A method for detecting contents of multiple index chemical components in spina date seed

Abstract

The application provides a detection method of multi-index chemical component content in spina date seed, and relates to the technical field of analysis and detection. The application establishes a method for simultaneously detecting the multi-index chemical component content in spina date seed medicinal materials based on HPLC-MS / MS, adopts 70-100% low-grade alcohol as an extraction solvent, detects through HPLC-MS / MS, the response of the chromatographic peak is stronger, and after adding 0.08-0.12 vol% formic acid in the mobile phase, the peak tailing phenomenon can be well improved, and the simultaneous determination of the content of more than 10 kinds of multi-index chemical components in spina date seed medicinal materials is realized. The detection method provided by the application is simple, efficient, high in sensitivity and strong in specificity, has good precision, repeatability and stability, can reflect the content difference of the measured chemical components in different batches of spina date seed medicinal materials, and can provide a reference for the quality evaluation and efficacy material basis research of spina date seed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and in particular to a method for detecting the content of multi-index chemical components in Zizyphus jujuba Mill. BACKGROUND

[0002] Zizyphus jujuba Mill. is the dry mature seed of Ziziphus jujuba Mill. Var. spinosa (Bunge) Hu ex H.F. Chou, and its chemical components are complex, containing flavonoids, saponins and triterpenoids, alkaloids, organic acids, fatty oils, various amino acids and trace elements. Modern pharmacological studies have shown that Zizyphus jujuba Mill. has the pharmacological effects of sedation, anti-anxiety, anti-depression, hypoglycemic, anti-dementia and immune enhancement.

[0003] Due to the differences in ecological environment and cultivation management methods of different producing areas, the chemical components of Zizyphus jujuba Mill. are significantly different, which leads to different qualities of the medicinal materials. In order to ensure the quality of Zizyphus jujuba Mill. and establish a solid good agricultural practice (GAP) foundation, it is particularly important to deeply study the quality differences of Zizyphus jujuba Mill. in different regions. Therefore, it is necessary to establish a high-efficiency and accurate multi-component content determination method to provide a basis for the quality evaluation of Zizyphus jujuba Mill. The prior art discloses a method for simultaneously detecting 9 chemical components, including epiberberine, magnoflorine, vachelin II, spinosin, wedelolactone, kaempferol-3-O-rutinoside, 6''-feruloylspinosin, zizyphin A and zizyphin B, in Zizyphus jujuba Mill. However, the components detected are few, and the quality control of Zizyphus jujuba Mill. cannot be comprehensive. Therefore, it is of great significance to establish a high-efficiency and accurate multi-component content determination method for the quality evaluation of Zizyphus jujuba Mill. SUMMARY

[0004] Therefore, the present application aims to provide a method for detecting the content of multi-index chemical components in Zizyphus jujuba Mill. The detection method provided by the present application can simultaneously realize accurate and high-sensitivity detection of the content of multi-index chemical components in Zizyphus jujuba Mill., and thus realize quality monitoring of Zizyphus jujuba Mill.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a method for detecting the content of multi-index chemical components in Zizyphus jujuba Mill., comprising the following steps:

[0007] The extractant is used to extract the Zizyphus jujuba Mill. powder to be detected to obtain a sample solution to be detected; the extractant comprises a lower alcohol or a lower alcohol aqueous solution; the volume fraction of the lower alcohol in the extractant is 70-100%;

[0008] The sample solution to be detected is detected by high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS) to obtain the detection results of the contents of multiple index chemical components in Zizyphus jujuba Mill.

[0009] The multiple index chemical components include at least 10 of Zizyphus jujuba Mill saponin A, Zizyphus jujuba Mill saponin B, Zizyphus jujuba Mill saponin D, spinosin, 6''-feruloylspinosin, vicenin II, magnoflorine, wongaiine, fructus armeniaceae leaf base, nuciferine, proto-nuciferine, isofarnetin, oroxindin, rutin, quercetin, kaempferol-3-O-rutinoside, isoquercitrin, ferulic acid, naringin, hesperidin, naringenin, camellin B and epicatechin.

[0010] The high performance liquid chromatography tandem mass spectrometry detection includes high performance liquid chromatography separation and mass spectrometry detection.

[0011] The high performance liquid chromatography separation condition includes that a mobile phase A is 0.08-0.12 vol% formic acid aqueous solution, a mobile phase B is acetonitrile, an elution mode is gradient elution, and a program of the gradient elution is as follows: 0-3 min, a volume fraction of the mobile phase B is increased from 20% to 32%; 3-5 min, the volume fraction of the mobile phase B is increased from 32% to 41%; 5-6 min, the volume fraction of the mobile phase B is increased from 41% to 55%; and 6-8 min, the volume fraction of the mobile phase B is increased from 55% to 90%.

[0012] Preferably, the low alcohol includes methanol and / or ethanol.

[0013] Preferably, a solid-liquid ratio of the sample Zizyphus jujuba Mill powder to the extraction agent is 0.4-0.6 g:10 mL.

[0014] Preferably, the extraction includes ultrasonic extraction.

[0015] Preferably, the ultrasonic extraction has a power of 280-320 W, a frequency of 30-50 kHz, and a time of 20-40 min.

[0016] Preferably, a chromatographic column used in the high performance liquid chromatography separation is C 18 The chromatographic column has a column temperature of 28-32 ℃.

[0017] Preferably, an injection amount of the high performance liquid chromatography separation is 1.8-2.2 μL.

[0018] Preferably, a flow rate of the mobile phase of the high performance liquid chromatography separation is 0.28-0.32 mL / min.

[0019] Preferably, the mass spectrometry detection condition comprises: the ion source is an electrospray ion source, the detection mode is a multiple reaction ion monitoring, the scanning mode is a positive and negative ion scanning mode, the gas temperature is 320℃; the gas flow rate is 10 L / min, the atomizer pressure is 40 psig, the fragmentation voltage is 85-148 V, and the collision energy is 10-50 V.

[0020] Preferably, the quantitative analysis ion pairs of the magnolialide, magnoflorine, honokiol, vachelin II, spinosin, jujuboside A, jujuboside B, jujuboside D, isoquercitrin, kaempferol-3-O-rutinoside, ferulic acid, 6'''-feruloylspinosin, naringin, hesperidin, quercetin, fructus armeniaceae leaf base, naringenin, rutin, isofisetin, oroxinidin, epicatechin and camelliatin B are 286.3 / 107.1, 343.3 / 266.2, 296.2 / 250.1, 282.1 / 235.1, 593.5 / 353.3, 607.4 / 427.3, 1205.3 / 1073.8, 1043.3 / 911.1, 1205.3 / 1073.8, 463.2 / 300.1, 593.5 / 285.3, 193.0 / 134.0, 783.5 / 427.2, 579.3 / 271.2, 609.3 / 301.2, 301.2 / 151.3, 533.9 / 247.5, 271.0 / 119.2, 609.6 / 300.3, 431.5 / 311.2, 445.3 / 282.4, 289.0 / 125.1 and 725.2 / 283.8, respectively.

[0021] The present application establishes a method for simultaneously determining the contents of multiple index chemical components in Zizyphi Semen Praeparatae by HPLC-MS / MS. The response of chromatographic peaks is stronger by using 70-100% low alcohol as the extraction solvent and detecting by HPLC-MS / MS. The peak tailing phenomenon is well improved by adding 0.08-0.12 vol% formic acid in the mobile phase, and the contents of more than 10 (up to 23) chemical components in Zizyphi Semen Praeparatae, such as jujuboside A, jujuboside B, jujuboside D, spinosin, 6'''-feruloylspinosin, vachelin II, magnolialide, magnoflorine, fructus armeniaceae leaf base, honokiol, honokiol, isofisetin, oroxinidin, rutin, quercetin, kaempferol-3-O-rutinoside, isoquercitrin, ferulic acid, naringin, hesperidin, naringenin, camelliatin B and epicatechin, are simultaneously determined. The detection method provided by the present application is simple, efficient, high in sensitivity and specificity, and has good precision, repeatability and stability, and can reflect the content differences of the measured chemical components in different batches of Zizyphi Semen Praeparatae, thereby providing a reference for the quality evaluation and efficacy material basis research of Zizyphi Semen Praeparatae. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 MRM chromatograms of standard (left) and sample (right), wherein 1 is kaempferol-3-O-rutinoside, 2 is 6'''-ferulic acid spinosin, 3 is jujuboside B, 4 is rutin, 5 is hesperidin, 6 is spinosin, 7 is vicenin II, 8 is frangufolin, 9 is isoquercitrin, 10 is quercetin, 11 is onjisapin, 12 is nuciferine, 13 is protopine, 14 is magnoflorine, 15 is safrole, 16 is ferulic acid, 17 is jujuboside A, 18 is jujuboside B, 19 is isovitexin, 20 is naringenin, 21 is epicatechin, 22 is camellatin B, and 23 is naringin. DETAILED DESCRIPTION

[0023] The present application provides a method for detecting the content of multiple index chemical components in Zizyphus jujuba Mill., comprising the following steps:

[0024] The Zizyphus jujuba Mill. powder to be tested is extracted by an extracting agent to obtain a sample solution to be tested; the extracting agent comprises a lower alcohol or a lower alcohol aqueous solution; the volume fraction of the lower alcohol in the extracting agent is 70-100%;

[0025] The sample solution to be tested is detected by high performance liquid chromatography tandem mass spectrometry to obtain the detection result of the content of multiple index chemical components in Zizyphus jujuba Mill.;

[0026] The multiple index chemical components comprise at least 10 of jujuboside A, jujuboside B, jujuboside D, spinosin, 6'''-ferulic acid spinosin, vicenin II, safrole, magnoflorine, frangufolin, nuciferine, protopine, isovitexin, onjisapin, rutin, quercetin, kaempferol-3-O-rutinoside, isoquercitrin, ferulic acid, naringin, hesperidin, naringenin, camellatin B and epicatechin;

[0027] The high performance liquid chromatography tandem mass spectrometry comprises high performance liquid chromatography separation and mass spectrometry detection;

[0028] The high performance liquid chromatography separation condition comprises: the mobile phase A is 0.08-0.12 vol% formic acid aqueous solution, the mobile phase B is acetonitrile, the elution mode is gradient elution, and the program of the gradient elution is as follows: 0-3 min, the volume fraction of the mobile phase B is increased from 20% to 32%; 3-5 min, the volume fraction of the mobile phase B is increased from 32% to 41%; 5-6 min, the volume fraction of the mobile phase B is increased from 41% to 55%; and 6-8 min, the volume fraction of the mobile phase B is increased from 55% to 90%.

[0029] The present application extracts the to-be-tested Semen Ziziphi Spinosae powder with an extracting agent to obtain a to-be-tested sample solution; the extracting agent comprises a lower alcohol or a lower alcohol aqueous solution; the volume fraction of the lower alcohol in the extracting agent is 70-100%.

[0030] In the present application, the to-be-tested Semen Ziziphi Spinosae powder is preferably obtained by crushing Semen Ziziphi Spinosae and sieving, and the sieving is preferably sieving through a No. 4 sieve.

[0031] In the present application, the volume fraction of the lower alcohol in the extracting agent is preferably 70-90%, more preferably 70-80%, and further preferably 70-75%; the lower alcohol comprises methanol and / or ethanol, and is more preferably methanol.

[0032] In the present application, the solid-liquid ratio of the to-be-tested Semen Ziziphi Spinosae powder (dry weight) to the extracting agent is preferably 0.4-0.6 g:10 mL, and in specific embodiments, can be 0.4 g:10 mL, 0.45 g:10 mL, 0.5 g:10 mL, 0.55 g:10 mL, or 0.6 g:10 mL.

[0033] In the present application, the extraction preferably comprises ultrasonic extraction; the power of the ultrasonic extraction is preferably 280-320 W, more preferably 290-310 W, and further preferably 300 W; the frequency of the ultrasonic extraction is preferably 30-50 kHz, more preferably 35-45 kHz, and further preferably 40 kHz; and the time of the ultrasonic extraction is preferably 20-40 min, more preferably 25-35 min, and further preferably 30 min.

[0034] After the extraction is completed, the present application preferably further comprises: centrifuging the obtained extraction system, and filtering the obtained extraction liquid through a 0.22 μm microporous filter membrane to obtain a test sample solution. In the present application, the rotation speed of the centrifugation is preferably 10000-20000 r / min, and more preferably 14000-15000 r / min; and the time of the centrifugation is preferably 5-15 min, and more preferably 10 min. In the present application, the microporous filter membrane is preferably a 0.22 μm microporous filter membrane.

[0035] After the to-be-tested sample solution is obtained, the present application detects the to-be-tested sample solution by high performance liquid chromatography tandem mass spectrometry to obtain a detection result of the content of multiple index chemical components in Semen Ziziphi Spinosae.

[0036] In the present application, the multi-index chemical components include at least 10 of jujuboside A, jujuboside B, jujuboside D, spinosin, 6''-feruloylspinosin, vicenin II, magnoflorine, magnoliavine, frangulanine, nuciferine, nuciferine, isofarnetin, oroxylin, rutin, quercetin, kaempferol-3-O-rutinoside, isoquercitrin, ferulic acid, naringin, hesperidin, naringenin, camellatin B and epicatechin.

[0037] In the present application, the high performance liquid chromatography tandem mass spectrometry detection includes high performance liquid chromatography separation and mass spectrometry detection.

[0038] In the present application, the high performance liquid chromatography separation conditions include: the chromatographic column is preferably a C 18 The chromatographic column is more preferably a Waters CORTECS C18 chromatographic column; the column temperature is preferably 28-32℃, more preferably 29-31℃, and further preferably 30℃; the mobile phase A is 0.08-0.12vol% formic acid aqueous solution, more preferably 0.09-0.11vol% formic acid aqueous solution, and further preferably 0.1vol% formic acid aqueous solution; the mobile phase B is acetonitrile; the flow rate of the mobile phase is preferably 0.28-0.32mL / min, more preferably 0.29-0.31mL / min, and further preferably 0.3mL / min; the elution mode is gradient elution, and the program of the gradient elution is as follows: 0-3min, the volume fraction of the mobile phase B is increased from 20% to 32%; 3-5min, the volume fraction of the mobile phase B is increased from 32% to 41%; 5-6min, the volume fraction of the mobile phase B is increased from 41% to 55%; 6-8min, the volume fraction of the mobile phase B is increased from 55% to 90%; and the injection amount is preferably 1.8-2.2μL, more preferably 1.9-2.1μL, and further preferably 2μL.

[0039] In the present application, the mass spectrometry detection conditions preferably include: the ion source is an electrospray ion source (ESI), the detection mode is multiple reaction ion monitoring (MRM), the scanning mode is positive and negative ion scanning mode, the gas temperature (Gas Temp) is 320℃; the gas flow rate (Gas Flow) is 10L / min, the nebulizer pressure is 40psig, the fragmentation voltage is 85-148V, the collision energy is 10-50V, and the collision gas is preferably high-purity nitrogen; the atomizing gas is preferably high-purity nitrogen; the quantitative analysis ion pair, mass spectrometry parameters and ion mode are shown in Table 1.

[0040] Table 1 mass spectrometry parameters of 23 chemical components in Semen Ziziphi Spinosae

[0041]

[0042]

[0043] In the present application, the method for obtaining the multi-index chemical component content is preferably a standard curve method, specifically, a mixed control substance linear solution is detected according to the conditions of ultra-high performance liquid chromatography tandem mass spectrometry detection, the chromatographic peak area is obtained, the concentration of the mixed control substance linear solution is taken as the independent variable, and the chromatographic peak area is taken as the dependent variable, a standard curve is drawn, and a standard curve regression equation is obtained; the chromatographic peak area of the sample liquid to be detected is brought into the standard curve regression equation, and the detection result of the multi-index chemical component content is obtained.

[0044] In the present application, the preparation method of the mixed control substance linear solution preferably comprises the following steps: preparing a standard control substance stock solution, diluting the control substance stock solution to obtain a mixed control substance solution; and stepwise diluting the mixed control substance solution to obtain a mixed control substance linear solution.

[0045] In the present application, the preparation method of the control substance stock solution preferably comprises the following steps: dissolving the control substances of zizyphin A, zizyphin B, zizyphin D, spinosin, 6'' '-ferulic acid spinosin, vicenin II, magnoflorine, phillyrin, frangulanin, nuciferine, proto-nuciferine, isofukinianin, onjisapin, rutin, quercetin, kaempferol-3-O-rutinoside, isoquercitrin, ferulic acid, naringin, hesperidin, naringenin, camellianin B and epicatechin in methanol to prepare 23 single control substance control substance stock solutions with a concentration of 1 mg / mL.

[0046] In the present application, the preparation method of the mixed control substance solution preferably comprises the following steps: precisely measuring each control substance stock solution into the same volumetric flask, diluting with methanol to obtain a mixed control substance solution; the concentration of magnoflorine in the mixed control substance solution is 55 μg / mL, the concentrations of 6'' '-ferulic acid spinosin and spinosin are 30 μg / mL, the concentrations of zizyphin A and zizyphin D are both 15 μg / mL, the concentrations of vicenin II and magnoflorine are both 10 μg / mL, the concentrations of camellianin B, epicatechin, zizyphin B and frangulanin are all 8 μg / mL, the concentrations of isofukinianin, kaempferol-3-O-rutinoside and ferulic acid are all 5 μg / mL, the concentrations of rutin, onjisapin and proto-nuciferine are all 1 μg / mL, the concentrations of isoquercitrin, naringenin, quercetin and hesperidin are all 0.5 μg / mL, and the concentrations of naringin and nuciferine are both 0.2 μg / mL.

[0047] In the present application, the preparation method of the mixed control linear solution preferably comprises the following steps: diluting the mixed control solution 2 times with methanol to obtain mixed control linear solution 1; continuing to dilute the mixed control linear solution 1 2.5 times with methanol to obtain mixed control linear solution 2; continuing to dilute the mixed control linear solution 2 2 times with methanol to obtain mixed control linear solution 3; continuing to dilute the mixed control linear solution 3 2 times with methanol to obtain mixed control linear solution 4; continuing to dilute the mixed control linear solution 4 2 times with methanol to obtain mixed control linear solution 5; continuing to dilute the mixed control linear solution 5 2 times with methanol to obtain mixed control linear solution 6; continuing to dilute the mixed control linear solution 6 2.5 times with methanol to obtain mixed control linear solution 7; continuing to dilute the mixed control linear solution 7 2 times with methanol to obtain mixed control linear solution 8; and continuing to dilute the mixed control linear solution 8 2 times with methanol to obtain mixed control linear solution 9.

[0048] In order to further illustrate the present application, the detection method of the content of multiple index chemical components in Suanzaoren provided by the present application is described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0049] In the examples of the present application, the specific instruments are as follows: Agilent 1200 type high performance liquid chromatograph (Agilent Company, USA); Agilent 6430 triple quadrupole tandem mass spectrometer (Agilent Company, USA); Agilent MassHunter analysis software (Agilent Company, USA); Milli-Q IQ 7005 ultrapure water preparation instrument (Millipore Company); 5424R type high-speed centrifuge (Eppendorf Company, Germany); AS 60 / 220.R2 type one hundredth balance (Radwag Company, Poland); G3KT 18273 type vortex mixer (Thermo Fisher Scientific Company).

[0050] The standard controls are as follows: Jujuboside A (batch number: DST231025-058), Jujuboside B (batch number: DST220608-059), Jujuboside D (batch number: DST231115-119), Spinonin (batch number: DST230060-0020), 6'' '-feruloyl spinonin (batch number: DST230720-013), Vicenin II (batch number: DST201104-023), Magnoflorine (batch number: DST220723-216), Kadsurin (batch number: DST190625-004), Frangulanine (batch number: DST231114-023), Nuciferine (batch number: DSTDH005102), Nor-nuciferine (batch number: DST200722-238), Isostrictinin (batch number: DSTDY005401), Swertisin (batch number: DST200817-084), Rutin (batch number: DST190901-017), Quercetin (batch number: 190926-029), Kaempferol-3-O-rutinoside (batch number: DST18110-075), Isoquercitrin (batch number: DSTDY000601), Ferulic acid (batch number: DSTDF008101), Naringin (batch number: DSTDY009904), Hesperidin (batch number: DSTDC003803), Naringenin (batch number: DST211116-100), Camellin B (batch number: DST131573-90-5), and Epicatechin (batch number: DST200711-035) are all purchased from Chengdu Desite Biotechnology Co., Ltd., and the purity of each is greater than 98%. Methanol, acetonitrile (chromatographic grade) are purchased from Fisher Company, USA; formic acid is purchased from ROE Company, USA; ultrapure water is prepared by a Milli-Q ultrapure water preparation instrument. The specific source information of the jujube sample is shown in Table 2.

[0051] Table 2 Specific source information of jujube medicinal materials

[0052] No. Origin Batch No. No. Origin Batch No. S1 Hebei 20231005 S8 Henan 20231024 S2 Hebei 20231110 S9 Henan 20231014 S3 Hebei 20231125 S10 Shaanxi 20231107 S4 Hebei 20231116 S11 Shaanxi 20231207 S5 Shandong 20231103 S12 Shaanxi 20231210 S6 Shandong 20231002 S13 Liaoning 20231207 S7 Shandong 20231121 S14 Liaoning 20231214

[0053] The HPLC chromatographic conditions are as follows:

[0054] The chromatographic column was Waters CORTECS C18 (2.1 x 50 mm, 2.7 μm; Waters); the mobile phase A was 0.1 vol% formic acid-water, and the mobile phase B was acetonitrile; gradient elution, the elution gradient program was as follows: 0-3 min, the volume fraction of the mobile phase B was increased from 20% to 32%; 3-5 min, the volume fraction of the mobile phase B was increased from 32% to 41%; 5-6 min, the volume fraction of the mobile phase B was increased from 41% to 55%; 6-8 min, the volume fraction of the mobile phase B was increased from 55% to 90%; the flow rate of the mobile phase was 0.3 mL / min; the column temperature was 30°C; the injection volume was 2.0 μL.

[0055] The mass spectrometry conditions were as follows: the ion source was an electrospray ion source (ESI); the detection mode was multiple reaction ion monitoring (MRM); the scan mode was a positive and negative ion scan mode; the gas temperature (Gas Temp) was 320°C; the gas flow rate (Gas Flow) was 10 L / min; the nebulizer pressure (Nebulizer) was 40 psig; the collision gas was preferably high-purity nitrogen; the nebulizing gas was preferably high-purity nitrogen; the quantitative ion pair, the mass spectrometry parameters, and the ion mode are shown in Table 1.

[0056] Example 1

[0057] Methodology Investigation-Standard Curve Regression Equation

[0058] Preparation of the reference substance stock solution: the reference substances of jujuboside A, jujuboside B, jujuboside D, spinosin, 6'''-feruloylspinosin, vicenin II, magnoflorine, wongaienine, frangulanine, nuciferine, proto-nuciferine, isofukinianin, onjisapogenin, rutin, quercetin, kaempferol-3-O-rutinoside, isoquercitrin, ferulic acid, naringin, hesperidin, naringenin, camellatin B, and epicatechin were respectively dissolved in methanol to prepare 23 single reference substance reference substance stock solutions with a concentration of 1 mg / mL.

[0059] Preparation of mixed control solution: precisely pipette each control stock solution into the same volumetric flask, dilute with methanol to obtain the mixed control solution; the concentration of magnoflorine in the mixed control solution is 55 μg / mL, the concentrations of 6'''-feruloylsppinostanol and spinoosin are 30 μg / mL, the concentrations of jujuboside A and jujuboside D are 15 μg / mL, the concentrations of vicenin II and liriodendrin are 10 μg / mL, the concentrations of camelliatin B, epicatechin, jujuboside B and frangulanin are 8 μg / mL, the concentrations of isofukin, kaempferol-3-O-rutinoside and ferulic acid are 5 μg / mL, the concentrations of rutin, onogeral, and norlaudanosine are 1 μg / mL, the concentrations of isoquercitrin, naringenin, quercetin and hesperidin are 0.5 μg / mL, and the concentrations of naringin and laudanosine are 0.2 μg / mL.

[0060] Preparation of mixed control linear solution: dilute the mixed control solution with methanol by 2 times to obtain mixed control linear solution 1; continue to dilute the mixed control linear solution 1 with methanol by 2.5 times to obtain mixed control linear solution 2; continue to dilute the mixed control linear solution 2 with methanol by 2 times to obtain mixed control linear solution 3; continue to dilute the mixed control linear solution 3 with methanol by 2 times to obtain mixed control linear solution 4; continue to dilute the mixed control linear solution 4 with methanol by 2 times to obtain mixed control linear solution 5; continue to dilute the mixed control linear solution 5 with methanol by 2 times to obtain mixed control linear solution 6; continue to dilute the mixed control linear solution 6 with methanol by 2.5 times to obtain mixed control linear solution 7; continue to dilute the mixed control linear solution 7 with methanol by 2 times to obtain mixed control linear solution 8; continue to dilute the mixed control linear solution 8 with methanol by 2 times to obtain mixed control linear solution 9.

[0061] Preparation of sample solution: precisely weigh 500.0 mg of crude powder of Zizyphi Semen pretreated with 70 vol% methanol aqueous solution, and add 70 vol% methanol aqueous solution to the mark line, ultrasonic treat for 30 min under the condition of 300 W and 40 kHz, cool, make up the weight loss, shake well, centrifuge at 14000 r / min for 10 min, and then pass through a 0.22 μm microporous filter membrane to obtain the sample solution.

[0062] Take each of the above solutions, and inject HPLC-MS / MS analysis. Use weighted least squares method for regression calculation, the abscissa (X) is the concentration of the measured substance, the ordinate (Y) is the peak area of the measured substance, and the weight coefficient is 1 / X 2, jujuboside A, jujuboside B, jujuboside D, spinosin, 6'' '-feruloyl spinosin, vicenin II, magnoflorine, wuweizisu A, fructus armeniaceae leaf base, nuciferine, nuciferine, isofukinianin, oroxindin, rutin, quercetin, kaempferol-3-O-rutinoside, isoquercitrin, ferulic acid, naringin, hesperidin, naringenin, camellatin B and epicatechin. The concentration of the control sample calculated when the signal-to-noise ratio (S / N) is 10 is taken as the lowest limit of quantification (LLOQ), and the results are shown in Table 3.

[0063] Table 3 Standard curve regression equations and the lowest limit of quantification (LLOQ) of 23 chemical components in Zizyphus jujuba

[0064]

[0065]

[0066] Figure 1 The MRM graphs of the standard control sample (left) and the test sample (right) are shown in Figure 1, in which 1 is kaempferol-3-O-rutinoside, 2 is 6'' '-feruloyl spinosin, 3 is jujuboside B, 4 is rutin, 5 is hesperidin, 6 is spinosin, 7 is vicenin II, 8 is fructus armeniaceae leaf base, 9 is isoquercitrin, 10 is quercetin, 11 is oroxindin, 12 is nuciferine, 13 is nuciferine, 14 is wuweizisu A, 15 is magnoflorine, 16 is ferulic acid, 17 is jujuboside A, 18 is jujuboside B, 19 is isofukinianin, 20 is naringenin, 21 is epicatechin, 22 is camellatin B, and 23 is naringin. It can be seen from Figure 1 that the peak shapes of the compounds are good and do not interfere with each other. Figure 1

[0067] Example 2

[0068] Methodology investigation - precision test

[0069] Precision (intra-day): The test sample solution was prepared according to Example 1. HPLC-MS / MS analysis was performed 6 times in succession, and the RSD values of the peak areas of the compounds were calculated. The results are shown in Table 4, which indicates that the intra-day precision of the instrument is good. In Tables 4-6, 1-6 represent the 1st-6th sample analysis.

[0070] Precision (inter-day): The test sample solution was prepared according to Example 1. HPLC-MS / MS analysis was performed twice in succession for 3 days, and the RSD values of the peak areas of the compounds were calculated. The results are shown in Table 5, which indicates that the inter-day precision of the instrument is good.

[0071] Table 4 Intra-day precision results (n = 6) of 23 chemical components in Zizyphus jujuba

[0072] ​

[0073] Table 5 Inter-day precision results of 23 chemical components in Semen Ziziphi Spinosae (n = 6)

[0074]

[0075]

[0076] Example 3

[0077] Methodology investigation - repeatability test

[0078] According to Example 1, 6 test sample solutions were prepared in parallel, and were respectively injected into HPLC-MS / MS for analysis, and the RSD values of the concentrations of each compound were calculated. The results are shown in Table 6, indicating that the method has good repeatability.

[0079] Table 6 Repeatability results of 23 chemical components in Semen Ziziphi Spinosae (n = 6, ng / mL)

[0080]

[0081]

[0082] Example 4

[0083] Methodology investigation - stability test

[0084] According to Example 1, the test sample solution was prepared, and was respectively injected into HPLC-MS / MS for analysis at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h, and the RSD values of the peak areas of each compound were calculated. The results are shown in Table 7, indicating that each compound has good stability.

[0085] Table 7 Stability results of 23 chemical components in Semen Ziziphi Spinosae (n = 6)

[0086]

[0087]

[0088] Example 5

[0089] Methodology investigation - spike recovery test

[0090] Preparation of spiked test sample solution: 250.0 mg of Semen Ziziphi Spinosae crude powder (passed through a No. 4 sieve) was accurately weighed into a 10 mL volumetric flask, and different amounts (see Table 8) of the mixed reference solution prepared in Example 1 were added, and 70 vol% methanol aqueous solution was added to the mark line, and was ultrasonically treated at 300 W and 40 kHz for 1 h. After cooling, the weight loss was made up, and was shaken well, and was filtered through a 0.22 μm microporous filter membrane to obtain a spiked test sample solution.

[0091] The spiked sample solutions were respectively injected into HPLC-MS / MS for analysis 6 times, and the recovery rates of the compounds were calculated, and the results were shown in Table 8.

[0092] Table 8: Recovery rates of 23 chemical components in Zizyphi Spinosae Semen (n = 6)

[0093]

[0094]

[0095] Example 6

[0096] Content determination

[0097] The crude powder of Zizyphi Spinosae Semen of each batch (S1-S14) was precisely weighed, and the sample solution was prepared according to Example 1, and was injected into HPLC-MS / MS for analysis, and the concentrations of 23 compounds were recorded and the content was calculated, and the results were shown in Table 9.

[0098] Table 9: Contents of 23 chemical components in Zizyphi Spinosae Semen of different batches (μg / g, n = 3)

[0099]

[0100]

[0101] In summary, the method for simultaneously determining the contents of 23 chemical components in Zizyphi Spinosae Semen was established based on HPLC-MS / MS, and was applied to the content determination of Zizyphi Spinosae Semen of different batches. The analysis method has high sensitivity and strong specificity, and has good precision, repeatability and stability, and can reflect the content differences of the measured chemical components in Zizyphi Spinosae Semen of different batches, which can provide reference for the quality evaluation and efficacy material basis research of Zizyphi Spinosae Semen.

[0102] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A method for detecting the content of multiple index chemical components in Semen Ziziphi Spinosae, comprising the following steps: ultrasonic extraction of a to-be-tested Semen Ziziphi Spinosae powder with an extracting agent to obtain a to-be-tested sample solution; the extracting agent is a lower alcohol or a lower alcohol aqueous solution; the volume fraction of the lower alcohol in the extracting agent is 70-100%; the solid-liquid ratio of the to-be-tested Semen Ziziphi Spinosae powder to the extracting agent is 0.4-0.6 g:10 mL; high performance liquid chromatography tandem mass spectrometry detection of the to-be-tested sample solution to obtain the detection results of the content of multiple index chemical components in Semen Ziziphi Spinosae; the multiple index chemical components are Semen Ziziphi Spinosae saponin A, Semen Ziziphi Spinosae saponin B, Semen Ziziphi Spinosae saponin D, spinosin, 6‴-feruloylspinosin, vicenin II, magnoflorine, magnoliflorine, fructus padus leaf base, nuciferine, proto-nuciferine, isofraxidin, oroxindin, rutin, quercetin, kaempferol-3-O-rutinoside, isoquercitrin, ferulic acid, naringin, hesperidin, naringenin, camellin B and epicatechin; the high performance liquid chromatography tandem mass spectrometry detection comprises high performance liquid chromatography separation and mass spectrometry detection; the high performance liquid chromatography separation conditions comprise: the chromatographic column is a Waters CORTECS C18, 2.1*50 mm, 2.7 μm; the mobile phase A is 0.08-0.12 vol% formic acid aqueous solution, the mobile phase B is acetonitrile, the elution mode is gradient elution, and the program of the gradient elution is as follows: 0-3 min, the volume fraction of the mobile phase B is increased from 20% to 32%; 3-5 min, the volume fraction of the mobile phase B is increased from 32% to 41%; 5-6 min, the volume fraction of the mobile phase B is increased from 41% to 55%; 6-8 min, the volume fraction of the mobile phase B is increased from 55% to 90%; the quantitative analysis ion pairs of magnoflorine, magnoliflorine, proto-nuciferine, nuciferine, vicenin II, spinosin, Semen Ziziphi Spinosae saponin A, Semen Ziziphi Spinosae saponin B, Semen Ziziphi Spinosae saponin D, isoquercitrin, kaempferol-3-O-rutinoside, ferulic acid, 6‴-feruloylspinosin, naringin, hesperidin, quercetin, fructus padus leaf base, naringenin, rutin, isofraxidin, oroxindin, epicatechin and camellin B are 286.3 / 107.1, 343.3 / 266.2, 296.2 / 250.1, 282.1 / 235.1, 593.5 / 353.3, 607.4 / 427.3, 1205.3 / 1073.8, 1043.3 / 911.1, 1205.3 / 1073.8, 463.2 / 300.1, 593.5 / 285.3, 193.0 / 134.0, 783.5 / 427.2, 579.3 / 271.2, 609.3 / 301.2, 301.2 / 151.3, 533.9 / 247.5, 271.0 / 119.2, 609.6 / 300.3, 431.5 / 311.2, 445.3 / 282.4, 289.0 / 125.1 and 725.2 / 283.8, respectively.

2. The detection method according to claim 1, characterized in that, The low alcohol includes methanol and / or ethanol.

3. The method of claim 1, wherein, The ultrasonic extraction is performed at a power of 280-320 W, a frequency of 30-50 kHz, and a time of 20-40 min.

4. The method of claim 1, wherein The high performance liquid chromatography separation column temperature is 28-32 DEG C.

5. The method of claim 1, wherein The high performance liquid chromatography separation sample injection volume is 1.8-2.2 muL.

6. The detection method according to claim 1, 4 or 5, characterized in that, The high performance liquid chromatography separation mobile phase flow rate is 0.28-0.32 mL / min.

7. The method of claim 1, wherein, The mass spectrometry detection conditions include: the ion source is an electrospray ion source, the detection mode is multiple reaction ion monitoring, the scanning mode is positive and negative ion scanning mode, the gas temperature is 320 DEG C; the gas flow rate is 10 L / min, the atomizer pressure is 40 psig, the fragmentation voltage is 85-148 V, and the collision energy is 10-50 V.

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