A pretreatment method for compound donkey-hide gelatin slurry and a detection method for oligosaccharides in compound donkey-hide gelatin slurry

Abstract

The present invention provides a pretreatment method of compound donkey-hide glue slurry and a method for detecting oligosaccharides in compound donkey-hide glue slurry, and relates to the technical field of analysis and detection. The present invention first uses a first alcohol solvent for precipitation to remove part of monosaccharides, reduces the response value of monosaccharides, and improves the characteristic spectrum of oligosaccharides; uses a mixed solvent of water and a second alcohol with a volume ratio of 1:1-1.5 for precipitation to remove the interference of proteins and polysaccharides and protect oligosaccharides from destruction; uses a 50-70% acetonitrile aqueous solution for dissolution, thereby improving the stability of the sample liquid to be tested; HPLC detection uses an ELSD detector with good solvent compatibility; uses a specific mobile phase system and gradient system program, and the separation of each component in the obtained oligosaccharide characteristic spectrum is good, without interference from impurity peaks, and the detection result of mannotriose content has high accuracy, precision and stability, which fills the gap in the oligosaccharide characteristic spectrum of compound donkey-hide glue slurry and the method for determining the content of mannotriose in the Chinese Pharmacopoeia.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a pretreatment method of compound donkey-hide gelatin slurry and a detection method of oligosaccharides in the compound donkey-hide gelatin slurry. Background Art

[0002] Compound donkey-hide gelatin paste is a traditional Chinese medicine formula made from Codonopsis pilosula, red ginseng, Rehmannia glutinosa, hawthorn and donkey-hide gelatin through modern processing. Its main function is to nourish qi and blood, and it is used for qi and blood deficiency, dizziness, palpitations and insomnia. Compound donkey-hide gelatin paste has the functions of auxiliary anti-cancer, anti-fatigue, treatment of gynecological diseases and blood nourishment. The 2020 edition of the Chinese Pharmacopoeia stipulates the total nitrogen and n-butanol extract content of compound donkey-hide gelatin paste, and also makes qualitative provisions for its characteristic amino acids, but there is no characteristic spectrum item.

[0003] Modern pharmacological studies have found that Codonopsis oligosaccharides can regulate immunity by regulating the MAPKs signaling pathway, and Rehmannia oligosaccharides have physiological activities such as enhancing immunity, enhancing hematopoiesis, improving glucose metabolism disorders and protecting oxidative liver damage. Therefore, oligosaccharides are an important material basis for the compound donkey-hide glue to exert its core effect of replenishing qi and nourishing blood. As an important component in the compound donkey-hide glue, sugars account for about 40wt% of the total extract content. However, previous basic research on the chemical substances of the four botanical drugs in the compound donkey-hide glue has mostly focused on saponins, flavonoids, polyphenols, etc., and there are few studies on sugars in the compound donkey-hide glue. Therefore, it is of great significance to construct a simple analysis method for oligosaccharides in the compound donkey-hide glue for qualitative and quantitative research on the oligosaccharide components of the compound donkey-hide glue. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a pretreatment method for compound donkey-hide gelatin slurry and a detection method for oligosaccharides in compound donkey-hide gelatin slurry. The impurity content of the oligosaccharides in the compound donkey-hide gelatin slurry to be tested obtained by the pretreatment method provided by the present invention is small, and the purity of the oligosaccharides is high. The detection method provided by the present invention can realize the qualitative and accurate quantitative detection of the oligosaccharide components of the compound donkey-hide gelatin slurry.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a pre-treatment method of compound donkey-hide gelatin paste, comprising the following steps:

[0007] Mixing the compound donkey-hide gelatin slurry with a first alcohol solvent, performing a first precipitation, and obtaining an alcohol precipitate;

[0008] The alcohol precipitate, water and a second alcohol solvent are mixed to perform a second precipitation to obtain a liquid component; the volume ratio of the water to the second alcohol solvent is 1:(1-1.5);

[0009] The liquid component is concentrated to obtain the compound donkey-hide gelatin syrup oligosaccharide to be tested.

[0010] 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.

[0011] Preferably, the volume ratio of the compound donkey-hide gelatin slurry to water is 1:(2-50).

[0012] Preferably, the first alcohol solvent and the second alcohol solvent independently include ethanol and / or methanol.

[0013] Preferably, the time for the first precipitation and the second precipitation is independently 12 to 18 hours.

[0014] The present invention also provides a method for detecting oligosaccharides in the compound donkey-hide gelatin paste, comprising the following steps:

[0015] The oligosaccharide of compound donkey-hide glue to be tested obtained by the pretreatment method described in the above technical scheme is dissolved in an acetonitrile aqueous solution to obtain a sample solution to be tested; the volume fraction of acetonitrile in the acetonitrile aqueous solution is 50-70%;

[0016] The sample solution to be tested is subjected to high performance liquid chromatography to obtain the test result of oligosaccharides in the compound donkey-hide glue paste; the test result includes the oligosaccharide characteristic spectrum and / or the content of mannotriose; the oligosaccharides include glucose, fructose, sucrose, melibiose, trisaccharide, tetrasaccharide, mannotriose and stachyose;

[0017] The detection conditions of the high performance liquid phase include:

[0018] The mobile phase system includes mobile phase A and mobile phase B, wherein the mobile phase A includes 0.1-0.3 v / v% triethylamine aqueous solution, and the mobile phase B includes acetonitrile; the gradient elution program is:

[0019] 0~5min, the volume fraction of mobile phase B is 78%,

[0020] From 5 to 18 minutes, the volume fraction of mobile phase B decreases from 78% to 70% at a constant rate.

[0021] 18-28min, the volume fraction of mobile phase B is uniformly reduced from 70% to 60%.

[0022] 28~32min, mobile phase B volume fraction is 60%,

[0023] 32-33min, the volume fraction of mobile phase B increased from 60% to 78% at a constant rate.

[0024] 33-40min: the volume fraction of mobile phase B is 78%;

[0025] The detector is an evaporative light scattering detector, and the conditions of the evaporative light scattering detector include: drift tube temperature 85-95°C, air flow rate 2.0-2.5L·min -1 .

[0026] Preferably, the volume ratio of the acetonitrile aqueous solution to the compound donkey-hide gelatin slurry used to prepare compound donkey-hide gelatin slurry oligosaccharides is 3 to 5:1.

[0027] Preferably, the detection conditions of the HPLC also include: the chromatographic column is Waters XBridge BEHAmide; the column temperature is 25-40°C; and the injection volume is 5-10 μL.

[0028] Preferably, the conditions of the evaporative light scattering detector also include a gain value of 1-3.

[0029] The present invention provides a pretreatment method of compound donkey-hide gelatin paste, comprising the following steps: mixing the compound donkey-hide gelatin paste with a first alcohol solvent, performing a first precipitation, and obtaining an alcohol precipitate; mixing the alcohol precipitate, water, and a second alcohol solvent, performing a second precipitation, and obtaining a liquid component; the volume ratio of the water to the second alcohol solvent is 1: (1 to 1.5); concentrating the liquid component to obtain the compound donkey-hide gelatin paste oligosaccharide to be tested. The pretreatment method provided by the present invention first uses the first alcohol solvent for precipitation, which can remove part of the monosaccharide, thereby reducing the response value of the monosaccharide in the subsequent high-performance liquid phase detection process and improving the oligosaccharide characteristic spectrum; and then using a mixed solvent with a volume ratio of water to the second alcohol solvent of 1: (1 to 1.5) for precipitation, which not only removes the interference of protein and polysaccharide components, but also protects the higher molecular weight oligosaccharide components from damage. Moreover, the pretreatment method provided by the present invention is simple to operate, and the compound donkey-hide gelatin slurry oligosaccharide sample to be tested obtained by pretreatment is detected by HPLC. The components in the characteristic spectrum of the obtained oligosaccharides (glucose, fructose, sucrose, trisaccharides, melibiose, tetrasaccharides, mannotriose and stachyose) have good separation, no interference from impurity peaks, and the detection accuracy, precision and stability of the mannotriose content are high.

[0030] The present invention also provides a method for detecting oligosaccharides in compound donkey-hide glue slurry. The present invention dissolves the oligosaccharides of the compound donkey-hide glue slurry to be tested obtained by pretreatment in an acetonitrile aqueous solution with an acetonitrile volume fraction of 50 to 70%. The sample liquid to be tested is not easy to stratify, has high stability, and has high accuracy of the test result; an evaporative light scattering detector is used to improve the solvent compatibility of the detection method; a specific mobile phase system and gradient system program are used to obtain a good separation of the components in the oligosaccharide characteristic spectrum, without interference from impurity peaks, and the detection results of the oligosaccharide content have high accuracy, precision and stability. The detection method provided by the present invention fills the gaps in the oligosaccharide characteristic spectrum of compound donkey-hide glue slurry and the method for determining the content of mannotriose in the Chinese Pharmacopoeia (2020 edition). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the HPLC chromatogram of the test solution;

[0032] Figure 2 It is the HPLC chromatogram of the mixed reference solution;

[0033] Figure 3 It is the HPLC overlay diagram of blank solvent, negative sample solution and test solution, where a is blank solvent, b is negative sample without Rehmannia glutinosa, c is negative sample without Codonopsis pilosula, d is negative sample without Red Ginseng, e is negative sample without Crataegus pinnatifida, and f is the test solution;

[0034] Figure 4 HPLC chromatograms of 16 batches of test solution;

[0035] Figure 1 to Figure 4 In, 1-glucose, 2-fructose, 3-sucrose, 4-triose, 5-melibiose, 6-tetraose, 7-mannotriose, 8-stachyose;

[0036] Figure 5 is the HPLC chromatogram of blank solvent;

[0037] Figure 6 It is the HPLC chromatogram of the oligosaccharide test solution of compound donkey-hide glue, in which 7-mannotriose;

[0038] Figure 7 It is the HPLC chromatogram of the mannotriose reference substance, in which 7-mannotriose;

[0039] Figure 8 It is the HPLC chromatogram of compound donkey-hide glue syrup oligosaccharide solution (the mobile phase is water (A)-acetonitrile (B));

[0040] Fig. 9 This is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution (the mobile phase is 0.2% triethylamine (A)-acetonitrile (B);

[0041] Fig.10 is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution in step 5.2.1;

[0042] Fig.11 is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution in step 5.2.2;

[0043] Fig.12 The HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution in 5.2.3;

[0044] Fig.13 The HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution in 5.2.4;

[0045] Fig.14 It is the HPLC chromatogram of compound donkey-hide gelatin oligosaccharide solution (80% ethanol precipitation);

[0046] Fig.15 It is the HPLC chromatogram of compound donkey-hide glue syrup oligosaccharide solution (80% ethanol precipitation + ethyl acetate-n-butanol extraction);

[0047] Fig.16 It is the HPLC chromatogram of compound donkey-hide gelatin oligosaccharide solution (98% ethanol precipitation + 60% ethanol precipitation);

[0048] Fig.17 This is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution when the column temperature is 30°C;

[0049] Fig.18 This is the HPLC chromatogram of compound donkey-hide gelatin oligosaccharide solution when the column temperature is 35°C. DETAILED DESCRIPTION

[0050] The present invention provides a pre-treatment method of compound donkey-hide gelatin paste, comprising the following steps:

[0051] Mixing the compound donkey-hide gelatin slurry with a first alcohol solvent, performing a first precipitation, and obtaining an alcohol precipitate;

[0052] The alcohol precipitate, water and a second alcohol solvent are mixed to perform a second precipitation to obtain a liquid component; the volume ratio of the water to the second alcohol solvent is 1:(1-1.5);

[0053] The liquid component is concentrated to obtain the compound donkey-hide gelatin syrup oligosaccharide to be tested.

[0054] In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.

[0055] The present invention mixes the compound donkey-hide gelatin slurry with a first alcohol solvent, performs a first precipitation, and obtains an alcohol precipitate.

[0056] In the present invention, the first alcohol solvent preferably includes anhydrous ethanol and / or methanol, and is more preferably an anhydrous alcohol solvent. In the present invention, the volume of the first alcohol solvent is preferably 95-98% of the total volume of the compound donkey-hide gelatin syrup and the first alcohol solvent, more preferably 96-98%, and further preferably 97-98%.

[0057] The present invention has no particular limitation on the mixing method, as long as the raw materials can be mixed evenly, such as stirring and mixing; the mixing temperature is preferably room temperature.

[0058] In the present invention, the temperature of the first precipitation is preferably room temperature, 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 static conditions.

[0059] After the first precipitation is completed, the present invention preferably further comprises solid-liquid separation of the obtained first precipitation system to obtain an alcohol precipitate. The present invention has no special limitation on the solid-liquid separation method, and the solid-liquid separation method well known to those skilled in the art can be used to concentrate to constant weight, such as centrifugal separation; the speed of the centrifugal separation is preferably 4000-6000rpm, more preferably 4500-5500rpm, and further preferably 5000rpm; the time of the centrifugal separation is preferably 10-15min, more preferably 11-14min, and further preferably 12-13min.

[0060] After obtaining the alcohol precipitate, the present invention mixes the alcohol precipitate, water and a second alcohol solvent to perform a second precipitation to obtain a liquid component.

[0061] In the present invention, the second alcohol solvent preferably includes anhydrous ethanol and / or methanol, and is more preferably an anhydrous alcohol solvent. In the present invention, the volume ratio of water to the second alcohol solvent is 1: (1-1.5), preferably 1: (1.1-1.4), and more preferably 1: (1.2-1.3). In the present invention, the volume ratio of the compound donkey-hide gelatin slurry to water is preferably 1: (2-50), more preferably 1: (5-40), further preferably 1: (10-30), and most preferably 1: (20-25).

[0062] The present invention has no particular limitation on the mixing method, and the raw materials can be mixed uniformly, such as stirring and mixing; the mixing temperature is preferably room temperature. In a specific embodiment of the present invention, the mixing is preferably to dissolve the alcohol precipitate in water, and then add the second alcohol solvent for mixing.

[0063] In the present invention, the temperature of the second precipitation is preferably room temperature, 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 static conditions.

[0064] After completing the second precipitation, the present invention preferably further comprises subjecting the obtained second precipitation system to solid-liquid separation to obtain an alcohol precipitate. The present invention has no particular limitation on the solid-liquid separation method, and the solid-liquid separation method well known to those skilled in the art can be used to concentrate to constant weight, such as centrifugal separation; the rotation speed of the centrifugal separation is preferably 4000-6000rpm, more preferably 4500-5500rpm, and further preferably 5000rpm; the time of the centrifugal separation is preferably 10-15min, more preferably 11-14min, and further preferably 12-13min.

[0065] After obtaining the liquid component, the present invention concentrates the liquid component to obtain the compound donkey-hide gelatin oligosaccharide to be tested. The present invention has no special limitation on the concentration method, and the concentration method well known to those skilled in the art can be used to concentrate to a constant weight, such as reduced pressure distillation; the concentration temperature is preferably 40 to 70°C, more preferably 50 to 60°C.

[0066] The present invention also provides a method for detecting oligosaccharides in the compound donkey-hide gelatin paste, comprising the following steps:

[0067] The oligosaccharide of compound donkey-hide glue to be tested obtained by the pretreatment method described in the above technical scheme is dissolved in an acetonitrile aqueous solution to obtain a sample solution to be tested; the volume fraction of acetonitrile in the acetonitrile aqueous solution is 50-70%;

[0068] The sample solution to be tested is subjected to high performance liquid chromatography to obtain the test result of oligosaccharides in the compound donkey-hide glue paste; the test result includes the oligosaccharide characteristic spectrum and / or the content of mannotriose; the oligosaccharides include glucose, fructose, sucrose, melibiose, trisaccharide, tetrasaccharide, mannotriose and stachyose;

[0069] The detection conditions of the high performance liquid phase include:

[0070] The mobile phase system includes mobile phase A and mobile phase B, wherein the mobile phase A includes 0.1-0.3 v / v% triethylamine aqueous solution, and the mobile phase B includes acetonitrile; the gradient elution program is:

[0071] 0~5min, the volume fraction of mobile phase B is 78%,

[0072] From 5 to 18 minutes, the volume fraction of mobile phase B decreases from 78% to 70% at a constant rate.

[0073] 18-28min, the volume fraction of mobile phase B is uniformly reduced from 70% to 60%.

[0074] 28~32min, mobile phase B volume fraction is 60%,

[0075] 32-33min, the volume fraction of mobile phase B increased from 60% to 78% at a constant rate.

[0076] 33-40min: the volume fraction of mobile phase B is 78%;

[0077] The detector is an evaporative light scattering detector, and the conditions of the evaporative light scattering detector include: drift tube temperature 85-95°C, air flow rate 2.0-2.5L·min -1 .

[0078] The present invention dissolves the compound donkey-hide glue slurry oligosaccharide to be tested obtained by the pretreatment method described in the above technical scheme in an acetonitrile aqueous solution to obtain a sample solution to be tested. In the present invention, the volume fraction of acetonitrile in the acetonitrile aqueous solution is preferably 50-70%, more preferably 50-65%, and further preferably 55-60%. In the present invention, the volume ratio of the acetonitrile aqueous solution to the compound donkey-hide glue slurry for preparing compound donkey-hide glue slurry oligosaccharide is preferably 3-5:1, more preferably 3.5-4.5:1, and further preferably 4:1.

[0079] After obtaining the sample liquid to be tested, the present invention performs high performance liquid phase detection on the sample liquid to be tested to obtain the detection result of oligosaccharides in the compound donkey-hide gelatin slurry.

[0080] In the present invention, the detection conditions of the high performance liquid phase include:

[0081] The mobile phase system includes mobile phase A and mobile phase B, wherein the mobile phase A includes 0.1-0.3 v / v% triethylamine aqueous solution, preferably 0.2 v / v% triethylamine aqueous solution, and the mobile phase B includes acetonitrile; the gradient elution program is:

[0082] 0~5min, the volume fraction of mobile phase B is 78%,

[0083] From 5 to 18 minutes, the volume fraction of mobile phase B decreases from 78% to 70% at a constant rate.

[0084] 18-28min, the volume fraction of mobile phase B is uniformly reduced from 70% to 60%.

[0085] 28~32min, mobile phase B volume fraction is 60%,

[0086] 32-33min, the volume fraction of mobile phase B increased from 60% to 78% at a constant rate.

[0087] 33-40min: the volume fraction of mobile phase B is 78%;

[0088] The chromatographic column is preferably Waters XBridge BEHAmide;

[0089] The column temperature is preferably 25 to 40°C, more preferably 30 to 35°C;

[0090] The detector is an evaporative light scattering detector, and the conditions of the evaporative light scattering detector include: drift tube temperature 85-95°C, air flow rate 2.0-2.5L·min -1 ; The gain value is preferably 1 to 3, more preferably 1 to 2, and further preferably 1;

[0091] The injection volume is preferably 5 to 10 μL.

[0092] In the present invention, the test results include the oligosaccharide characteristic spectrum and / or the content of mannotriose; the oligosaccharides include glucose, fructose, sucrose, melibiose, trisaccharide, tetrasaccharide, mannotriose and stachyose.

[0093] In the present invention, the high performance liquid chromatography detection preferably comprises the following steps:

[0094] (1) dissolving a glucose reference substance, a fructose reference substance, a sucrose reference substance, a melibiose reference substance, a mannotriose reference substance and a stachyose reference substance in an acetonitrile aqueous solution to obtain a mixed reference substance solution; the volume fraction of acetonitrile in the acetonitrile aqueous solution is 50 to 70%;

[0095] (2) subjecting the mixed reference solution to high performance liquid chromatography to obtain a reference chromatogram; and obtaining reference retention times of six oligosaccharides according to the reference chromatogram;

[0096] (3) subjecting the sample solution to be tested to high performance liquid chromatography to obtain a sample chromatogram; and obtaining the sample retention times and peak areas of 8 oligosaccharides in the sample to be tested according to the sample chromatogram;

[0097] (4) taking the retention time of mannotriose in the retention time of the reference substance as the reference peak, calculating the relative retention times of the other seven oligosaccharides to obtain an oligosaccharide characteristic spectrum; the relative retention time is within ±5%;

[0098] (5) calculating the content of mannotriose in the sample solution to be tested according to the mannotriose peak area and a predetermined standard curve; the standard curve is a standard curve of the chromatographic peak area (Y) of mannotriose and the logarithm of the concentration (X);

[0099] There is no chronological order between step (2) and step (3), and there is no chronological order between step (4) and step (5).

[0100] The present invention dissolves a glucose reference substance, a fructose reference substance, a sucrose reference substance, a melibiose reference substance, a mannotriose reference substance and a stachyose reference substance in an acetonitrile aqueous solution to obtain a mixed reference substance solution. In the present invention, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 50-70%, preferably 50-65%, and more preferably 55-60%. In the present invention, the concentration of each reference substance in the mixed reference substance solution is preferably 0.3-0.6 mg / mL, and more preferably 0.4-0.45 mg / mL. In the present invention, the English name of the glucose reference substance is D-Glucose, and the molecular formula is C6H 12 O6, IUPAC named (3R,4S,5S,6R)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol, CAS 2280-44-6. In the present invention, the fructose reference substance is named D-Fructose in English, and its molecular formula is C6H 12 O6, CAS 6347-01-9, IUPAC named (3S,4R,5R)-2-(hydroxymethyl)oxane-2,3,4,5-tetrol. In the present invention, the English name of sucrose is Sucrose, and the molecular formula is C 12 H 22 O 11 , CAS number is 57-50-1, and IUPAC name is (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol. In the present invention, the English name of the melibiose reference substance is Melibiose, and the molecular formula is C 12 H 22 O 11 , IUPAC name is (3R,4S,5S,6R)-6-[[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxane-2,3,4,5-tetrol, CAS number is 5340-95-4. In the present invention, the English name of the mannotriose reference substance is Manninotriose, and the molecular formula is C 18 H 32 O 16, CAS number is 13382-86-0, IUPAC name 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-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-2-yl]oxyhexanal. In the present invention, the English name of the stachyose reference substance is Stachyose, and the molecular formula is C 24 H 42 O 21 , CAS 470-55-3, IUPAC named (2S,3R,4S,5R,6R)-2-[[(2R,3R,4S,5R,6S)-6-[[(2R,3S,4S,5R,6R)-6-[(2S,3S,4S,5R,6R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methoxy]-3,4,5-trihydroxyoxan-2-yl]methoxy]-6-(hydroxymethyl)oxane-3,4,5-triol. In the embodiment of the present invention, the 6 kinds of oligosaccharide reference substances glucose, fructose, sucrose, melibiose, mannotriose and stachyose are preferably purchased from Chengdu Desite Biotechnology Co., Ltd., and the purity of the 8 kinds of reference substances is preferably ≥98%. In the present invention, the structural formulas of the 6 oligosaccharide reference substances are as follows:

[0101]

[0102] After obtaining the mixed reference solution, the present invention performs high performance liquid chromatography on the mixed reference solution to obtain a reference chromatogram; and obtains the reference retention times of the six oligosaccharides according to the reference chromatogram. In the present invention, the conditions for the high performance liquid chromatography detection are preferably the same as the above-mentioned high performance liquid chromatography detection conditions, which will not be repeated here.

[0103] The present invention performs high performance liquid chromatography detection on the sample liquid to obtain a sample chromatogram; and obtains the sample retention time and mannotriose peak area of ​​8 oligosaccharides in the sample to be tested according to the sample chromatogram. In the present invention, the conditions of the high performance liquid chromatography detection are the same as those of the above-mentioned high performance liquid chromatography detection, and are not described in detail here.

[0104] After obtaining the reference substance retention time, the present invention uses the retention time of mannotriose in the reference substance retention time as the reference peak to calculate the relative retention time of the other seven oligosaccharides, and the relative retention time is within ±5%.

[0105] After obtaining the mannotriose peak area, the present invention calculates the content of mannotriose in the sample solution to be tested according to the mannotriose peak area and a predetermined standard curve; the standard curve is a standard curve of the logarithm of the chromatographic peak area (X) and the logarithm of the concentration (Y) of mannotriose.

[0106] In the present invention, the method for drawing the standard curve preferably comprises the following steps:

[0107] Prepare a series of standard solutions of mannotriose reference substances;

[0108] The series of reference standard solutions were subjected to high performance liquid chromatography detection to obtain the chromatographic peak area of ​​mannotriose, and the chromatographic peak area was linearly fitted with the concentration to obtain a standard curve.

[0109] The present invention prepares a series of reference standard solutions of mannotriose, preferably comprising the following steps:

[0110] Adding acetonitrile aqueous solution to mannotriose to obtain a mannotriose reference substance stock solution;

[0111] Acetonitrile aqueous solution was added to the mannotriose reference substance stock solution for stepwise dilution to obtain a series of mannotriose reference substance standard solutions.

[0112] In the present invention, the volume fraction of acetonitrile in the acetonitrile aqueous solution is preferably 50-70%, more preferably 50-65%, and further preferably 55-60%. In the present invention, the concentration of the mannotriose reference substance stock solution is preferably 3-6 mg / mL, more preferably 4-4.5 mg / mL.

[0113] The present invention preferably performs high performance liquid chromatography detection on the series of mannotriose reference standard solutions according to the detection conditions of the above-mentioned high performance liquid chromatography to obtain the peak area of ​​the series of reference standard solutions of mannotriose of corresponding concentrations, and uses the logarithm of the peak area (X) as the abscissa and the logarithm of the mass concentration (Y) as the ordinate to perform linear regression using the partial least squares method to obtain a standard curve, and the result is shown in formula (1):

[0114] Y = 1.5748X + 3.606, r = 1.0000 Formula (1), the linear range is 10.88 ~ 544.0 ng / mL.

[0115] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0116] The materials and instruments used in the embodiments of the present invention are as follows: (1) Materials: Compound donkey-hide gelatin slurry (purchased from Dong-E-E-Jiao Co., Ltd., batch numbers are 2106029, 2106030, 2106031, 2106032, 2106033, 2106034, 2106035, 2106036, 2106037, 2106038, 200421, 2011008, 2101027, 2103023, 2104003 and 2106013, numbered as S1 to S16, respectively); acetonitrile (HPLC grade), triethylamine (purity 99.0%, analytical grade), anhydrous ethanol (analytical grade); sucrose reference substance (CAS No.: 57-50-1, purity ≥ 98%), glucose reference (CAS No.: 50-99-7, purity ≥ 98%), fructose reference (CAS No.: 57-48-7, purity ≥ 98%), mannotriose reference (CAS No. 13382-86-0, purity ≥ 98%) were all purchased from Baoji Chenguang Biotechnology Co., Ltd.; stachyose reference (CAS No. 54261-98-2, purity ≥ 98%), melibiose reference (CAS No. 585-99-9, purity ≥ 98%) were all purchased from Chengdu Desit Biotechnology Co., Ltd.; (2) Instrument: Analytical 1260 InfinityⅡHPLC system (ELSD 6000 evaporative light scattering detector), analytical balance (Sartorius Scientific Instruments Beijing Co., Ltd.), CR22GⅡ floor-standing centrifuge, RE-2000A rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), SHZ-Ⅲ circulating water vacuum pump (Shanghai Yarong Biochemical Instrument Factory).

[0117] Example 1

[0118] Determination of characteristic spectrum of compound donkey-hide gelatin solution

[0119] 1.1 Preparation of test solution

[0120] The compound donkey-hide gelatin slurry (S1-S16) was shaken to dissolve the precipitate at the bottom, 1 mL of the compound donkey-hide gelatin slurry (S1-S16) was accurately measured and put into 50 mL centrifuge tubes, anhydrous ethanol was added to an ethanol volume concentration of 98%, and the mixture was allowed to stand for alcohol precipitation at room temperature for 12 h, followed by centrifugation. The supernatant was discarded, and the obtained alcohol precipitate was redissolved in 2 mL of distilled water, and 1.5 times the volume of anhydrous ethanol was added. The mixture was allowed to precipitate at room temperature for 12 h, and centrifuged at 4000 rpm for 15 min. The obtained clear liquid was concentrated by rotary evaporation to constant weight, and then diluted to 4 mL with 50% acetonitrile, filtered through a 0.22 μm filter membrane, and 16 batches of test solution with a concentration of 25% were obtained.

[0121] 1.2 Preparation of negative sample solution

[0122] The negative sample solution was prepared according to the preparation method of step 1.1. The difference from step 1.1 was that the negative sample of Rehmannia glutinosa, the negative sample of Codonopsis pilosula, the negative sample of Ginseng rubrum and the negative sample of Crataegus pinnatifida were missing, respectively, to obtain the negative sample solution without Rehmannia glutinosa, the negative sample solution without Codonopsis pilosula, the negative sample solution without Ginseng rubrum and the negative sample solution without Crataegus pinnatifida.

[0123] 1.3 Preparation of mixed reference solution

[0124] Glucose reference substance, fructose reference substance, sucrose reference substance, melibiose reference substance, mannotriose reference substance and stachyose reference substance were dissolved in 50% acetonitrile and mixed, and filtered through a 0.22 μm filter membrane to obtain a mixed reference substance solution with a concentration of 0.4 mg / mL for each reference substance.

[0125] 1.4 Determination

[0126] Accurately pipette 10 μL of blank solvent (50% acetonitrile), test solution, mixed reference solution and negative sample solution into a liquid chromatograph for HPLC determination. The HPLC determination conditions are as follows:

[0127] The chromatographic column used was Waters XBridge BEHAmide (5 μm, 4.6×250 mm); the column temperature was 35° C.; the mobile phase system was mobile phase A (0.2 v / v% triethylamine) and mobile phase B (acetonitrile); the gradient elution program was:

[0128] 0~5min, the volume fraction of mobile phase B is 78%,

[0129] From 5 to 18 minutes, the volume fraction of mobile phase B decreases from 78% to 70% at a constant rate.

[0130] 18-28min, the volume fraction of mobile phase B is uniformly reduced from 70% to 60%.

[0131] 28~32min, mobile phase B volume fraction is 60%,

[0132] 32-33min, the volume fraction of mobile phase B increased from 60% to 78% at a constant rate.

[0133] 33-40min: the volume fraction of mobile phase B is 78%;

[0134] ELSD conditions: drift tube temperature 90 °C, air flow rate 2.3 L min -1 , gain value is 1, injection volume is 10μL.

[0135] Figure 1 is the HPLC chromatogram of the test solution; Figure 2 It is the HPLC chromatogram of the mixed reference solution; Figure 3 It is the HPLC overlay diagram of blank solvent, negative sample solution and test solution, where a is blank solvent, b is negative sample without Rehmannia glutinosa, c is negative sample without Codonopsis pilosula, d is negative sample without Red Ginseng, e is negative sample without Crataegus pinnatifida, and f is the test solution; Figure 4 HPLC chromatograms of 16 batches of test solution; Figure 1 to Figure 4 In the sample, 1-glucose, 2-fructose, 3-sucrose, 4-triose, 5-melibiose, 6-tetraose, 7-mannotriose, and 8-stachyose.

[0136] Peaks with basically consistent elution time and larger peak area in the characteristic spectra of 16 test samples were selected as common peaks, and a total of 8 common peaks were calibrated. The sum of the peak areas of these 8 peaks accounted for more than 80% of the total peak area. Among them, peaks 1, 2, 3, 5, 7 and 8 were consistent with the retention times of glucose, fructose, sucrose, melibiose, mannotriose and stachyose in the mixed reference, respectively. Among them, peaks 3, 7 and 8 were confirmed by secondary mass spectrometry comparison; by comparing the liquid chromatograms of each negative sample solution with the test sample solution, it was found that the source of oligosaccharides in the compound donkey-hide glue syrup was mainly Rehmannia glutinosa.

[0137] The AIA data of the chromatograms of the oligosaccharides of compound donkey-hide glue syrup of 16 batches were sequentially imported into the "Similarity Evaluation System of Chromatographic Characteristic Spectra of Traditional Chinese Medicine" (2012 Edition) promulgated by the National Pharmacopoeia Committee. The sample S1 spectrum was used as the reference spectrum. The median method was used with a time window width of 0.10min to perform multi-point correction on the chromatographic peaks, automatically match them, and generate a reference spectrum. A total of 8 chromatographic peaks were calibrated. The similarities between the chromatograms of samples S1 to S16 and the reference spectrum were 0.997, 0.997, 0.997, 0.997, 0.995, 0.996, 0.997, 0.997, 0.997, 0.996, 0.996, 0.966, 0.999, 0.999, 0.986 and 0.905, respectively, indicating that the characteristic spectra of the oligosaccharides of compound donkey-hide glue syrup of 16 batches were highly similar to the reference spectrum.

[0138] There should be 8 characteristic peaks in the characteristic spectrum of the test sample, among which peak No. 7 should have the same retention time as the corresponding reference peak (mannotriose). Taking mannotriose as the S peak, calculate the relative retention time of other characteristic peaks. The specified values ​​are shown in Table 1, and their relative retention times should be within ±5% of the specified values.

[0139] Table 1 Relative retention time of oligosaccharides in compound donkey-hide gelatin

[0140]

[0141] Example 2

[0142] Methodological investigation of feature maps

[0143] 2.1 Precision

[0144] According to the HPLC detection conditions of Example 1, the same test solution (batch number: 2106035) was sampled and tested 5 times, and the test results showed that the RSD values ​​of the common logarithm of the peak area (logarithm with base 10) were 0.07%, 0.100%, 0.07%, 0.68%, 0.13%, 0.05%, 0.02%, and 0.73% respectively; the RSD values ​​of the common logarithm of the retention time were 0.75%, 0.69%, 0.52%, 0.44%, 0.32%, 0.17%, 0.12%, and 0.08% respectively. This shows that the instrument precision of the detection method provided by the present invention is good.

[0145] 2.2 Stability

[0146] According to the HPLC detection conditions of Example 1, the same test solution (batch number: 2106035) was taken and sampled after the test solution was placed for 0, 2, 4, 6, 8, 12 and 24 hours. The test results showed that the RSD values ​​of the common logarithms of the peak areas were 0.12%, 0.14%, 0.38%, 1.40%, 2.13%, 0.39%, 0.08% and 0.84% ​​respectively; the RSD values ​​of the common logarithms of the retention times were 0.09%, 0.11%, 0.09%, 0.09%, 0.13%, 0.11%, 0.08% and 0.10% respectively. It shows that the sample solution treated by the pretreatment method provided by the present invention has good stability within 24 hours.

[0147] 2.3 Repeatability

[0148] Five portions of the same sample (batch number: 2106035) were accurately measured and prepared into a test solution, and the test was performed according to the HPLC test conditions of Example 1. Test results: the RSD values ​​of the common logarithms of the peak areas were 0.59%, 0.71%, 0.82%, 1.42%, 0.56%, 0.47%, 0.37%, and 0.67%, respectively; the RSD values ​​of the common logarithms of the retention times were 0.09%, 0.09%, 0.09%, 0.08%, 0.07%, 0.05%, 0.04%, and 0.03%, respectively. This shows that the detection method provided by the present invention has good repeatability.

[0149] Example 3

[0150] Determination of Mannotriose Content in Compound Donkey-hide Glue

[0151] 3.1 Preparation of Mannotriose Reference Solution

[0152] Accurately weigh the mannotriose reference substance, dissolve it in distilled water, and filter it through a 0.22 μm filter membrane to obtain a mannotriose reference substance solution with a concentration of 4.164 mg / mL.

[0153] 3.2 Testing

[0154] According to the HPLC detection conditions of Example 1, the mannotriose reference solution and each test solution prepared in Example 1 were injected for detection, wherein the injection volume was 5 μL.

[0155] The test results of each test solution are shown in Table 2:

[0156] Table 2 Determination results of each test solution (n=3)

[0157]

[0158] As shown in Table 2, the content of mannotriose in 16 batches of compound donkey-hide gelatin solution ranged from 8.63 to 12.29 mg·mL -1 , each 1mL of compound donkey-hide gelatin syrup contains an average of 11.17mg. Therefore, it is preliminarily stipulated that the content of mannotriose in compound donkey-hide gelatin syrup shall not be less than 60% of the average value, that is, the content of mannotriose in each 1mL of compound donkey-hide gelatin syrup shall not be less than 6.7mg.

[0159] Example 4

[0160] Methodological investigation of content determination

[0161] 4.1 Specificity Investigation

[0162] According to the HPLC detection conditions of Example 1, the blank solvent and mannotriose reference substance in Example 3 and the test solution prepared in Example 1 (batch number: 2106035) were respectively taken for determination.

[0163] Figure 5 is the HPLC chromatogram of blank solvent, Figure 6 This is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide test solution. Figure 7 The HPLC chromatogram of mannotriose reference substance is shown in Figure 2. Figure 5 to Figure 7 It can be seen that there is no interference from other components near mannotriose, and the separation is good.

[0164] 4.2 Preparation of standard curve

[0165] Take the mannotriose reference substance stock solution in Example 3.

[0166] Accurately measure 50, 80, 120, 160 and 200 μL of the reference substance stock solution, dilute them to 200 μL with water, shake them ultrasonically, and obtain a series of reference substance standard solutions with concentrations of 1.0410, 1.6656, 2.4984, 3.3312, and 4.1640 mg / mL, respectively.

[0167] The series of reference standard solutions were tested according to the HPLC test conditions of Example 1, with the common logarithm (base 10) of the peak area of ​​the reference standard (X) as the abscissa and the mass concentration of mannotriose (mg·mL -1 ) was plotted as the ordinate, and linear regression was performed using the partial least squares method to obtain the standard curve Y = 1.5748X + 3.606 (r = 1.0000), indicating a good linear relationship in the range of 1.04 to 4.16 mg / mL. The detection limit and quantification limit of mannotriose were calculated to be 0.32 μg and 0.53 μg, respectively, when the signal-to-noise ratio was 3 and 10, respectively.

[0168] 4.3 Precision

[0169] According to the HPLC detection conditions of Example 1, the mannotriose reference solution prepared in Example 3 was repeatedly sampled and tested 5 times, and the RSD value of the common logarithm of the mannotriose peak area was 1.08%, and the RSD value of the common logarithm of the retention time was 0.05%, indicating that the detection method provided by the present invention has good instrument precision.

[0170] 4.4 Stability

[0171] The test solution prepared in Example 1 (batch number: 2106036) was sampled and tested at 0, 2, 4, 6, 8, 12 and 24 hours after preparation, and the test results showed that the RSD value of the common logarithm of the mannotriose peak area was 1.20%; the RSD value of the common logarithm of the retention time was 0.08%, indicating that the sample solution treated by the pretreatment method provided by the present invention had good stability within 24 hours.

[0172] 4.5 Repeatability

[0173] Five portions of the same compound donkey-hide gelatin syrup (batch: 2106036) were accurately measured to prepare a test solution, and the test was performed according to the HPLC detection conditions of Example 1. Test results: The RSD value of the common logarithm of the mannotriose peak area was 0.87%, and the RSD of the common logarithm of the retention time was 0.04%. This shows that the detection method provided by the present invention has good repeatability.

[0174] 4.6 Sample recovery rate

[0175] The method of Example 1 was used to prepare 9 concentrations of test solution in parallel (the batch of compound donkey-hide glue was 2106036, and the content of mannotriose was shown in Table 3), and 0.5 mL of the test solution was taken from each, and three different concentration levels of mannotriose reference solution were added, namely, low (80% of mannotriose content), medium (100% of mannotriose content) and high (120% of mannotriose content), to obtain mannotriose loading solution, and each concentration was paralleled for 3 times. The mannotriose loading solution was tested according to the HPLC detection conditions of Example 1, and the test results are shown in Table 3:

[0176] Table 3 Mannotriose sample recovery test results (n = 9)

[0177]

[0178] As shown in Table 3, the average recovery rate of mannotriose is 99.94% and the RSD is 2.15% (n=9), indicating that the detection method provided by the present invention has a good recovery rate.

[0179] Example 5

[0180] The compound donkey-hide gelatin slurry (batch number 2106036) was shaken to dissolve the precipitate at the bottom, 1 mL of the compound donkey-hide gelatin slurry was accurately measured and put into a 50 mL centrifuge tube, anhydrous ethanol was added to a volume concentration of 98% ethanol, and the mixture was allowed to stand for 12 h at room temperature and then centrifuged. The supernatant was discarded, and the obtained alcohol precipitate was redissolved in 2 mL of distilled water, 1.5 times the volume of anhydrous ethanol was added, and the mixture was allowed to precipitate for 12 h at room temperature. The mixture was centrifuged at 4000 rpm for 15 min, and the obtained clear liquid was concentrated by rotary evaporation to constant weight, and then diluted to 4 mL with 50% acetonitrile, and filtered through a 0.22 μm filter membrane to obtain a compound donkey-hide gelatin slurry oligosaccharide solution.

[0181] 5.1 Effect of mobile phase composition on the detection of oligosaccharide solution in compound donkey-hide gelatin

[0182] 5.1.1 The compound donkey-hide gelatin oligosaccharide solution was tested according to the HPLC test conditions of Example 1.

[0183] 5.1.2 Perform detection according to the method in step 5.1.2. The difference from step 5.1.2 is that mobile phase A is water.

[0184] Figure 8 This is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution (the mobile phase is water (A)-acetonitrile (B)). Fig. 9 The HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution (mobile phase is 0.2% triethylamine (A)-acetonitrile (B). Figure 8-Figure 9 It can be seen that when acetonitrile and water are used as the mobile phase system, the peak shape of oligosaccharides is poor and forked peaks appear. Adding 0.2% triethylamine can significantly improve the peak shape.

[0185] 5.2 Effect of elution mode on the detection of oligosaccharide solution of compound donkey-hide gelatin

[0186] 5.2.1 The compound donkey-hide gelatin oligosaccharide solution was tested according to the HPLC detection conditions of step 5.1.1. The difference from step 5.1.1 is that the elution method is isocratic elution and the volume ratio of mobile phase A to mobile phase B is 25:75.

[0187] 5.2.2 The compound donkey-hide gelatin oligosaccharide solution was tested according to the HPLC test conditions of step 5.1.1. The difference from step 5.1.1 is that the gradient elution procedure is:

[0188] 0~12min, the volume fraction of mobile phase B is 80%,

[0189] 12-22min, the volume fraction of mobile phase B is uniformly reduced from 80% to 65%.

[0190] 22~26min, the volume fraction of mobile phase B is 65%,

[0191] From 26 to 30 min, the volume fraction of mobile phase B increased uniformly from 65% to 80%.

[0192] 5.2.3 The compound donkey-hide gelatin oligosaccharide solution was tested according to the HPLC test conditions of step 5.1.1. The difference from step 5.1.1 is that the gradient elution procedure is:

[0193] 0~12min, the volume fraction of mobile phase B is 80%,

[0194] 12-25min, the volume fraction of mobile phase B is uniformly reduced from 80% to 65%.

[0195] 25~28min, mobile phase B volume fraction is 65%,

[0196] From 28 to 30 min, the volume fraction of mobile phase B increased uniformly from 65% to 80%.

[0197] 5.2.4 The compound donkey-hide gelatin oligosaccharide solution was tested according to the HPLC test conditions of step 5.1.1. The difference from step 5.1.1 is that the gradient elution procedure is:

[0198] 0~5min, the volume fraction of mobile phase B is 78%,

[0199] From 5 to 18 minutes, the volume fraction of mobile phase B decreases from 78% to 70% at a constant rate.

[0200] 18-28min, the volume fraction of mobile phase B is uniformly reduced from 70% to 60%.

[0201] 28~32min, mobile phase B volume fraction is 60%,

[0202] 32-33min, the volume fraction of mobile phase B increased from 60% to 78% at a constant rate.

[0203] The volume fraction of mobile phase B from 33 to 40 min is 78%.

[0204] Fig.10 is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution in step 5.2.1, Fig.11 is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution in step 5.2.2, Fig.12 This is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution in 5.2.3. Fig.13 The HPLC chromatogram of the oligosaccharide solution of compound donkey-hide gelatin in 5.2.4. Figure 10 to Figure 13It can be seen that the separation effect of compound donkey-hide glue slurry oligosaccharides is poor under isocratic elution, and baseline separation cannot be achieved. By changing the ratio of organic phase to aqueous phase, the optimal elution gradient program is finally obtained: 0-5min, the volume fraction of mobile phase B is 78%, 5-18min, the volume fraction of mobile phase B is uniformly reduced from 78% to 70%, 18-28min, the volume fraction of mobile phase B is uniformly reduced from 70% to 60%, 28-32min, the volume fraction of mobile phase B is 60%, 32-33min, the volume fraction of mobile phase B is uniformly increased from 60% to 78%, 33-40min The volume fraction of mobile phase B is 78%. Under this gradient elution program, the separation of the peaks of compound donkey-hide glue slurry oligosaccharides is good, and baseline separation can be achieved.

[0205] 5.3 Effect of pretreatment methods on the detection of compound donkey-hide gelatin oligosaccharide solution

[0206] 5.3.1 Shake the compound donkey-hide gelatin slurry (batch number 2106035) to dissolve the precipitate at the bottom. Accurately measure 1 mL of the compound donkey-hide gelatin slurry into 50 mL centrifuge tubes, add anhydrous ethanol to a volume concentration of 80%, let stand for 12 h at room temperature, then centrifuge and discard the supernatant. Dilute the resulting alcohol precipitate with 50% acetonitrile to 4 mL, filter through a 0.22 μm filter membrane, and obtain the compound donkey-hide gelatin slurry oligosaccharide solution (80% ethanol precipitate) to be tested.

[0207] 5.3.2 Shake the compound donkey-hide gelatin slurry (batch number 2106035) to dissolve the precipitate at the bottom, accurately measure 1 mL of the compound donkey-hide gelatin slurry into 50 mL centrifuge tubes, add anhydrous ethanol to a volume concentration of 80% ethanol, let it stand at room temperature for 12 h and then centrifuge to separate. Extract the supernatant with an equal volume of ethyl acetate-n-butanol mixed solvent (the volume ratio of ethyl acetate to n-butanol is 1:1), concentrate the ethyl acetate-n-butanol extract by rotary evaporation to constant weight, then dilute to 4 mL with 50% acetonitrile, and filter through a 0.22 μm filter membrane to obtain the compound donkey-hide gelatin slurry oligosaccharide solution to be tested (80% ethanol precipitation + ethyl acetate-n-butanol extraction).

[0208] 5.3.3 Shake the compound donkey-hide gelatin slurry (batch number 2106035) to dissolve the precipitate at the bottom, accurately measure 1 mL of the compound donkey-hide gelatin slurry into 50 mL centrifuge tubes, add anhydrous ethanol to an ethanol volume concentration of 98%, let it stand at room temperature for 12 hours, and then centrifuge. The obtained supernatant is extracted with an equal volume of ethyl acetate, and the obtained alcohol precipitate is redissolved in 2 mL of distilled water, and anhydrous ethanol is added to an ethanol volume concentration of 60%, and alcohol precipitation is carried out at room temperature for 12 hours. Centrifuge at 4000 rpm for 15 min, and the obtained clear liquid is concentrated by rotary evaporation to constant weight, and then diluted to 4 mL with 50% acetonitrile, and filtered through a 0.22 μm filter membrane to obtain the compound donkey-hide gelatin slurry oligosaccharide solution to be tested (98% ethanol precipitation + 60% ethanol precipitation).

[0209] The compound donkey-hide gelatin oligosaccharide solutions obtained in steps 5.3.1 to 5.3.3 were tested according to the HPLC detection conditions of step 5.1.1.

[0210] Fig.14 This is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution (80% ethanol precipitation). Fig.15 This is the HPLC chromatogram of the compound donkey-hide gelatin oligosaccharide solution (80% ethanol precipitation + ethyl acetate-n-butanol extraction). Fig.16 The HPLC chromatogram of compound donkey-hide gelatin oligosaccharide solution (98% ethanol precipitation + 60% ethanol precipitation). Figure 14 to Figure 16 It can be seen that the oligosaccharide solution of compound donkey-hide gelatin prepared directly by 80% ethanol precipitation has a high monosaccharide content and a flat peak; and after the supernatant is extracted with ethyl acetate-n-butanol, some oligosaccharides are lost; and the pretreatment method of removing some monosaccharides by 98% anhydrous ethanol precipitation and then removing polysaccharides and proteins by 60% ethanol precipitation is simple and easy, the spectrum is clear, and the loss of oligosaccharides is extremely small.

[0211] The compound donkey-hide glue slurry oligosaccharide solution obtained in step 5.1.1 was tested according to the HPLC detection conditions of step 5.1.1. The difference from step 5.1.1 is that the column temperatures are 30°C and 35°C respectively. The effect of column temperature on the liquid chromatography spectrum of the compound donkey-hide glue slurry oligosaccharide solution is as follows: Figures 17-18 As shown, Fig.17 This is the HPLC spectrum of the compound donkey-hide gelatin oligosaccharide solution when the column temperature is 30°C. Fig.18 The HPLC spectrum of the compound donkey-hide gelatin oligosaccharide solution at a column temperature of 35°C is shown in FIG. Figures 17-18 It can be seen that the column temperature has little effect on the liquid chromatography spectrum of compound donkey-hide gelatin oligosaccharide solution.

[0212] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pretreatment method for compound donkey-hide gelatin paste, comprising the following steps: The compound donkey-hide gelatin slurry is mixed with a first alcohol solvent, wherein the volume of the first alcohol solvent is 95-98% of the total volume of the compound donkey-hide gelatin slurry and the first alcohol solvent, and a first precipitation is performed, and the obtained first precipitation is subjected to solid-liquid separation to obtain an alcohol precipitate, and the solid-liquid separation method is centrifugal separation, the speed of the centrifugal separation is 4000-6000 rpm, and the time of the centrifugal separation is 10-15 min; The alcohol precipitate, water and a second alcohol solvent are mixed, wherein the volume ratio of the compound donkey-hide gelatin slurry to water is 1:(2-10), and a second precipitation is performed to obtain a liquid component; wherein the volume ratio of the water to the second alcohol solvent is 1:(1-1.5); Concentrating the liquid component to obtain the compound donkey-hide gelatin syrup oligosaccharide to be tested; The first alcohol solvent and the second alcohol solvent independently include ethanol and / or methanol, the ethanol is anhydrous ethanol, and the time of the first precipitation and the second precipitation is independently 12 to 18 hours.

2. A method for detecting oligosaccharides in compound donkey-hide gelatin paste, comprising the following steps: Dissolve the compound donkey-hide glue slurry oligosaccharide to be tested obtained by the pretreatment method of claim 1 in an acetonitrile aqueous solution, wherein the volume ratio of the acetonitrile aqueous solution to the compound donkey-hide glue slurry used to prepare the compound donkey-hide glue slurry oligosaccharide to be tested is 3-5:1, to obtain a sample solution to be tested; the volume fraction of acetonitrile in the acetonitrile aqueous solution is 50-70%; The sample solution to be tested is subjected to high performance liquid chromatography to obtain the test result of oligosaccharides in the compound donkey-hide glue paste; the test result includes the characteristic spectrum of oligosaccharides and / or the content of mannotriose; the oligosaccharides include glucose, fructose, sucrose, melibiose, mannotriose and stachyose; The detection conditions of the high performance liquid phase include: The mobile phase system includes mobile phase A and mobile phase B, wherein the mobile phase A includes a 0.1-0.3 v / v% triethylamine aqueous solution, The mobile phase B includes acetonitrile; the gradient elution program: 0~5min, the volume fraction of mobile phase B is 78%, From 5 to 18 minutes, the volume fraction of mobile phase B was uniformly reduced from 78% to 70%. From 18 to 28 minutes, the volume fraction of mobile phase B was uniformly reduced from 70% to 60%. 28~32min, mobile phase B volume fraction is 60%, From 32 to 33 minutes, the volume fraction of mobile phase B increased from 60% to 78% at a constant rate. From 33 to 40 min, the volume fraction of mobile phase B was 78%; The chromatographic column was Waters XBridge BEH Amide; the column temperature was 25-40°C; the injection volume was 5-10 μL, The detector is an evaporative light scattering detector, and the conditions of the evaporative light scattering detector include: drift tube temperature 85~95℃, air flow rate 2.0~2.5L·min -1 , the gain value is 1~3.

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