Determination of sugar components in Quassima by HPLC-CAD

Abstract

The present invention relates to a method for detecting the content of sugar components in a bitter wood slice or preparation, and more particularly to a method for simultaneously determining the content of fructose, glucose and sucrose using an HPLC-CAD method. The method uses an amino-bonded silica gel chromatographic column, an electrospray detector (CAD), and acetonitrile-water as a mobile phase. The method is simple, rapid, accurate, and reliable, has good chromatographic separation, can make fructose, glucose and sucrose present good separation, and is suitable for simultaneously determining the content of fructose, glucose and sucrose.

Description

Technical Field

[0001] The invention relates to a method for detecting the content of sugar components in Quassinus quinata decoction pieces or preparations, and in particular to a method for simultaneously determining the contents of fructose, glucose and sucrose in Quassinus quinata by using an HPLC-CAD method. Background Art

[0002] Quassioides is the dried branches, stems and leaves of Picrasma quassioides (D. Don) Benn. of the genus Picrasma of the family Quassioides. It is cold in nature, bitter in taste, has the effects of clearing away heat, detoxifying and removing dampness, and is mainly used for wind-heat cold, sore throat, damp-heat diarrhea, snake and insect bites and other symptoms. The main chemical components in Quassioides are alkaloid components, followed by bitter components. Content determination is a key content of drug quality control standards. In the current quality standards for Quassioides medicinal materials, there are no relevant requirements for carbohydrate components. In the study of the material basis of Quassioides, it was found that the content of carbohydrate components in Quassioides medicinal materials is relatively high, which is an important part of its material basis. In recent years, many scholars have found that the research on carbohydrate components in traditional Chinese medicine has certain pharmacological effects. Based on this, the inventor carried out research on its carbohydrate components.

[0003] According to existing literature, the determination of sugar components is mostly carried out by colorimetric method (phenol-sulfuric acid method, etc.), HPLC-RI method and HPLC-ELSD method. The literature "Determination of polysaccharide content in arrowhead by phenol-sulfuric acid method" reported by Wang Jing et al. uses polysaccharide dehydration under sulfuric acid conditions to generate furfural or its derivatives, and then condenses with phenol to generate a substance with special color with ultraviolet absorption. The obtained test solution has no maximum absorption at 485nm, which may be due to the low sugar content and cannot reach its detection limit. The HPLC-RI method for determining the content of fructose, glucose and sucrose is included in the honey content determination of the 2015 edition of the "Chinese Pharmacopoeia". However, the differential refractive index detector (RI) has only moderate sensitivity, cannot be used for trace analysis, cannot be used for gradient elution, is sensitive to temperature changes, and requires effective heat exchange for determination. The fructose, glucose and sucrose in the test solution cannot be completely separated from adjacent chromatographic peaks. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention provides a method for simultaneously determining the contents of fructose, glucose and sucrose in quassia by HPLC-CAD. The method is simple, rapid, accurate and reliable, has good chromatographic separation, can make fructose, glucose and sucrose present good separation, and is suitable for detecting fructose, glucose and sucrose in quassia.

[0005] The present invention provides a method for determining the carbohydrate components in bitter wood slices or preparations by HPLC-CAD, characterized in that the method comprises the following steps: respectively analyzing the test solution and the reference solution by high performance liquid chromatography, recording the chromatogram, and the conditions of the high performance liquid chromatography are: using Shodex Asahipak NH2P-504E or Grace PrevailCarbohydrate ES as the chromatographic column, using water as the mobile phase A, using acetonitrile as the mobile phase B, and gradient elution, and the gradient elution program is as follows:

[0006] Time (min) A％ B％ 0～5 20～23 80～77 5～15 23～30 77～70 15～35 20 80

[0007] In the table, the total volume of the mobile phase is taken as 100%.

[0008] Preferably, the preparation method of the test solution is: take an appropriate amount of bitter wood powder, add ethanol, ultrasonically treat and filter, and remove the filtrate as the test solution; more preferably, the ethanol concentration is 10% to 100% (volume ratio), preferably 80%; and / or, ultrasonic treatment is performed for 10 to 60 minutes, preferably 30 minutes.

[0009] Preferably, the preparation method of the reference substance stock solution or the reference substance solution is: weigh appropriate amounts of fructose reference substance, glucose reference substance, and sucrose reference substance, place them in a volumetric flask, add water to dissolve and dilute to the scale, shake well, use it as the reference substance stock solution, and then dilute with water to obtain the reference substance solution; more preferably, the concentration ratio of the fructose reference substance, glucose reference substance, and sucrose reference substance is 4-8:3-7:12-20; more preferably, the concentration ratio of the fructose reference substance, glucose reference substance, and sucrose reference substance is 6:5:16.

[0010] Preferably, the preparation method of the reference substance solution is: taking the reference substance stock solution, diluting it with water and making up to the scale, and shaking it well to obtain the reference substance solution;

[0011] Preferably, the high performance liquid chromatograph needs to be equipped with an electrospray detector.

[0012] Preferably, the flow rate of the mobile phase is 0.8-1.2 mL / min.

[0013] Preferably, the column temperature of the chromatographic column is set at 20-30°C.

[0014] Preferably, the injection volume is set to 2 μL to 8 μL.

[0015] Preferably, the elution time is 15 min to 35 min.

[0016] Preferably, the preparation of the test solution requires filtration through a 0.22 μm or 0.45 μm filter membrane.

[0017] The beneficial effects of the present invention are as follows:

[0018] 1. The present invention adopts the HPLC-CAD method. Compared with the prior art HPLC-RI, the sensitivity of CAD is 10 to 100 times higher, and the detection limit can reach 1 to 100 ng.

[0019] 2. The applicant investigated chromatographic columns of different types and manufacturers, such as Phenomenex LunaNH2 (4.6×250mm, 5μm), Phenomenex ACE Excel 5NH2 (4.6×250mm, 5μm), Grace PrevailCarbohydrate ES (4.6×250mm, 5μm), Ultimate Hilic (4.6×250mm, 3μm), ShodexAsahipak NH2P-50 4E (4.6×250mm, 5μm), a total of five chromatographic columns. Among them, Phenomenex Luna NH2, Phenomenex ACE Excel 5NH2, and Ultimate Hilic had large column bleed, resulting in large baseline noise, affecting the determination of the target component. However, Shodex Asahipak NH2P-504E and Grace Prevail CarbohydrateES had small column bleed and could well separate the target component from other components.

[0020] 3. The technical solution of the present invention is accurate and reliable, with high sensitivity and good repeatability. It can simply and quickly determine the content of fructose, glucose and sucrose in Quassin, providing a basis for comprehensively and effectively controlling the quality of Quassin and improving its quality control level. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Chromatogram of reference solution.

[0022] Figure 2 : Chromatogram of test solution. Specific implementation methods

[0023] The claims are further explained below in conjunction with specific embodiments, but do not constitute any limitation to the present invention. Any technical solution obtained by making limited modifications to the present invention still falls within the scope of protection of the present invention.

[0024] Example 1

[0025] 1. Instruments and reagents

[0026] Agilent high performance liquid chromatograph (Agilent Technologies, Inc., USA), Thermo Fisher CoronaUltra electrospray detector (Thermo Fisher Scientific, Inc., USA);

[0027] The chromatographic column was Shodex Asahipak NH2P-504E (4.6 × 250 mm, 5 μm);

[0028] Acetonitrile was of chromatography grade (Merck GmbH, Germany, batch number: SHBK2103);

[0029] Water was ultrapure water (Merck Mili-Q homemade);

[0030] Fructose reference substance (batch number 100231-201606, content 99.7%, China Food and Drug Inspection Institute); glucose reference substance (batch number LRAB1913, content 100%, Sigma-Aldrich); sucrose reference substance (batch number 111507-201704, content 100%, China Food and Drug Inspection Institute);

[0031] Quassia sinensis (Jiangxi Qingfeng Pharmaceutical Co., Ltd., batch number: 20180402).

[0032] 2 Methods and Results

[0033] 2.1 Chromatographic conditions

[0034] The analysis was performed by high performance liquid chromatography. The detection parameters were as follows: chromatographic column: Shodex Asahipak NH2P-504E (4.6×250 mm, 5 μm); flow rate: 1.0 mL·min -1 ; Column temperature: 25℃; Injection volume: 5μL; Mobile phase: water-acetonitrile, Post-run: 20min. The parameters of the electrospray detector are as follows: Evaporation temperature: 35℃; Sampling frequency: 5Hz; Filter: 5S.

[0035] The model of the high performance liquid chromatograph was: Agilent 1260 high performance liquid chromatograph, equipped with a Thermo Fisher Corona Ultra electrospray detector.

[0036] The volume ratio of water to acetonitrile in the mobile phase was 20:80, and the linear gradient changed to 23:77 and then to 30:70 over time.

[0037] The mobile phase was added by gradient elution, and the specific parameters were as follows:

[0038] The total elution time was 35 min;

[0039] From 0 to 5 min, the volume ratio of water to acetonitrile in the mobile phase was 20–23:80–77;

[0040] From 5 to 15 min, the volume ratio of water to acetonitrile in the mobile phase was 23–30:77–70;

[0041] From 15 to 35 min, the volume ratio of water to acetonitrile in the mobile phase was 20:80.

[0042] 2.2 Preparation of test solution: Take about 1.0g of quassin powder, weigh it accurately, put it in a stoppered conical flask, add 25mL of 80% ethanol accurately, weigh it, treat it with ultrasound for 30min, let it cool to room temperature, weigh it again, make up the lost weight with 80% ethanol, shake it well, filter it, and remove the filtrate to obtain the solution;

[0043] 2.3 Preparation of reference substance stock solution: Accurately weigh 30 mg of fructose reference substance, 25 mg of glucose reference substance, and 80 mg of sucrose, place in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain;

[0044] 2.4 Preparation of reference solution: Accurately pipette 5 mL of reference stock solution into a 25 mL volumetric flask, dilute with water and make up to the mark, shake well, and the solution is ready.

[0045] 2.5 Determination method

[0046] Accurately pipette 5 μL of the mixed reference solution of fructose, glucose and sucrose and the test solution respectively, inject them into the liquid chromatograph, measure, record the chromatogram, and calculate, and then obtain: Figure 1 and Figure 2 .

[0047] 2.6 Specificity test

[0048] 5 μL of blank (i.e. ultrapure water) was accurately pipetted and injected into the liquid chromatograph for determination and the chromatogram was recorded. Results: Ultrapure water had no interference with the detection.

[0049] 2.7 Linear relationship test

[0050] Accurately weigh 75 mg of fructose, glucose, and sucrose respectively, place in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well. Accurately pipette 1.0 mL, 1.0 mL, 1.0 mL, and 10.0 mL of the above solutions respectively into 100 mL, 25 mL, 10 mL, and 25 mL volumetric flasks, dilute to the scale with water, shake well, and obtain 5 concentrations of reference solution. Accurately pipette 5 μL in order from low to high concentration, inject into the liquid chromatograph, measure according to the method, and record the chromatogram. Use the reference concentration as the horizontal axis and the peak area as the vertical axis to draw a standard curve and perform linear regression. It is obtained that fructose, glucose, and sucrose are concentrated at 0.03 mg·mL -1~3.0mg·mL -1 The range has a good linear relationship with the peak area, and the regression equations are Y 果糖 =1495.8X+38.259(r=0.9993), Y 葡萄糖 =1101.3X+68.158(r=0.9993), Y 蔗糖 =2342.6X-348.9 (r=0.9994).

[0051] 2.8 Precision test

[0052] The same reference solution was accurately drawn and sampled 6 times continuously. The peak areas were recorded and the RSDs of fructose, glucose and sucrose were calculated. The RSDs of fructose, glucose and sucrose were all less than 2.0%. The results showed that the instrument had good precision.

[0053] Table 1 Precision test results

[0054]

[0055] 2.9 Repeatability test

[0056] Take 6 portions of the same quassin powder and prepare it according to the test solution method in "2.2". According to the method, the average fructose content was calculated to be 3.09 mg·g -1 (RSD = 1.0%), the average glucose content was 2.78 mg g -1 (RSD = 1.1%), the average sucrose content was 8.09 mg g -1 (RSD = 0.4%). The results showed that this method has good repeatability.

[0057] Table 2 Fructose repeatability test results

[0058]

[0059] Table 3 Glucose repeatability test results

[0060]

[0061] Table 4 Sucrose repeatability test results

[0062]

[0063] 2.10 Stability test

[0064] Take the reference solution and measure it at 0h, 2h, 4h, 8h, 12h, and 16h respectively. The peak area is measured. The RSD of the fructose peak area is 1.2%, the RSD of the glucose peak area is 3.1%, and the RSD of the sucrose peak area is 0.8%. According to the data requirements of the repeatability test in the General Rules 9101 of the Fourth Part of the 2015 Edition of the Chinese Pharmacopoeia, the content of the component to be determined is 0.1%, and the RSD should be less than 3%. The reference solution is placed at room temperature for 16 hours. The RSD of fructose and sucrose is less than 3%, which is stable within 16 hours; the RSD of glucose is greater than 3.0%, indicating that the reference solution is relatively stable within 12 hours at room temperature.

[0065] Table 5 Stability test results

[0066]

[0067] 2.11 Sample recovery test

[0068] Take 0.5g of bitter wood sample, weigh accurately, put it in a 100mL stoppered conical flask, weigh 9 portions in parallel, add 1.25mL, 2.5mL, and 3.75mL of the "2.3" reference substance stock solution, prepare 3 portions in parallel, accurately add 80% ethanol to 25mL, weigh the weight, ultrasonically treat for 30 minutes, let it stand at room temperature, weigh it again, make up the lost weight with 80% ethanol, shake well, filter, remove the filtrate, and determine according to the law. According to the data requirements of the accuracy of the fourth general rule 9101 of the 2015 edition of the "Chinese Pharmacopoeia", the content of the component to be determined is 0.1%, and the sample recovery rate should be 90% 108%, RSD should be less than 6%. The recovery rates of fructose, glucose and sucrose meet the requirements, with the RSD of glucose recovery rate being 2.9%, fructose recovery rate being 2.7%, and sucrose recovery rate being 1.5%, all meeting the requirements.

[0069] Table 6 Fructose sample recovery test results

[0070]

[0071] Table 7 Glucose sample recovery test results

[0072]

[0073] Table 8 Sucrose sample recovery test results

[0074]

[0075] Sample determination

[0076] Prepare the sample according to the preparation method described in "2.2", accurately pipette 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine according to the method to obtain the result.

[0077] Table 9 Determination results of fructose, glucose and sucrose content in quassia

[0078]

[0079] Example 2

[0080] Preparation of test solution: Take about 1.0g of three batches of quassin powder, weigh accurately, put it in a stoppered conical flask, add 25mL of 100% ethanol accurately, weigh it, treat it with ultrasound for 10min, let it cool to room temperature, weigh it again, make up the lost weight with 100% ethanol, shake it well, filter it, and remove the filtrate to obtain the solution;

[0081] Preparation of reference substance stock solution: accurately weigh 30 mg of fructose reference substance, 25 mg of glucose reference substance, and 80 mg of sucrose, place in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain;

[0082] Preparation of reference solution: accurately pipette 5 mL of reference stock solution into a 25 mL volumetric flask, dilute with water and make up to the mark, shake well, and the solution is ready.

[0083] Determination method: Accurately pipette 8 μL of fructose, glucose and sucrose mixed reference solution and test solution respectively, inject into liquid chromatograph, and determine. The calculation results are shown in the table below.

[0084]

[0085] Example 3

[0086] Preparation of test solution: Take about 1.0g of three batches of quassin powder, weigh accurately, put it in a stoppered conical flask, add 25mL of 10% ethanol accurately, weigh it, treat it with ultrasound for 60min, let it cool to room temperature, weigh it again, make up the lost weight with 10% ethanol, shake it well, filter it, and remove the filtrate to obtain the solution;

[0087] Preparation of reference substance stock solution: accurately weigh 30 mg of fructose reference substance, 25 mg of glucose reference substance, and 80 mg of sucrose, place in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain;

[0088] Preparation of reference solution: accurately pipette 5 mL of reference stock solution into a 25 mL volumetric flask, dilute with water and make up to the mark, shake well, and the solution is ready.

[0089] Determination method: Accurately pipette 5 μL of fructose, glucose and sucrose mixed reference solution and test solution respectively, inject into liquid chromatograph, and determine. The calculation results are shown in the table below.

[0090]

[0091] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining the carbohydrate components in Quassia strychnifolia decoction pieces by HPLC-CAD, characterized in that: The method comprises the following steps: respectively analyzing the test solution and the reference solution by high performance liquid chromatography, recording the chromatogram, and the conditions of the high performance liquid chromatography are as follows: chromatographic column: Shodex Asahipak NH2P-504E, specification: 4.6×250mm, 5μm; flow rate: 1.0mL / min; column temperature: 25°C; injection volume: 5μL; using water as mobile phase A and acetonitrile as mobile phase B, running: 20min, gradient elution, and the gradient elution program is as follows: In the table, the total volume of the mobile phase is taken as 100%; The preparation method of the test solution is as follows: take an appropriate amount of bitter wood powder, add ethanol solution, ultrasonically treat and filter, and take the filtrate as the test solution; the concentration of the ethanol solution is 80% by volume, and the ultrasonic treatment is performed for 30 minutes; the preparation of the test solution requires filtration through a 0.22μm or 0.45μm filter membrane; The preparation method of the reference substance solution is as follows: weigh appropriate amounts of fructose reference substance, glucose reference substance, and sucrose reference substance, place them in a volumetric flask, add water to dissolve and dilute to the scale, shake well, use as the reference substance stock solution, and then dilute with water to obtain the reference substance solution.

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