] In a second aspect, the present invention provides a method for determining the content of multiple components of Siyu Baotai Pill, wherein a sample of Siyu Baotai Pill is taken, and the content of components in the sample is calculated according to the above-mentioned method for establishing the fingerprint spectrum of Siyu Baotai Pill.

[0022] As a preferred option, the contents of paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin and magnolol in the sample were calculated respectively.

[0023] Compared with the prior art, the beneficial technical effects of the present invention are as follows: 1. This invention establishes a technical means to characterize the quality of Siyu Baotai Pill by integrating fingerprint spectrum similarity and multi-component content determination, providing a scalable quality control template for traditional Chinese medicine compound preparations and promoting the upgrading of industry standards.

[0024] 2. The fingerprint spectrum establishment and multi-component content determination method established by this invention has good versatility and can be applied to the quality control of different batches of Siyu Baotai Pills. It is also applicable to the detection of intermediate products in the production process of Siyu Baotai Pills, providing strong support for quality assurance in the entire production process.

[0025] 3. This invention overcomes the problems of low efficiency and high cost of traditional methods, significantly shortens the detection time, and covers all the ingredients of Siyu Baotai Pill, thus completing the construction of a high-throughput detection system for complex Chinese medicine compositions.

[0026] 4. This invention is the first to achieve accurate simultaneous content determination of multiple key active ingredients (paeoniflorin, hyperoside, ferulic acid, etc.) in Siyu Baotai Pill, covering the main effective components that enable the drug to exert its fertility and pregnancy-preserving effects, and providing a more comprehensive and scientific basis for the quality evaluation of the drug.

[0027] 5. This invention uses a UHPLC system to construct characteristic fingerprint spectra, which improves the separation and detection sensitivity of multiple components in traditional Chinese medicine compositions, reduces the use of organic solvents and waste of raw materials, and improves detection efficiency.

[0028] 6. The present invention uses optimized chromatographic conditions, which can present the characteristic fingerprint spectrum of Siyu Baotai Pill more clearly and comprehensively, improve the resolution and repeatability of the fingerprint spectrum, and make the quality control of different batches of Siyu Baotai Pill more accurate and reliable. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 This is a flowchart illustrating the technical process for establishing the fingerprint spectrum and determining the content of multiple components of the fertility-preserving pill of the present invention.

[0031] Figure 2 The fingerprint chromatograms (S1-S3) of different batches of the fertility-preserving pills in Example 3 and the control fingerprint chromatogram (R) are shown.

[0032] Figure 3 This is the chromatographic peak assignment diagram of the UHPLC fingerprint of the "Siyu Baotai Pill" in Example 4.

[0033] Figure 4 The chromatogram is of the mixed reference standard (A) and sample (B) in Example 6. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the formulations involved in the following examples are all commercially available products that can be purchased from the market.

[0036] The present invention will be further described in detail below with reference to the illustrations and embodiments: To comprehensively reflect the quality of Siyu Baotai Pills and improve detection efficiency and drug quality controllability, this invention first utilizes the advantages of ultra-high performance liquid chromatography (UHPLC)—high sensitivity and high resolution—to optimize chromatographic conditions. This includes examining the baseline, peak shape, and separation effect of chromatographic peaks under different columns; the number of chromatographic peaks and the separation of each peak at different wavelengths (230 nm, 254 nm, 280 nm, 320 nm) in Siyu Baotai Pills samples; and the influence of temperature on peak shape and separation effect, thus selecting the optimal chromatographic detection conditions for Siyu Baotai Pills. Furthermore, addressing the issue that simultaneously extracting multiple components makes it difficult to simultaneously achieve optimal extraction conditions for different components, affecting overall detection accuracy, further investigation was conducted on factors such as extraction solvent, ultrasonic time, and solid-liquid ratio to optimize sample extraction conditions. Based on this, a characteristic fingerprint chromatogram of Siyu Baotai Pills was established, common peaks and identifying components were identified, and its methodology was evaluated.

[0037] Based on the formulation characteristics of Siyu Baotai Pill and the traditional Chinese medicine theory of "principal, assistant, adjuvant, and guide" in drug formulation, and combining the main active ingredients and chromatographic results of each medicinal material, the components contained in the formula were identified. Seven active ingredients in Siyu Baotai Pill were determined using optimized screening conditions, and their methodology was investigated. Thus, this invention successfully established a fingerprint chromatogram of Siyu Baotai Pill and a method for the simultaneous determination of seven components, enabling a systematic quantitative analysis of Siyu Baotai Pill and providing a relatively comprehensive reflection of its intrinsic quality.

[0038] Example 1 (1) Preparation of mixed reference solution: Weigh appropriate amounts of chlorogenic acid, paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin, magnolol, and atractylodes lactone I reference standards, respectively. Accurately weigh each, add 75% (v / v) methanol aqueous solution, mix well, and make up to the corrected volume. Store these solutions at 4°C in the dark. Accurately measure appropriate amounts of each reference standard stock solution to prepare a mixed reference solution containing 5 µg chlorogenic acid, 30 µg paeoniflorin, 10 µg hyperoside, 5 µg ferulic acid, 5 µg glycyrrhizin, 100 µg naringin, 50 µg neohesperidin, 3.5 µg magnolol, and 23 µg atractylodes lactone I per mL. Vortex to mix.

[0039] (2) Preparation of the test solution: Accurately weigh 0.5 g of raw powder of Siyu Baotai Pill (purchased from Beijing Tongrentang Pharmaceutical Factory, batch number 19100101098), place it in a stoppered conical flask, accurately add 25 mL of 75% methanol aqueous solution, weigh the flask, seal it tightly, and extract by sonication (140W power, 40KHz frequency) for 30 min. After cooling, make up the mass, shake well, take the supernatant, and filter it through a 0.22 µm microporous membrane to obtain the test solution.

[0040] (3) Establishment of fingerprint spectrum: The mixed reference solution and the test solution were determined by UHPLC, the chromatograms were recorded and compared, the common peaks were identified, and the fingerprint spectrum for fertility and pregnancy maintenance was established. The chromatographic conditions for the UHPLC are as follows: Chromatographic column: Waters ACQUITY UPLC BEH C8, 2.1 mm × 100 mm, 1.7 mm; Mobile phase: Acetonitrile as phase A, and 0.1% trifluoroacetic acid aqueous solution as phase B, for gradient elution; Detection wavelength: 230 nm; Flow rate: 0.4 mL / min; Column temperature: 30℃; Injection volume: 2 μL; The gradient elution procedure is shown in Table 1 below: Table 1 Gradient elution program

[0041] Example 2 Fingerprint mapping methodology investigation (1) Precision test Six portions of the test solution (S1) from Example 1 were accurately weighed and injected six times to examine the instrument's precision. Using peak 13 (neohesperidin) as the reference peak, the results showed that the relative retention time RSD of all 24 common peaks was less than 0.14%, and the relative peak area RSD was less than 3.00%, indicating that the instrument's precision was good and met the requirements of fingerprint spectroscopy technology.

[0042] (2) Repeatability test Six samples (numbered S1) were accurately weighed and prepared according to the method for preparing the test solution in Example 1. The samples were then injected under the chromatographic conditions of Example 1 to examine the repeatability of the method. The results showed that, using peak 13 (neohesperidin) as the reference peak, the relative retention time RSD of all 24 common peaks was less than 0.15%, and the relative peak area RSD was less than 4.03%, indicating good repeatability and meeting the requirements of fingerprint chromatographic technology.

[0043] (3) Stability test The same test solution (numbered S1) prepared in Example 1 was injected and measured at 0, 1, 2, 4, 6, 8, 12 and 24 h after preparation, according to the method in Example 1. Peak 13 (neohesperidin) was used as the reference peak. The results showed that the relative retention time RSD of the 24 common peaks was less than 0.29% and the relative peak area RSD was less than 4.68%, indicating that the test solution was stable within 24 h.

[0044] Example 3 Fingerprint mapping and similarity evaluation Three batches of raw powder samples of Siyu Baotai Pills (S1~S3) were collected. Test solutions were prepared according to the method in Example 1, and the samples were injected according to the method in Example 1. Chromatograms were recorded. The three batches of samples were analyzed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012). After multi-point correction, Mark peak matching was performed (time window of 0.1 min). Fingerprint chromatograms and control fingerprint chromatograms were generated using the mean method. See [link to relevant documentation]. Figure 2After Mark peak matching, 24 common peaks were identified, and the similarity results were calculated. The results showed that the similarity results of S1 to S3 were 0.999, 0.999, and 0.999, respectively. The similarity results of the three batches of samples were all greater than 0.990. A total of 24 common peaks were identified, and 9 of them were identified. Peak 1 is chlorogenic acid, peak 3 is paeoniflorin, peak 5 is hyperoside, peak 6 is ferulic acid, peak 7 is glycyrrhizin, peak 11 is naringin, peak 13 is neohesperidin, peak 23 is magnolol, and peak 24 is atractylodes lactone I.

[0045] This indicates that the chemical composition of different batches of Siyu Baotai Pills is highly consistent, and the overall quality difference is not significant. The established fingerprint spectrum is representative and can be used for the quality control of Siyu Baotai Pills.

[0046] Example 4 Attribution of common peaks in fingerprint spectrum Peak components were confirmed by comparing the chromatogram with that of the reference standard. Figure 3 ,Depend on Figure 3 It can be seen that peak 1 is chlorogenic acid, peak 3 is paeoniflorin, peak 5 is hyperoside, peak 6 is ferulic acid, peak 7 is glycyrrhizin, peak 11 is naringin, peak 13 is neohesperidin, peak 23 is magnolol, and peak 24 is atractylodes lactone I.

[0047] Example 5 This embodiment provides a method for determining the content of multiple components in a pregnancy-preserving pill, including the following steps: (1) Preparation of mixed reference solution: Accurately weigh appropriate amounts of paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin, and magnolol reference standards, respectively. Add 75% (v / v) methanol aqueous solution, mix well, and make up the correction volume to obtain the reference standard solutions. Store these solutions at 4°C in the dark. Accurately measure appropriate amounts of each reference standard solution to prepare a mixed reference standard solution containing 30 µg paeoniflorin, 10 µg hyperoside, 5 µg ferulic acid, 5 µg glycyrrhizin, 100 µg naringin, 50 µg neohesperidin, and 7 µg magnolol per mL. Vortex to mix well to obtain the mixed reference standard solution.

[0048] (2) Preparation of the test solution: Accurately weigh 0.5 g of the raw powder of Siyu Baotai Pill and place it in a stoppered conical flask. Accurately add 25 mL of methanol aqueous solution with a volume percentage concentration of 75%, weigh the sample, stopper the flask tightly, and extract by ultrasonication (power 140W, frequency 40KHz) for 30 min. After cooling, make up the mass, shake well, take the supernatant, and filter it through a 0.22 µm microporous membrane to obtain the test solution.

[0049] (3) Determination of component content: The component content of the test solution was determined by UHPLC; The chromatographic conditions for the UHPLC are as follows: Chromatographic column: Waters ACQUITY UPLC BEH C8, 2.1 mm × 100 mm, 1.7 mm; Mobile phase: Acetonitrile as phase A, and 0.1% trifluoroacetic acid aqueous solution as phase B, for gradient elution; Detection wavelength: 230 nm; Flow rate: 0.4 mL / min; Column temperature: 30℃; Injection volume: 2 μL; The gradient elution procedure is the same as that in Table 1 of Example 1.

[0050] Example 6 Methodological investigation (1) Specificity examination Samples of the original powder of Siyu Baotai Pill and reference standards were prepared and analyzed according to the method in Example 5. The results showed that the seven chromatographic peaks were well separated (peak 3 was paeoniflorin, peak 5 was hyperoside, peak 6 was ferulic acid, peak 7 was glycyrrhizin, peak 11 was naringin, peak 13 was neohesperidin, and peak 23 was magnolol). See [link to sample]. Figure 4 This method has good specificity.

[0051] (2) Examination of linear relationships Accurately take 200 µL of each reference solution (excluding naringin) (paeoniflorin), 80 µL of hyperoside, 20 µL of ferulic acid, 25 µL of glycyrrhizin, 250 µL of neohesperidin, and 40 µL of magnolol), place them in the same centrifuge tube, dilute with 75% methanol aqueous solution to 1 mL, shake well, and gradually dilute to the lower limit of quantitation for each component, shaking well to obtain a series of mixed reference solutions; accurately pipette 500 µL of naringin reference solution, gradually dilute to its lower limit of quantitation, shaking well to obtain a series of naringin solutions, accurately pipette 100 µL of each reference solution, and inject for determination according to the chromatographic conditions of Example 5 above.

[0052] With concentration (X, µg·mL) -1 Using the concentration (x) as the abscissa and the peak area (y) as the ordinate, a linear regression was performed to plot the standard curves for each component. The regression equations and correlation coefficients are shown in Table 2. The results show that the concentration of each component and the peak area have a good linear relationship within the experimental range.

[0053] Table 2. Regression equations and linear ranges for the seven components.

[0054] (3) Precision test The same test solution prepared in Example 5 was injected six times repeatedly according to the method in Example 5, and the peak area data of paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin, and magnolol were recorded. The RSDs of their peak areas were 3.03%, 4.69%, 4.58%, 2.86%, 0.40%, 1.42%, and 0.88%, respectively. n =6), indicating that the instrument has good precision.

[0055] (4) Repeatability test Six portions of the same batch of test sample (number S1) were accurately weighed. Samples were prepared according to the method described in Example 5, and injected according to the method described in Example 5. Chromatograms were recorded, and the peak areas of the seven components in the Siyu Baotai Pill were calculated. The contents and their RSDs were also calculated. The average contents of paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin, and magnolol were 1484.3 µg·g⁻¹. -1 260.65 µg·g -1 129.5 µg·g -1 236.5 µg·g -1 4227.4 µg·g -1 2349.95 µg·g -1 266.8 µg·g -1 The RSDs were 0.93%, 1.26%, 1.84%, 1.67%, 0.94%, 0.60%, and 1.83% (n=6), indicating that the method had good repeatability.

[0056] (5) Stability test The same test sample solution (number S1) prepared in Example 5 was taken and injected for determination at 1, 2, 4, 8, 10, 12 and 24 h after preparation, according to the method in Example 5. The peak area data of paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin and magnolol were recorded. The RSD value of each peak area was between 1.93% and 3.91%, indicating that the test sample solution had good stability within 24 h.

[0057] (6) Spiking recovery test Accurately weigh 0.25 g of the same batch of test sample (No. S1) raw powder and place it in six stoppered conical flasks. Add 25 mL of freshly prepared mixed reference solution (equivalent to 50% of the original mass fraction) to each flask according to the method in Example 5. Prepare samples according to the test sample solution preparation method in Example 5 and inject and determine according to the method in Example 5. Record the chromatograms. Calculate the recovery rate and RSD of each component based on the measured amount and the amount added. The results show that the average recoveries of paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin, and magnolol are 105.2%, 104.3%, 105.8%, 107.3%, 96.2%, 98.6%, and 97.1%, respectively, with RSDs of 1.86%, 2.27%, 3.81%, 1.89%, 1.25%, 1.51%, and 2.0%, respectively. n =6, measured 6 times). The RSD values ​​obtained by the above method are all less than 3%, which meets the requirements of the guidelines of Part IV of the 2020 edition of the Chinese Pharmacopoeia, indicating that the method has good repeatability.

[0058] Table 3 Results of the recovery test of 7 components

[0059] Example 7 Sample content determination Three batches of Siyu Baotai Pills from different batches were collected, and the peak area data of paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin, and magnolol were recorded. The contents were calculated using the external standard method, and the results are shown in Table 4 (average of two measurements). The results indicate that the contents of each component in the three different batches of Siyu Baotai Pills did not differ significantly.

[0060] Table 4. Content of 7 components of Siyu Baotai Pill (µg·g) -1 ) Measurement (n=2)

[0061] This invention provides a method for establishing a fingerprint spectrum of a pregnancy-preserving pill and a method for determining the content of multiple components (the process is as follows). Figure 1As shown in the diagram, the combined strategy of "fingerprint chromatographic overall control + multi-index component quantification" can solve existing technical problems and provide a technical solution for Siyu Baotai Pill that can comprehensively reflect the overall quality of traditional Chinese medicine preparations. Based on this, this invention applies ultra-high performance liquid chromatography (UHPLC) technology to the quality analysis of Siyu Baotai Pill for the first time: constructing a fingerprint chromatogram, identifying 24 common peaks and identifying 9 components to achieve overall quality monitoring; simultaneously determining 7 components (paeoniflorin, ferulic acid, hyperoside, glycyrrhizin, naringin, neohesperidin, and magnolol), covering multiple traditional Chinese medicines in the formula, which can make up for the deficiencies of existing standards. This method can provide key technical support for improving the quality standards of Siyu Baotai Pill and ensuring clinical efficacy.

[0062] Given that existing technological deficiencies have hindered the development of a quality evaluation system for Siyu Baotai Pills, this invention provides a method for determining the content of Siyu Baotai Pills that simultaneously analyzes multiple components, features efficient and simple pretreatment, and accurately optimizes detection conditions. This method improves detection efficiency and accuracy, comprehensively reflecting the quality of the preparation.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of a pregnancy-preserving pill, characterized in that, Includes the following steps: (1) Preparation of mixed reference solution: Prepare a mixed reference solution containing chlorogenic acid, paeoniflorin, hyperoside, ferulic acid, glycyrrhizin, naringin, neohesperidin, magnolol and atractylodes lactone I with methanol. (2) Preparation of test solution: Weigh Dingsiyu Baotai Pills, add methanol, and extract by ultrasonication to obtain the test solution; (3) Establishment of fingerprint spectrum: The mixed reference solution and the test solution were determined by UHPLC, the chromatograms were recorded and compared, the common peaks were identified, and the fingerprint spectrum for fertility and pregnancy maintenance was established. The chromatographic conditions for the UHPLC are as follows: Chromatographic column: Waters ACQUITY UPLC BEH C8, 2.1 mm × 100 mm, 1.7 mm; Mobile phase: Acetonitrile as phase A, and 0.1% trifluoroacetic acid aqueous solution as phase B, for gradient elution; Detection wavelength: 230 nm; Flow rate: 0.4 mL / min; Column temperature: 30℃; Injection volume: 2 μL.

2. The method for establishing the fingerprint spectrum of the fertility-preserving pill according to claim 1, characterized in that, In step (1), each 1 mL of the mixed reference solution contains 5 µg of chlorogenic acid, 30 µg of paeoniflorin, 10 µg of hyperoside, 5 µg of ferulic acid, 5 µg of glycyrrhizin, 100 µg of naringin, 50 µg of neohesperidin, 3.5 µg of magnolol, and 23 µg of atractylodes lactone I.

3. The method for establishing the fingerprint spectrum of the fertility-preserving pill according to claim 1 or 2, characterized in that, In step (2), the specific conditions for preparing the test solution are as follows: accurately weigh the Siyu Baotai Pill, accurately add methanol, weigh the mass, extract by ultrasound, cool and make up the mass, shake well, take the supernatant, filter with a filter membrane, and the test solution is obtained.

4. The method for establishing the fingerprint spectrum of the fertility-preserving pill according to claim 1 or 3, characterized in that, The power of the ultrasound is 130~150W, preferably 135~145W; the frequency of the ultrasound is 35~45 KHz.

5. The method for establishing the fingerprint spectrum of the fertility-preserving pill according to claim 3, characterized in that, The filter membrane has a pore size of 0.22 µm.

6. The method for establishing the fingerprint spectrum of the fertility-preserving pill according to claim 1 or 5, characterized in that, The methanol in steps (1) and (2) is each independently a methanol solution with a volume percentage concentration of 70% to 75%.

7. The method for establishing the fingerprint spectrum of the fertility-preserving pill according to claim 6, characterized in that, The mass ratio of the fertility-preserving pill to the volume ratio of the methanol solution is 1g:(45~55)mL.

8. The method for establishing the fingerprint spectrum of the fertility-preserving pill according to claim 1 or 7, characterized in that, In step (3), the gradient elution procedure is as follows: 0~2 min, A 5%→14%; 2~5 min, A 14%→16%; 5~8 min, A 16%→30%; 8~10 min, A 30%→40%; 10~12 min, A 40%→70%; 12~13 min, A 70%→85%; 13~15 min, A 85%.

9. The method for establishing the fingerprint spectrum of the fertility-preserving pill according to claim 1 or 8, characterized in that, There are 24 common peaks, among which, 。 10. A method for determining the content of multiple components in a pregnancy-preserving pill, characterized in that, Take a sample of Siyu Baotai Pill and calculate the content of its components according to the fingerprint spectrum establishment method of Siyu Baotai Pill as described in any one of claims 1-9.

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