Methods for determining the content of unsaturated fatty acids in Sichuan pepper and its preparations

Abstract

This invention relates to the field of traditional Chinese medicine detection technology, specifically providing a method for determining the content of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations. The chromatographic column is an ACQUITY UPLC CSH column. TM C18, using an aqueous mobile phase consisting of acetonitrile and acetic acid, employs gradient elution. Extensive experimental screening has shown that using acetonitrile and acetic acid as the mobile phase, under specific elution procedures and with a specific chromatographic column, the separation of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations is achieved. The peaks exhibit good symmetry, without shoulder peaks, bifurcation peaks, or peak engulfment. Liquid chromatography is used to determine the chromatograms of the test solution and standard solution, respectively, and the content of unsaturated fatty acids in the test solution can be calculated. This allows for accurate determination of unsaturated fatty acid content and enables quality control.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically to a method for determining the content of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations. Background Technology

[0002] Sichuan pepper seeds, also known as pepper mortars, are the mature, dried seeds of Sichuan pepper. They are nearly round, black, smooth, and glossy. Sichuan pepper seeds are classified as a warming medicine, with pungent and warm properties. They enter the spleen, stomach, and kidney meridians and have the effects of warming the middle jiao to relieve pain, killing parasites and relieving itching, and promoting diuresis and reducing swelling.

[0003] Sichuan pepper seeds can be used clinically to treat the following conditions: 1. This herb can be used to treat spleen and stomach cold syndrome. For abdominal pain and vomiting caused by spleen and stomach deficiency and cold, it can be used in combination with dried ginger and codonopsis. For abdominal pain, vomiting, and diarrhea caused by cold and dampness obstructing the middle jiao, it can be used in combination with atractylodes and magnolia bark. 2. This herb can be used to treat abdominal pain due to ascariasis, eczema, itching, vulvar itching, and other diseases. For abdominal pain due to intestinal parasites and cold extremities, it can be used in combination with dried plum and phellodendron. For eczema, itching, and vulvar itching, it can be used in combination with sophora flavescens and phellodendron. When using this herb, it is important to note that it is pungent, hot, and drying; the dosage should not be too large, and prolonged use can easily damage yang and promote fire. It is not suitable for patients with excess heat syndrome, yin deficiency with fire excess, or deficiency of body fluids and blood. Pregnant patients should use it with caution.

[0004] Zanthoxylum is not listed in the Chinese Pharmacopoeia, but it is included in the Zhejiang Provincial Standards for Processing Traditional Chinese Medicine. Current research has found that the main components of Zanthoxylum include organic acids such as α-linolenic acid, linoleic acid, and oleic acid, as well as alkaloids such as hydroxy-α-sanshool. Among these, α-linolenic acid, linoleic acid, and oleic acid are all unsaturated fatty acids, and their content is significantly higher than other components. Furthermore, α-linolenic acid, linoleic acid, and oleic acid are unsaturated fatty acids that the human body cannot synthesize and must be obtained from external sources. α-linolenic acid, linoleic acid, and oleic acid have effects such as promoting absorption, anti-cancer properties, improving memory, and improving sleep, similar to the medicinal effects of Zanthoxylum. Currently, there are no methods for determining the content of α-linolenic acid, linoleic acid, and oleic acid. Summary of the Invention

[0005] This invention provides a method for determining the content of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations. It achieves the separation of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations, with good peak symmetry and no shoulder peaks, bifurcated peaks, or enclosed peaks. It can accurately determine the content of unsaturated fatty acids and achieve quality control.

[0006] This invention discloses a method for determining the content of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations, comprising the following steps:

[0007] (1) Preparation of test solution and standard solution;

[0008] (2) Determination by liquid chromatography: The test solution and standard solution were determined by liquid chromatography, and the content of unsaturated fatty acids in the test solution was calculated; the chromatographic column was ACQUITY UPLC CSH. TM C18; Gradient elution was performed using acetonitrile and acetic acid aqueous solution as the mobile phase. The gradient elution program included: 0 → 20 min → 23 min → 25 min, with the volume percentage of acetonitrile in the mobile phase being 70% → 76% → 100% → 70%.

[0009] Further, in step (1), the method for preparing the test solution includes:

[0010] 1) Take the test sample, add solvent to extract, and obtain the extract;

[0011] 2) Separate the solid and liquid components of the extract and take the liquid, which is the test solution.

[0012] Further, the solvent is selected from one or more of water, methanol, and ethanol; and / or, the mass ratio of the test sample to the volume of the solvent is 0.5g:5-20mL; and / or, the extraction method is ultrasonic extraction, the extraction time is 20min-40min, and the ultrasonic power is 250-520W; and / or, the solid-liquid separation method is centrifugation or filtration; and / or, the test sample is selected from one or more of Zanthoxylum bungeanum medicinal material, Zanthoxylum bungeanum processed slices, Zanthoxylum bungeanum granules, Zanthoxylum bungeanum decoction, and Zanthoxylum bungeanum freeze-dried powder.

[0013] Further, in step (1), the method for preparing the standard solution includes dissolving the standard in a solvent to obtain the solution; preferably, the solvent is selected from one or more of methanol, ethanol, and water.

[0014] Furthermore, the standard is an unsaturated fatty acid; preferably, the unsaturated fatty acid is selected from one or more of α-linolenic acid, linoleic acid, and oleic acid.

[0015] Further, in step (2), the volume percentage of acetic acid in the acetic acid aqueous solution is 0.09-0.12%.

[0016] Further, in step (2), the column temperature is 39-41℃; and / or the detection wavelength is 202-204nm; and / or the flow rate is 0.23-0.27mL / min; and / or the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.7μm.

[0017] Further, in step (1), N standard solutions with concentrations of 0.0024-0.6 mg / mL are prepared, where N is an integer ≥3; preferably, N is an integer ≥5.

[0018] Furthermore, the concentration of the α-linolenic acid standard solution is 0.0024-0.04 mg / mL; and / or, the concentration of the linoleic acid standard solution is 0.035-0.6 mg / mL; and / or, the concentration of the oleic acid standard solution is 0.011-0.2 mg / mL.

[0019] The technical solution of this invention has the following advantages:

[0020] 1. The method for determining the content of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations provided by this invention uses an ACQUITY UPLC CSH column. TM C18, using an aqueous mobile phase consisting of acetonitrile and acetic acid, employs gradient elution. Extensive experimental screening has shown that using acetonitrile and acetic acid as the mobile phase, under specific elution procedures and with a specific chromatographic column, the separation of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations is achieved. The peaks exhibit good symmetry, without shoulder peaks, bifurcation peaks, or peak engulfment. Liquid chromatography is used to determine the chromatograms of the test solution and standard solution, respectively, and the content of unsaturated fatty acids in the test solution can be calculated. This allows for accurate determination of unsaturated fatty acid content and enables quality control.

[0021] 2. The method for determining the content of unsaturated fatty acids in Sichuan pepper and its preparations provided by the present invention further improves the accuracy of unsaturated fatty acid content determination by limiting the conditions in the preparation method of the test solution or chromatographic conditions such as column temperature, flow rate, and detection wavelength.

[0022] 3. The method for determining the content of unsaturated fatty acids in Zanthoxylum bungeanum and its preparations provided by the present invention is applicable to one or more of Zanthoxylum bungeanum medicinal materials, Zanthoxylum bungeanum slices, Zanthoxylum bungeanum granules, Zanthoxylum bungeanum decoctions, and Zanthoxylum bungeanum freeze-dried powders. It has a wide range of applications and is characterized by high precision, good stability, good repeatability, high accuracy, and fast analysis rate. Attached Figure Description

[0023] 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.

[0024] Figure 1 This is the chromatogram of the test solution in Example 1.

[0025] Figure 2 This is a chromatogram of the test solution and the reference solution in Comparative Example 1.

[0026] Figure 3The chromatogram is shown for the test solution in Comparative Example 2.

[0027] Figure 4 The chromatogram is shown for the test solution in Comparative Example 3.

[0028] Figure 5 The chromatogram is shown for the test solution in Comparative Example 4.

[0029] Figure 6 The chromatogram of the test solution in Comparative Example 5 is shown between 0 and 10.5 min.

[0030] Figure 7 The chromatograms of the test solution in Comparative Example 5 are taken between 3 and 25 min.

[0031] Figure 8 The graph shows the linear relationship of α-linolenic acid reference standard in Experiment Example 2.

[0032] Figure 9 The graph shows the linear relationship of linoleic acid reference standard in Experiment Example 2.

[0033] Figure 10 The graph shows the linear relationship of oleic acid reference standard in Experiment Example 2.

[0034] Figure 11 This is a chromatogram overlay of the specificity test results from Example 2, consisting of the test solution, α-linolenic acid reference solution, linoleic acid reference solution, and excipient test solution.

[0035] in, Figure 1 , 3 In the -7 peak, peak 1 is α-linolenic acid, peak 2 is linoleic acid, and peak 3 is oleic acid. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] The main experimental instruments and reagents used in the following examples, comparative examples, and experimental cases are as follows:

[0039] Instruments: Analytical balance (Sartorius, SQP QUINTIX224-1CN, Germany), Waters high-performance liquid chromatograph (equipped with TUV detector, temperature-controlled column oven, and temperature-controlled autosampler, USA), high-speed benchtop centrifuge (SIGMA, 1-14, Germany), ultrasonic cleaner (Tianjin Autoshine Instruments Co., Ltd., AS20500BT, China).

[0040] Test drug: Zanthoxylum bungeanum extract granules (batch numbers 19044601, 19044621, 20040571), prepared by Beijing Kangrentang Pharmaceutical Co., Ltd.; the preparation method of Zanthoxylum bungeanum extract granules is as follows: Zanthoxylum bungeanum extract was taken and extracted twice by heating and reflux. For the first extraction, 12 times the weight of Zanthoxylum bungeanum extract was added to water and the mixture was heated and refluxed for 1 hour. After filtration, for the second extraction, 11 times the weight of Zanthoxylum bungeanum extract was added to water and the mixture was heated and refluxed for 1 hour. After filtration, the filtrates were combined and concentrated to a relative density of 1.06 g / mL (measured at 60℃). Maltodextrin was added as an excipient (5% of the Zanthoxylum bungeanum extract amount) and spray-dried. Maltodextrin was added to the spray-dried powder (the amount of maltodextrin added and the total weight of the spray-dried powder accounted for 21% of the Zanthoxylum bungeanum extract amount), and the mixture was mixed evenly and then dry-granulated to make granules.

[0041] Alpha-linolenic acid (batch number: 111631-202006, China National Institutes for Food and Drug Control);

[0042] Linoleic acid (batch number: 111622-202105, China National Institutes for Food and Drug Control);

[0043] Oleic acid (batch number: 111621-202108, China National Institutes for Food and Drug Control);

[0044] Reagents: Acetonitrile, methanol, and acetic acid were all of chromatographic grade.

[0045] Example 1

[0046] This embodiment provides a method for determining the content of Zanthoxylum bungeanum and its preparations, including the following:

[0047] Chromatographic conditions using octadecylsilane-bonded silica gel as the stationary phase (ACQUITY UPLC CSH) TM A C18 column (100 mm length, 2.1 mm inner diameter, 1.7 μm particle size) was used. Acetonitrile was used as mobile phase A, and 0.1% aqueous acetic acid solution was used as mobile phase B. Gradient elution was performed as follows: flow rate 0.25 mL / min; column temperature 40 °C; detection wavelength 203 nm. The theoretical plate number, calculated based on the α-linolenic acid peak, should be no less than 5000.

[0048] Table 1 Gradient elution program

[0049]

[0050] Preparation of reference solution: Take appropriate amounts of α-linolenic acid, linoleic acid and oleic acid reference standards, accurately weigh them, and add methanol to prepare a solution containing 15 μg of α-linolenic acid, 70 μg of linoleic acid and 40 μg of oleic acid per 1 ml.

[0051] Preparation of the test solution: Take an appropriate amount of Sichuan pepper granules, grind them finely, weigh about 0.5g accurately, place them in a stoppered conical flask, add 10ml of methanol, stopper tightly, weigh, sonicate (power 520W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0052] The assay involves precisely pipetting 2 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0053] The results are shown in Table 2 below. Figure 1 As shown.

[0054] Table 2 Experimental Results

[0055] batch number Alpha-linolenic acid content (mg / g) Linoleic acid content (mg / g) Oleic acid content (mg / g) 19044601 0.16 2.09 0.99

[0056] As shown in the table and figure above, the peaks corresponding to α-linolenic acid, linoleic acid and oleic acid in the test solution were well separated (resolution was 3.0, 4.1 and 10.0 respectively) and had good peak shapes (symmetry factors were 1.2, 1.2 and 1.2 respectively). The method in Example 1 can accurately determine the content of α-linolenic acid, linoleic acid and oleic acid.

[0057] Comparative Example 1

[0058] This comparative example provides a method for determining the content of Zanthoxylum bungeanum and its preparations, including the following:

[0059] Chromatographic conditions: Column: Kromasil C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: acetonitrile-1% acetic acid solution (90:10); Detection wavelength: 205 nm; Flow rate: 1.0 mL / min -1 Column temperature: 25℃; Injection volume: 10μL. The theoretical plate number, calculated based on the α-linolenic acid peak, should be no less than 4000.

[0060] Preparation of reference solutions: Take appropriate amounts of α-linolenic acid, oleic acid, and linoleic acid reference standards, weigh them accurately, and add ethanol to prepare solutions containing 0.2 mg of α-linolenic acid, oleic acid, or linoleic acid per 1 mL.

[0061] Preparation of the test solution: Take an appropriate amount of Sichuan pepper granules, grind them finely, and accurately weigh approximately 1g of the powder. Place the powder in a stoppered conical flask, add 50mL of petroleum ether (60-90℃) successively, and extract twice with ultrasonication (300W power, 40kHz frequency), 30min each time. Filter the solution into a round-bottom flask, combine the filtrates, and recover the petroleum ether under reduced pressure. Then, add 10mL of 0.5mol / L potassium hydroxide ethanol solution to the round-bottom flask, reflux for 30min, cool, add 3 drops of phenolphthalein indicator, and add 0.5mol / L hydrochloric acid solution until the red color just disappears. Transfer the solution to a 50mL volumetric flask, wash the round-bottom flask with ethanol, add the washings to the volumetric flask, add ethanol to the mark, and shake well. Accurately measure 1mL into a 10mL volumetric flask, add ethanol to the mark, and shake well to obtain the test solution.

[0062] Test results are shown Figure 2 As can be seen from the figure, the chromatographic peaks of α-linolenic acid and oleic acid in the test solution exhibit peak encapsulation at their leading edges, making them unsuitable for content calculation.

[0063] Comparative Example 2

[0064] This comparative example provides a method for determining the content of Zanthoxylum bungeanum and its preparations. The only difference between this method and the method in Example 1 is the gradient elution program and the chromatographic column. The gradient elution program used in this comparative example is shown in Table 3 below. The chromatographic column is an ACQUITY UPLC BEHSheild RP18, and the column specifications are the same as those in Example 1.

[0065] Table 3 Gradient elution program

[0066]

[0067] The test solution was prepared and tested according to the method described in this comparative example. The test results are shown in the figure. Figure 3 As can be seen from the figure, peaks 1 and 3 exhibit a peak-enclosing phenomenon, making them unsuitable for content calculation.

[0068] Comparative Example 3

[0069] This comparative example provides a method for determining the content of Zanthoxylum bungeanum and its preparations. The only difference between this method and the method in Example 1 is the gradient elution program and the chromatographic column. The gradient elution program used in this comparative example is shown in Table 4 below. The chromatographic column is an ACQUITY UPLC BEHSheild RP18, and the column specifications are the same as those in Example 1.

[0070] Table 4 Gradient elution program

[0071]

[0072] The test solution was prepared and tested according to the method described in this comparative example. The test results are shown in the figure. Figure 4As can be seen from the figure, peaks 1 and 3 exhibit a peak-enclosing phenomenon, making them unsuitable for content calculation.

[0073] Comparative Example 4

[0074] This comparative example provides a method for determining the content of Zanthoxylum bungeanum and its preparations. The only difference between this method and the method in Example 1 is the gradient elution program and the chromatographic column. The gradient elution program used in this comparative example is shown in Table 5 below. The chromatographic column is an ACQUITY UPLC BEHSheild RP18, and the column specifications are the same as those in Example 1.

[0075] Table 5 Gradient elution program

[0076]

[0077] The test solution was prepared and tested according to the method described in this comparative example. The test results are shown in the figure. Figure 5 As can be seen from the figure, the separation of peak 1 is poor, with a separation of 1.3, which is less than 1.5.

[0078] Comparative Example 5

[0079] This comparative example provides a method for determining the content of Zanthoxylum bungeanum and its preparations. The only difference between this method and the method in Example 1 is the gradient elution program and the chromatographic column. The gradient elution program used in this comparative example is shown in Table 6 below. The chromatographic column is an ACQUITY UPLC BEHSheild RP18, and the column specifications are the same as those in Example 1.

[0080] Table 6 Gradient elution program

[0081]

[0082] The test solution was prepared and tested according to the method described in this comparative example. The test results are shown in the figure. Figure 6 and Figure 7 As can be seen from the figure, peak 1 has good separation, while peak 3 exhibits a peak engulfing phenomenon and cannot be used for content calculation.

[0083] Experimental Example 1

[0084] This experimental example investigated the preparation method of the test solution, including the following:

[0085] 1. Investigation of extraction solvents

[0086] The granules of Sichuan pepper were weighed and the test solution was prepared according to the method described in Example 1. The only difference was that the extraction solvents used were water, 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 100% methanol, 30% ethanol aqueous solution, 50% ethanol aqueous solution, 70% ethanol aqueous solution, and 100% ethanol, respectively. The ultrasonic treatment power was 390W and the frequency was 40kHz. The prepared test solution was tested according to the method described in Example 1, and the results are shown in Table 7 below.

[0087] Table 7 Results of the investigation of extraction solvents

[0088] Extraction solvent Alpha-linolenic acid content / mg / g Linoleic acid content / mg / g Oleic acid content / mg / g 30% methanol aqueous solution 0.028 0.27 0.73 50% methanol aqueous solution 0.100 1.07 0.77 70% methanol aqueous solution 0.173 2.08 0.95 100% methanol 0.181 2.09 0.99 30% ethanol aqueous solution 0.043 0.51 0.68 50% ethanol aqueous solution 0.171 2.03 0.75 70% ethanol aqueous solution 0.163 2.02 0.80 100% ethanol 0.157 2.00 0.90 water 0.011 0.29 1.20

[0089] As shown in the table above, compared with other solvents, methanol is preferred as the solvent because it contains higher levels of α-linolenic acid, linoleic acid, and oleic acid, and is also more cost-effective.

[0090] 2. Investigation into the amount of extraction solvent used

[0091] Based on the extraction solvent determined above, the granules of Sichuan pepper were weighed and the test solution was prepared according to the preparation method of the test solution in Example 1. The only difference was that the amount of extraction solvent used was 5 ml, 10 ml, and 20 ml, and the ultrasonic treatment power was 390 W and the frequency was 40 kHz. The prepared test solution was tested according to the method in Example 1, and the results are shown in Table 8 below.

[0092] Table 8 Results of the investigation on the amount of extraction solvent used

[0093] Amount of extraction solvent Alpha-linolenic acid content / mg / g Linoleic acid content / mg / g Oleic acid content / mg / g 5ml 0.161 2.08 0.99 10mL 0.163 2.09 0.99 20mL 0.161 2.09 0.98 RSD / % 0.71 0.28 0.59

[0094] As shown in the table above, when using 5ml, 10ml, and 20ml of methanol as the extraction solvent, the RSD of the results for the content of α-linolenic acid, linoleic acid, and oleic acid is less than 3%. Therefore, different amounts of extraction solvent have no significant difference in the content of α-linolenic acid, linoleic acid, and oleic acid. When the amount is 10ml, the content of α-linolenic acid, linoleic acid, and oleic acid is moderate and more economical in terms of solvent. Therefore, 10ml is preferred as the amount of extraction solvent.

[0095] 3. Examination of extraction time

[0096] Based on the extraction solvent and its dosage determined above, the granules of Sichuan pepper were weighed and the test solution was prepared according to the method described in Example 1. The only difference was that the extraction times were 20 min, 30 min, and 40 min, and the ultrasonic treatment power was 390 W with a frequency of 40 kHz. The prepared test solutions were tested according to the method described in Example 1, and the results are shown in Table 9 below.

[0097] Table 9 Results of the investigation on extraction time

[0098] Extraction time Alpha-linolenic acid content / mg / g Linoleic acid content / mg / g Oleic acid content / mg / g 20min 0.163 2.08 0.98 30min 0.163 2.09 0.99 40min 0.167 2.09 0.98 RSD / % 1.4 0.28 0.59

[0099] As shown in the table above, there was no significant difference in the content of α-linolenic acid, linoleic acid, and oleic acid at different extraction times, and the RSD value of their content results was less than 3%. Taking all factors into consideration, the optimal extraction time is 30 min.

[0100] 4. Examination of ultrasonic power

[0101] Based on the extraction solvent, solvent dosage, and extraction time determined above, the granules of Sichuan pepper were weighed and the test solution was prepared according to the method described in Example 1. The only difference was that the ultrasonic power used in this study was 50%, 75%, and 100% of the rated ultrasonic power (rated ultrasonic power is 520W, 40KHz). The prepared test solutions were tested according to the method described in Example 1, and the results are shown in Table 10 below.

[0102] Table 10 Results of Ultrasonic Power Evaluation

[0103] Ultrasonic power Alpha-linolenic acid content / mg / g Linoleic acid content / mg / g Oleic acid content / mg / g 50% of rated ultrasonic power 0.161 2.08 0.97 75% of rated ultrasonic power 0.163 2.09 0.98 100% rated ultrasonic power 0.163 2.09 0.99 RSD / % 0.71 0.28 1.02

[0104] As shown in the table above, the content of α-linolenic acid, linoleic acid, and oleic acid under different ultrasonic powers are not significantly different. Therefore, 100% of the rated ultrasonic power is preferred as the ultrasonic power in the preparation process of the test solution.

[0105] In summary, the preparation method of the test solution is as follows: Take 0.5g of Sichuan pepper granules, accurately weigh them, place them in a stoppered conical flask, accurately add 10ml of methanol, seal tightly, weigh, sonicate (power 520W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0106] Experiment Example 2

[0107] The methodological examination of the method in Example 1 includes the following:

[0108] 1. System Applicability

[0109] Weigh out α-linolenic acid reference standard and prepare reference solution according to the method in Example 1. Repeat the injection of the same reference solution 5 times according to the liquid chromatography method in Example 1. Record the peak area of ​​α-linolenic acid and calculate the relative standard deviation. The results are shown in Table 11 below.

[0110] Table 11 System Adaptability Test Results

[0111] serial number α-Linolenic acid peak area mAU*min 1 1077870 2 1068326 3 1068911 4 1075415 5 1074297 Peak area RSD / % 0.39

[0112] As shown in the table above, the RSD value of the peak area of ​​the α-linolenic acid reference solution after 5 repeated injections was 0.39%, which meets the system suitability requirements.

[0113] 2. Repeatability

[0114] Six test solutions were prepared by weighing the granules of Sichuan pepper and preparing them according to the method in Example 1. The results are shown in Table 12 below.

[0115] Table 12 Results of Repeatability Tests

[0116] serial number Alpha-linolenic acid content (mg / g) Linoleic acid content (mg / g) Oleic acid content (mg / g) 1 0.159 2.09 0.99 2 0.161 2.09 0.98 3 0.157 2.08 0.99 4 0.161 2.09 0.99 5 0.167 2.08 0.98 6 0.159 2.09 0.99 RSD / % 2.14 0.25 0.52

[0117] As shown in the table above, the reproducibility of the six test solutions is good, which meets the requirements of the Guidelines for Validation of Analytical Methods for Drug Quality Standards (General Chapter 9101, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0118] 3. Accuracy

[0119] Accuracy refers to the degree to which the results measured using this method are close to the true or reference values, and is generally expressed as recovery rate (%).

[0120] Weigh out the granulated Sichuan pepper formula and prepare 6 test solutions according to the method in Example 1, and take 2 μL for detection. Add the reference standard to the test solution at a ratio of 1:0.5 or 1:1 to prepare the sample after spiking, and perform detection. Calculate the recovery rate according to the following formula: Recovery rate = (Measured value of the analyte in the sample after spiking - Measured value of the analyte in the test solution) / Mass of added reference standard × 100%.

[0121] The recovery rate was calculated using the average value, as shown in Table 13-15 below.

[0122] Table 13 Accuracy Test Results - α-Linolenic Acid

[0123]

[0124] Table 14 Accuracy Test Results - Linoleic Acid

[0125]

[0126]

[0127] Table 15 Accuracy Test Results - Oleic Acid

[0128]

[0129] As shown in the table above, the recovery rates of α-linolenic acid ranged from 100.77% to 106.79%, linoleic acid from 98.08% to 101.92%, and oleic acid from 101.48% to 104.36%, which meet the requirements of the Guidelines for Validation of Analytical Methods for Drug Quality Standards (General Chapter 9101, Part IV, Chinese Pharmacopoeia 2020 Edition), indicating good accuracy.

[0130] 4. Linear

[0131] Linearity and range of α-linolenic acid: Take an appropriate amount of α-linolenic acid reference standard, accurately weigh it, and add methanol to prepare a standard solution containing 30 μg of α-linolenic acid per 1 ml; the actual weighing was 39.69 μg, and a standard solution with a concentration of 0.03969 mg / mL was prepared.

[0132] Methanol was added to the standard solution to dilute it, resulting in α-linolenic acid standard solutions with concentrations of 6.25%, 12.5%, 25%, and 50% of the standard solution concentration. The concentrations after dilution were 0.002481 mg / mL, 0.004961 mg / mL, 0.009923 mg / mL, and 0.019845 mg / mL, respectively. The standard solutions were then analyzed using the liquid chromatography method described in Example 1. The α-linolenic acid standard curve was obtained by fitting the peak area as the x-axis and the concentration as the y-axis: y = 27125495.1682x - 924.1250, R0 2 =1.0000, see results Figure 8 .

[0133] Linearity and range of linoleic acid: Take an appropriate amount of linoleic acid reference standard, accurately weigh it, and add methanol to prepare a standard solution containing 0.6 mg of linoleic acid per 1 ml; the actual weighing was 573.30 μg, and a standard solution with a concentration of 0.5733 mg / mL was prepared.

[0134] Methanol was added to the standard solution to dilute it, resulting in linoleic acid standard solutions with concentrations of 6.25%, 12.5%, 25%, and 50% of the standard solution concentration. The concentrations after dilution were 0.035831 mg / mL, 0.071663 mg / mL, 0.143325 mg / mL, and 0.28665 mg / mL, respectively. The standard solutions were then analyzed using the liquid chromatography method described in Example 1. A standard curve for linoleic acid was obtained by fitting the peak area as the x-axis and the concentration as the y-axis: y = 4730837.9880x - 18198.0000, R0 2 =0.9999, see results Figure 9 .

[0135] Linearity and range of oleic acid: Take an appropriate amount of oleic acid reference standard, accurately weigh it, and add methanol to prepare a standard solution containing 0.2 mg of oleic acid per 1 ml; the actual weighing was 182.9 μg, and a standard solution with a concentration of 0.1829 mg / mL was prepared.

[0136] Methanol was added to the standard solution to dilute it, resulting in oleic acid standard solutions with concentrations of 6.25%, 12.5%, 25%, and 50% of the standard solution concentration. The concentrations after dilution were 0.01143125 mg / mL, 0.0228625 mg / mL, 0.045725 mg / mL, and 0.09145 mg / mL, respectively. The standard solutions were then analyzed using the liquid chromatography method described in Example 1. A standard curve for oleic acid was fitted with peak area as the x-axis and concentration as the y-axis: y = 2604548.6398x - 1361.1271, R0 2 =1.0000, see results Figure 10 .

[0137] The results above show that α-linolenic acid exhibits good linearity in the range of 2.48–39.69 μg, linoleic acid in the range of 35.83–573.30 μg, and oleic acid in the range of 11.43–182.90 μg.

[0138] 5. Exclusivity

[0139] The test solution, α-linolenic acid, linoleic acid, and oleic acid reference solutions were prepared and tested according to the method in Example 1; the excipients in the pepper formula granules were taken and the excipient test solution was prepared according to the method for preparing the test solution in Example 1 and tested.

[0140] See results Figure 11 As can be seen from the figure, the peaks in the spectrum of the test solution correspond to the peaks of α-linolenic acid, linoleic acid and oleic acid in turn. There are no interfering peaks at the negative position of the pepper formula granules, indicating good specificity for content determination.

[0141] 6. Durability

[0142] 6.1 Stability assessment:

[0143] Weigh the peppercorn granules and prepare the test solution according to the method in Example 1. The solution was injected and measured at 0h, 2h, 4h, 8h, 12h and 24h respectively. The peak area was used as an indicator to examine the stability of the sample within 24h. The results are shown in Table 16 below.

[0144] Table 16 Results of the stability study of the test solution

[0145]

[0146]

[0147] As shown in the table above, the RSD value of the peak area is less than 2.6%, indicating that the test solution has good stability within 24 hours after preparation.

[0148] 6.2 Investigation at different column temperatures:

[0149] The granules of Sichuan pepper were weighed and the test solution was prepared according to the method in Example 1. The test was basically performed according to the liquid chromatography method in Example 1, with the only difference being that the column temperatures used were 39℃, 40℃, and 41℃. The results are shown in Table 17 below.

[0150] Table 17 Results of content determination of test sample solutions at different column temperatures

[0151] Column temperature (°C) Alpha-linolenic acid content / mg / g Linoleic acid content / mg / g Oleic acid content / mg / g 39 0.164 2.10 0.99 40 0.164 2.09 1.01 41 0.162 2.09 0.99 RSD / % 0.7 0.3 1.2

[0152] In summary, the RSD values ​​of α-linolenic acid, linoleic acid, and oleic acid in the chromatograms obtained at different column temperatures were all less than 2.0%, indicating that the method in Example 1 has good robustness at different column temperatures.

[0153] 6.3 Investigation of different flow velocities:

[0154] The granules of Sichuan pepper were weighed and the test solution was prepared according to the method in Example 1. The test was basically performed according to the liquid chromatography method in Example 1, with the only difference being that the flow rates used were 0.23 ml / min, 0.25 ml / min, and 0.27 ml / min. The results are shown in Table 18 below.

[0155] Table 18 Results of content determination of test sample solutions at different flow rates

[0156] Flow rate (ml / min) Alpha-linolenic acid content / mg / g Linoleic acid content / mg / g Oleic acid content / mg / g 0.23 0.160 2.09 0.98 0.25 0.164 2.09 1.01 0.27 0.162 2.08 0.99 RSD / % 1.2 0.3 1.5

[0157] In summary, the RSD values ​​of α-linolenic acid, linoleic acid, and oleic acid in the chromatograms obtained at different flow rates were all less than 2.0%, indicating that the method in Example 1 has good robustness at different flow rates.

[0158] 7. Intermediate precision

[0159] Six test samples were prepared from the granulated Sichuan pepper formula according to the method in Example 1 and tested using instruments different from those in Example 1. The RSD value was calculated by combining the repeatability experimental data and the data obtained from this test to examine the intermediate precision. The results are shown in Table 19 below.

[0160] Table 19 Results of Intermediate Precision Test

[0161]

[0162] As shown in the table above, the RSD value of the content is less than 5%, indicating good intermediate precision.

[0163] 8. Limit of detection and limit of quantitation

[0164] The detection limit refers to the lowest concentration of a substance in a sample that can be qualitatively detected but does not need to be accurately quantified. In chromatographic analysis, the signal-to-noise ratio (S / N) is generally used to define the detection limit. The S / N is the ratio of the response value of the target analyte in the sample to the baseline noise. It is generally considered that an S / N greater than or equal to 3 can be used for qualitative analysis, and an S / N greater than or equal to 10 can be used for quantitative analysis.

[0165] When the response value of a sample component is greater than or equal to 3 times the baseline noise (i.e., S / N ≥ 3), the concentration of the sample is the instrument's lowest qualitative detection limit for the target compound. When the response value of a sample component is greater than or equal to 10 times the baseline noise (i.e., S / N ≥ 10), the concentration of the sample is the instrument's lowest quantitative limit for the target compound.

[0166] The test solution was prepared according to the method in Example 1, and the test solution was diluted stepwise and detected. The signal-to-noise ratio (S / N) of the target analyte was measured, and the results are shown in Table 20 below.

[0167] Table 20 Results of Limit of Detection and Limit of Quantification Experiments

[0168]

[0169] As shown in the table above, the detection limit for α-linolenic acid in this method is 0.919 μg / ml, and the quantitation limit is 3.063 μg / ml; the detection limit for linoleic acid is 6.852 μg / ml, and the quantitation limit is 22.841 μg / ml.

[0170] 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 determining the content of unsaturated fatty acids in Sichuan pepper and its preparations, characterized in that, Includes the following steps: (1) Preparation of test solution and standard solution; the test solution is prepared by taking the test sample, adding solvent to extract, obtaining extract, separating solid and liquid of the extract, and taking the liquid, which is the test solution; the solvent is selected from methanol and / or ethanol; the test sample is selected from one or more of Zanthoxylum bungeanum medicinal material, Zanthoxylum bungeanum slices, Zanthoxylum bungeanum granules, Zanthoxylum bungeanum decoction, and Zanthoxylum bungeanum freeze-dried powder; the standard is an unsaturated fatty acid, and the unsaturated fatty acid is α-linolenic acid, linoleic acid and oleic acid; (2) Determination by liquid chromatography: The test solution and standard solution were determined by liquid chromatography, and the content of unsaturated fatty acids in the test solution was calculated. The chromatographic column was ACQUITY UPLC CSHTM C18. Acetonitrile and acetic acid aqueous solution were used as the mobile phase, and gradient elution was performed. The gradient elution program included: 0 → 20 minutes → 23 minutes → 25 minutes. The volume percentage of acetonitrile in the mobile phase was 70% → 76% → 100% → 70%. The volume percentage of acetic acid in the acetic acid aqueous solution was 0.09-0.12%. The detection wavelength was 202-204 nm. The column length was 100 mm, the inner diameter was 2.1 mm, and the particle size was 1.7 μm.

2. The content determination method according to claim 1, characterized in that, The mass ratio of the test sample to the volume of the solvent is 0.5 g: 5-20 mL; and / or, the extraction method is ultrasonic extraction, the extraction time is 20 min-40 min, and the ultrasonic power is 250-520 W; and / or, the solid-liquid separation method is centrifugation or filtration.

3. The content determination method according to claim 1 or 2, characterized in that, In step (1), the method for preparing the standard solution includes dissolving the standard in a solvent to obtain the solution; the solvent is selected from one or more of methanol, ethanol, and water.

4. The content determination method according to claim 1 or 2, characterized in that, In step (2), the column temperature is 39-41℃; and / or the flow rate is 0.23-0.27 mL / min.

5. The content determination method according to claim 1 or 2, characterized in that, In step (1), N standard solutions with concentrations of 0.0024-0.6 mg / mL are prepared, where N is an integer ≥3.

6. The content determination method according to claim 5, characterized in that, The concentration of the α-linolenic acid standard solution is 0.0024-0.04 mg / mL; and / or, the concentration of the linoleic acid standard solution is 0.035-0.6 mg / mL; and / or, the concentration of the oleic acid standard solution is 0.011-0.2 mg / mL.

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