A quantitative detection method for external use pain relief ointment

Abstract

The present invention relates to a quantitative detection method of an externally applied pain-relieving plaster for traumatic injuries. The detection method processes a test sample by a suitable pretreatment method, and can effectively elute and separate impurities such as rubber in the externally applied pain-relieving plaster for traumatic injuries, thereby better protecting a liquid chromatography column, improving the durability of the chromatographic column, and greatly reducing the inspection cost. At the same time, a series of research and optimization are conducted on the detection chromatographic conditions of the effective ingredient gardenia glycoside in the detection method, effectively realizing the quantitative detection of gardenia glycoside in the externally applied pain-relieving plaster for traumatic injuries by high performance liquid chromatography, and proposing a method for quantitatively detecting the externally applied pain-relieving plaster for traumatic injuries with strong specificity, and the method has good detection accuracy, high sensitivity, good peak shape of the detection result, and few impurities. Thus, the quality control of the externally applied pain-relieving plaster for traumatic injuries can be better realized, which is beneficial to the safety of the drug.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a quantitative detection method for an external traumatic analgesic plaster. Background Art

[0002] The external analgesic ointment for traumatic injuries is a compound preparation of traditional Chinese medicine. The main ingredients in the prescription include: earthworm, Dipsacus asper, Saposhnikovia divaricata, Dragon bone, Strychnos nux vomica, Sanguisorba officinalis, Gardenia jasminoides, Taraxacum officinale, Scutellaria baicalensis, Honeysuckle, Polygonum cuspidatum, Rhubarb, Myrrh, Catechu, Safflower, Hibiscus, Drynaria fortunei, menthol, methyl salicylate, borneol, and camphor, a total of 22 herbs. Its functions and indications: promote bone healing, promote blood circulation and remove blood stasis, reduce swelling, and relieve pain.

[0003] As an important Chinese medicine compound preparation, the quality inspection of external-use pain-relieving plaster is very important. However, the current quality inspection standard for external-use pain-relieving plaster is only contained in the National Drug Standard WS3-B-3796-98-2016 of the State Food and Drug Administration, and the standard only contains the items of [properties], [identification], and [volatile oil], and there is no content determination item. In addition, as a rubber plaster, the external-use pain-relieving plaster has complex components and high viscosity, which makes it difficult to separate and control the quality. In particular, it contains a large amount of rubber matrix, which is composed of rubber, rosin, vaseline, liquid paraffin and other substances. It is difficult to extract its effective ingredients using conventional extraction methods, and there is currently no solution to control its quality based on the content of a certain effective ingredient.

[0004] Therefore, it is urgent to provide a method that can carry out quantitative detection for external-use traumatic analgesic ointment.

[0005] Based on this, the inventors found in their previous studies that the main drug dragon bone, earthworm and the auxiliary drug myrrh had no content determination items for effective ingredient detection. When studying the content determination method of the auxiliary drug safflower, it was found that when the preliminary test of the content determination of kaempferol in safflower was carried out, the test results also failed to detect kaempferol. Then, the content determination of the adjuvant catechu in the prescription was studied and it was found that when the content of catechin and epicatechin was determined, the content of catechin in the finished product was high, but the content of epicatechin was very low in the test results. It may be that the two were converted during the preparation of this product or the preparation of the test solution. The preparation and concentration of the test sample could not be taken into account at the same time, and the mobile phase of this method was not stable enough, so the content determination of catechu in the finished product could not be used as a method that can quantitatively detect the external traumatic analgesic plaster. Summary of the invention

[0006] Based on this, one of the objects of the present invention is to provide a quantitative detection method for external traumatic analgesic ointment.

[0007] Including the following technical solutions:

[0008] A quantitative detection method for external-use pain-relieving plaster, which uses high performance liquid chromatography to detect the external-use pain-relieving plaster test solution obtained by pre-treatment;

[0009] The pretreatment of the external analgesic plaster for traumatic injuries is as follows: taking the external analgesic plaster for traumatic injuries, removing the cover lining, cutting into strips, mixing to obtain the mixed external analgesic plaster for traumatic injuries, adding ethanol for extraction, heating and refluxing for 60 minutes to 180 minutes, supplementing the lost weight with ethanol, shaking, filtering, and obtaining a filtrate.

[0010] In order to find a method capable of quantitatively detecting an external-use pain-relieving plaster for traumatic injuries, the present invention detects multiple components in the external-use pain-relieving plaster for traumatic injuries and, based on the dosage form characteristics of the external-use pain-relieving plaster for traumatic injuries, finds that in the preparation of a test solution of the external-use pain-relieving plaster for traumatic injuries, a suitable pretreatment method is used to treat the test sample, so that impurities such as rubber in the external-use pain-relieving plaster can be effectively eluted and separated, thereby better protecting a liquid chromatography column and improving the durability of the chromatography column; moreover, the inventor finds that when acetonitrile-water (11:89) is used, a peak of gardenia glycoside and a peak of impurities in a chromatogram of the test solution can be well separated, and when the entire detection method is coordinated in an orderly manner, the quantitative detection of gardenia glycoside in the external-use pain-relieving plaster for traumatic injuries by high-performance liquid chromatography is effectively realized, thereby achieving a method with strong specificity and capable of quantitatively detecting the external-use pain-relieving plaster for traumatic injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the result of HPLC detection using chromatographic column No. 1 in Example 3, wherein A is the gardenia glycoside reference substance, B is the test substance (batch number: XC006), and C is the negative control substance lacking gardenia.

[0012] Figure 2 This is the result of HPLC detection using chromatographic column II in Example 3, wherein A is the gardenia glycoside reference substance, B is the test substance (batch number: XC006), and C is the negative control substance lacking gardenia.

[0013] Figure 3 The figure is the result of HPLC detection using chromatographic column III in Example 3, wherein A is the gardenia glycoside reference substance, B is the test substance (batch number: XC006), and C is the negative control substance lacking gardenia.

[0014] Figure 4 The specificity verification test results of Example 4 were obtained by using the detection method of Example 1 of the present invention, wherein A is the Gardenia glycoside reference substance, B is the test substance (Batch No.: XC006), and C is the negative control substance lacking Gardenia jasminoides.

[0015] Figure 5 This is a standard curve diagram of the linear detection results using the detection method of Example 1 of the present invention in Example 4.

[0016] Figure 6 This is the detection result when the ratio of acetonitrile to water is 15:85 according to the detection method of Example 1.

[0017] Figure 7 Schematic diagram of the comparison results of different elution solvents in Example 2, wherein 1 is the gardenoside control solution, 2 is the test solution 1, 3 is the test solution 2, 4 is the test solution 3, and 5 is the test solution 4. DETAILED DESCRIPTION

[0018] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0019] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0020] Throughout the specification and claims, the following terms have the meanings clearly associated with this document unless the context clearly dictates otherwise. The phrase "in one embodiment" used in the present invention does not necessarily refer to the same embodiment, although it may be. In addition, the phrase "in another embodiment" used in the present invention does not necessarily refer to a different embodiment, although it may be. Therefore, as described below, the various embodiments of the present invention can be easily combined without departing from the scope or spirit of the present invention.

[0021] When the technicians of the present invention conducted in-depth research on the detection method of the external pain-relieving plaster for traumatic injuries, they found that when the preparation of the test solution and the chromatographic conditions were optimized closely around the recovery rate of gardenia glycoside, the obtained method had good detection accuracy and high sensitivity, good peak shape of the detection result, and few impurities, which could better achieve the quality control of the external pain-relieving plaster for traumatic injuries and was beneficial to the safety of the drug.

[0022] Some embodiments of the present invention provide a quantitative detection method for external traumatic analgesic plaster, which uses high performance liquid chromatography to detect the external traumatic analgesic plaster sample solution obtained by pre-treatment; the pre-treatment of the external traumatic analgesic plaster is: taking the external traumatic analgesic plaster, removing the cover lining, cutting it into strips, mixing it, adding an extracting solution for extraction, filtering, and obtaining a filtrate. The extracting solution includes ethanol, and the extraction method is to heat the external traumatic analgesic plaster for 30 minutes to 300 minutes, more preferably, 60 minutes to 180 minutes.

[0023] This detection method has good specificity and high sensitivity, and can better achieve quality control of external analgesic ointment, which is beneficial to drug safety.

[0024] In some embodiments, the preparation steps of the above-mentioned filtrate are as follows: take several pieces of external-use analgesic plaster, remove the cover lining, cut into strips, mix, and accurately weigh as a test sample; add accurately weighed ethanol, heat and reflux, take out and cool, and make up the lost weight with ethanol, shake well, and filter with dry filter paper to obtain.

[0025] In some embodiments, the above-mentioned external pain relief plaster for traumatic injuries refers to a plaster suitable for promoting bone healing, promoting blood circulation and removing blood stasis, reducing inflammation, reducing swelling and relieving pain. It is used for external plasters for closed fractures, sprains and contusions and other orthopedic diseases and rheumatoid arthritis, and further refers to an external plaster containing gardenia glycoside as an effective ingredient, preferably Wuyang brand pain relief plaster for traumatic injuries.

[0026] In some embodiments, the external traumatic pain relief plaster is heated and refluxed with ethanol for 60 to 180 minutes. More preferably, the heating and reflux time is within 120 minutes, at which time the content of geniposide in the sample extract increases with time.

[0027] In some of the embodiments, the ratio of the added ethanol to the mixed external traumatic analgesic plaster is 50ml:2g-3g, preferably 50ml:2.g-2.6g, and more preferably 50ml:2.5g.

[0028] In some of the embodiments, the preparation of the above-mentioned external analgesic plaster test solution also includes a purification step, and further, the purification step is enrichment and purification through an alumina column.

[0029] In some of the embodiments, 10 ml of the filtrate is loaded onto an alumina chromatography column and eluted with 25-35 ml of ether, 45-55 ml of ethanol, and 45-55 ml of 20±1% ethanol in sequence, the 20±1% ethanol eluate is collected and evaporated to dryness, the residue is dissolved in methanol solution, transferred to a 5 ml volumetric flask and diluted to the mark, shaken to obtain the product.

[0030] In some embodiments, the methanol concentration in the methanol solution is 15%-25% by volume, and more preferably 20% by volume.

[0031] Ether can be used to remove impurities with low polarity and residual rubber, petrolatum and other oil-soluble impurities, and ethanol is then used to elute the impurities with high polarity and water-soluble impurities. Finally, 50 ml of 20% ethanol is used for elution, which can achieve the effect of complete elution of gardenia glycoside.

[0032] During the study, it was found that Gardenia glycoside has a high polarity and is easily adsorbed by neutral alumina. The use of a solvent with a high polarity for elution results in a small volume of elution solvent and a relatively complete elution. The detection method of the present invention is examined based on the recovery rate.

[0033] In some embodiments, the alumina column used in the preparation of the test solution is a neutral alumina column, the inner diameter of the column is 2.0±0.2 cm, the filler particle size is 100-200 mesh, and the filler amount is 5-15 g. Selecting the appropriate alumina column in combination with the appropriate elution method described in the present invention can remove impurities such as acids, flavonoids, phenols, tannins, etc. to the greatest extent.

[0034] In some embodiments, the mobile phase of the HPLC method is acetonitrile-water solution, and further, acetonitrile-water (11:89) is used as the mobile phase.

[0035] In some embodiments, the chromatographic conditions of the above-mentioned high performance liquid chromatography include:

[0036] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0037] Detection wavelength: 238nm;

[0038] Injection volume: 10 μl;

[0039] Flow rate: 1.0ml / min;

[0040] Column temperature: 20-30°C;

[0041] The theoretical plate number calculated based on the Gardenia jasminoides peak should be no less than 10,000.

[0042] In some embodiments, the chromatographic column of the HPLC method is selected from any one of Kromasil 100-5C18, Agilent Eclipse XDB-C18, and Hypersil ODS2.

[0043] Compared with other methods, the detection method of the present invention does not use toxic reagents, uses less reagents, and has good prospects for practical application and promotion.

[0044] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0045] The present invention is further described in detail below with reference to specific embodiments.

[0046] Example 1

[0047] Instruments and reagents

[0048] Instrument: Waters 2695 liquid chromatograph;

[0049] Reagents: Acetonitrile was of chromatographic grade, and other reagents were of analytical grade.

[0050] Reference substance: Gardenia jasminoides (batch number 110749-200714, for content determination), purchased from China Food and Drug Inspection Institutes.

[0051] Test samples: Wuyang brand analgesic ointment, batch numbers: VC011, VC003, UC05, UC02, TC04, XC005, VC004, UC04, UC03, TC03, XC006.

[0052] Gardenia glycoside negative control: According to the prescription and preparation method of this product, a negative control sample lacking Gardenia jasminoides was prepared. Take an amount equivalent to the test sample and prepare a negative control solution according to the preparation method of the test sample solution.

[0053] Chromatographic conditions:

[0054] Chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as filler;

[0055] Mobile phase: acetonitrile-water, isocratic elution, initial ratio (11:89).

[0056] Flow rate: 1.0 ml / min; column temperature: 20-30°C; injection volume: 10 μl; detection wavelength: 238 nm; theoretical plate number calculated based on the gardenia glycoside peak is not less than 10,000.

[0057] Preparation of reference solution: Accurately weigh the Gardenia glycoside reference substance and add 20% methanol to make a solution containing 50 μg per 1 ml.

[0058] Preparation of test solution:

[0059] Take 10 pieces of external pain relief plaster (each piece is larger than 35cm 2 Should be cut into 35cm 2), remove the cover lining, accurately weigh, cut into strips, mix, take about 2.5g, accurately weigh, accurately add 50ml of ethanol, weigh the weight, heat and reflux for 2 hours, take out, cool, weigh again, make up the lost weight with ethanol, shake well, filter, accurately measure 10ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 10g, inner diameter 2.0cm), elute with 30ml of ether, 50ml of ethanol, and 50ml of 20% ethanol in turn, collect the 20% ethanol eluate, evaporate to dryness, add 20% methanol to dissolve the residue, transfer to a 5ml volumetric flask and make up to the scale, shake well, and you have it.

[0060] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. 2 Contains Gardenia jasminoides and geniposide (C 17 H 24 O 10 ) shall not be less than 0.70 mg.

[0061] The test sample should show a chromatographic peak with the same retention time as the chromatographic peak of the gardenia glycoside reference substance; and the chromatographic peak area of ​​the test sample should not be less than the chromatographic peak area of ​​the gardenia glycoside reference substance.

[0062] The inventors found in experiments that when the ratio of acetonitrile to water was 15:85, the peak of Gardenia glycoside in the chromatogram of the test solution contained impurity peaks (see Figure 6 ), and when the inventors adjusted the ratio to acetonitrile-water (11:89), it was found that the peak of gardenia glycoside and the peak of impurities in the chromatogram of the test solution could be well separated (see Figure 1-4 ).

[0063] Example 2 Optimization of test solution preparation process

[0064] 1.1 Comparison of extraction solvents and time

[0065] Take this product (batch number: XC006), remove the cover lining, cut into strips, take about 2.5g, take 2 portions each time, accurately weigh, accurately add (Group 1: 50ml of ethanol; Group 2: 50ml of 75% ethanol; Group 3: 30ml of ethanol; Group 4: 50ml of 50% ethanol) and weigh the weight, heat and reflux for 2 hours respectively, take out, cool, weigh again, make up the lost weight with ethanol, shake well, filter, accurately measure 10ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 10g, inner diameter 2.0cm), elute with 30ml of ether, 50ml of ethanol, and 50ml of 20% ethanol in turn, collect the 20% ethanol eluate, evaporate to dryness, add 20% methanol to dissolve the residue, transfer to a 5ml volumetric flask and make up to the scale, shake well, and obtain.

[0066] The test results are summarized in Table 1 below.

[0067] Table 1 Comparison of different extraction solvents (n=2)

[0068]

[0069] Result description: Using 50ml of ethanol as the extraction solvent, the content of gardenia glycoside is the highest and the detection effect is the best.

[0070] 1.2 Comparison of extraction time

[0071] Take this product (batch number: XC006), remove the cover lining, cut into strips, take about 2.5g, take 2 portions each time, accurately weigh, accurately add 50ml of ethanol respectively, weigh the weight, heat and reflux for 30min, 1 hour, 2 hours, and 3 hours respectively, take out, cool, weigh again, make up the lost weight with ethanol, shake well, filter, accurately measure 10ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 10g, inner diameter 2.0cm), elute with 30ml of ether, 50ml of ethanol, and 50ml of 20% ethanol in turn, collect the 20% ethanol eluate, evaporate to dryness, add 20% methanol to dissolve the residue, transfer to a 5ml volumetric flask and make up to the scale, shake well, and obtain.

[0072] The test results are summarized in Table 2 below. The content of gardeniaposide increases with time when the heating reflux time is within 120 minutes, but there is no significant difference between the content of gardeniaposide measured by heating reflux for 180 minutes and that measured by heating reflux for 120 minutes, so the heating reflux time is selected as 120 minutes (2 hours).

[0073] Table 2 Comparison of extraction time (n=2)

[0074]

[0075] 1.3 Comparison of elution solvent volumes

[0076] Take this product (batch number: XC006), remove the cover lining, cut into strips, take about 2.5g, accurately weigh, accurately add 50ml of ethanol, weigh the weight, heat and reflux for 2 hours, take out, cool, weigh again, make up the lost weight with ethanol, shake well, and filter.

[0077] Group 1: Accurately measure 10 ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 10 g, inner diameter 2.0 cm), and elute with 30 ml of ether, 50 ml of ethanol, and 50 ml of 20% ethanol in sequence; collect the final eluate of each group, evaporate to dryness, add 20% methanol to dissolve the residue, transfer the eluted portion to a 5 ml volumetric flask and make up to the scale.

[0078] Then elute with 30 ml of 20% ethanol, collect the last eluent, evaporate to dryness, dissolve the residue in 1 ml of methanol, transfer to a 1 ml volumetric flask and dilute to the mark for later use (test solution 1).

[0079] Group 2: Accurately measure 10 ml of the filtrate, add it to a neutral alumina column (same as Group 1), and elute with 30 ml of ether, 50 ml of ethanol, and 50 ml of 50% ethanol in sequence; collect the final eluate of each group, evaporate to dryness, add 20% methanol to dissolve the residue, transfer the eluted portion to a 5 ml volumetric flask and make up to the scale.

[0080] Then elute with 30 ml of 20% ethanol, collect the last eluent, evaporate to dryness, dissolve the residue in 1 ml of methanol, transfer to a 1 ml volumetric flask and dilute to the mark for later use (test solution 2).

[0081] Group 3: Accurately measure 10 ml of the filtrate, add it to a neutral alumina column (same as Group 1), and elute with 30 ml of ether, 50 ml of 20% ethanol, and 50 ml of ethanol in that order; collect the final eluate of each group, evaporate to dryness, add 20% methanol to dissolve the residue, transfer the eluted portion to a 5 ml volumetric flask and make up to the mark.

[0082] Then elute with 30 ml of 20% ethanol, collect the last eluent, evaporate to dryness, dissolve the residue in 1 ml of methanol, transfer to a 1 ml volumetric flask and dilute to the mark for later use (test solution 3).

[0083] Group 4: Accurately measure 10 ml of the filtrate and add it to a neutral alumina column (same as group 1), and elute with 30 ml of ether, 50 ml of ethanol, and 45 ml of 20% ethanol in sequence.

[0084] The final eluate was evaporated to dryness, and the residue was dissolved in 20% methanol. The eluted portion was transferred to a 5 ml volumetric flask and made up to the mark.

[0085] Then elute with 30 ml of 20% ethanol, collect the last eluent, evaporate to dryness, dissolve the residue in 1 ml of methanol, transfer to a 1 ml volumetric flask and dilute to the mark for later use (test solution 4).

[0086] Take an appropriate amount of Gardenia glycoside (batch number: 110749-201718) reference substance, add methanol to make a solution containing 1 mg of this component per 1 ml as the reference solution. Take 1ul of the reference substance and 2ul of the test solution of group 1, group 2, group 3, and group 4 respectively, and spot them on the same silica gel H thin layer plate, use butyl acetate-formic acid-water (7:2.5:2.5 upper layer solution as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm).

[0087] Results: Under the same conditions, the aluminum hydroxide columns of Group 1 and Group 4 were eluted with 30 ml of 20% ethanol respectively, and no gardeniain was detected in the elution area. However, under the same conditions, the aluminum hydroxide columns of Group 2 and Group 3 were eluted with 30 ml of 20% ethanol respectively, and a small amount of gardeniain was still detected in the elution area. Figure 7 .

[0088] The results showed that the third use of 20% ethanol 50ml peroxide aluminum column could completely elute.

[0089] No gardeniain was detected in the elution area with 45 ml of 20% ethanol, indicating that 50 ml of 20% ethanol can completely elute it.

[0090] Example 3

[0091] (1) Comparison between degreasing with ether and not degreasing:

[0092] Degreasing by adding ether: Take this product (batch number: XC006), remove the cover lining, take about 2.5g, accurately weigh, add 30ml of ether, shake lightly, pour out the supernatant after standing, repeat the operation 3 times, evaporate the ether, accurately add 50ml of ethanol, weigh the weight, heat and reflux for 2 hours, take out, cool, weigh again, make up the lost weight with ethanol, shake well, filter, accurately measure 10ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 10g, inner diameter 2.0cm), elute with 30ml of ether, 50ml of ethanol, and 50ml of 20% ethanol in turn, collect the 20% ethanol eluate, evaporate to dryness, add 20% methanol to dissolve the residue, transfer to a 5ml volumetric flask and make up to the scale, shake well, and you have it.

[0093] Without defatting: Take this product (batch number: XC006), remove the cover lining, take about 2.5g, accurately weigh, accurately add 50ml of ethanol, weigh the weight, heat and reflux for 2 hours, take out, cool, weigh again, make up the lost weight with ethanol, shake well, filter, accurately measure 10ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 10g, inner diameter 2.0cm), elute with 30ml of ether, 50ml of ethanol, and 50ml of 20% ethanol in sequence, collect the 20% ethanol eluate, evaporate to dryness, add 20% methanol to dissolve the residue, transfer to a 5ml volumetric flask and make up to the scale, shake well, and obtain.

[0094] The test results are summarized in Table 3 below. There is no significant difference between the two, so the preparation method without degreasing is adopted.

[0095] Table 3 Comparison of degreasing with ether and without degreasing

[0096] Degreasing with ether (n=2) No skimming (n=1) Gardenia jasminoides content (mg / g) 0.48 0.48

[0097] The inventors found in the experiment that the test results showed that the content of gardenia glycoside was the same and there was no significant difference between the two methods when degreasing with ether and degreasing without ether, and the experiment could achieve better results without performing this step. The main reason was that in the subsequent operation steps, ether was first used for elution when passing through the neutral alumina column, which could also remove fat-soluble impurities. Therefore, the previous operation steps adopted the preparation method without degreasing.

[0098] (2) Comparison of different chromatographic columns

[0099] Two portions of one batch of samples (batch number: XC006) were taken and determined according to the content determination method using three brands of chromatographic columns: No. Ⅰ: Kromasil100-5C18 (200×4.6mm), column number: E103072; No. Ⅱ: Agilent Eclipse XDB-C18 5μm (4.6×150mm); column number: 993967-902; No. Ⅲ: Elite Hypersil ODS2 (5μm4.6mm×150mm), column number: E2523888.

[0100] Table 4 Determination results of different chromatographic columns

[0101] batch number Column Ⅰ content Column II content Column III content average value RSD% XC006 0.494mg / g 0.507mg / g 0.510mg / g 0.50mg / g 1.7

[0102] Combined with Table 4 and the test results Figure 1-4 It can be seen that the separation degrees of the three brands of chromatographic columns all reached above 1.5, the theoretical plate numbers reached above 10,000, and RSD=1.7%.

[0103] It can be seen from the above experimental results that the pretreatment method described in the present application can obtain good test samples without the need for an additional defatting step, and the obtained test samples have strong adaptability to the chromatographic column.

[0104] Example 4 Performance study of the detection method described in Example 1 of the present invention

[0105] (1) Accuracy

[0106] Take 6 samples of the same batch of known content (batch number: XC006, gardenia glycoside content 0.49 mg / g), each sample size is about 1.25g, accurately add gardenia glycoside reference substance (0.625mg) at a ratio of 1:1 of the current sampling content, and determine according to the content determination method to calculate the recovery rate of gardenia glycoside. Results: The average recovery rate of gardenia glycoside is 95.4%, RSD = 0.6% (see Table 5).

[0107] Table 5 Gardenia glycoside accuracy test results (n = 6)

[0108]

[0109]

[0110] (2) Repeatability

[0111] Six samples of the same batch of test products (batch number: XC006) were taken and tested according to the content determination method. The results were sorted and statistically analyzed as shown in Table 6 below, indicating that the test repeatability was good.

[0112] Table 6 Repeatability test results (n=6)

[0113]

[0114] (3) Exclusivity

[0115] The specificity of detecting the content of geniposide in external traumatic analgesic ointment by the method described in Example 1 of the present invention was studied.

[0116] 1. Instruments and reagents

[0117] Instrument: Waters 2695 liquid chromatograph;

[0118] Reference substance: Gardenia jasminoides (batch number 110749-200714, for content determination), purchased from China Food and Drug Inspection Institutes.

[0119] Reagents: Acetonitrile was of chromatographic grade, and other reagents were of analytical grade.

[0120] Chromatographic conditions:

[0121] Chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as filler;

[0122] Mobile phase: acetonitrile-water, isocratic elution, initial ratio (11:89).

[0123] Flow rate: 1.0 ml / min; column temperature: 20℃-30℃; injection volume: 10 μl; detection wavelength: 238 nm; theoretical plate number calculated based on the gardenia glycoside peak is not less than 10,000.

[0124] 2. Sample preparation

[0125] Reference solution:

[0126] Accurately weigh the Gardenia glycoside reference substance and add 20% methanol to prepare a solution containing 50 μg per 1 ml.

[0127] Test solution:

[0128] Take 10 pieces of external analgesic plaster (each piece with an area greater than 35cm2 should be cut into 35cm2), remove the cover lining, accurately weigh, cut into strips, mix, take about 2.5g, accurately weigh, accurately add 50ml of ethanol, weigh the weight, heat and reflux for 2 hours, take out, cool, weigh again, make up the lost weight with ethanol, shake well, filter, accurately measure 10ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 10g, inner diameter 2.0cm), elute with 30ml of ether, 50ml of ethanol, and 50ml of 20% ethanol in turn, collect the 20% ethanol eluate, evaporate to dryness, add 20% methanol to dissolve the residue, transfer to a 5ml volumetric flask and make up to the scale, shake well, and obtain.

[0129] Accurately weigh the negative control substance lacking gardenia glycoside, add 20% methanol to make a solution containing 50 μg per 1 ml.

[0130] Accurately pipette 10 μl of each reference solution, test solution and negative control solution, inject them into the liquid chromatograph, and determine according to the content determination method. Results In the negative control solution, there is no chromatographic peak interference at the corresponding position of the gardenia glycoside reference substance (see Figure 4 ).

[0131] The results of the above-mentioned methodological validation test showed that the established test method had no effect on the determination within a small range of variation and was suitable for the determination of the content of Gardenia jasminoides in this product.

[0132] (4) Linear investigation

[0133] 1 μl, 2 μl, 8 μl, 14 μl, 20 μl, 26 μl and 32 μl of the 0.0517 mg / ml Gardenia glycoside reference solution were accurately drawn and injected into a liquid chromatograph. The standard curve was plotted with the injection amount of the reference solution (μg) as the abscissa and the measured peak area as the ordinate. The regression equation of Gardenia glycoside was obtained as follows: A=1535.8953x-1.4580, r=0.99999. The results showed that the injection amount of the Gardenia glycoside reference solution between 0.0517 μg and 1.6544 μg had a good linear relationship with its peak area (see Table 7, Figure 5 ).

[0134] Table 7 Linearity test results

[0135]

[0136] Example 5 Determination of content limit of test sample

[0137] Instruments and reagents

[0138] Instrument: Waters 2695 liquid chromatograph;

[0139] Reagents: Acetonitrile was of chromatographic grade, and other reagents were of analytical grade.

[0140] Reference substance: Gardenia jasminoides (batch number 110749-200714, for content determination), purchased from China Food and Drug Inspection Institutes.

[0141] Test samples: Wuyang brand analgesic ointment, batch numbers: VC011, VC003, UC05, UC02, TC04, XC005, VC004, UC04, UC03, TC03, XC006.

[0142] Chromatographic conditions:

[0143] Chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as filler;

[0144] Mobile phase: acetonitrile-water, isocratic elution, initial ratio (11:89).

[0145] Flow rate: 1.0 ml / min; column temperature: 20℃-30℃; injection volume: 10 μl; detection wavelength: 238 nm; theoretical plate number calculated based on the gardenia glycoside peak is not less than 10,000.

[0146] Preparation of reference solution: Accurately weigh the Gardenia glycoside reference substance and add 20% methanol to make a solution containing 50 μg per 1 ml.

[0147] Preparation of test solution:

[0148] Take 10 pieces of external pain relief plaster (each piece is larger than 35cm 2 Should be cut into 35cm 2 ), remove the cover lining, accurately weigh, cut into strips, mix, take about 2.5g, accurately weigh, accurately add 50ml of ethanol, weigh the weight, heat and reflux for 2 hours, take out, cool, weigh again, make up the lost weight with ethanol, shake well, filter, accurately measure 10ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 10g, inner diameter 2.0cm), elute with 30ml of ether, 50ml of ethanol, and 50ml of 20% ethanol in turn, collect the 20% ethanol eluate, evaporate to dryness, add 20% methanol to dissolve the residue, transfer to a 5ml volumetric flask and make up to the scale, shake well, and you have it.

[0149] Determination of content limit: Calculated according to the prescription of this product, every 100cm 2 Contains 0.06433g of Gardenia jasminoides. According to the limit of "Content Determination" in the 2010 edition of the Chinese Pharmacopoeia, the content of geniposide in Gardenia jasminoides should be no less than 1.8%. The content in the finished product should be calculated based on the transfer rate of 100%, which should be per 100cm 2Contains not less than 1.15794 mg of Gardenia glycoside. According to the measured value, considering the loss in production and inspection, it is proposed that this product should be 2 Contains Gardenia jasminoides and geniposide (C 17 H 24 O 10 ) shall not be less than 0.70 mg.

[0150] Eleven batches of the above test samples were taken and tested according to the above content limit test method. The results are shown in Table 8 below.

[0151] Table 8 Test sample content determination results

[0152]

[0153]

[0154] Combined with the test data in Table 8 above, it can be seen that among the 11 batches of external traumatic analgesic ointment samples, every 100cm 2 Contains Gardenia jasminoides and geniposide (C 17 H 24 O 10 ) were all greater than 0.70 mg, meeting the requirements for content limit determination.

[0155] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A quantitative detection method for external traumatic analgesic ointment, characterized in that: The HPLC method was used to detect the Gardenia glycoside in the pre-treated external pain relief ointment test solution. The pretreatment of the external pain-relieving plaster for traumatic injuries is as follows: taking the external pain-relieving plaster for traumatic injuries, removing the cover lining, cutting into strips, mixing, obtaining the mixed external pain-relieving plaster for traumatic injuries, adding ethanol for extraction, heating and refluxing for 60 minutes to 180 minutes, supplementing the lost weight with ethanol, shaking, filtering, obtaining a filtrate; the ratio of the added ethanol to the mixed external pain-relieving plaster for traumatic injuries is 50 ml: 2.5 g; The preparation of the external-use pain-relieving plaster test solution also includes enrichment and purification by an alumina column, wherein the alumina column enrichment and purification includes: loading 10 ml of the filtrate onto an alumina chromatography column, eluting with 25-35 ml of ether, 45-55 ml of ethanol, and 45-55 ml of 20±1% ethanol in sequence, collecting the 20±1% ethanol eluate, evaporating to dryness, dissolving the residue in methanol solution, transferring to a 5 ml volumetric flask and diluting to the mark, shaking well, and obtaining the product; The alumina column used in the preparation of the test solution is a neutral alumina column with an inner diameter of 2.0±0.2 cm, a filler particle size of 100-200 mesh, and a filler amount of 8-12 g; The mobile phase of the high performance liquid chromatography is an acetonitrile aqueous solution, with the ratio of acetonitrile to water being 11:89 as the mobile phase.

2. The quantitative detection method according to claim 1, characterized in that: The external analgesic plaster was heated and refluxed with ethanol for 90 to 140 minutes.

3. The quantitative detection method according to claim 1, characterized in that: In the preparation of the test solution, the elution volumes of ether, ethanol and 20% ethanol are 30 ml, 50 ml and 50 ml respectively.

4. The quantitative detection method according to claim 3, characterized in that: The methanol concentration in the methanol solution is 15%-25% by volume.

5. The quantitative detection method according to claim 4, characterized in that: The methanol concentration in the methanol solution is 20% by volume.

6. The quantitative detection method according to claim 1, characterized in that: The alumina column used in the preparation of the test solution is a neutral alumina column with an inner diameter of 2.0 cm, a filler particle size of 100-200 meshes, and a filler amount of 10 g.

7. The quantitative detection method according to any one of claims 1 to 6, characterized in that: The chromatographic conditions of the high performance liquid chromatography include: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Detection wavelength: 238nm; Injection volume: 10 μl; Flow rate: 1.0ml / min; Column temperature: 20℃-30℃; The theoretical plate number calculated based on the Gardenia jasminoides peak should be no less than 10,000.

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