A characteristic map detection method and quality control method for garlic medicinal materials or pharmaceutical preparations thereof

Abstract

This invention relates to the field of traditional Chinese medicine detection technology, specifically a method for detecting the characteristic chromatograms of garlic medicinal materials or their pharmaceutical preparations. The method employs high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and 0.02-0.04% phosphoric acid solution as mobile phase B. The gradient elution program includes: 0-12 min, mobile phase A to mobile phase B volume ratio of 2%:98%; 12-15 min, mobile phase A to mobile phase B volume ratio of 2%:98% → 17%:83%; 15-25 min, mobile phase A to mobile phase B volume ratio of 17%:83%; 25-30 min, mobile phase A to mobile phase B volume ratio of 17%:83% → 40%:60%; 30-35 min, mobile phase A to mobile phase B volume ratio of 40%:60%. This method enables more comprehensive and specific quality and content detection of garlic medicinal materials or their pharmaceutical preparations.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically to a method for detecting and controlling the quality of characteristic spectra of garlic medicinal materials or their pharmaceutical preparations. Background Technology

[0002] Garlic is the bulb of *Allium sativum* L., a plant in the Liliaceae family. It is harvested in summer when the leaves wither, the fibrous roots and dirt are removed, and it is then air-dried until the outer skin is dry. Garlic has the effects of relieving indigestion, killing bacteria and insects, as recorded in herbal books such as *Xinxiu Bencao* and *Bencao Gangmu*.

[0003] The 2020 edition of the Chinese Pharmacopoeia includes a method for determining the content of garlic medicinal materials. However, this method can only determine the content of allicin in garlic and cannot comprehensively detect and control the quality of garlic and its pharmaceutical preparations.

[0004] Currently, there are few reports on the characteristic chromatograms of garlic. Relevant literature uses Fourier transform infrared spectroscopy or HPLC-UV-PITC pre-column derivatization for analysis, such as in the study "Comparative Study of FTIR Spectra of Garlic from Different Geographical Populations." However, the sample preparation methods in these methods are complex and cumbersome, and the instrument detection time is long. Chinese patent document CN105158370B discloses a method for establishing HPLC fingerprint chromatograms of garlic enzymatic hydrolysates induced from callus culture. The sample in this document is the enzymatic hydrolysate of garlic induced from callus culture, not garlic medicinal material or its pharmaceutical preparations. Therefore, it is not specific to garlic medicinal material or its pharmaceutical preparations, and it only detects two chemical components, resulting in incomplete detection. Furthermore, the two chemical components identified are poorly separated, failing to achieve effective separation and making identification difficult. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is the detection method and quality control method of characteristic chromatograms of garlic medicinal materials or their pharmaceutical preparations. The chromatograms obtained by the method have rich identification chromatographic peak information and good separation. The method is also simpler and faster to operate, and is more comprehensive and specific for the quality detection of garlic medicinal materials or their pharmaceutical preparations.

[0006] Therefore, the present invention provides the following technical solution:

[0007] A method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation, comprising detection by high performance liquid chromatography (HPLC), wherein the chromatographic conditions are: using octadecylsilane-bonded silica gel as the stationary phase, methanol as mobile phase A, and 0.02-0.04 v / v% phosphoric acid solution as mobile phase B, and the gradient elution program includes:

[0008] From 0 to 12 min, the volume ratio of mobile phase A to mobile phase B was 2%:98%;

[0009] Over 12-15 minutes, the volume ratio of mobile phase A to mobile phase B changed from 2%:98% to 17%:83%.

[0010] For 15-25 minutes, the volume ratio of mobile phase A to mobile phase B is 17%:83%.

[0011] After 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 17%:83% to 40%:60%.

[0012] For 30-35 minutes, the volume ratio of mobile phase A to mobile phase B is 40%:60%.

[0013] Optionally, the chromatographic conditions include at least one of the following conditions:

[0014] 1) The detection wavelength is 225nm;

[0015] 2) The flow rate is 0.55-0.65 ml / min;

[0016] 3) Column temperature is 28-32℃;

[0017] 4) The chromatographic column specifications are 4.6mm×150mm, 2.7μm; optionally, the chromatographic column is selected from Poroshell 120AQ-C18 or Poroshell 120EC-C18;

[0018] 5) The injection volume is 1-10 μL.

[0019] Optionally, the preparation of the test solution may also be included: take the test sample, add it to the extraction solvent for extraction, filter, and take the filtrate;

[0020] Optionally, the extraction solvent is water or a 25-75 v / v% methanol solution;

[0021] Optionally, the extraction time is 15-45 minutes;

[0022] Optionally, the mass-to-volume ratio of the test sample to the extraction solvent is 0.05-0.4 g / 10 ml.

[0023] Optionally, the preparation of a reference solution may also be included: γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​reference standard is added to a solvent to prepare a solution;

[0024] Optionally, the solvent is water or a 25-75 v / v% methanol solution;

[0025] Optionally, the concentration of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​in the reference solution is 0.1 mg / ml.

[0026] A method for determining the content of garlic medicinal material or its pharmaceutical preparation, comprising:

[0027] Take the test solution and the reference solution, and perform high performance liquid chromatography (HPLC) according to the detection method of the characteristic spectrum of garlic medicinal material or its pharmaceutical preparation as described in any one of claims 1-4.

[0028] The application of the method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation or the method for determining the content of garlic medicinal material or its pharmaceutical preparation in the quality testing of garlic medicinal material or its pharmaceutical preparation.

[0029] A method for quality testing of garlic medicinal material or its pharmaceutical preparation, comprising:

[0030] The steps are as follows: obtaining the characteristic spectrum of the test sample according to the detection method of the characteristic spectrum of garlic medicinal material or its pharmaceutical preparation, and comparing the characteristic spectrum with the control characteristic spectrum;

[0031] The control feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of garlic medicinal materials or their pharmaceutical preparations as standards according to the detection method of the feature spectrum of garlic medicinal materials or their pharmaceutical preparations.

[0032] Optionally, the reference characteristic spectrum includes 8 characteristic peaks, wherein peak 7 should correspond to the retention time of the γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​reference peak, and the peak corresponding to the γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​reference peak is the S peak. The relative retention times of peaks 1 to 6, peak 8 and peak S are calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are 0.28 for peak 1, 0.35 for peak 2, 0.67 for peak 3, 0.86 for peak 4, 0.92 for peak 5, 0.98 for peak 6 and 1.17 for peak 8.

[0033] Optionally, peak 1 corresponds to L-pyroglutamic acid; peak 2 corresponds to L-tyrosine; peak 3 corresponds to guanosine; peak 5 corresponds to tryptophan; and peak 7 corresponds to γ-glutamyl-S-(trans-1-propenyl)-L-cysteine.

[0034] The method for detecting the characteristic spectrum of garlic medicinal material or its pharmaceutical preparation, the method for determining the content of garlic medicinal material or its pharmaceutical preparation, or the method for quality testing of garlic medicinal material or its pharmaceutical preparation, wherein the garlic medicinal material or its pharmaceutical preparation includes garlic medicinal material, standard decoction of garlic slices, or garlic formula granules.

[0035] The technical solution of this invention has the following advantages:

[0036] 1. The present invention provides a method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation, comprising detection by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows: using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and 0.02-0.04 v / v% phosphoric acid solution as mobile phase B. The gradient elution program includes: 0-12 min, the volume ratio of mobile phase A to mobile phase B is 2%:98%; 12-15 min, the volume ratio of mobile phase A to mobile phase B is 2%:98% → 17%:83%; 15-25 min, the volume ratio of mobile phase A to mobile phase B is 17%:83%; 25-30 min, mobile phase A... The volume ratio of mobile phase A to mobile phase B is 17%:83% → 40%:60%; after 30-35 minutes, the volume ratio of mobile phase A to mobile phase B is 40%:60%. The chromatogram obtained by this method has rich information on the identifying chromatographic peaks, which can reflect the characteristic components of garlic or its pharmaceutical preparations. Five known components can be identified from the characteristic peaks. L-pyroglutamic acid, L-tyrosine, guanosine, tryptophan, and γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​can be determined simultaneously with good separation. The method is also simpler and faster to operate. In summary, the above method can perform more comprehensive and specific quality and content detection on garlic medicinal materials or its pharmaceutical preparations. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 These are the characteristic chromatograms of three batches of garlic formula granules in Example 1 of this invention;

[0039] Figure 2 These are comparative characteristic chromatograms of garlic standard decoction (freeze-dried powder) and garlic formulation granules in Example 1 of the present invention;

[0040] Figure 3 This is a characteristic spectrum of the garlic formula granule extract in Example 1 of the present invention;

[0041] Figure 4 This is a characteristic spectrum of the garlic formula granules in Embodiment 1 of the present invention;

[0042] Figure 5 This is a reference standard positioning diagram of the garlic standard decoction (freeze-dried powder) in Example 1 of the present invention;

[0043] Figure 6The characteristic spectrum is obtained under the condition of a flow rate of 0.55 ml / min in Example 2 of this invention;

[0044] Figure 7 This is the characteristic spectrum under the condition of a flow rate of 0.60 ml / min in Example 2 of the present invention;

[0045] Figure 8 This is the characteristic spectrum under the condition of a flow rate of 0.65 ml / min in Example 2 of the present invention;

[0046] Figure 9 This is a characteristic spectrum under the condition of column temperature of 28℃ in Embodiment 3 of the present invention;

[0047] Figure 10 This is a characteristic spectrum under the condition of column temperature of 30℃ in Embodiment 3 of the present invention;

[0048] Figure 11 This is a characteristic spectrum under the condition of column temperature of 32℃ in Embodiment 3 of the present invention;

[0049] Figure 12 This is a characteristic spectrum of the phosphoric acid solution with a concentration of 0.02% in Example 4 of the present invention;

[0050] Figure 13 This is a characteristic spectrum of the phosphoric acid solution with a concentration of 0.03% in Example 4 of the present invention;

[0051] Figure 14 This is a characteristic spectrum of the phosphoric acid solution with a concentration of 0.04% in Example 4 of the present invention;

[0052] Figure 15 This is a characteristic chromatogram under the conditions of the Poroshell 120AQ-C18 (serial number 68) chromatographic column in Example 5 of the present invention;

[0053] Figure 16 This is a characteristic chromatogram under the conditions of the Poroshell 120AQ-C18 (serial number 70) chromatographic column in Example 5 of the present invention;

[0054] Figure 17 This is a characteristic chromatogram under the conditions of the Poroshell 120EC-C18 column (serial number 23) in Example 5 of the present invention;

[0055] Figure 18 This is the characteristic spectrum of the maltodextrin excipient in "1. Specificity Investigation" of Embodiment 6 of the present invention;

[0056] Figure 19 This is the characteristic chromatogram of the reference standard in "1. Specificity Examination" of Embodiment 6 of the present invention;

[0057] Figure 20This is the characteristic spectrum of the garlic formula granule test sample in "1. Specificity Examination" of Example 6 of the present invention;

[0058] Figure 21 This is a characteristic spectrum of the precision experiment in Embodiment 6 of the present invention;

[0059] Figure 22 This is a feature map of the repeatability experiment in Embodiment 6 of the present invention;

[0060] Figure 23 These are characteristic maps of intermediate precision experiments conducted by different personnel in Embodiment 6 of the present invention;

[0061] Figure 24 These are characteristic spectra of intermediate precision experiments using different instruments in Embodiment 6 of the present invention;

[0062] Figure 25 This is a characteristic spectrum of the stability test experiment in Embodiment 6 of the present invention. Detailed Implementation

[0063] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0064] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0065] Instruments and equipment

[0066] Table 1. Statistics on Instruments and Equipment

[0067] Instrument Name model factory One ten-thousandth balance ME104 Mettler Toledo International Trading (Shanghai) Co., Ltd. One-hundred-thousandth balance MS105DU / A Mettler Toledo International Trading (Shanghai) Co., Ltd. One-millionth balance XPR2 Mettler Toledo International Trading (Shanghai) Co., Ltd. CNC ultrasonic instrument KQ-300DB Kunshan Ultrasonic Instruments Co., Ltd. High Performance Liquid Chromatography Waters H-class Waters Corporation High Performance Liquid Chromatography Waters e 2695 Waters Corporation High Performance Liquid Chromatography Agilent 1290 Agilent Technologies Chromatographic column (4.6*150mm, 2.7μm) Poroshell 120Aq-C18 Agilent Technologies Chromatographic column (4.6*150mm, 2.7μm) Poroshell 120EC-C18 Agilent Technologies

[0068] Reagents and reagents

[0069] Table 2. Statistics of Reagents and Test Items

[0070]

[0071] The batch numbers of the 15 batches of garlic standard decoction (freeze-dried powder) are 2103001Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103009Y, 2103010Y, 2103011Y, 2103012Y, 2103013Y, 2103014Y, 2103015Y, 2209001Y, 2209002Y, 2209003Y, 2306001Y, 2306002Y, and 2306003Y.

[0072] The batch numbers of the three batches of garlic formula granules are 2306001Y, 2306002Y, and 2306003Y, respectively.

[0073] The garlic formula granule extract was provided by China Resources Sanjiu Modern Chinese Medicine Pharmaceutical Co., Ltd., and the preparation method is as follows:

[0074] Take an appropriate amount of garlic slices, decoct them twice with water, filter them through a 200-mesh filter cloth, combine the filtrates, concentrate them into a paste, and spray dry them to obtain garlic formula granule extract.

[0075] Example 1

[0076] This embodiment provides a method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparations, including detection by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows: a Poroshell 120Aq-C18 column (4.6 mm × 150 mm, 2.7 μm) is used; methanol is used as mobile phase A, and 0.03 v / v% phosphoric acid solution is used as mobile phase B, with gradient elution as specified in the table below; the flow rate is 0.6 ml per minute; the column temperature is 30 °C; the detection wavelength is 225 nm; and the theoretical plate number, calculated based on the γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​peak, should not be less than 10,000.

[0077] Table 3. Chromatographic conditions

[0078] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 2 98 12～15 2→17 98→83 15～25 17 83 25～30 17→40 83→60 30～35 40 60

[0079] Preparation of reference solution: Take an appropriate amount of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​reference standard, accurately weigh it, and add 25 v / v% methanol aqueous solution to prepare a solution containing 0.1 mg of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​per 1 ml.

[0080] Preparation of the test solution: Weigh approximately 0.1 g of the powder accurately and place it in a stoppered conical flask. Accurately add 10 ml of 25 v / v% methanol aqueous solution, weigh the solution, sonicate for 30 minutes (300 W power, 40 kHz frequency), cool, weigh the solution again, replenish the lost weight with 25% methanol aqueous solution, shake well, filter, and collect the filtrate to obtain the test solution.

[0081] Determination method: Accurately pipette 3 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0082] Fifteen batches of standard garlic decoction (lyophilized powder) and three batches of garlic granules were tested according to the aforementioned detection method for the characteristic chromatograms of garlic medicinal materials or their pharmaceutical preparations. The test results are shown in the table below. Figure 1 This indicates that the relative retention times of each characteristic peak of 15 batches of standard decoction (freeze-dried powder) and 3 batches of garlic formula granules are all within ±10% of the specified values.

[0083] Table 4. Results of relative retention time determination of characteristic spectra of 15 batches of standard garlic decoctions and 3 batches of formulated granules.

[0084]

[0085]

[0086] Table 5. Results of relative peak area determination of characteristic spectra of 15 batches of standard garlic decoctions and 3 batches of formulated granules.

[0087]

[0088]

[0089] The detection results of characteristic chromatograms of multiple batches of test samples were analyzed. The fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission was used, and the "multi-point correction, MARK peak matching" mode was employed to fit and generate control characteristic chromatograms. The obtained HPLC characteristic chromatograms of 15 batches of garlic standard decoction (lyophilized powder) and three batches of formulation granules contained a total of 8 chromatographic peaks (e.g., ...). Figure 2 (As shown). Compare the control characteristic chromatograms of garlic standard decoction (lyophilized powder) and three batches of formulation granules, and the characteristic chromatograms of garlic formulation granule extract (detected according to the above methods, see...). Figure 3 Characteristic spectrum of garlic formula granules (see) Figure 4 The results indicate that the garlic standard decoction, garlic formula granule extract, and garlic formula granule characteristic spectrum have a good correlation, thus demonstrating that the garlic formula granules of the present invention are of qualified quality.

[0090] right Figure 2The S peak in the sample was selected. Literature review revealed that the main active components of garlic include sulfur-containing compounds, polysaccharides, and polyphenols. Garlic polysaccharides possess physiological activities such as liver protection, immune regulation, antioxidation, anticoagulation, myocardial protection, antiviral activity, lipid-lowering, and intestinal flora balance regulation; however, these substances are difficult to detect using HPLC-UV methods. Sulfur-containing compounds in garlic include flavor precursors before cell disruption and flavor components after cell disruption. The main flavor precursors in garlic include γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​(GSPC), γ-glutamyl-S-allyl-L-cysteine ​​(GSMC), γ-glutamyl-5-allyl-L-cysteine ​​(GMAC), and S-alkyl-L-cysteine. After garlic cells are disrupted, enzymatic reactions occur, generating thiosulfinate compounds such as allicin. Compositional analysis of the garlic standard decoction (freeze-dried powder) revealed that it contained almost no allicin (presumably because the high temperature during garlic decoction destroyed the activity of alliinase, preventing allicin formation). However, it was rich in garlic flavor precursors, such as γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​(peak 7) and γ-glutamyl-S-allyl-L-cysteine ​​(peak 4). Among these, γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​(peak 7) was present in the highest concentration. Therefore, γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​was chosen as the reference for the characteristic chromatogram and also as the indicator component for content determination. Thus, the peak corresponding to the γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​reference peak was designated as peak S. The relative retention times of peaks 1-6 and 8 with peak S were calculated.

[0091] right Figure 2 The common chromatographic peaks were identified and located. Through LC-MS / MS analysis and reference standard localization, peak 1 was determined to be L-pyroglutamic acid, peak 2 to be L-tyrosine, peak 3 to be guanosine, peak 5 to be tryptophan, and peak 7 to be γ-glutamyl-S-(trans-1-propenyl)-L-cysteine. Furthermore, through literature review and analysis, the structures and components of peaks 4, 6, and 8 were inferred. The inferred results are: peak 4 to be γ-L-glutamyl-S-allyl-L-cysteine, peak 6 to be γ-glutamyl-5-allyl-L-cysteine, and peak 8 to be γ-glutamyl-phenylalanine. The LC / MS / MS analysis results are shown in the table below. Figure 5 .

[0092] Table 6. LC / MS / MS Analysis Results of Garlic

[0093]

[0094] Note: Because mass spectrometry is incompatible with phosphoric acid solution, 0.05% formic acid solution was used to replace the original 0.03% phosphoric acid solution in the mobile phase during UPLC-Q-TOF-MS analysis. Therefore, the retention time and elution order of each characteristic peak are different from the original spectrum. The compounds corresponding to each characteristic peak were determined by comparison with reference standards and spectral analysis.

[0095] Therefore, in comparison with the characteristic map ( Figure 2 In the above calculation, peak 7 should correspond to the retention time of the reference peak of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine. The peak corresponding to the reference peak of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​is peak S. Calculate the relative retention times of peaks 1 to 6, peak 8, and peak S. The relative retention times should be within ±10% of the specified values, which are 0.28 for peak 1, 0.35 for peak 2, 0.67 for peak 3, 0.86 for peak 4, 0.92 for peak 5, 0.98 for peak 6, and 1.17 for peak 8. Peak 1 corresponds to L-pyroglutamic acid; peak 2 corresponds to L-tyrosine; peak 3 corresponds to guanosine; peak 5 corresponds to tryptophan; and peak 7 corresponds to γ-glutamyl-S-(trans-1-propenyl)-L-cysteine.

[0096] Therefore, the characteristic chromatogram of the test sample should show 8 characteristic peaks, among which peak 7 should correspond to the retention time of the reference peak of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine. The peak corresponding to the reference peak of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​is the S peak. The relative retention times of peaks 1 to 6 and peak 8 with peak S should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are 0.28 (peak 1), 0.35 (peak 2), 0.67 (peak 3), 0.86 (peak 4), 0.92 (peak 5), 0.98 (peak 6), and 1.17 (peak 8). Peak 1 corresponds to L-pyroglutamic acid; peak 2 corresponds to L-tyrosine; peak 3 corresponds to guanosine; peak 5 corresponds to tryptophan; and peak 7 corresponds to γ-glutamyl-S-(trans-1-propenyl)-L-cysteine.

[0097] Furthermore, when using the method of this embodiment to conduct quality testing on garlic medicinal materials or their pharmaceutical preparations, the test sample is tested according to the aforementioned characteristic chromatogram detection method for garlic medicinal materials or their pharmaceutical preparations. The obtained characteristic chromatogram of the test sample is compared with the reference characteristic chromatogram. If at least 16 characteristic peaks identical to those in the reference characteristic chromatogram appear in the characteristic chromatogram of the test sample, the peak corresponding to the reference peak of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​is designated as the S peak. The relative retention times of peaks 1 to 6, peak 8, and the S peak are calculated. If the relative retention times are within ±10% of the specified value, the quality of the garlic medicinal material or its pharmaceutical preparation is qualified; otherwise, it is unqualified. The pharmaceutical preparation includes standard garlic decoction or garlic formula granules. Using the above method for quality testing, the obtained chromatograms provide rich information on identifying chromatographic peaks, more comprehensive detection, and good separation. The operation is also simpler and faster.

[0098] Example 2

[0099] The difference between this embodiment and Example 1 is that the flow rates in the chromatographic conditions were set to 0.55 ml / min, 0.6 ml / min, and 0.65 ml / min, respectively. The same formulation particles (batch number: 2306001Y) were measured, and the relative retention times and relative peak areas of each characteristic peak were calculated. The results showed that the relative retention times of each characteristic peak and the reference peak S were all less than 5%, and the RSD values ​​of the relative peak areas were all less than 10%. This indicates that small variations in flow rate have little impact on the relative retention times of the characteristic peaks, and the detection method has good robustness to different flow rates. Detailed results are shown in the table below. Figures 6-8 .

[0100] Table 7. Comparison of Relative Retention Times for Different Flow Rates

[0101]

[0102]

[0103] Table 8. Comparison of relative peak area results for different flow velocities

[0104]

[0105] Example 3

[0106] The difference between this embodiment and Example 1 is that the column temperature in the chromatographic conditions was set to 28℃, 30℃, and 32℃, respectively, for the detection of the same formulation particles (batch number: 2306001Y). The relative retention time and relative peak area of ​​each characteristic peak were calculated. The results showed that the relative retention time of each characteristic peak and the reference peak S were all less than 5%, and the RSD value of the relative peak area was all less than 10%. This indicates that small changes in column temperature have little effect on the relative retention time of the characteristic peaks, and the detection method has good robustness to column temperature. Detailed results are shown in the table below. Figures 9-11 .

[0107] Table 9. Comparison of relative retention times at different column temperatures

[0108]

[0109] Table 10. Comparison of relative peak areas at different column temperatures

[0110]

[0111] Example 4

[0112] The difference between this embodiment and Example 1 is that the concentration of phosphoric acid solution in mobile phase B was set to 0.02%, 0.03%, and 0.04% in the chromatographic conditions. The same formulation particles were analyzed, and the relative retention times and relative peak areas of each characteristic peak were calculated. The results showed that the RSD values ​​of the relative retention times of each characteristic peak and the reference peak S were all less than 5.98%, but the relative peak area of ​​peak 1 and the reference peak S exceeded 5%. This indicates that even small changes in the acid concentration of the mobile phase have a certain impact on the relative retention times of the characteristic peaks. Therefore, a phosphoric acid concentration of 0.03% is optimal. Detailed results are shown in the following table and... Figures 12-14 .

[0113] Table 11. Comparison of Retention Times with Different Mobile Phase Acid Concentrations

[0114]

[0115] Table 12. Comparison of relative peak areas for different mobile phase acid concentrations

[0116]

[0117] Example 5

[0118] The difference between this embodiment and Example 1 is that the chromatographic columns used in this embodiment are Poroshell 120AQ-C18 2.7μm 150×4.6mm (serial numbers 68 and 70) and Poroshell 120EC-C18 2.7μm 150×4.6mm (serial number 23), respectively. The results show that Poroshell 120Aq-C18 has no significant effect on the characteristic peaks, while replacing it with a Poroshell 120EC-C18 column has little effect on the relative retention times of the characteristic peaks. The results are shown in the following table and... Figures 15-17 .

[0119] Table 13. Comparison of relative retention times for different chromatographic columns

[0120]

[0121]

[0122] Table 14. Comparison of relative peak areas for different chromatographic columns

[0123]

[0124] Example 6 Methodological Validation

[0125] 1. Specificity assessment

[0126] The test solution and reference solution were prepared according to the preparation methods described in Example 1. HPLC analysis was performed under the chromatographic conditions described in Example 1 to investigate the chromatographic conditions and system suitability of the garlic formulation granules, and to examine whether the maltodextrin excipient would cause interference. Chromatograms were recorded. The results are shown in the table below. Figures 18-20 The results show that maltodextrin excipients do not interfere with the characteristic chromatogram of the test sample, and the chromatographic method has good system suitability and specificity, and can be used as a method for detecting the characteristic chromatogram of garlic formulation granules.

[0127] Table 15. Suitability Parameters of Reference Standard System

[0128]

[0129] Table 16. System Suitability Parameters for Test Samples

[0130] Serial Number Retention time Peak area Peak height Theoretical number of plates Tail factor Resolution 1 7.058 35.907 4.639 19985 1.10 2.59 2 8.946 92.012 10.560 24298 1.03 1.61 3 16.539 18.717 3.598 234616 0.99 41.47 4 21.068 63.277 8.070 172778 1.08 26.68 5 22.497 109.578 12.616 150614 1.04 6.57 6 23.956 17.489 1.596 116738 1.12 5.70 7(S) 24.545 280.003 25.583 114161 1.01 2.08 8 28.814 29.117 2.686 159488 1.03 4.73

[0131] 2. Precision Experiment

[0132] A sample solution of the same garlic granule formula (batch number: 2306001Y) was injected six times consecutively under the chromatographic conditions described in Example 1. The relative retention time and relative peak area of ​​each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, and the RSD of the relative peak area of ​​each characteristic peak was less than 5%, indicating good instrument precision. Detailed results are shown in the table below. Figure 21 .

[0133] Table 17. Results of Instrument Precision Relative Retention Time Test

[0134]

[0135] Table 18. Results of Instrument Precision Relative Peak Area Test

[0136]

[0137] 3. Repeatability experiment

[0138] Six test solutions were prepared repeatedly using the same batch of garlic granules (batch number: 2306001Y) according to the test solution preparation method in Example 1. The solutions were then analyzed under chromatographic conditions, and the relative retention times and relative peak areas of each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, and the RSD of the relative peak area of ​​each characteristic peak was less than 5%, indicating good repeatability of the method. Detailed results are shown in the table below. Figure 22 .

[0139] Table 19. Results of Repeatable Relative Retention Time Tests

[0140]

[0141] Table 20. Results of Repeatability Relative Peak Area Test

[0142]

[0143] 4. Intermediate precision

[0144] 4.1 Intermediate precision among different personnel

[0145] Following the sample preparation method in Example 1, three personnel (A, B, and C) each prepared two sample solutions. These solutions were then analyzed on the same instrument under the same chromatographic conditions. The relative retention times and relative peak areas of each characteristic peak were calculated. The results showed that the RSD of the relative retention times of each characteristic peak was less than 2%, and the RSD of the relative peak areas of each characteristic peak was less than 5%, indicating good intermediate precision among different personnel. Detailed results are shown in the table below. Figure 23 .

[0146] Table 21. Results of Intermediate Precision Relative Retention Time Test for Different Personnel

[0147]

[0148] Table 22. Results of Intermediate Precision Relative Peak Area Test for Different Personnel

[0149]

[0150] 4.2 Intermediate precision of different instruments

[0151] Two researchers prepared six test solutions each at different times according to the test solution preparation method in Example 1. The solutions were then analyzed using different instruments. The relative retention time and relative peak area of ​​each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 5%, and the RSD of the relative peak area of ​​each characteristic peak was less than 10%, indicating good intermediate precision across different instruments. Detailed results are shown in the table below. Figure 24 .

[0152] Table 23. Test results of relative retention time for intermediate precision of different instruments

[0153]

[0154]

[0155] Table 24. Results of Intermediate Precision Relative Peak Area Tests for Different Instruments

[0156]

[0157]

[0158] 5. Stability test

[0159] The same sample solution was prepared according to the chromatographic conditions in Example 1 and injected at 0, 2, 4, 8, 12, 18, 24, 36, and 48 hours for analysis. The relative retention time and relative peak area of ​​each characteristic peak were calculated. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 2%, and the RSD values ​​of the relative peak areas of each characteristic peak were all less than 5%, indicating that the sample solution was stable within 48 hours and met the determination requirements. See the table below for details. Figure 25 .

[0160] Table 25. Results of relative retention time tests for stability assessment

[0161]

[0162] Table 26. Results of relative peak area test for stability assessment

[0163]

[0164]

[0165] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation, characterized in that, The detection was performed using high-performance liquid chromatography (HPLC), with the following chromatographic conditions: octadecylsilane-bonded silica gel as the packing material; a column size of 4.6 mm × 150 mm, 2.7 μm; mobile phase A: methanol; mobile phase B: 0.02-0.04 v / v% phosphoric acid solution; detection wavelength: 225 nm; gradient elution program including: From 0 to 12 minutes, the volume ratio of mobile phase A to mobile phase B was 2%:98%. Over 12-15 minutes, the volume ratio of mobile phase A to mobile phase B changed from 2%:98% to 17%:83%. For 15-25 minutes, the volume ratio of mobile phase A to mobile phase B is 17%:83%. After 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 17%:83% to 40%:60%. For 30-35 minutes, the volume ratio of mobile phase A to mobile phase B is 40%:60%. Preparation of the test solution: Take the test sample and add it to the extraction solvent for extraction, filter, and take the filtrate; the extraction solvent is water or 25-75 v / v% methanol solution; In the characteristic spectrum, peak 1 corresponds to L-pyroglutamic acid; peak 2 corresponds to L-tyrosine; peak 3 corresponds to guanosine; peak 5 corresponds to tryptophan; and peak 7 corresponds to γ-glutamyl-S-(trans-1-propenyl)-L-cysteine.

2. The method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation according to claim 1, characterized in that, The chromatographic conditions include at least one of the following conditions: 1) The flow rate is 0.55-0.65 ml / min; 2) Column temperature is 28-32℃; 3) The chromatographic columns were selected from Poroshell 120 AQ-C18 and Poroshell 120 EC-C18; 4) The injection volume is 1-10 μL.

3. The method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation according to claim 1 or 2, characterized in that, In the preparation of the test solution, the extraction time is 15-45 min; And / or, the mass-to-volume ratio of the test sample to the extraction solvent is 0.05-0.4 g / 10 ml.

4. The method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation according to claim 1 or 2, characterized in that, It also includes the preparation of a reference solution: γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​reference standard is added to a solvent to prepare a solution.

5. The method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation according to claim 4, characterized in that, The solvent is water or a 25-75 v / v% methanol solution; And / or, the concentration of γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​in the reference solution is 0.1 mg / ml.

6. The method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation according to any one of claims 1-5, characterized in that, The garlic medicinal preparations include standard garlic decoctions or garlic granules.

7. The method for detecting the characteristic chromatogram of garlic medicinal material or its pharmaceutical preparation as described in any one of claims 1-5 is used in the quality testing of garlic medicinal material or its pharmaceutical preparation.

8. A method for quality testing of garlic medicinal material or its pharmaceutical preparation, characterized in that, include: The method for detecting the characteristic spectrum of garlic medicinal material or its pharmaceutical preparation according to any one of claims 1-5 is used to obtain the characteristic spectrum of the test sample and to compare the characteristic spectrum with the control characteristic spectrum. The control feature spectrum is obtained by fitting the feature spectrum obtained by the detection method of the feature spectrum of garlic medicinal materials or their pharmaceutical preparations according to any one of claims 1-5 using at least a batch of standard garlic medicinal materials or their pharmaceutical preparations.

9. The method for quality testing of garlic medicinal material or its pharmaceutical preparation according to claim 8, characterized in that, The reference characteristic spectrum includes 8 characteristic peaks, among which peak 7 should correspond to the retention time of the γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​reference peak. The peak corresponding to the γ-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​reference peak is the S peak. The relative retention times of peaks 1 to 6, peak 8 and S peak are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are 0.28 for peak 1, 0.35 for peak 2, 0.67 for peak 3, 0.86 for peak 4, 0.92 for peak 5, 0.98 for peak 6 and 1.17 for peak 8.

10. The method for quality testing of garlic medicinal material or its pharmaceutical preparation according to any one of claims 8-9, characterized in that, The garlic medicinal preparations include standard garlic decoctions or garlic granules.

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