CN108872441B - Method for measuring glucosamine and chondroitin sulfate - Google Patents
Method for measuring glucosamine and chondroitin sulfate Download PDFInfo
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Abstract
The invention relates to the technical field of analytical chemistry, in particular to a method for simultaneously determining glucosamine and chondroitin sulfate in a health food. The method adopts an evaporative light scattering detector to improve the sensitivity of glucosamine and chondroitin sulfate, adopts an amide chromatographic column with moderate retention time, good separation degree, good chromatographic peak separation of each color, stable baseline, good peak shape and good repeatability, has good linear relation, precision, stability and recovery rate, can accurately and simultaneously determine the content of glucosamine and chondroitin sulfate in health-care food, is suitable for determining the glucosamine and chondroitin sulfate in the health-care food, and is also suitable for determining raw materials of glucosamine or chondroitin sulfate.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for determining glucosamine and chondroitin sulfate in food or medicines.
Background
Glucosamine is an hexosamine extracted from natural carapace, is a natural component of glycoprotein in joint tissue of human body and animal, and has antiinflammatory, analgesic, and joint cartilage tissue repairing effects. Currently, glucosamine and hydrochloride, sodium sulfate or potassium sulfate thereof are commonly used in the market. Chondroitin sulfate is an acidic mucopolysaccharide extracted from animal cartilage, belongs to a biopolymer compound, and is mainly used for treating nervous headache, angina pectoris, coronary heart disease and osteoarticular diseases. In recent years, more and more reports are made on the treatment of osteoarthritis by using chondroitin sulfate, aminodextran, sodium hyaluronate and other medicines in a combined manner, and the chondroitin sulfate is not only widely used in health-care food, but also used in the fields of medicines, daily products and the like, and attracts more and more attention, so that a corresponding detection method is also needed to carry out effective quality control on the products.
Currently, commonly used detection methods for glucosamine include an ultraviolet spectrophotometry (national drug standard WS1- (X-090) -2005Z), an HPLC-UV method (high performance liquid chromatography-ultraviolet detection) and an HPLC-ELSD method (high performance liquid chromatography-evaporative light scattering detection). The ultraviolet spectrophotometry sample needs derivatization treatment, and has the disadvantages of complex operation, poor stability and poor repeatability; if the HPLC-UV method does not derive, the ultraviolet absorption is weak, the sensitivity is low, and the derivation operation is fussy, the period is long, and the stability is poor; the HPLC-ELSD has high sensitivity and overcomes the characteristic of weak ultraviolet absorption, but the chromatographic column mostly adopts a C18 column, almost no retention exists, peaks appear about 2min, the separation effect is poor, and an amino chromatographic column is also adopted in research, but the service life is short. Common methods for detecting chondroitin sulfate include an Elson-Morgan method, a carbazole method, a titration method (United states and European pharmacopoeias), an HPLC-enzymatic hydrolysis method (2015, second division of Chinese pharmacopoeia) and an HPLC-ELSD method. The first three methods have obvious limitations, are complicated to operate and have poor specificity and accuracy; the HPLC-enzymolysis method has good specificity and accuracy, but the sample treatment is complicated, the time consumption is long, the enzyme is expensive, and the sensitivity is low by adopting a UV detector. The simultaneous determination of two target components in the current health food taking glucosamine and chondroitin sulfate as raw materials is mainly based on GB/T20365-; the mobile phase adopts ion pair reagent, and the preparation is complicated.
An Evaporative Light Scattering Detector (ELSD) is used as a general detector, does not depend on the optical characteristics of compounds, has good response to non-volatile chemical components, and is widely applied to the quality control of traditional Chinese medicine components such as saponins, terpenoids, alkaloids, saccharides and the like. The glucosamine and the chondroitin sulfate almost have no ultraviolet absorption, can be directly measured without derivatization, and have simple and convenient operation and high sensitivity.
HILIC (hydrophilic interaction chromatography) is suitable for separating polar compounds which are not retained on a reversed phase chromatographic column, especially has strong retention capacity on strong polar alkaline compounds, becomes the first choice for researching drug metabolism, drug discovery and combined chemical science, and is especially suitable for HPLC-MS and HPLC-ELSD. The Amide chromatographic column is the first-choice chromatographic column of HILIC series, has the advantages of stable bonding phase, strong retention on high-polarity compounds, wide pH resistance, high temperature resistance, capability of combining epimers without losing reducing sugar and the like, and is suitable for measuring water-soluble glucosamine and chondroitin sulfate.
The existing domestic and foreign standards, patents and papers do not well solve the problem of measuring glucosamine and chondroitin sulfate, so that an analysis method which is simple and convenient to operate, high in sensitivity, good in repeatability and accurate in result needs to be developed.
Disclosure of Invention
In view of the above, the present invention is directed to a qualitative or quantitative method for determining glucosamine and chondroitin sulfate, comprising: and (3) carrying out HPLC detection on a test solution prepared from glucosamine or chondroitin sulfate by using an evaporative light scattering detector, wherein a chromatographic column adopts an amide chromatographic column, and acetonitrile and a salt containing a weak base are used as mobile phases.
Preferably, the test flow rate of the test solution is 0.7-1.0mL/min, preferably 0.8mL/min, and the column temperature is 40 ℃.
Specifically, the salt is one or more of ammonium formate, ammonium acetate and ammonium carbonate.
Specifically, the weak base is selected from ammonia, methylamine, ethylamine and triethylamine; preferably 0.6% triethylamine, i.e. 0.6% triethylamine by volume in the salt solution.
Further, the Amide column is selected from a Waters Xbridge Amide column, Shimadzu Inertsil Amide column or
Amide chromatographic column.
The invention also provides a qualitative or quantitative method for determining glucosamine and chondroitin sulfate in food or medicine, wherein the food can be health food, and the method is characterized by adopting an evaporative light scattering detector as an HPLC detector.
Further, the method adopts an amide chromatographic column, and acetonitrile and salt containing weak base are used as mobile phases.
The acetonitrile-ammonium acetate-triethylamine water system is a mobile phase system of the method; the mobile phase acetonitrile-triethylamine water (containing ammonium acetate) ratio is preferably 77-73: 23-27.
Specifically, in the method, the preparation process of the reference solution is as follows: accurately weighing appropriate amount of glucosamine and chondroitin sulfate reference substances, adding 2ml acetonitrile, oscillating to disperse uniformly, adding water, and ultrasonically dissolving to obtain mixed low concentration standard solution containing 0.2mg glucosamine and chondroitin sulfate per 1 ml;
the preparation process of the test solution comprises the following steps: grinding the food or medicine to be tested, taking 100mg of powder, precisely weighing, placing in a 50mL measuring flask, adding 5mL acetonitrile, ultrasonically dispersing for 1min, adding water to approximate scale, ultrasonically dispersing for 10min, adding water to constant volume to scale, shaking up, and centrifuging to obtain the final product.
Further, the detection flow rate of the method was 0.8mL/min, and the column temperature was 40 ℃. The salt is one or more of ammonium formate, ammonium acetate and ammonium carbonate. The weak base is selected from ammonia, methylamine, ethylamine and triethylamine. The Amide chromatographic column is selected from Waters Xbridge Amide chromatographic column, Shimadzu Inertsil Amide chromatographic column or
amide chromatography column.
Preferably, the salt is ammonium acetate, preferably 50mmol/L ammonium acetate; the weak base is 0.6% of triethylamine; waters
Amide (4.6X 150mm, 3.5 μm) as a column.
The invention provides a method for simultaneously measuring glucosamine and chondroitin sulfate, which is suitable for measuring glucosamine and chondroitin sulfate in food or medicine and is also suitable for measuring raw materials of glucosamine or chondroitin sulfate, and has the following advantages:
1. the method for simultaneously measuring two target components of glucosamine and chondroitin sulfate by adopting the evaporative light scattering detector has the advantages of high sensitivity, high efficiency and strong specificity, and is not only suitable for preparations taking the glucosamine and the chondroitin sulfate as raw materials, but also suitable for measuring the raw materials.
2. The amide chromatographic column has wide pH resistance, high temperature resistance, stable bonding phase and less loss, and can separate compounds with proper retention time.
3. The weak base is added into the mobile phase to adjust the retention time and the peak shape of the chondroitin sulfate, and the weak base can enable the peak shape of the glucosamine to be sharp and symmetrical.
4. The methodological verification of the system proves that the method is simple and quick, has good specificity, accurate result and good repeatability, and has certain popularization.
Drawings
FIG. 1 shows Waters Atlantis T of the present invention3 C18Chromatogram of the mixed reference solution of the chromatographic column;
FIG. 2 shows Waters Atlantis T3C of the present invention18Chromatographic column health food sample atlas;
FIG. 3 is a chromatogram of a mixed control solution of an amide chromatographic column of the present invention;
FIG. 4 is a sample chromatogram of an amide chromatographic column health food according to the present invention;
FIG. 5 is a diagram of acetonitrile-water as the mobile phase of the present invention;
FIG. 6 is a diagram of acetonitrile-formic acid water as a mobile phase according to the present invention;
FIG. 7 is a diagram of acetonitrile-triethylamine water as a mobile phase according to the present invention;
FIG. 8 is a diagram of acetonitrile-ammonium acetate water as a mobile phase according to the present invention;
FIG. 9 is a diagram of acetonitrile-ammonium acetate-triethylamine water as a mobile phase according to the present invention;
FIG. 10 is a graph of the mobile phase of the present invention containing 0.2% triethylamine;
FIG. 11 is a graph of the mobile phase of the present invention containing 0.4% triethylamine;
FIG. 12 is a graph of the mobile phase of the present invention containing 0.6% triethylamine;
FIG. 13 is a graph of the mobile phase of the present invention containing 0.8% triethylamine;
FIG. 14 is a diagram of a mobile phase of the present invention containing 30mmol/L ammonium acetate;
FIG. 15 is a diagram of a mobile phase of the present invention containing 40mmol/L ammonium acetate;
FIG. 16 is a diagram of a mobile phase of the present invention containing 50mmol/L ammonium acetate;
FIG. 17 is a spectrum of the present invention with a column temperature of 35 ℃;
FIG. 18 is a graph of the column temperature of the present invention at 40 ℃;
FIG. 19 is a spectrum of the present invention with a column temperature of 45 ℃;
FIG. 20 is a graph showing a column temperature of 50 ℃ in accordance with the present invention;
FIG. 21 is a plot of mobile phase ratio 80:20 according to the present invention;
FIG. 22 is a graph of the present invention at a mobile phase ratio of 77: 23;
FIG. 23 is a graph of the present invention at a mobile phase ratio of 75: 25;
FIG. 24 is a graph of the present invention at a mobile phase ratio of 73: 27;
FIG. 25 is a graph of the present invention at a mobile phase ratio of 70: 30;
FIG. 26 is a graph of the flow rate of 0.7ml/min according to the present invention;
FIG. 27 is a graph of a flow rate of 0.8ml/min according to the present invention;
FIG. 28 is a graph of the flow rate of 0.9ml/min according to the present invention;
FIG. 29 is a graph of the flow rate of 1.0ml/min according to the present invention;
FIG. 30 is an HPLC-ELSD map of a blank solvent of the present invention;
FIG. 31 is an HPLC-ELSD map of a mixed control solution of the present invention;
FIG. 32 is an HPLC-ELSD spectrum of a potassium glucosamine sulfate source in accordance with the present invention;
FIG. 33 is an HPLC-ELSD chromatogram of chondroitin sulfate starting material of the present invention;
FIG. 34 is an HPLC-ELSD spectrum of the health food of the present invention;
FIG. 35 is an HPLC-ELSD profile of a negative sample of a nutraceutical of the present invention;
FIG. 36 is an HPLC-UV chart of the mixed control solution of GB/T20365-2006 method;
FIG. 37 is an HPLC-UV chart of the health food obtained by GB/T20365-2006 method.
Detailed Description
The invention discloses a method for measuring the content of glucosamine and chondroitin sulfate in food by taking glucosamine and chondroitin sulfate as raw materials. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The medicines, reagents and instruments used in the method for measuring the content of glucosamine and chondroitin sulfate provided by the invention are all conventional products which can be obtained commercially without indicating manufacturers. Those who do not specify the particular experimental conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer.
The instrument comprises the following steps: high performance liquid chromatography (Agilent 1260); an ELSD detector (Alltech 2000 ES);
electronic balance (METTLER TOLEDO MS-105DU, METTLER TOLEDO XP6, Sartorius BSA 224S-CW); ultra pure water machine (Milli-Q Academic U.S. MiLLIPOE); a numerical control ultrasonic cleaner (KQ-500DB, ultrasonic instruments Co., Ltd., Kunshan); CIMODHG-91438S-III electrothermal constant temperature air-blast drying oven (Shanghai Xinmiao medical instruments manufacturing Co., Ltd.).
Reagent: acetonitrile (chromatographically pure TEDIA,17050287 batches); triethylamine (chromatographically pure TRDIA, 16050721);
ammonium acetate (chromatographic grade laddin,1704005 batches); the water was 18 Ω ultrapure water prepared by an ultrapure water meter.
Control and sample: d-glucose hydrochloride control (lot No. 140649-201304) and chondroitin sulfate control (lot No. 140792-201702) were provided by the Chinese food and drug testing institute. D-glucosamine potassium sulfate raw material (0220170355, Jiangsu Olympic engineering Co., Ltd.), and chondroitin sulfate (ox bone, 0720170209, Jiangsu Olympic engineering Co., Ltd.). Samples of Yinyi tablets (lot numbers 20180201, 20180202, 20180203) as a health food were produced by Jiangsu Kangyuan pharmaceutical Co.
Embodiments of the present invention will be described in detail below with reference to examples:
example 1 selection of chromatographic conditions
Preparation of control solutions: weighing 5mg of chondroitin sulfate and D-glucosamine hydrochloride which are dried for 4 hours at 105 ℃ into a 10ml measuring flask, adding 1ml of acetonitrile, shaking to uniformly disperse the mixture, adding water, dissolving the mixture by ultrasonic treatment, and fixing the volume to the scale by using water to obtain 0.5mg of mixed reference substance solution containing glucosamine and chondroitin sulfate per 1 ml.
Preparation of a test solution: 20180201 batches of samples are taken, film coated tablets are removed, the samples are ground, 100mg of powder is taken, the powder is precisely weighed and placed in a 50mL measuring flask, 5mL of acetonitrile is added, ultrasonic dispersion is carried out for 1min, water is added until the volume is close to the scale, ultrasonic treatment is carried out for 10min, the volume is fixed to the scale by water, shaking up is carried out, and centrifugation is carried out, thus obtaining the product.
1. Selection of chromatography columns
First, the most commonly used C18 column (Watersaltlis T3C)18) Pre-testing is carried out, and the mobile phase is acetonitrile: the column temperature was 30 ℃ as usual, the flow rate was 0.6mL/min, the ELSD detector, the drift tube temperature was 105 ℃ and the N2 flow rate was 2.5mL/min, with 0.1% triethylamine being 10: 90. The results are shown in FIGS. 1 and 2.
As can be seen from the figure, the two target peaks appeared too early, and chondroitin sulfate almost coincided with the peripheral impurity peaks, because the C18 column hardly retained the compounds having large polarity, and even if the ratio was changed, the retention time and the separation effect were not significantly changed. Therefore, it is better to select an amino column and a hydrophilic column which have completely different packing properties from C18 and are suitable for separating the large polar compounds, wherein the amino column has short service life, so the first-choice column amide column of the hydrophilic series is selected for examination. Column was Waters Xbridge amide (4.6X 150mm, 3.5 μm), acetonitrile: the flow rate of 0.8mL/min with 0.1% triethylamine 75:25 was the same as "selection of column 1", and the results are shown in fig. 3 and fig. 4.
As can be seen from fig. 3 and 4, the compound on the amide column exhibited a completely different chromatographic behavior from C18, an epimer of glucosamine was present, and chondroitin sulfate was not peaked, although the effect was not ideal, but could be optimized from the mobile phase side, so the amide column was selected for the next optimization.
2. Selection of mobile phase species
And respectively eluting by using acetonitrile-water, acetonitrile-formic acid water, acetonitrile-triethylamine water, acetonitrile-ammonium acetate water and acetonitrile-ammonium acetate-triethylamine water systems, and primarily optimizing the types of the mobile phases by mixing reference substance solutions, wherein the results are shown in the figures 5-9. As can be seen from the figure, neither compound in the acetonitrile-water system has a peak, glucosamine in the acetonitrile-formic acid water system has a split peak and has a poor peak shape width, chondroitin sulfate has no peak, glucosamine in the acetonitrile-triethylamine water system has a peak but has a split peak, and chondroitin sulfate has no peak; both acetonitrile-ammonium acetate systems peak, but glucosamine cracks, and almost coincides with the chondroitin sulfate peak; the acetonitrile-ammonium acetate-triethylamine water system has proper retention time and relatively better peak type, so the acetonitrile-ammonium acetate-triethylamine water system is preferably used as the mobile phase system of the method.
3. Selection of triethylamine concentration in mobile phase
The carbohydrate is easy to epimerize on an amide column, and the problem can be solved by adding triethylamine with proper concentration and raising the column temperature, so that the influence of triethylamine with different concentrations (0.2%, 0.4%, 0.6% and 0.8%) on the separation effect of the target compound is examined, and the result is 10-13. As can be seen from the figure, the concentration of triethylamine has a large influence on the peak shape of glucosamine, and has a certain influence on the retention time of chondroitin sulfate, as the concentration of triethylamine is increased, the better the glucosamine isomers are combined, but the peak appearance time of chondroitin sulfate is delayed, and under the condition of 0.6% of triethylamine, the peak shapes of glucosamine and chondroitin sulfate are better, and the retention time is moderate, so that 0.6% of triethylamine is preferred.
4. Selection of ammonium acetate concentration in mobile phase
The effect of different concentrations of ammonium acetate (30, 40, 50mmol/L) on the separation effect of the target compound was examined, and the results are shown in FIGS. 14-16. As can be seen from the figure, the effect of ammonium acetate concentration on chondroitin sulfate is large, the retention time of chondroitin sulfate is reduced with the increase of ammonium acetate concentration, chondroitin sulfate does not peak at 30mmol/L, and chondroitin sulfate does not peak at 40mmol/L, but the peak type is wide, and 50mmol/L ammonium acetate is preferred in consideration of the separation effect and the tolerance of the apparatus to salt.
5. Selection of column temperature
Increasing the column temperature will promote the combination of glucosamine and isomers, so the effect of different column temperatures (35 ℃, 40 ℃, 45 ℃, 50 ℃) on the separation effect of the target compound is examined, as shown in fig. 17-20, it can be seen from the figure that the separation degree of the target compound is greater than 1.5 at different temperatures, which indicates that the durability of the temperature is better, and the separation effect, the peak shape and the service life of the column are comprehensively considered, preferably 40 ℃.
6. Investigation of mobile phase ratio
The effect of different mobile phase ratios on the separation effect of the target compound, namely acetonitrile-triethylamine water (80:20, 77:23, 75:25, 73:27, 70:30) (containing 50mmol/L ammonium acetate), was examined as shown in FIGS. 21-25. Through examination of different mobile phase ratios, the chondroitin sulfate has late peak emergence time and wider peak form under the condition of 80:20, the separation degree of glucosamine and chondroitin sulfate in a sample is poor under the condition of 70:30, the separation degree under the conditions of 77:23, 75:25 and 73:27 is more than 1.5, the peak form symmetry is good, the content is not obviously different, and the preferable ratio is 75: 25.
7. Investigation of flow Rate
The effect of different flow rates (0.7, 0.8, 0.9 and 1.0mL/min) on the separation effect is examined, the results are shown in FIGS. 26-29, and as can be seen from the figures, the separation effect is better at each flow rate, which shows that the method has better durability on the flow rate, and the flow rate range is selected to be 0.8mL/min in consideration of the tolerance of the amide column.
In summary, the optimal chromatographic conditions are: chromatography column Waters Xbridge Amide (4.6X 150mm, 3.5 μm); mixing acetonitrile: 50mmol of ammonium acetate water (containing 0.6% triethylamine) 75:25, isocratic elution is carried out on the mobile phase; the flow rate is 0.8 mL/min; the column temperature was 40 ℃; the temperature of the evaporative light drift tube was 105 ℃ and the nitrogen flow rate was 2.5 ml/min.
Example 2 HPLC-ELSD quantitative determination method of glucosamine and chondroitin sulfate in health food
1. Preparation of Mixed control solutions
Taking 5mg of glucosamine and chondroitin sulfate reference substances which are dried for 4 hours at 105 ℃, accurately weighing the glucosamine and chondroitin sulfate reference substances in a 25ml measuring flask, adding 2ml of acetonitrile, oscillating to uniformly disperse the acetonitrile, adding water, ultrasonically dissolving the water, cooling to room temperature, fixing the water to a certain volume to scale, and shaking up to obtain mixed low-concentration standard solutions with 0.2mg of glucosamine and chondroitin sulfate in each 1 ml; a mixed low-concentration standard solution containing 0.6mg of glucosamine and chondroitin sulfate per 1ml was prepared in the same manner.
2. Preparation of test solution
Taking the product, removing the film coated tablet, grinding, taking 100mg of powder, precisely weighing, placing in a 50mL measuring flask, adding 5mL acetonitrile, ultrasonically dispersing for 1min, adding water to approximate scale, ultrasonically dispersing for 10min, fixing volume with water to scale, shaking, and centrifuging to obtain the final product.
3. Chromatographic conditions
Waters Xbridge Amide (4.6X 150mm, 3.5 μm) as column; mixing acetonitrile: 50mmol of ammonium acetate water (containing 0.6% triethylamine) 75:25, isocratic elution is carried out on the mobile phase; the flow rate is 0.8 mL/min; the column temperature was 40 ℃; the temperature of the evaporative light drift tube was 105 ℃ and the nitrogen flow rate was 2.5 ml/min.
4. Measurement of
Precisely sucking 5 μ l of the mixed reference solution and sample solution respectively, injecting into liquid chromatograph, measuring, and calculating according to the following formula.
In the formula: the content of D-glucosamine potassium sulfate in the X-sample is g/100 g;
c-the glucosamine concentration in the sample, mg/mL, as determined from the standard curve;
v, determining the volume of the sample to be the volume mL;
m-weight of the sample, g;
f-conversion factor of glucosamine hydrochloride and glucosamine sulfate potassium salt or sodium salt.
In the formula: the content of chondroitin sulfate in the X-sample is g/100 g;
c, the concentration of chondroitin sulfate in the sample, mg/mL, is obtained by a standard curve;
v, determining the volume of the sample to be the volume mL;
m-weight of sample, g.
5. Results
Three batches of health food Yinyi tablets (batch numbers 20180201, 20180202 and 20180203 respectively) were taken for measurement, and the results are shown in Table 1. The theoretical value of glucosamine potassium sulfate in the sample is 28 percent, the theoretical value of chondroitin sulfate is 23 percent, and the table shows that the measured value is closer to the theoretical value, thereby indicating that the result is more accurate.
TABLE 1 results of the assay of the three samples
EXAMPLE 3 HPLC-ELSD quantitative determination method of glucosamine and chondroitin sulfate in raw materials
Respectively taking 20mg of glucosamine potassium sulfate salt or chondroitin sulfate as raw materials, precisely weighing, placing in a 50mL measuring flask, adding 5mL of acetonitrile, ultrasonically dispersing for 1min, adding water to approximate scale, ultrasonically dispersing for 10min, adding water to constant volume to scale, shaking up, and centrifuging to obtain the final product. The remainder was conducted in the same manner as in example 2, whereby the content of glucosamine chondroitin sulfate potassium salt as a raw material was 95.37% and 97.80%.
Example 4 method verification
1. Specialization inspection
Preparation of control solutions: the mixed control solution of example 2 was prepared according to the method described above.
Preparation of a test solution: the test solution was prepared according to the method of example 2.
Preparation of negative test solution: taking a negative sample without glucosamine potassium sulfate salt and chondroitin sulfate, and preparing the negative sample according to the preparation method of the test solution.
And (3) taking a blank solvent, a mixed reference substance solution, a test sample solution and a negative test sample solution, and injecting samples respectively as shown in the figure (30-35). As can be seen from the figure, the chromatogram of the test sample shows a chromatographic peak which is consistent with the retention time of the main peak of the chromatogram of the reference sample, and the chromatograms of the blank solvent and the negative test sample basically have no impurity peak at the retention time of the component to be detected. The method has good specificity.
2. Investigation of linear relationships
Preparation of standard stock solutions: taking 40mg of glucosamine and chondroitin sulfate reference substances dried for 4 hours at 105 ℃, precisely weighing the glucosamine and chondroitin sulfate reference substances in a 10mL volumetric flask, adding 1mL of acetonitrile, oscillating to uniformly disperse the acetonitrile, adding water, ultrasonically dissolving the acetonitrile, cooling to room temperature, fixing the volume to the scale with water, and shaking up to obtain mixed standard stock solutions containing 4mg of glucosamine and chondroitin sulfate in each 1 mL.
Precisely measuring 0.4 mL, 0.6mL, 0.8mL, 1.0mL, 1.2 mL, 1.4 mL and 1.6mL of the mixed standard stock solution in a 10mL volumetric flask, adding water to dilute the solution to a scale, preparing mixed reference solutions with 7 series of concentrations, and drawing a standard curve according to example 2 by taking the natural logarithm of the average value of the chromatographic peak area as the abscissa and the natural logarithm of the mass as the ordinate.
The linear equation of glucosamine is 1.3625x +6.12, and r is 0.9999; the linear equation for chondroitin sulfate is 1.3986x +5.0758, and r is 0.9994. The result shows that the linear relation of glucosamine in 0.862-3.449 mug is good, and the linear relation of chondroitin sulfate in 0.843-2.952 mug is good.
3. Precision test
Taking linear low, medium and high concentration mixed reference substance solution, continuously injecting for 6 times according to example 2, measuring peak area, calculating RSD, and obtaining RSD of 0.73%, 0.66% and 1.38% respectively for low, medium and high concentrations of glucosamine, and RSD of 0.73%, 0.66% and 1.38% respectively for low, medium and high concentrations of chondroitin sulfate: 2.47%, 2.09% and 1.57%, all of which are less than 3%, indicating that the precision of the instrument is good.
4. Stability test
The same reference solution and the same test solution were respectively taken and measured according to the method of example 2 at 0, 2, 4, 6, 8, 10, and 12h, and RSD was calculated, and as a result, glucosamine RSD was 0.96% and 1.97% respectively, and chondroitin sulfate RSD was 1.65 and 3.15% respectively, indicating that the reference solution and the test solution had good stability within 12 h.
5. Repeatability test
The same batch of samples was sampled, 6 parts of test solutions were prepared by the method of example 2, and the content and RSD value of each component were calculated by the method of example 2. The results show that the average glucosamine content is 27.02% and the RSD is 1.95%; the average chondroitin sulfate content was 23.16% and RSD was 0.32%, indicating good reproducibility of the method.
6. Sample application recovery test
50mg of the product (with glucosamine content of 27.02% and chondroitin sulfate content of 23.16%) is precisely weighed in a 50mL volumetric flask, the ratio of the addition amount of the reference substance to the amount of the component to be measured in the sample is 1:1 (added in solid form), 6 parts of sample solution is prepared according to the method of example 2, and the results are shown in tables 1-2 by the method of example 2. As a result, the average recovery rate of glucosamine was 95.19%, the average recovery rate of RSD was 1.14%, the average recovery rate of chondroitin sulfate was 103.72%, and the average recovery rate of RSD was 2.84%. The average recovery rate is between 95% and 105%, and the RSD is less than 3%, which indicates that the method has better accuracy.
TABLE 1 glucosamine sample recovery test
TABLE 2 chondroitin sulfate sample application recovery test
Example 5 comparative example-determination of glucosamine and chondroitin sulfate content in health food Yinyi tablet by GB/T20365-
1. Preparation of control solutions
Weighing 5mg of chondroitin sulfate and D-glucosamine hydrochloride which are dried for 4 hours at 105 ℃ into a 10ml measuring flask, adding 1ml of acetonitrile, shaking to uniformly disperse the mixture, adding water, dissolving the mixture by ultrasonic treatment, and fixing the volume to the scale by using water to obtain 0.5mg of mixed reference substance solution containing glucosamine and chondroitin sulfate per 1 ml.
2. Preparation of test solution
Taking 20180201 batches of samples in example 2, removing film coated tablets, grinding, taking 100mg of powder, precisely weighing, placing in a 50mL measuring flask, adding 5mL of acetonitrile, ultrasonically dispersing for 1min, adding water to approach the scale, ultrasonically treating for 10min, fixing the volume to the scale with water, shaking up, and centrifuging to obtain the product.
3. Chromatographic conditions
NUCLEODUR C18 Pyramid (4.6 mm. times.250 mm, 5 μm) as chromatographic column; acetonitrile + pentane sodium sulfonate (10+90) is used as a mobile phase for isocratic elution; the flow rate is 0.8 mL/min; the column temperature was 25 ℃; the wavelength was 192 nm.
4. Measurement of
Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring to obtain a chromatogram shown in fig. 36-37. As can be seen from the figure, chondroitin sulfate and glucosamine show peaks at about 2min, the peak type tailing is serious, the separation degree of two peaks and the middle impurity peak in the test solution is less than 1.5, the separation effect is poor, and the quantification is inaccurate. In addition, the ion pair reagent and the strong anion chromatographic column are adopted, the balance can be realized for at least 2 hours, the time consumption is long, and the stability and the repeatability are poor.
Therefore, the invention adopts an evaporative light scattering detection detector, improves the sensitivity of glucosamine and chondroitin sulfate, adopts an amide chromatographic column with moderate retention time, good separation degree, symmetrical peak pattern and good repeatability. The method is verified by systematic methodology to have good linear relation, precision, stability and recovery rate, can accurately determine the content of glucosamine and chondroitin sulfate in the health food, is simple and convenient to operate, has short period, is suitable for determining the glucosamine and chondroitin sulfate in the health food, and is also suitable for determining raw materials of the glucosamine or chondroitin sulfate.
The above embodiments are not intended to limit the present invention, and the scope of the present invention is defined by the appended claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.
Claims (5)
1. A method for determining glucosamine and chondroitin sulfate, the method comprising: carrying out HPLC detection on a test solution prepared from glucosamine and chondroitin sulfate, wherein the HPLC detection uses an evaporative light scattering detector, and the chromatographic conditions of the detection are as follows: amide chromatographic column Waters
Amide: 4.6X 150mm, 3.5 μm; mixing acetonitrile: 50mmol/L ammonium acetate water = 75: isocratic elution is carried out on 25 as a mobile phase, and 50mmol/L ammonium acetate contains 0.6 percent of triethylamine.
2. The method of claim 1, wherein the test solution has a detection flow rate of 0.7 to 1.0mL/min and a column temperature of 40 ℃.
3. A method for determining glucosamine and chondroitin sulfate in food or medicine, characterized in that an evaporative light scattering detector is used as an HPLC detector, and the chromatographic conditions for detection are as follows: amide chromatographic column Waters
Amide: 4.6X 150mm, 3.5 μm; mixing acetonitrile: 50mmol/L ammonium acetate water = 75: isocratic elution is carried out on 25 as a mobile phase, and 50mmol/L ammonium acetate contains 0.6 percent of triethylamine.
4. The method of claim 3, wherein the control solution is prepared by: accurately weighing appropriate amount of glucosamine and chondroitin sulfate reference substances, adding 2ml acetonitrile, oscillating to disperse uniformly, adding water, and ultrasonically dissolving to obtain mixed low concentration standard solution containing 0.2mg glucosamine and chondroitin sulfate per 1 ml; the preparation process of the test solution comprises the following steps: grinding the food or medicine to be tested, taking 100mg of powder, precisely weighing, placing in a 50mL measuring flask, adding 5mL acetonitrile, ultrasonically dispersing for 1min, adding water to approximate scale, ultrasonically dispersing for 10min, adding water to constant volume to scale, shaking up, and centrifuging to obtain the final product.
5. The method of claim 3, wherein the method has a detection flow rate of 0.8mL/min and a column temperature of 40 ℃.
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