CN115219628A - Pretreatment method of compound donkey-hide gelatin slurry and detection method of oligosaccharide in compound donkey-hide gelatin slurry - Google Patents
Pretreatment method of compound donkey-hide gelatin slurry and detection method of oligosaccharide in compound donkey-hide gelatin slurry Download PDFInfo
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Abstract
The invention provides a pretreatment method of compound donkey-hide gelatin slurry and a detection method of oligosaccharide in the compound donkey-hide gelatin slurry, and relates to the technical field of analysis and detection. According to the invention, the first alcohol solvent is firstly used for precipitation to remove part of monosaccharide, so that the response value of the monosaccharide is reduced, and the oligosaccharide characteristic map is improved; the volume ratio is 1: 1-1.5 of water and a second alcohol mixed solvent are precipitated to remove the interference of protein and polysaccharide and protect oligosaccharide from being damaged; the stability of the sample solution to be detected is improved by using 50-70% acetonitrile water solution for dissolution; an ELSD detector is adopted for HPLC detection, and the compatibility of a solvent is good; by adopting a specific mobile phase system and a gradient system program, the separation degree of each component in the obtained oligosaccharide characteristic spectrum is good, no impurity peak interference exists, the accuracy, the precision and the stability of the detection result of the content of the manninotriose are high, and the blank of the oligosaccharide characteristic spectrum of the compound donkey-hide gelatin syrup in Chinese pharmacopoeia and the method for measuring the content of the manninotriose is made up.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a pretreatment method of compound donkey-hide gelatin slurry and a detection method of oligosaccharide in the compound donkey-hide gelatin slurry.
Background
The compound donkey-hide gelatin syrup is a Chinese medicinal prescription preparation prepared by modern processing of codonopsis pilosula, red ginseng, prepared rhizome of rehmannia, hawthorn and donkey-hide gelatin, has the main effects of tonifying qi and nourishing blood, and is used for treating deficiency of both qi and blood, dizziness, palpitation, insomnia and the like. The compound colla Corii Asini slurry has effects of adjuvant resisting cancer, relieving fatigue, treating gynecological diseases and replenishing blood. The 2020 version of Chinese pharmacopoeia stipulates the content of total nitrogen and n-butanol extract in the quality standard of compound colla Corii Asini slurry, and also qualitatively stipulates its characteristic amino acids, but has no characteristic spectrum item.
Modern pharmacological research finds that the codonopsis pilosula oligosaccharide can regulate immunity by regulating MAPKS signal channels, and the rehmannia oligosaccharide has the physiological activities of enhancing immunity, enhancing hematopoiesis, improving glycometabolism disorder, protecting oxidative liver injury and the like, so that the oligosaccharide is an important material basis for the compound donkey-hide gelatin syrup to play the core effects of tonifying qi and nourishing blood. The saccharide is used as an important component in the compound donkey-hide gelatin syrup, the content of the saccharide is about 40wt% of the total extract content, however, the prior basic researches on the four plant medicinal chemicals of the compound donkey-hide gelatin syrup mostly concentrate on saponins, flavonoids, polyphenols and the like, and the research on the saccharide in the compound donkey-hide gelatin syrup is less. Therefore, the method for analyzing the oligosaccharide in the compound donkey-hide gelatin syrup is simple and convenient to construct and has important significance for qualitative and quantitative research on oligosaccharide components in the compound donkey-hide gelatin syrup.
Disclosure of Invention
In view of the above, the present invention aims to provide a pretreatment method of compound donkey-hide gelatin slurry and a detection method of oligosaccharide in compound donkey-hide gelatin slurry, the compound donkey-hide gelatin slurry oligosaccharide to be detected obtained by the pretreatment method provided by the present invention has the advantages of low impurity content and high oligosaccharide purity, and the detection method provided by the present invention can realize qualitative and accurate quantitative detection of oligosaccharide component in compound donkey-hide gelatin slurry.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a pretreatment method of compound donkey-hide gelatin syrup, which comprises the following steps:
mixing the compound donkey-hide gelatin slurry with a first alcohol solvent, and performing first precipitation to obtain an alcohol precipitate;
mixing the alcohol precipitate, water and a second alcohol solvent, and performing second precipitation to obtain a liquid component; the volume ratio of the water to the second glycol solvent is 1: (1-1.5);
and concentrating the liquid component to obtain the compound donkey-hide gelatin syrup oligosaccharide to be detected.
Preferably, the volume of the first alcohol solvent is 95-98% of the total volume of the compound donkey-hide gelatin syrup and the first alcohol solvent.
Preferably, the volume ratio of the compound donkey-hide gelatin syrup to water is 1: (2 to 50).
Preferably, the first alcohol solvent and the second alcohol solvent independently comprise ethanol and/or methanol.
Preferably, the time of the first precipitation and the second precipitation is independently 12 to 18 hours.
The invention also provides a method for detecting oligosaccharide in the compound donkey-hide gelatin syrup, which comprises the following steps:
dissolving the compound donkey-hide gelatin syrup oligosaccharide to be detected obtained by the pretreatment method in the technical scheme in acetonitrile aqueous solution to obtain sample liquid to be detected; the volume fraction of acetonitrile in the acetonitrile water solution is 50-70%;
performing high performance liquid detection on the sample liquid to be detected to obtain a detection result of oligosaccharide in the compound donkey-hide gelatin syrup; the detection result comprises an oligosaccharide characteristic map and/or the content of mannotriose; the oligosaccharide comprises glucose, fructose, sucrose, melibiose, trisaccharide, tetrasaccharide, mannotriose and stachyose;
the detection conditions of the high-performance liquid phase comprise:
the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises 0.1-0.3 v/v% triethylamine aqueous solution, and the mobile phase B comprises acetonitrile; gradient elution procedure:
0-5 min, the volume fraction of the mobile phase B is 78%,
the volume fraction of the mobile phase B is reduced from 78 percent to 70 percent at a constant speed within 5 to 18min,
the volume fraction of the mobile phase B is reduced from 70 percent to 60 percent at a constant speed for 18 to 28min,
28-32 min, the volume fraction of the mobile phase B is 60 percent,
the volume fraction of the mobile phase B is increased from 60 percent to 78 percent at a constant speed within 32-33 min,
the volume fraction of the mobile phase B is 78 percent in 33-40 min;
the detector is an evaporative light scattering detector, and the conditions of the evaporative light scattering detector comprise: the drift tube temperature is 85-95 ℃, and the air flow rate is 2.0-2.5 L.min -1 。
Preferably, the volume ratio of the acetonitrile aqueous solution to the compound donkey-hide gelatin syrup for preparing the compound donkey-hide gelatin syrup oligosaccharide is 3-5: 1.
preferably, the detection conditions of the high performance liquid phase further include: the chromatographic column is Waters Xbridge BEHAmide; the column temperature is 25-40 ℃; the sample injection amount is 5-10 mu L.
Preferably, the conditions of the evaporative light scattering detector further include a gain value of 1 to 3.
The invention provides a pretreatment method of compound donkey-hide gelatin syrup, which comprises the following steps: mixing the compound donkey-hide gelatin slurry with a first alcohol solvent, and performing first precipitation to obtain an alcohol precipitate; mixing the alcohol precipitate, water and a second alcohol solvent, and performing second precipitation to obtain a liquid component; the volume ratio of the water to the second glycol solvent is 1: (1-1.5); and concentrating the liquid component to obtain the compound donkey-hide gelatin syrup oligosaccharide to be detected. According to the pretreatment method provided by the invention, the first alcohol solvent is used for precipitation, so that part of monosaccharide can be removed, the response value of monosaccharide in the subsequent high-performance liquid detection process is reduced, and the oligosaccharide characteristic map is improved; then, adopting the volume ratio of water to the second glycol solvent as 1: the mixed solvent of (1-1.5) is used for precipitation, thereby not only removing the interference of protein and polysaccharide components, but also protecting oligosaccharide components with higher molecular weight from being damaged. Moreover, the pretreatment method provided by the invention is simple to operate, the compound donkey-hide gelatin syrup oligosaccharide sample to be detected obtained by pretreatment is detected by HPLC, the separation degree of each component in the characteristic map of the obtained oligosaccharide (glucose, fructose, sucrose, trisaccharide, melibiose, tetrasaccharide, mannotriose and stachyose) is good, no impurity peak interference exists, and the detection accuracy, the precision and the stability of the mannotriose content are high.
The invention also provides a detection method of oligosaccharide in the compound donkey-hide gelatin syrup. According to the invention, the compound donkey-hide gelatin syrup oligosaccharide to be detected obtained by pretreatment is dissolved in acetonitrile aqueous solution with the acetonitrile volume fraction of 50-70%, so that the sample solution to be detected is not easy to delaminate, the stability is high, and the detection result is high in accuracy; an evaporative light scattering detector is adopted, so that the solvent compatibility of the detection method is improved; by adopting a specific mobile phase system and a gradient system program, the separation degree of each component in the obtained oligosaccharide characteristic map is good, no impurity peak interference exists, and the accuracy, precision and stability of the detection result of the oligosaccharide content are high. The detection method provided by the invention fills the blank of the oligosaccharide characteristic map of compound donkey-hide gelatin syrup and the method for measuring the content of mannotriose in Chinese pharmacopoeia (2020 edition).
Drawings
FIG. 1 is an HPLC chromatogram of a test solution;
FIG. 2 is an HPLC chromatogram of a mixed control solution;
FIG. 3 is an HPLC overlay chart of a blank solvent, a negative sample solution and a sample solution, wherein a is the blank solvent, b is a negative sample without rehmannia glutinosa, c is a negative sample without Codonopsis pilosula, d is a negative sample without Ginseng radix Rubri, e is a negative sample without crataegi fructus, and f is the sample;
FIG. 4 is an HPLC chromatogram of 16 batches of test solution;
in FIGS. 1 to 4, 1-glucose, 2-fructose, 3-sucrose, 4-trisaccharide, 5-melibiose, 6-tetrasaccharide, 7-mannotriose, and 8-stachyose;
FIG. 5 is an HPLC chromatogram of a blank solvent;
FIG. 6 is an HPLC chromatogram of a test solution of compound colla Corii Asini paste oligosaccharide, wherein 7-mannotriose;
FIG. 7 is an HPLC chromatogram of a mannotriose control, in which 7-mannotriose;
FIG. 8 is an HPLC chromatogram of a compound donkey-hide gelatin syrup oligosaccharide solution (water (A) -acetonitrile (B) as the mobile phase);
FIG. 9 is an HPLC chromatogram of a compound donkey-hide gelatin syrup oligosaccharide solution (mobile phase of 0.2% triethylamine (A) -acetonitrile (B));
FIG. 10 is an HPLC chromatogram of the oligosaccharide solution of compound colla Corii Asini slurry of step 5.2.1;
fig. 11 is an HPLC chromatogram of the compound colla corii asini syrup oligosaccharide solution in step 5.2.2;
FIG. 12 is an HPLC chromatogram of the oligosaccharide solution of Compound colla Corii Asini in 5.2.3;
FIG. 13 is an HPLC chromatogram of the oligosaccharide solution of Compound donkey-hide gelatin syrup in 5.2.4;
FIG. 14 is an HPLC chromatogram of a FUFANGAIJIANG oligosaccharide solution (80% ethanol precipitation);
FIG. 15 is an HPLC chromatogram of a compound colla Corii Asini oligosaccharide solution (80% ethanol precipitation + ethyl acetate-n-butanol extraction);
FIG. 16 is an HPLC chromatogram of a compound donkey-hide gelatin syrup oligosaccharide solution (98% ethanol precipitation +60% ethanol precipitation);
FIG. 17 is an HPLC chromatogram of a complex donkey-hide gelatin syrup oligosaccharide solution at a column temperature of 30 ℃;
FIG. 18 is an HPLC chromatogram of a complex donkey-hide gelatin syrup oligosaccharide solution at a column temperature of 35 ℃.
Detailed Description
The invention provides a pretreatment method of compound donkey-hide gelatin syrup, which comprises the following steps:
mixing the compound donkey-hide gelatin slurry with a first alcohol solvent, and performing first precipitation to obtain an alcohol precipitate;
mixing the alcohol precipitate, water and a second alcohol solvent, and performing second precipitation to obtain a liquid component; the volume ratio of the water to the second glycol solvent is 1: (1-1.5);
and concentrating the liquid component to obtain the compound donkey-hide gelatin syrup oligosaccharide to be detected.
In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.
The invention mixes the compound donkey-hide gelatin slurry with a first alcohol solvent, and carries out first precipitation to obtain an alcohol precipitate.
In the present invention, the first alcohol solvent preferably includes absolute ethanol and/or methanol, and more preferably an absolute alcohol solvent. In the present invention, the volume of the first alcohol solvent is preferably 95 to 98%, more preferably 96 to 98%, and even more preferably 97 to 98% of the total volume of the compound donkey-hide gelatin syrup and the first alcohol solvent.
The mixing mode is not particularly limited, and the raw materials can be uniformly mixed, such as stirring and mixing; the temperature of the mixing is preferably room temperature.
In the present invention, the temperature of the first precipitation is preferably room temperature, and the time of the first precipitation is preferably 12 to 18 hours, more preferably 13 to 17 hours, further preferably 14 to 16 hours, and most preferably 15 hours; the first precipitation is preferably carried out under standing conditions.
After the first precipitation is completed, the method preferably further comprises the step of carrying out solid-liquid separation on the obtained first precipitation system to obtain an alcohol precipitate. The solid-liquid separation mode is not particularly limited, and the solid-liquid separation mode known to a person skilled in the art is adopted for concentration until the weight is constant, such as centrifugal separation; the rotation speed of the centrifugal separation is preferably 4000 to 6000rpm, more preferably 4500 to 5500rpm, and further preferably 5000rpm; the time for the centrifugal separation is preferably 10 to 15min, more preferably 11 to 14min, and still more preferably 12 to 13min.
After the alcohol precipitate is obtained, the alcohol precipitate, water and a second alcohol solvent are mixed for second precipitation to obtain a liquid component.
In the present invention, the second alcohol solvent preferably includes absolute ethanol and/or methanol, and more preferably an absolute alcohol solvent. In the present invention, the volume ratio of the water to the second glycol solvent is 1: (1 to 1.5), preferably 1: (1.1 to 1.4), more preferably 1: (1.2-1.3). In the invention, the volume ratio of the compound donkey-hide gelatin syrup to water is preferably 1: (2 to 50), more preferably 1: (5 to 40), more preferably 1: (10-30), most preferably 1: (20 to 25).
The mixing mode is not particularly limited, and the raw materials can be uniformly mixed, such as stirring and mixing; the temperature of the mixing is preferably room temperature. In the embodiment of the present invention, the mixing is preferably performed by dissolving the alcohol precipitate in water and then adding the second glycol solvent.
In the present invention, the temperature of the second precipitation is preferably room temperature, and the time of the second precipitation is preferably 12 to 18 hours, more preferably 13 to 17 hours, further preferably 14 to 16 hours, and most preferably 15 hours; the second precipitation is preferably carried out under stationary conditions.
After the second precipitation is completed, the method preferably further comprises the step of carrying out solid-liquid separation on the obtained second precipitation system to obtain an alcohol precipitate. The solid-liquid separation mode is not particularly limited, and the solid-liquid separation mode known to a person skilled in the art is adopted for concentration until the weight is constant, such as centrifugal separation; the rotation speed of the centrifugal separation is preferably 4000 to 6000rpm, more preferably 4500 to 5500rpm, and further preferably 5000rpm; the time for the centrifugal separation is preferably 10 to 15min, more preferably 11 to 14min, and still more preferably 12 to 13min.
After the liquid component is obtained, the liquid component is concentrated to obtain the compound donkey-hide gelatin syrup oligosaccharide to be detected. The concentration method is not particularly limited in the invention, and the concentration method known to those skilled in the art can be adopted to concentrate the solution to constant weight, such as reduced pressure distillation; the temperature of the concentration is preferably 40 to 70 ℃, more preferably 50 to 60 ℃.
The invention also provides a method for detecting oligosaccharide in the compound donkey-hide gelatin syrup, which comprises the following steps:
dissolving the compound donkey-hide gelatin syrup oligosaccharide to be detected obtained by the pretreatment method in the technical scheme in acetonitrile aqueous solution to obtain a sample solution to be detected; the volume fraction of acetonitrile in the acetonitrile water solution is 50-70%;
performing high performance liquid detection on the sample liquid to be detected to obtain a detection result of oligosaccharide in the compound donkey-hide gelatin slurry; the detection result comprises an oligosaccharide characteristic map and/or the content of mannotriose; the oligosaccharide comprises glucose, fructose, sucrose, melibiose, trisaccharide, tetrasaccharide, mannotriose and stachyose;
the detection conditions of the high-performance liquid phase comprise:
the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises 0.1-0.3 v/v% triethylamine aqueous solution, and the mobile phase B comprises acetonitrile; gradient elution procedure:
0-5 min, the volume fraction of the mobile phase B is 78%,
the volume fraction of the mobile phase B is reduced from 78 percent to 70 percent at a constant speed within 5 to 18min,
the volume fraction of the mobile phase B is reduced from 70 percent to 60 percent at a constant speed for 18 to 28min,
28-32 min, the volume fraction of the mobile phase B is 60 percent,
the volume fraction of the mobile phase B is increased from 60 percent to 78 percent at a constant speed within 32-33 min,
the volume fraction of the mobile phase B is 78 percent in 33-40 min;
the detector is an evaporative light scattering detector, and the conditions of the evaporative light scattering detector comprise: the drift tube temperature is 85-95 ℃, and the air flow rate is 2.0-2.5L/min -1 。
The compound donkey-hide gelatin syrup oligosaccharide to be detected obtained by the pretreatment method in the technical scheme is dissolved in acetonitrile aqueous solution to obtain sample liquid to be detected. In the present invention, the volume fraction of acetonitrile in the acetonitrile aqueous solution is preferably 50 to 70%, more preferably 50 to 65%, and still more preferably 55 to 60%. In the invention, the volume ratio of the acetonitrile aqueous solution to the compound donkey-hide gelatin syrup for preparing the compound donkey-hide gelatin syrup oligosaccharide is preferably 3-5: 1, more preferably 3.5 to 4.5:1, more preferably 4:1.
after the sample liquid to be detected is obtained, the invention carries out high performance liquid detection on the sample liquid to be detected to obtain the detection result of oligosaccharide in the compound donkey-hide gelatin syrup.
In the present invention, the detection conditions of the high performance liquid phase include:
the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises 0.1-0.3 v/v% of triethylamine aqueous solution, preferably 0.2v/v% of triethylamine aqueous solution, and the mobile phase B comprises acetonitrile; gradient elution procedure:
0-5 min, the volume fraction of the mobile phase B is 78%,
5-18 min, the volume fraction of the mobile phase B is reduced from 78 percent to 70 percent at a constant speed,
the volume fraction of the mobile phase B is reduced from 70 percent to 60 percent at a constant speed for 18 to 28min,
28-32 min, the volume fraction of the mobile phase B is 60 percent,
the volume fraction of the mobile phase B is increased from 60 percent to 78 percent at a constant speed within 32-33 min,
the volume fraction of the mobile phase B is 78 percent in 33-40 min;
the chromatographic column is preferably a Waters XBridge BEHAmide;
the column temperature is preferably 25 to 40 ℃, and more preferably 30 to 35 ℃;
the detector is an evaporative light scattering detector, and the conditions of the evaporative light scattering detector comprise: the drift tube temperature is 85-95 ℃, and the air flow rate is 2.0-2.5L/min -1 (ii) a The gain value is preferably 1 to 3, more preferably 1 to 2, and still more preferably 1;
the amount of the sample is preferably 5 to 10. Mu.L.
In the present invention, the detection result includes oligosaccharide profile and/or mannotriose content; the oligosaccharide comprises glucose, fructose, sucrose, melibiose, trisaccharide, tetrasaccharide, mannotriose and stachyose.
In the present invention, the high performance liquid chromatography detection preferably comprises the steps of:
(1) Dissolving a glucose reference substance, a fructose reference substance, a sucrose reference substance, a melibiose reference substance, a manninotriose reference substance and stachyose reference substance in an acetonitrile aqueous solution to obtain a mixed reference substance solution; the volume fraction of acetonitrile in the acetonitrile water solution is 50-70%;
(2) Performing high performance liquid chromatography detection on the mixed reference solution to obtain a reference chromatogram; obtaining the retention time of 6 oligosaccharide contrast products according to the chromatogram of the contrast product;
(3) Performing high performance liquid chromatography detection on the sample liquid to be detected to obtain a sample chromatogram; respectively obtaining the sample retention time and the mannotriose peak area of 8 oligosaccharides in the sample to be detected according to the sample chromatogram;
(4) Calculating the relative retention time of other 7 kinds of oligosaccharides by taking the retention time of the mannotriose in the retention time of the reference substance as a reference peak to obtain an oligosaccharide characteristic map; the relative retention time is within ± 5%;
(5) Calculating the content of the mannotriose in the sample liquid to be detected according to the peak area of the mannotriose and a preset standard curve; the standard curve is a standard curve of chromatographic peak area (Y) and concentration logarithm (X) of mannotriose;
the step (2) and the step (3) have no time sequence, and the step (4) and the step (5) have no time sequence.
The invention compares glucoseDissolving the product, fructose reference substance, sucrose reference substance, melibiose reference substance, mannotriose reference substance and stachyose reference substance in acetonitrile aqueous solution to obtain mixed reference substance solution. In the present invention, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 50 to 70%, preferably 50 to 65%, and more preferably 55 to 60%. In the present invention, the concentration of each control in the mixed control solution is preferably 0.3 to 0.6mg/mL, more preferably 0.4 to 0.45mg/mL. In the invention, the Glucose reference substance has the English name of D-Glucose and the molecular formula of C 6 H 12 O 6 IUPAC is named (3R, 4S,5S, 6R) -6- (hydroxyymethyl) oxane-2,3,4,5-tetrol, CAS is 2280-44-6. In the invention, the Fructose reference substance is named as D-Fructose in English, and the molecular formula is C 6 H 12 O 6 CAS is 6347-01-9, IUPAC named (3S, 4R, 5R) -2- (hydroxymethy) oxane-2,3,4,5-tetrol. In the invention, the Sucrose English name is Sucross, and the molecular formula is C 12 H 22 O 11 CAS is 57-50-1, the IUPAC name being (2R, 3R,4S,5S, 6R) -2- [ (2S, 3S,4S, 5R) -3,4-dihydroxy-2,5-bis (hydroxymethy) oxolan-2-yl]oxy-6- (hydroxymethyl) oxide-3, 4,5-triol. In the invention, the Melibiose reference substance is named as Melibiose in English, and the molecular formula is C 12 H 22 O 11 IUPAC is named (3R, 4S,5S, 6R) -6- [ [ (2S, 3R,4S,5R, 6R) -3,4,5-trihydroxy-6- (hydroxymethy) oxan-2-yl]oxymethyl]oxane-2,3,4,5-tetrol, CAS, 5340-95-4. In the invention, the mannotriose reference substance is named Manninotriose in English and has a molecular formula of C 18 H 32 O 16 CAS is 13382-86-0, the IUPAC designation is (2R, 3S,4R, 5R) -2,3,4,5-tetrahydroxy-6- [ (2S, 3R,4S,5R, 6R) -3,4,5-trihydroxy-6- [ [ (2S, 3R,4S,5R, 6R) -3,4,5-trihydroxy-6- (hydroxyymethyl) oxan-2-yl]oxymethyl]oxan-2-yl]oxyhexanal. In the invention, the Stachyose reference substance has the English name of Stachyose and the molecular formula of C 24 H 42 O 21 CAS is 470-55-3, the IUPAC name is (2S, 3R,4S,5R, 6R) -2- [ [ (2R, 3R,4S,5R, 6S) -6- [ [ (2R, 3S,4S,5R, 6R) -6- [ (2S, 3S,4S, 5R) -3,4-dihydroxy-2,5-bis (hydroxymethy) oxolan-2-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methoxy]-3,4,5-trihydroxyoxan-2-yl]methoxy]-6- (hydroxymethy) oxane-3,4,5-triol. In the present example, 6 oligosaccharide control products glucose, fructose, sucrose, melibiose, mannotriose and stachyose are preferably purchased from WUDSTE Biotechnology Limited, and the purity of 8 control products is preferably more than or equal to 98%. In the present invention, the 6 oligosaccharide reference substances have the following structural formulas:
after obtaining a mixed reference substance solution, carrying out high performance liquid chromatography detection on the mixed reference substance solution to obtain a reference substance chromatogram; and obtaining the retention time of the 6 kinds of oligosaccharide contrast products according to the chromatogram of the contrast product. In the present invention, the conditions of the high performance liquid chromatography detection are preferably the same as the conditions of the high performance liquid chromatography detection, and are not described herein again.
Carrying out high performance liquid chromatography detection on the sample liquid to be detected to obtain a sample chromatogram; and respectively obtaining the sample retention time and the mannotriose peak area of 8 oligosaccharides in the sample to be detected according to the sample chromatogram. In the present invention, the conditions of the high performance liquid chromatography detection are the same as the conditions of the high performance liquid chromatography detection, and are not described herein again.
After the retention time of the control is obtained, the relative retention time of other 7 oligosaccharides is calculated by taking the retention time of the mannotriose in the retention time of the control as a reference peak, and the relative retention time is within +/-5%.
After the peak area of the mannotriose is obtained, the content of the mannotriose in the sample liquid to be detected is calculated according to the peak area of the mannotriose and a preset standard curve; the standard curve is a standard curve of the logarithm of chromatographic peak area (X) and the logarithm of concentration (Y) of the mannotriose.
In the present invention, the method for drawing the standard curve preferably includes the following steps:
preparing a series of reference substance standard solutions of manninotriose;
and performing high performance liquid detection on the series of reference substance standard solutions to obtain the chromatographic peak area of the mannotriose, and performing linear fitting on the chromatographic peak area and the concentration to obtain a standard curve.
The invention prepares a series of reference substance standard solutions of mannotriose, preferably comprising the following steps:
adding acetonitrile aqueous solution into mannotriose to obtain mannotriose reference substance stock solution;
and respectively adding acetonitrile aqueous solution into the stock solutions of the mannotriose reference substances for gradual dilution to obtain serial standard solutions of the mannotriose reference substances.
In the present invention, the volume fraction of acetonitrile in the acetonitrile aqueous solution is preferably 50 to 70%, more preferably 50 to 65%, and still more preferably 55 to 60%. In the present invention, the concentration of the stock solution of the mannotriose control is preferably 3 to 6mg/mL, more preferably 4 to 4.5mg/mL.
Preferably, the high performance liquid chromatography detection is carried out on the series of mannotriose reference substance standard solutions according to the detection conditions of the high performance liquid chromatography to obtain peak areas of the series of mannotriose reference substance standard solutions with corresponding concentrations, linear regression is carried out by using a partial least square method by using a peak area logarithm (X) as a horizontal coordinate and a mass concentration logarithm (Y) as a vertical coordinate to obtain a standard curve, and the result is shown as a formula (1):
y =1.5748X +3.606, r =1.0000 formula (1), linear range is 10.88-544.0 ng/mL.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The materials and instruments used in the examples of the invention are as follows: (1) materials: compound donkey-hide gelatin syrup (purchased from east donkey-hide gelatin limited company, with the batch numbers 2106029, 2106030, 2106031, 2106032, 2106033, 2106034, 2106035, 2106036, 2106037, 2106038, 200421, 2011008, 2101027, 2103023, 2104003 and 2106013, and the numbers are respectively marked as S1-S16); acetonitrile (HPLC grade), triethylamine (purity 99.0%, analytical grade), absolute ethanol (analytical grade); sucrose reference substance (CAS No. 57-50-1, purity is more than or equal to 98%), glucose reference substance (CAS No. 50-99-7, purity is more than or equal to 98%), fructose reference substance (CAS No. 57-48-7, purity is more than or equal to 98%), mannotriose reference substance (CAS No. 13382-86-0, purity is more than or equal to 98%), all purchased from Baojichen optical biotechnology limited company; stachyose reference substances (CAS No. 54261-98-2, purity is more than or equal to 98%), and melibiose reference substances (CAS No. 585-99-9, purity is more than or equal to 98%) are purchased from Kyodsite biotech GmbH; (2) apparatus: analytical 1260Infinity II HPLC system (ELSD 6000 evaporative light scattering detector), analytical balance (Sedous science instruments, inc.), CR22 GII floor centrifuge, RE-2000A rotary evaporator (Shanghai Yangrong Biochemical instruments plant), SHZ-III circulating water vacuum pump (Shanghai Yangrong Biochemical instruments plant).
Example 1
Determination of characteristic spectrum of compound donkey-hide gelatin syrup
1.1 preparation of test solutions
Shaking compound donkey-hide gelatin slurry (S1-S16) to dissolve bottom sediment, respectively precisely measuring 1mL to 50mL of compound donkey-hide gelatin slurry (S1-S16) in a centrifuge tube, adding absolute ethyl alcohol until the volume concentration of the ethanol is 98%, standing at room temperature for alcohol precipitation for 12h, then performing centrifugal separation, pouring off supernatant, redissolving the obtained alcohol sediment in 2mL of distilled water, adding 1.5 times of volume of absolute ethyl alcohol, performing alcohol precipitation for 12h at room temperature, centrifuging at 4000rpm for 15min, performing rotary evaporation and concentration on the obtained clear liquid to constant weight, then diluting the obtained solution to 4mL by 50% acetonitrile, and filtering by a 0.22 mu m filter membrane to obtain 16 batches of sample solution with the concentration of 25%.
1.2 preparation of negative sample solution
Preparing a negative sample solution according to the preparation method of step 1.1, which is different from step 1.1 in that a radix rehmanniae preparata-free negative sample solution, a radix codonopsis-free negative sample solution, a red ginseng-free negative sample solution and a hawthorn-free negative sample solution are obtained in the absence of the radix rehmanniae preparata negative sample, the absence of the radix codonopsis-free negative sample, the absence of the red ginseng negative sample and the absence of the hawthorn-free negative sample, respectively.
1.3 preparation of Mixed control solutions
Dissolving glucose reference substance, fructose reference substance, sucrose reference substance, melibiose reference substance, mannotriose reference substance and stachyose reference substance in 50% acetonitrile, mixing, and filtering with 0.22 μm filter membrane to obtain mixed reference substance solution with each reference substance concentration of 0.4 mg/mL.
1.4 determination
Precisely sucking 10 mu L of blank solvent (50% acetonitrile), test solution, mixed reference solution and negative sample solution into a liquid chromatograph respectively, and performing HPLC determination, wherein the HPLC determination conditions are as follows:
the chromatographic column uses Waters Xbridge BEHAmide (5 μm, 4.6X 250 mm); the column temperature was 35 ℃; mobile phase system: mobile phase a (0.2 v/v% triethylamine) and mobile phase B (acetonitrile); gradient elution procedure:
0-5 min, the volume fraction of the mobile phase B is 78%,
the volume fraction of the mobile phase B is reduced from 78 percent to 70 percent at a constant speed within 5 to 18min,
the volume fraction of the mobile phase B is reduced from 70 percent to 60 percent at a constant speed within 18 to 28min,
28-32 min, the volume fraction of the mobile phase B is 60 percent,
the volume fraction of the mobile phase B is increased from 60 percent to 78 percent at a constant speed within 32-33 min,
the volume fraction of the mobile phase B is 78 percent in 33-40 min;
ELSD conditions: the drift tube temperature is 90 ℃, and the air flow rate is 2.3 L.min -1 The gain value is 1, and the sample size is 10 μ L.
FIG. 1 is an HPLC chromatogram of a test solution; FIG. 2 is an HPLC chromatogram of a mixed control solution; FIG. 3 is an HPLC overlay chart of a blank solvent, a negative sample solution and a sample solution, wherein a is the blank solvent, b is a negative sample without rehmannia glutinosa, c is a negative sample without Codonopsis pilosula, d is a negative sample without Ginseng radix Rubri, e is a negative sample without crataegi fructus, and f is the sample; FIG. 4 is an HPLC chromatogram of 16 batches of test solution; in FIGS. 1 to 4, 1-glucose, 2-fructose, 3-sucrose, 4-trisaccharide, 5-melibiose, 6-tetrasaccharide, 7-mannotriose, and 8-stachyose are used.
Selecting peaks with basically consistent peak emergence time and larger peak area in 16 test sample characteristic maps as common peaks, calibrating 8 common peaks, wherein the sum of the peak areas of the 8 peaks accounts for more than 80% of the total peak area, wherein the retention time of the No. 1, 2,3, 5, 7 and 8 peaks is respectively consistent with that of glucose, fructose, sucrose, melibiose, mannotriose and stachyose in a mixed reference sample, and the No. 3 peak, the No. 7 peak and the No. 8 peak are confirmed by comparison of a secondary mass spectrogram; comparing the liquid chromatogram of each negative sample solution with the test sample solution to find that the oligosaccharide in the compound colla Corii Asini slurry is mainly from radix rehmanniae Preparata.
And sequentially introducing the AIA data of the compound donkey-hide gelatin syrup oligosaccharide chromatograms of 16 batches into a traditional Chinese medicine chromatogram characteristic spectrum similarity evaluation system (2012 edition) issued by the national pharmacopoeia committee, taking the sample S1 chromatogram as a reference chromatogram, performing multi-point correction on a chromatogram peak by adopting a median method and a time window width of 0.10min, and automatically matching to generate a reference chromatogram. A total of 8 chromatographic peaks were normalized. The similarity between the chromatograms of the samples S1 to S16 and the contrast map is respectively 0.997, 0.995 0.996, 0.997, 0.996, 0.966, 0.999, 0.986 and 0.905, the similarity between the characteristic spectrum and the contrast spectrum of the compound donkey-hide gelatin syrup oligosaccharide of 16 batches is higher.
The test article should have 8 characteristic peaks, wherein the peak No. 7 should have the same retention time as the corresponding reference peak (mannotriose). And calculating the relative retention time of other characteristic peaks by taking the manninotriose as an S peak, wherein the specified value is shown in the table 1, and the relative retention time is within +/-5% of the specified value.
TABLE 1 relative Retention time of oligosaccharides in Compound colla Corii Asini slurry
Example 2
Methodological inspection of feature maps
2.1 precision
According to the HPLC detection conditions of example 1, the same sample solution (batch number: 2106035) is repeatedly injected and detected for 5 times, and the detection result is as follows: RSD values of common logarithm of peak area (base 10) of each peak were 0.07%,0.100%,0.07%,0.68%,0.13%,0.05%,0.02%,0.73%, respectively; the RSD values for the common logarithm of retention times were 0.75%,0.69%,0.52%,0.44%,0.32%,0.17%,0.12%,0.08%, respectively. The detection method provided by the invention has good instrument precision.
2.2 stability
The same test solution (lot number: 2106035) was used and subjected to measurement after the test solution was left for 0, 2, 4, 6, 8, 12 and 24 hours, respectively, according to the HPLC detection conditions of example 1, and the results of the measurement were as follows: RSD values of common logarithm of each peak area are 0.12%,0.14%,0.38%,1.40%,2.13%,0.39%,0.08% and 0.84% respectively; the RSD values of the common logarithm of retention times were 0.09%,0.11%,0.09%,0.09%,0.13%,0.11%,0.08%,0.10%, respectively. The sample solution treated by the pretreatment method provided by the invention has good stability within 24 hours.
2.3 repeatability
5 parts of the same sample (lot number: 2106035) were precisely measured and prepared as a test solution, which was measured under the HPLC detection conditions of example 1. And (3) detection results: the RSD values of common logarithm of the peak area of each peak are 0.59%,0.71%,0.82%,1.42%,0.56%,0.47%,0.37% and 0.67%, respectively; RSD values for the common logarithm of retention times were 0.09%,0.09%,0.09%,0.08%,0.07%,0.05%,0.04%,0.03%, respectively. The detection method provided by the invention has good repeatability.
Example 3
Content determination of manninotriose in compound donkey-hide gelatin syrup
3.1 preparation of Manitose reference solution
Precisely taking a designated mannotriose reference substance, adding distilled water for dissolving, and filtering through a 0.22 mu m filter membrane to obtain a mannotriose reference substance solution with the concentration of 4.164 mg/mL.
3.2 testing
According to the HPLC detection conditions of example 1, the mannotriose control solution and each test solution prepared in example 1 are injected and detected, wherein the injection amount is 5 μ L.
The measurement results of each test sample solution are shown in table 2:
TABLE 2 measurement results of each sample solution
(n＝3)
As shown in Table 2, the content of mannotriose in 16 batches of compound donkey-hide gelatin syrup ranges from 8.63 to 12.29 mg/mL -1 And each 1mL of compound donkey-hide gelatin syrup contains 11.17mg on average, so that the content of mannotriose in the compound donkey-hide gelatin syrup is preliminarily specified to be not lower than 60 percent of the average value, namely the content of mannotriose in each 1mL of compound donkey-hide gelatin syrup is not lower than 6.7mg.
Example 4
Methodological investigation of assay
4.1 specialization examination
The blank solvent and the mannotriose control in example 3 and the test solution prepared in example 1 (lot number: 2106035) were each measured under the HPLC test conditions in example 1.
FIG. 5 is an HPLC chromatogram of a blank solvent, FIG. 6 is an HPLC chromatogram of a compound donkey-hide gelatin syrup oligosaccharide test solution, and FIG. 7 is an HPLC chromatogram of a mannotriose reference substance. As is clear from FIGS. 5 to 7, the separation degree was good without interference of other components around mannotriose.
4.2 preparation of Standard Curve
Stock solutions of the mannotriose control from example 3 were taken.
Precisely measuring 50, 80, 120, 160 and 200 mu L of reference substance stock solutions respectively, adding water to dilute to 200 mu L respectively, and shaking up by ultrasound to obtain series reference substance solutions with concentrations of 1.0410, 1.6656, 2.4984, 3.3312 and 4.1640mg/mL respectively.
The standard solutions of the control were tested under the HPLC test conditions of example 1 with the common logarithm of the peak area of the control (base 10 logarithm) (X) as the abscissa and the mass concentration of mannotriose (mg. Multidot.mL) -1 ) The common logarithm (Y) of the standard curve is plotted as the ordinate, and linear regression is carried out by a partial least square method to obtain a standard curve Y =1.5748X +3.606 (r = 1.0000), which shows that the linear relation is good in the range of 1.04-4.16 mg/mL. The detection limit and the quantification limit of mannotriose were calculated to be 0.32. Mu.g and 0.53. Mu.g, respectively, at signal-to-noise ratios of 3 and 10, respectively.
4.3 precision
The mannotriose control solution prepared in example 3 was injected repeatedly for 5 times under the HPLC detection conditions of example 1, and the RSD value of the common logarithm of the peak area of mannotriose was 1.08%, and the RSD of the common logarithm of the retention time was 0.05%. The detection method provided by the invention has good instrument precision.
4.4 stability
The test solution prepared in example 1 (batch number: 2106036) was injected and measured 0, 2, 4, 6, 8, 12 and 24h after preparation, and the test results: the RSD value of the common logarithm of the peak area of the manninotriose is 1.20 percent; the RSD value for the common logarithm of retention time was 0.08%. The sample solution treated by the pretreatment method provided by the invention has good stability within 24 hours.
4.5 reproducibility
5 parts of the same compound donkey-hide gelatin slurry (batch: 2106036) are precisely measured to prepare a test solution, and the test solution is measured according to the HPLC detection conditions of the example 1. And (3) testing results: the RSD value for the common logarithm of the area of the mannotriose peak was 0.87%, and the RSD for the common logarithm of the retention time was 0.04%. The detection method provided by the invention has good repeatability.
4.6 recovery of sample
In example 1, 9 parts of test solution with a concentration (the batch of compound donkey-hide gelatin syrup is 2106036, and the content of mannotriose is shown in table 3) are prepared in parallel, 0.5mL of each test solution is added with 3 parts of mannotriose control solution with different concentration levels, namely low (80% of mannotriose content), medium (100% of mannotriose content) and high (120% of mannotriose content), to obtain a mannotriose sample solution, and each concentration is 3 parts in parallel. The mannotriose loading solution was tested according to the HPLC test conditions of example 1, and the test results are shown in Table 3:
TABLE 3 Mantotriose sample recovery test results (n = 9)
As can be seen from table 3, the average sample recovery rate of mannotriose was 99.94% and the RSD was 2.15% (n = 9), indicating that the sample recovery rate of the detection method of the present invention was good.
Example 5
Shaking compound donkey-hide gelatin slurry (batch number is 2106036) to dissolve a bottom precipitate, precisely weighing 1mL to 50mL of compound donkey-hide gelatin slurry in a centrifuge tube, adding absolute ethyl alcohol until the volume concentration of the ethanol is 98%, standing at room temperature for alcohol precipitation for 12h, then performing centrifugal separation, pouring off a supernatant, re-dissolving the obtained alcohol precipitate in 2mL of distilled water, adding 1.5 times of absolute ethyl alcohol by volume, performing alcohol precipitation for 12h at room temperature, centrifuging at 4000rpm for 15min, performing rotary evaporation and concentration on the obtained clear liquid to constant weight, diluting the obtained clear liquid to 4mL by using 50% acetonitrile, and filtering by using a 0.22 mu m filter membrane to obtain the compound donkey-hide gelatin slurry oligosaccharide solution.
5.1 Effect of mobile phase composition on detection of Compound donkey-hide gelatin syrup oligosaccharide solution
5.1.1 the detection of the compound donkey-hide gelatin syrup oligosaccharide solution is carried out according to the HPLC detection conditions of the example 1.
5.1.2 the assay was carried out according to step 5.1.2, differing from step 5.1.2 in that the mobile phase A was water.
Fig. 8 is an HPLC chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution (the mobile phase is water (a) -acetonitrile (B)), fig. 9 is an HPLC chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution (the mobile phase is 0.2% triethylamine (a) -acetonitrile (B)), it can be seen from fig. 8-9 that when acetonitrile and water are used as the mobile phase system, the oligosaccharide has poor peak shape and a forked peak appears, and the addition of 0.2% triethylamine can significantly improve the peak shape.
5.2 Effect of elution mode on Compound colla Corii Asini slurry oligosaccharide solution detection
5.2.1 detecting the compound donkey-hide gelatin syrup oligosaccharide solution according to the HPLC detection conditions of the step 5.1.1, and the difference from the step 5.1.1 is that the elution mode is isocratic elution, and the volume ratio of the mobile phase A to the mobile phase B is 25.
5.2.2 detecting the compound donkey-hide gelatin syrup oligosaccharide solution according to the HPLC detection conditions of the step 5.1.1, and the difference from the step 5.1.1 is that the gradient elution program comprises the following steps:
0-12 min, the volume fraction of the mobile phase B is 80 percent,
the volume fraction of the mobile phase B is reduced from 80 percent to 65 percent at a constant speed within 12 to 22min,
22-26 min, the volume fraction of the mobile phase B is 65 percent,
and (3) increasing the volume fraction of the mobile phase B from 65% to 80% at a constant speed for 26-30 min.
5.2.3 detecting the compound donkey-hide gelatin syrup oligosaccharide solution according to the HPLC detection conditions of the step 5.1.1, and the difference from the step 5.1.1 is that the gradient elution program comprises the following steps:
0-12 min, the volume fraction of the mobile phase B is 80 percent,
the volume fraction of the mobile phase B is reduced from 80 percent to 65 percent at a constant speed within 12 to 25min,
25-28 min, the volume fraction of the mobile phase B is 65%,
and (3) increasing the volume fraction of the mobile phase B from 65% to 80% at a constant speed for 28-30 min.
5.2.4 the compound donkey-hide gelatin syrup oligosaccharide solution is detected according to the HPLC detection conditions of the step 5.1.1, and the difference from the step 5.1.1 is that the gradient elution program:
0-5 min, the volume fraction of the mobile phase B is 78%,
the volume fraction of the mobile phase B is reduced from 78 percent to 70 percent at a constant speed within 5 to 18min,
the volume fraction of the mobile phase B is reduced from 70 percent to 60 percent at a constant speed for 18 to 28min,
28-32 min, the volume fraction of the mobile phase B is 60 percent,
the volume fraction of the mobile phase B is increased from 60 percent to 78 percent at a constant speed within 32-33 min,
the volume fraction of the mobile phase B is 78 percent in 33-40 min.
Fig. 10 is an HPLC chromatogram of the compound colla corii asini slurry oligosaccharide solution in step 5.2.1, fig. 11 is an HPLC chromatogram of the compound colla corii asini slurry oligosaccharide solution in step 5.2.2, fig. 12 is an HPLC chromatogram of the compound colla corii asini slurry oligosaccharide solution in step 5.2.3, and fig. 13 is an HPLC chromatogram of the compound colla corii asini slurry oligosaccharide solution in step 5.2.4. As can be seen from fig. 10 to 13, the compound donkey-hide gelatin syrup oligosaccharide has poor separation effect under isocratic elution, baseline separation cannot be realized, and the optimal elution gradient procedure obtained by changing the proportion of the organic phase and the aqueous phase is as follows: the volume fraction of the mobile phase B is reduced from 78% to 70% at a constant speed for 18-28 min, the volume fraction of the mobile phase B is reduced from 70% to 60% at a constant speed for 28-32 min, the volume fraction of the mobile phase B is 60% to 32-33 min, the volume fraction of the mobile phase B is increased from 60% to 78% at a constant speed, and the volume fraction of the mobile phase B is 78% at 33-40 min.
5.3 Effect of pretreatment method on Compound colla Corii Asini slurry oligosaccharide solution detection
5.3.1 shaking the compound donkey-hide gelatin slurry (batch No. 2106035) to dissolve the bottom precipitate, precisely measuring 1mL to 50mL of the compound donkey-hide gelatin slurry respectively, adding absolute ethyl alcohol until the volume concentration of the ethyl alcohol is 80%, standing at room temperature for alcohol precipitation for 12h, then carrying out centrifugal separation, pouring out the supernatant, diluting the obtained alcohol precipitate to 4mL with 50% acetonitrile, and filtering with a 0.22 mu m filter membrane to obtain the compound donkey-hide gelatin slurry oligosaccharide solution (80% ethanol precipitate) to be detected.
5.3.2 shake compound donkey-hide gelatin slurry (batch number 2106035) to dissolve the bottom precipitate, accurately measure compound donkey-hide gelatin slurry 1mL to 50mL respectively, add absolute ethanol until the volume concentration of ethanol is 80%, standing at room temperature for alcohol precipitation for 12h, then centrifugally separating, extracting the obtained supernatant with an equal volume of ethyl acetate-n-butanol mixed solvent (the volume ratio of ethyl acetate to n-butanol is 1), concentrating the obtained ethyl acetate-n-butanol extract by rotary evaporation to constant weight, then diluting with 50% acetonitrile to 4mL, and passing through a 0.22 μm filter membrane to obtain the compound donkey-hide gelatin slurry oligosaccharide solution to be detected (80% ethanol precipitation + ethyl acetate-n-butanol extraction).
5.3.3 shaking the compound donkey-hide gelatin slurry (batch number is 2106035) to dissolve the bottom precipitate, precisely measuring 1mL to 50mL of the compound donkey-hide gelatin slurry respectively, adding absolute ethyl alcohol until the volume concentration of the ethanol is 98%, standing at room temperature for alcohol precipitation for 12h, then carrying out centrifugal separation, extracting the obtained supernatant by using ethyl acetate with the same volume, redissolving the obtained alcohol precipitate in 2mL of distilled water, adding absolute ethyl alcohol until the volume concentration of the ethanol is 60%, carrying out alcohol precipitation for 12h at room temperature, centrifuging at 4000rpm for 15min, carrying out rotary evaporation and concentration on the obtained supernatant until the weight is constant, then diluting the obtained solution to 4mL by using 50% acetonitrile, and filtering the solution by using a 0.22 mu m filter membrane to obtain the compound donkey-hide gelatin slurry oligosaccharide solution to be detected (98% ethanol precipitate and 60% ethanol precipitate).
And (4) detecting each compound donkey-hide gelatin syrup oligosaccharide solution obtained in the step (5.3.1-5.3.3) according to the HPLC detection condition in the step (5.1.1).
Fig. 14 is an HPLC chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution (80% ethanol precipitation), fig. 15 is an HPLC chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution (80% ethanol precipitation + ethyl acetate-n-butanol extraction), and fig. 16 is an HPLC chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution (98% ethanol precipitation +60% ethanol precipitation). As can be seen from fig. 14-16, the compound colla corii asini pulp oligosaccharide solution prepared by directly precipitating with 80% ethanol has high monosaccharide content and flat peaks; after the supernatant is extracted by ethyl acetate-n-butyl alcohol, part of oligosaccharide is lost; the pretreatment method of removing part of monosaccharide by 98% absolute ethanol precipitation and then removing polysaccharide and protein by 60% ethanol precipitation is simple and easy to implement, the spectrogram is clear, and the loss of oligosaccharide is very small.
The compound donkey-hide gelatin syrup oligosaccharide solution obtained in the step 5.1.1 is detected according to the HPLC detection conditions in the step 5.1.1, and the difference from the step 5.1.1 is that the column temperatures are respectively 30 ℃ and 35 ℃. The influence of the column temperature on the liquid chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution is shown in fig. 17-18, wherein fig. 17 is an HPLC chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution at a column temperature of 30 ℃, fig. 18 is an HPLC chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution at a column temperature of 35 ℃, and fig. 17-18 show that the influence of the column temperature on the liquid chromatogram of the compound donkey-hide gelatin syrup oligosaccharide solution is small.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A pretreatment method of compound donkey-hide gelatin syrup comprises the following steps:
mixing the compound donkey-hide gelatin slurry with a first alcohol solvent, and performing first precipitation to obtain an alcohol precipitate;
mixing the alcohol precipitate, water and a second alcohol solvent, and performing second precipitation to obtain a liquid component; the volume ratio of the water to the second glycol solvent is 1: (1-1.5);
and concentrating the liquid component to obtain the compound donkey-hide gelatin syrup oligosaccharide to be detected.
2. The pretreatment method according to claim 1, wherein the volume of the first alcohol solvent is 95-98% of the total volume of the compound donkey-hide gelatin syrup and the first alcohol solvent.
3. The pretreatment method according to claim 1, wherein the volume ratio of the compound donkey-hide gelatin syrup to water is 1: (2 to 50).
4. The pretreatment method according to claim 1 or 2, wherein the first alcohol solvent and the second alcohol solvent independently comprise ethanol and/or methanol.
5. The pretreatment method according to claim 1, 2 or 3, wherein the time for the first precipitation and the time for the second precipitation are independently 12 to 18 hours.
6. A method for detecting oligosaccharide in compound donkey-hide gelatin syrup comprises the following steps:
dissolving compound donkey-hide gelatin syrup oligosaccharide to be detected obtained by the pretreatment method of any one of claims 1 to 5 in acetonitrile aqueous solution to obtain sample solution to be detected; the volume fraction of acetonitrile in the acetonitrile water solution is 50-70%;
performing high performance liquid detection on the sample liquid to be detected to obtain a detection result of oligosaccharide in the compound donkey-hide gelatin slurry; the detection result comprises an oligosaccharide characteristic map and/or the content of mannotriose; the oligosaccharide comprises glucose, fructose, sucrose, melibiose, trisaccharide, tetrasaccharide, mannotriose and stachyose;
the detection conditions of the high-performance liquid phase comprise:
the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises 0.1-0.3 v/v% triethylamine aqueous solution, and the mobile phase B comprises acetonitrile; gradient elution procedure:
0-5 min, the volume fraction of the mobile phase B is 78%,
the volume fraction of the mobile phase B is reduced from 78 percent to 70 percent at a constant speed within 5 to 18min,
the volume fraction of the mobile phase B is reduced from 70 percent to 60 percent at a constant speed within 18 to 28min,
28-32 min, the volume fraction of the mobile phase B is 60 percent,
the volume fraction of the mobile phase B is increased from 60 percent to 78 percent at a constant speed within 32-33 min,
the volume fraction of the mobile phase B is 78 percent in 33-40 min;
the detector is an evaporative light scattering detector, and the conditions of the evaporative light scattering detector comprise: the drift tube temperature is 85-95 ℃, and the air flow rate is 2.0-2.5 L.min -1 。
7. The detection method according to claim 6, wherein the volume ratio of the acetonitrile aqueous solution to the compound donkey-hide gelatin syrup for preparing the compound donkey-hide gelatin syrup oligosaccharide to be detected is 3-5: 1.
8. the detection method according to claim 6 or 7, wherein the detection conditions of the high-performance liquid phase further include: the chromatographic column is Waters Xbridge BEH Amide; the column temperature is 25-40 ℃; the sample amount is 5-10 mu L.
9. The method of claim 6 or 7, wherein the evaporative light scattering detector further comprises a gain value of 1 to 3.
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