CN103969385B - Five kinds of alkaloidal qualifications in the Bi roots of grass and pepper and content Simultaneous Determination method - Google Patents
Five kinds of alkaloidal qualifications in the Bi roots of grass and pepper and content Simultaneous Determination method Download PDFInfo
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Abstract
The invention discloses 5 kinds of alkaloidal qualifications in a kind of Bi roots of grass and pepper and content Simultaneous Determination method.The method adopts HPLC-ESI-IT-MS/MS method to identify 5 kinds of alkaloidal structures, and establishes the method for a kind of method of UFLC-ESI-MS/MS fast and accurately Simultaneously test 5 kinds of alkaloids first.5 kinds of alkaloidal linearly dependent coefficient R
2aMP.AMp.Amp gt; 0.955, lowest detection is limited to 0.02-0.03ng/mL, is minimumly quantitatively limited to 0.05-0.10ng/mL.5 kinds of alkaloid content determination method withinday precision RSD values are less than 9.30%, and day to day precision is less than 9.55%.The stability of the method, repeatability, average recovery all meet the requirements, and methodological study result shows that the method has good applicability.
Description
Technical field
The present invention relates to five kinds of alkaloidal qualifications in a kind of Bi roots of grass and pepper and content Simultaneous Determination method.
Background technology
The Bi roots of grass is the nearly ripe or ripening fruits of the drying of the Piperaceae plant Bi roots of grass (P.longumL.), is the conventional flavouring of south east asia.Pepper is the fruit of Piperaceae plant pepper (P.nigrumL.), can be divided into black pepper and white pepper, be widely used as flavoring.White pepper derives from the dry seed of pepper, and black pepper derives from the immature fruit of pepper.The Bi roots of grass is in China as a kind of conventional Chinese medicine, and be applied to the disease such as gastral cavity pain and vomiting clinically, and pepper is often applied to gastrofrigid vomiting clinically, stomachache is had loose bowels, poor appetite, and epilepsy phlegm waits disease.Modern study shows, main active in the Bi roots of grass and pepper is alkaloids, but also not yet have at present about while quick separating and measure the relevant report of pipering, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine and pellitorine in the Bi roots of grass and pepper.Therefore, need badly and set up one method of separating and assaying fast and accurately.
Summary of the invention
The object of this invention is to provide five kinds of alkaloidal qualifications in a kind of Bi roots of grass and pepper and content Simultaneous Determination method.
The invention provides a kind of method of isolating alkaloids from the Bi roots of grass and/or pepper, comprise the steps:
Testing sample is carried out liquid chromatography-tandem mass spectrometry detection, according to eluting peak retention time, Q
1and Q
3each eluting peak of character separation, namely obtains each alkaloid;
Described Q
1for molecular ion peak [M+H]
+quality (m/z), Q
3for the quality (m/z) of secondary fragment daughter ion;
Described testing sample is the Bi roots of grass and/or pepper;
Described alkaloid is selected from least one in pipering, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine and pellitorine;
The condition of described liquid chromatographic detection is as follows: chromatographic column used is PhenomenexGeminiC18 reverse-phase chromatographic column;
Eluent is made up of mobile phase A and Mobile phase B; Wherein, mobile phase A to be mass percentage concentration be 0.1% aqueous formic acid; Mobile phase B is the mixed liquor be made up of formic acid and acetonitrile, and the mass percentage concentration of formic acid is 0.1%;
Type of elution is gradient elution, and elution speed is 0.55mL/min;
Described gradient elution mode is as follows:
Within 0th minute, rise to the 0.49th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98:2;
Within 0.50th minute, rise to the 3.00th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98-2:2-98;
Within 3.10th minute, rise to the 3.99th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 2:98;
Within 4.00th minute, rise to the 4.00th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98-2:2-98;
Within 4.01st minute, rise to 5.49 minutes ends, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98:2;
Within 5.50th minute, rise, stop wash-out;
Described according to eluting peak retention time, Q
1and Q
3the method of each eluting peak of character separation is as follows:
Retention time is 3.78 minutes, Q
1be 258.9 and Q
3be 201.1 eluting peak be pipering;
Retention time is 3.73 minutes, Q
1be 273.9 and Q
3be 201.1 eluting peak be the bright peaceful alkali of the Bi roots of grass;
Retention time is 3.71 minutes, Q
1be 276.1 and Q
3be 134.9 eluting peak be the bright peaceful alkali of the dihydro Bi roots of grass;
Retention time is 3.76 minutes, Q
1be 288.0 and Q
3be 135.1 eluting peak be piperanine;
Retention time is 3.93 minutes, Q
1be 224.0 and Q
3be 80.9 eluting peak be pellitorine.
In said method, described pepper specifically can be black pepper or white pepper;
In the testing conditions of described liquid chromatography, the length of chromatographic column specifically can be 50 millimeters, and internal diameter specifically can be 2.0 millimeters, and the particle diameter of C18 specifically can be 5 μm;
Sample size is 10 μ L; Column temperature is room temperature.
In described Mass Spectrometer Method condition,
The ionization voltage (IonSpray) in electron spray ionisation source (ESI) is+5000V, and ion source temperature is 500 DEG C-600 DEG C, is specially 550 DEG C;
The pressure of atomization gas (GS1) is 40-60psi, is specially 50psi;
The pressure of auxiliary heating gas (GS2) is 40-60psi, is specially 50psi;
Gas curtain gas CUR pressure is 10-30psi, is specially 20psi;
The solution bunch voltage that often kind of alkaloid is corresponding, impact energy, collision cell exit potential (CXP) are as follows:
Pipering: separating bunch voltage is 71eV, and impact energy is 27eV, and collision cell exit potential (CXP) is 10V;
The bright peaceful alkali of the Bi roots of grass: separating bunch voltage is 56eV, and impact energy is 25eV, and collision cell exit potential (CXP) is 10V;
The bright peaceful alkali of the dihydro Bi roots of grass: separating bunch voltage is 66eV, and impact energy is 29eV, and collision cell exit potential (CXP) is 10V;
Pellitorine: separating bunch voltage is 96eV, and impact energy is 31eV, and collision cell exit potential (CXP) is 4V;
Piperanine: separating bunch voltage is 111eV, and impact energy is 39eV, and collision cell exit potential (CXP) is 10V.
In addition, said method also comprises the steps:
Before described liquid chromatography-tandem mass spectrometry detecting step, after testing sample and methyl alcohol are mixed with the ratio of 100mg:100mL, after filtering with microporous membrane, get filtrate, add 10 times wherein to the mixed liquor a of described filtrate volume, after vortex mixing, then add 19 times of mixed liquor vortexs be made up of isopyknic first alcohol and water to vortex gained liquor capacity wherein and mix;
Wherein, the aperture of described miillpore filter is specially 0.45 μm;
Described mixed liquor a is the solution of teldane; In described mixed liquor a, solvent is specially methyl alcohol and acetonitrile; The concentration of described teldane is specially 50ng/mL;
Described mixed liquor a is specifically obtained by following steps: teldane dissolved with methyl alcohol, and being mixed with concentration is after the methanol solution of the teldane of 1.00mg/mL, then to add isopyknic methyl alcohol and dilution in acetonitrile to the concentration of teldane be 50ng/mL, and obtains;
In described vortex mixing step, the time of vortex is specifically 10s.
Present invention also offers a kind of method measuring alkaloid in the Bi roots of grass and/or pepper, the method comprises the steps:
1) potpourri of the described alkaloidal reference substance of concentration known is separated according to preceding method, and the peak area that record often kind of alkaloid is corresponding; With often kind of alkaloidal concentration value for independent variable, with the peak area of its correspondence for dependent variable, obtain unary linear regression equation;
2) testing sample is separated according to preceding method, and the peak area that record often kind of alkaloid is corresponding;
3) by peak area substitution step 1 corresponding for often kind of alkaloid) middle unary linear regression equation, obtain each alkaloidal concentration in described testing sample;
Described testing sample is the Bi roots of grass and/or pepper;
Described alkaloid is selected from least one in pipering, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine and pellitorine.
In said method, described pepper specifically can be black pepper or white pepper;
The unary linear regression equation that various described alkaloid is corresponding is as follows:
Pipering: y=0.00353x-0.00046, independent variable concentration range is 10-10000ng/mL;
The bright peaceful alkali of the Bi roots of grass: y=0.00263x+0.00157, independent variable concentration range is 10-10000ng/mL;
The bright peaceful alkali of the dihydro Bi roots of grass: y=0.00362x+0.00670, independent variable concentration range is 10-10000ng/mL;
Piperanine: y=0.00327x-0.00184, independent variable concentration range is 10-10000ng/mL;
Pellitorine: y=0.00067x+0.00326, independent variable concentration range is 10-10000ng/mL.
In addition, said method also comprises the steps:
Before described liquid chromatography-tandem mass spectrometry detecting step, after testing sample and methyl alcohol are mixed with the ratio of 100mg:100mL, after filtering with microporous membrane, get filtrate, add 10 times wherein to the mixed liquor a of described filtrate volume, after vortex mixing, then add 19 times of mixed liquor vortexs be made up of isopyknic first alcohol and water to vortex gained liquor capacity wherein and mix;
Wherein, the aperture of described miillpore filter is specially 0.45 μm;
Described mixed liquor a is the solution of teldane; In described mixed liquor a, solvent is specially methyl alcohol and acetonitrile; The concentration of described teldane is specially 50ng/mL;
Described mixed liquor a is specifically obtained by following steps: teldane dissolved with methyl alcohol, and being mixed with concentration is after the methanol solution of the teldane of 1.00mg/mL, then to add isopyknic methyl alcohol and dilution in acetonitrile to the concentration of teldane be 50ng/mL, and obtains;
In described vortex mixing step, the time of vortex is specifically 10s.
The present invention's application high-efficient liquid is in series ion trap chromatogram (HPLC-ESI-IT-MS
n) method identifies 5 kinds of alkaloidal structures, and establish the content of pipering, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine, pellitorine 5 kinds of alkaloids compositions in a kind of method of UFLC-ESI-MS/MS fast and accurately Simultaneously test Bi roots of grass and pepper first.5 kinds of alkaloidal linearly dependent coefficient R
2>0.955, lowest detection is limited to 0.02-0.03ng/mL, is minimumly quantitatively limited to 0.05-0.10ng/mL.5 kinds of alkaloid withinday precision RSD values are less than 9.30%, and day to day precision is less than 9.55%.5 kinds of alkaloidal stability (RSD<2.53%), repeatability (RSD<2.58%), average recoveries (90.0-103.5%) all meet the requirements.Methodology result shows that the method has good applicability.Assay result shows, all containing these 5 kinds of alkaloids compositions in the Bi roots of grass and pepper, in Bi roots of grass total alkaloids, the content of these 5 kinds of alkaloid components is significantly higher than the Bi roots of grass and pepper.In Bi roots of grass medicinal material, content of piperine is lower than pepper, but the content of the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine is all higher than pepper.
Accompanying drawing explanation
Fig. 1 is the secondary full scan mass spectrogram that UFLC-MS/MS measures 5 kinds of alkaloids and interior mark teldane, wherein, and A: pipering (Piperine); The bright peaceful alkali (Piperlonguminine) of B: the Bi roots of grass; C: the bright peaceful alkali (Dihydropiperlonguminine) of the dihydro Bi roots of grass; D: pellitorine (Pellitorine); E: piperanine (Piperanine); F: teldane (interior mark);
Fig. 2 is that LC-ESI-MS/MS method measures 5 kinds of alkaloidal representative chromatograms, wherein, and A: blank solvent; B: standard solution (each compound concentration is 10ng/mL); C: Bi roots of grass total alkaloids (PLA200811); D: Bi roots of grass medicinal material (PL201105AG); E: pepper (WP201204AG).
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but the present invention is not limited to following examples.Described method is conventional method if no special instructions.Described starting material all can obtain from open commercial sources if no special instructions.
The preparation method of the pipering of product, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine, pellitorine is as follows in contrast:
By Bi roots of grass total alkaloids 43g, use isopyknic CHCl
3and CH
3the mixed liquor of OH dissolves, and mixes sample with 100-200 order silica gel under 50 DEG C of water bath condition, will mix the sample dry method loading after sample, and be separated through silica gel column chromatography.With methylene chloride-methanol (v:v=10:0 ~ 4:6) for mobile phase carries out gradient elution.According to thin-layer chromatography inspection knowledge situation, eluent is merged into Fr.1 ~ 13 part, recycling design.Fr.2 ~ 7 part is dissolved in appropriate methyl alcohol respectively, ultrasonic dissolution, after crossing the miillpore filter of 0.42 μm, adopts the method for reversed-phase silica gel column chromatography to be separated.RP-C18 silica gel chromatographic column balances through methanol-water (v:v=50:50) 500mL, wet method application of sample, with methanol-water (v:v=50:50 ~ 55:45 ~ 60:40 ~ 65:35) for eluent gradient wash-out, wash-out stream part is received by every part of 250mL, collect each concentration gradient eluent, obtain stream part Fr.1a ~ 40a.According to thin-layer chromatography inspection knowledge situation, after decompression and solvent recovery is carried out to Fr.10a, Fr.16a ~ 17a, Fr.18a ~ 19a, Fr.20a ~ 21a, Fr.22a ~ 25a and Fr.31a ~ 32a position, adopt the further separation and purification of means such as semi-preparative high performance liquid chromatography, preparative thin layer chromatography, recrystallization.Fr.18a ~ 19a position adopts the method for half preparative HPLC to carry out separation and purification, with water-methanol: 35:65 is mobile phase, flow velocity: 2mLmin
-1, determined wavelength: 275nm, column temperature: 25 DEG C, sample size: 200 μ L.Repeatedly sample introduction, collects eluent, after decompression and solvent recovery, obtains the bright peaceful alkali (130mg) of the Bi roots of grass.Fr.22a ~ 25a position obtains pipering (7000mg) by crystallization and recrystallization means.Respectively purification on normal-phase silica gel column chromatography Fr.1 part, reversed-phase silica gel column chromatography Fr.10a, Fr.16 ~ 17a, Fr.20a ~ 21a and Fr.31a ~ 32a part are adopted preparative thin layer chromatography separation and purification.After sample is dissolved with a small amount of methyl alcohol, strip point sample on preparative silica gel g thin-layer plate, Fr.1 position cyclohexane: ethyl acetate (v:v=3:1) solvent system ascending development, Fr.10a position cyclohexane: ethyl acetate: formic acid (v:v=3:2:0.05) solvent system ascending development, Fr.16a ~ 17a and Fr.20a ~ 21a cyclohexane: ethyl acetate (v:v=3:2) solvent system ascending development, Fr.31a ~ 32a position cyclohexane: ethyl acetate (v:v=2.5:1) solvent system ascending development, at wavelength 254nm, observe under the uviol lamp of 365nm, scrape the fluorescent belt of identical Rf value, collect the silica white scraped, dress post, complete by washed with methanol, collect eluent, after reduced pressure concentration, obtain the bright peaceful alkali (76mg) of the dihydro Bi roots of grass of purifying respectively, piperanine (200mg) and pellitorine (120mg).
Reference substance Structural Identification adopts ThermoLTQXLOrbitrap LC-MS instrument, utilizes high-efficient liquid to be in series ion trap mass spectrometry, and analyze 5 kinds of alkaloidal 1 grade and 2 grades of fragments, actual conditions is as follows:
Adopt DIONEXUItiMATE3000 liquid chromatograph (Dai An company of the U.S.) to carry out gradient elution, chromatographic column is AgilentEclipsePlusC18 reverse-phase chromatographic column (150mm*2.1mm, 5 μm);
Eluent is made up of mobile phase A and Mobile phase B; Wherein, mobile phase A to be mass percentage concentration be 0.25% aqueous formic acid; Mobile phase B is acetonitrile;
The condition of gradient elution is as follows:
Within 0th minute, rise to the 4th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 85:15;
Within 5th minute, rise to the 19th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 85-60:15-40;
Within 20th minute, rise to the 29th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 60-35:40-45;
Within 30th minute, rise to the 39th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 55-40:45-60;
Within 40th minute, rise to the 49th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 30-10:60-90;
Within 50th minute, rise to the 64th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 10:90;
Elution speed is 0.3mL/min; Column temperature is 40 DEG C; Sampling volume is 20 μ L.
Mass Spectrometer Method adopts ThermoLTQXLOrbitrap LC-MS instrument (Thermo Fischer Scient Inc. of the U.S.), ESI ionization source, positive ion scan pattern.Electron spray voltage is 4500V; Heated capillary temperature is 375 DEG C; Capillary voltage 40V; Sheath gas, assisted gas, sweep gas are nitrogen, and pressure is respectively 17arb, 3arb, 0arb.
Acquired results is shown in Fig. 1 and table 1;
Table 1HPLC-ESI-IT-MS/MS detects 5 kinds of alkaloid reference substance Structural Identification results and one-level, second order ms data
As from the foregoing, determined alkaloidal structure is errorless.
In addition, when preparing the pipering of above-mentioned product in contrast, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine, pellitorine, be used as raw material Bi roots of grass total alkaloids obtain as follows:
Take the Bi roots of grass fruit ear 3kg of drying and crushing, add the ethanol water 15L heating and refluxing extraction that mass percentage concentration is 85%, merging filtrate after extracting liquid filtering, reclaim ethanol and obtain long pepper extract.Adopt the ethanol water gradient elution of 0%, 20%, 40%, 60% and 85% through macroreticular resin after long pepper extract is water-soluble, ethanol is reclaimed after 60% ethanol elution stream part being merged, drying under reduced pressure obtains Bi roots of grass total alkaloids active component (PLA), and its total alkaloids massfraction of determined by ultraviolet spectrophotometry is 64.43%.
The Bi roots of grass of different batches, black pepper, white pepper purchased from Anguo medicinal material market, Tongrentang, cubic drink factory, medicinal material market, Guangxi province, concrete lot number and source are in table 2.
The source list of table 2 different batches sample
Wherein, Bi roots of grass total alkaloids-1 is obtained by the Bi roots of grass-1, and Bi roots of grass total alkaloids-2 is obtained by the Bi roots of grass-2, and Bi roots of grass total alkaloids-3 is obtained by the Bi roots of grass-3;
Teldane purchased from Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 100292-200201;
Methyl alcohol purchased from American Fisher company;
Acetonitrile purchased from American Fisher company; Methyl alcohol, acetonitrile are chromatographically pure.
Vortex device therefor is XH-C vortex mixer, purchased from Nuo Yi experimental apparatus factory of Jintan City;
Ultrasonic cleaning device therefor is Shu Mei KQ-500VDE numerical control double-frequency ultrasound washer, purchased from Kunshan Ultrasonic Instruments Co., Ltd..
Alkaloidal separation in embodiment 1, the Bi roots of grass and pepper
One, the preparation of reference substance solution
A, precision take pipering, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine, the pellitorine of product in contrast, after DMSO dissolves, are formulated as the reference substance storing solution of 1mg/mL respectively.Precision measures each 10 μ L of above-mentioned reference substance storing solution and is placed in same 1.5mLEP pipe respectively, then adds methanol dilution is 10.0 μ g/mL mixing reference substance storing solution to often kind of alkaloidal concentration.
B, get above-mentioned mixing reference substance storing solution, carry out serial dilution with methyl alcohol, be mixed with simultaneously containing the standard serial solution that 5 kinds of alkaloid concentrations are 10.0 μ g/mL, 5.0 μ g/mL, 2.0 μ g/mL, 1.00 μ g/mL, 0.5 μ g/mL, 0.2 μ g/mL, 0.1 μ g/mL, 0.05 μ g/mL, 0.02 μ g/mL, 0.01 μ g/mL.
It is appropriate that c, precision take teldane reference substance, dissolves and dilute, be mixed with the stock solution that concentration is 1.00mg/mL with methyl alcohol.Facing the used time adds isopyknic methyl alcohol and becomes with dilution in acetonitrile concentration to be the working solution of 50ng/mL.
Two, the preparation of need testing solution
Precision takes the Bi roots of grass-1100mg used in table 2 and is placed in 100mL volumetric flask respectively, and add methanol constant volume, ultrasonic 10min, methyl alcohol complements to scale, and 0.45 μm of filtering with microporous membrane, obtains original need testing solution.Before measuring, dilution 10 times is to suitable concentration, to be measured.
Get the above-mentioned need testing solution of 20 μ L before mensuration, the concentration accurately adding 200 μ L teldane methyl alcohol is the mixed liquor be made up of isopyknic teldane methyl alcohol and acetonitrile of 50ng/mL, vortex mixed 10s; Get 10 μ L vortex gained solution again, add the mixed liquor that 190 μ L are made up of isopyknic first alcohol and water wherein, vortex mixed 10s, to obtain final product.
Three, alkaloidal separation
Shimadzu LC-20A liquid chromatographic system (purchased from Japanese Shimadzu Corporation, comprising LC-20AD binary pump, CTO-20A column oven, SIL-20AC injector) and API4000Qtrap liquid chromatography mass spectrometric combined system (purchased from American Applied Biosystems, Inc.) is adopted by need testing solution to carry out liquid chromatography-tandem mass spectrometry detection.
Wherein, the actual conditions of liquid chromatographic detection is as follows:
Chromatographic column is PhenomenexGeminiC18 reverse-phase chromatographic column (particle diameter of 50mm*2.00mm, C18 is 5 μm);
Eluent is made up of mobile phase A and Mobile phase B; Wherein, mobile phase A to be mass percentage concentration be 0.1% aqueous formic acid; Mobile phase B is the mixed liquor be made up of formic acid and acetonitrile, and the mass percentage concentration of formic acid is 0.1%;
Type of elution is gradient elution, and elution speed is 0.55mL/min;
Described gradient elution mode is as follows:
Within 0th minute, rise to the 0.49th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98:2;
Within 0.50th minute, rise to the 3.00th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98-2:2-98;
Within 3.10th minute, rise to the 3.99th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 2:98;
Within 4.00th minute, rise to the 4.00th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98-2:2-98;
Within 4.01st minute, rise to 5.49 minutes ends, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98:2;
Within 5.50th minute, rise, stop wash-out;
The flow velocity of eluent is 0.55mL/min; Column temperature is room temperature; Sampling volume is 10 μ L.
The actual conditions of Mass Spectrometer Method is as shown in table 2, and experiment adopts ESI ionization source, and positive ion scans, MRM detecting pattern.
The solution bunch voltage that often kind of alkaloid is corresponding, impact energy, collision cell exit potential (CXP) are also as shown in table 3;
Table 3HPLC-ESI-MS/MS method detects 5 kinds of alkaloid mass spectrometry parameters used
Therefore retention time is 3.78 minutes, Q
1be 258.9 and Q
3be 201.1 eluting peak be pipering;
Retention time is 3.73 minutes, Q
1be 273.9 and Q
3be 201.1 eluting peak be the bright peaceful alkali of the Bi roots of grass;
Retention time is 3.71 minutes, Q
1be 276.1 and Q
3be 134.9 eluting peak be the bright peaceful alkali of the dihydro Bi roots of grass;
Retention time is 3.76 minutes, Q
1be 288.0 and Q
3be 135.1 eluting peak be piperanine;
Retention time is 3.93 minutes, Q
1be 224.0 and Q
3be 80.9 eluting peak be pellitorine.
The mensuration of embodiment 2, methodological study and alkaloid
One, the preparation of reference substance solution and need testing solution
Reference substance solution is prepared according to the step of embodiment 1;
Need testing solution is prepared according to the step of embodiment 1, only the Bi roots of grass-1 in embodiment 1 two is replaced with the sample of other lot numbers listed in table 1, and for the difference of total alkaloid content in different sample lots, by Sample Dilution in the range of linearity 10-10000ng/mL of alkaloid, as need testing solution, to be measured.
Two, methodological study
Methodology part contains selectivity to method, linearity and range, lowest detectable limit and minimum quantitative limit, precision, stability, repeatability, the investigation of average recovery.
1 selectivity
Get blank solvent, reference substance solution and need testing solution successively, according to the liquid chromatography in embodiment 1 and Mass Spectrometer Method condition sample introduction mensuration respectively, record gained chromatogram, contrasts each chromatogram, analyzes between each composition whether there is interference.
The selectivity of method measures through API4000Qtrap liquid chromatography mass spectrometric combined system, and the composition that coexists in result display matrix does not all disturb 5 kinds of alkaloidal mensuration, and 5 kinds of alkaloids do not interfere with each other each other, and the selectivity of method of proof is good.Gained chromatogram is shown in Fig. 2.
2 linearly investigate
Do linear regression with the ratio of often kind of alkaloid and interior mark peak area to its concentration respectively, weight factor is 1/X
2, obtain corresponding unary linear regression equation, gained unary linear regression equation is as shown in table 4:
After measured, the linear coefficient R of 5 kinds of alkaloid component regression equations
2be greater than 0.995, the range of linearity of independent variable concentration is 10-10000ng/mL.
3 lowest detectable limit and minimum quantitative limit
By gained reference substance solution according to the liquid chromatography in embodiment 1 and Mass Spectrometer Method condition respectively sample introduction measure the lowest detectable limit of each reference substance and minimum quantitative limit.
Sample introduction concentration when being about 10 times (10 times of signal to noise ratio (S/N ratio)) of baseline noise with peak response value is for quantitative limit, and sample introduction concentration when being about 3 times (3 times of signal to noise ratio (S/N ratio)) of baseline noise with peak response value is for detectability.
Acquired results is: lowest detectable limit and the minimum quantitative limit of the bright peaceful alkali of pipering, the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, pellitorine and piperanine are respectively 0.02-0.06ng/mL, 0.03-0.10ng/mL, 0.02-0.05ng/mL, 0.03-0.10ng/mL and 0.02-0.06ng/mL.
In 4 days, day to day precision
Reference substance solution and need testing solution is prepared according to the step of embodiment 1, only replace with basic, normal, high three concentration levels by mixing reference substance solution in a b, also namely 20,500,8000ng/mL, product solution in contrast, again according to the liquid chromatography in embodiment 1 and Mass Spectrometer Method condition, measuring with continuous three days sample introductions on the same day respectively, be recorded as in a few days, day to day precision.
Acquired results is: the withinday precision of the bright peaceful alkali of pipering, the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, pellitorine and piperanine is all lower than 9.30%, and day to day precision is all lower than 9.55%.
5 stability
The Bi roots of grass-1 in embodiment 1 two is replaced with the Bi roots of grass total alkaloids sample of listed three lot numbers in table 1, and precision takes in right amount, dilute 10 times to suitable concentration, as need testing solution, after obtained, namely lucifuge is placed at ambient temperature, respectively at the 0th, 2,5,12,24 hour sampling and measuring, record chromatogram.
Different time under lucifuge condition, the RSD value recording 5 kinds of compounds contents in sample is all less than 2.53%, show the bright peaceful alkali of pipering, the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, pellitorine and piperanine at ambient temperature lucifuge to place in 24 hours stable.
6 repeatability
Precision takes same batch 6 parts Bi roots of grass total alkaloids samples, prepares need testing solution respectively and measures wherein 5 kinds of alkaloidal content, investigate result as repeatability by the method for embodiment 1.
Acquired results is, the RSD value of 5 kinds of component contents is lower than 2.58%.
7 average recoveries
Precision takes the known Bi roots of grass total alkaloids-1 of content totally 9 parts respectively, accurately weighed, after preparing need testing solution by the method for embodiment 1, precision measures need testing solution 0.1mL, be placed in 100mL volumetric flask respectively, add basic, normal, high three kinds of concentration levels (20,500 respectively, 8000ng/mL) 5 kinds alkaloidal mixing reference substance solution are appropriate, then with methanol dilution to volume.Each concentration level 3 parts, then sample detection, calculates 5 kinds of alkaloidal average recoveries.
Acquired results is, 5 kinds of alkaloidal recovery are all between 90.0-103.5%.
Said method investigates detailed results in table 4.
Table 4HPLC-ESI-MS/MS method detects the methodological study result of 5 kinds of alkaloids
As shown in Table 4, the method has good repeatability and precision.
Three, liquid phase triple level Four bar mass spectrum of connecting is adopted to carry out the mensuration (all using teldane as internal standard compound) of alkaloid
1) according to the step of embodiment 1, only the Bi roots of grass-1 is replaced with the sample of table 1 other lot numbers listed, thus 5 kinds of alkaloids contained in each batch sample are separated, wherein:
Retention time is 3.78 minutes, Q
1be 258.9 and Q
3be 201.1 eluting peak be pipering;
Retention time is 3.73 minutes, Q
1be 273.9 and Q
3be 201.1 eluting peak be the bright peaceful alkali of the Bi roots of grass;
Retention time is 3.71 minutes, Q
1be 276.1 and Q
3be 134.9 eluting peak be the bright peaceful alkali of the dihydro Bi roots of grass;
Retention time is 3.76 minutes, Q
1be 288.0 and Q
3be 135.1 eluting peak be piperanine;
Retention time is 3.93 minutes, Q
1be 224.0 and Q
3be 80.9 eluting peak be pellitorine;
Record the peak area that in the sample of each batch, often kind of alkaloid is corresponding simultaneously, respectively peak area corresponding for often kind of alkaloid is substituted into the unary linear regression equation in table 4 again, thus obtain each alkaloidal concentration in each batch sample, also be alkaloidal content in each batch sample, as shown in table 5.
The measurement result of 5 kinds of alkaloids in table 5 different batches sample
As shown in Table 5,5 kinds of alkaloids all can be detected in the Bi roots of grass and pepper of listed various different batches.
In Bi roots of grass medicinal material, content of piperine is lower than pepper (P<0.05), but the content of the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine is all higher than pepper (P<0.05).
In addition, because Bi roots of grass total alkaloids sample is the long pepper extract of enriched biological alkali components, therefore contained 5 kinds of alkaloidal content are all far above the Bi roots of grass and pepper medicinal material sample.
As from the foregoing, the present invention establishes the content of pipering, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine, pellitorine 5 kinds of alkaloids compositions in a kind of method of UFLC-ESI-MS/MS fast and accurately Simultaneously test Bi roots of grass and pepper.It is accurate, reliable that methodological study result shows to utilize the data measured by the method.Sample determination result shows, in Bi roots of grass medicinal material, content of piperine is lower than pepper, but the content of the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine is all higher than pepper.The method can be used for the total quality control of the Bi roots of grass and pepper.
Claims (6)
1. the method for isolating alkaloids from the Bi roots of grass and/or pepper, comprises the steps:
Testing sample is carried out liquid chromatography-tandem mass spectrometry detection, according to eluting peak retention time, Q
1and Q
3each eluting peak of character separation, namely obtains each alkaloid;
Described Q
1for molecular ion peak [M+H]
+quality, Q
3for the quality of secondary fragment daughter ion;
Described testing sample is the Bi roots of grass and/or pepper;
Simultaneously test five kinds of alkaloids are pipering, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine and pellitorine;
The condition of described liquid chromatographic detection is as follows: chromatographic column used is PhenomenexGeminiC18 reverse-phase chromatographic column;
In the testing conditions of described liquid chromatography, the length of described chromatographic column is 50 millimeters, and internal diameter is 2.0 millimeters, and the particle diameter of C18 is 5 μm;
Sample size is 10 μ L;
Column temperature is room temperature;
Eluent is made up of mobile phase A and Mobile phase B; Wherein, mobile phase A to be mass percentage concentration be 0.1% aqueous formic acid; Mobile phase B is the mixed liquor be made up of formic acid and acetonitrile, and the mass percentage concentration of formic acid is 0.1%;
Type of elution is gradient elution, and elution speed is 0.55mL/min;
Described gradient elution mode is as follows:
Within 0th minute, rise to the 0.49th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98:2;
Within 0.50th minute, rise to the 3.00th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98-2:2-98;
Within 3.10th minute, rise to the 3.99th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 2:98;
Within 4.00th minute, rise to the 4.00th minute end, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98-2:2-98;
Within 4.01st minute, rise to 5.49 minutes ends, in eluent, the volume ratio of mobile phase A and Mobile phase B is 98:2;
Within 5.50th minute, rise, stop wash-out;
Described according to eluting peak retention time, Q
1and Q
3the method of each eluting peak of character separation is as follows:
Retention time is 3.78 minutes, Q
1be 285.9 and Q
3be 201.1 eluting peak be pipering;
Retention time is 3.73 minutes, Q
1be 273.9 and Q
3be 201.1 eluting peak be the bright peaceful alkali of the Bi roots of grass;
Retention time is 3.71 minutes, Q
1be 276.1 and Q
3be 134.9 eluting peak be the bright peaceful alkali of the dihydro Bi roots of grass;
Retention time is 3.76 minutes, Q
1be 288.0 and Q
3be 135.1 eluting peak be piperanine;
Retention time is 3.93 minutes, Q
1be 224.0 and Q
3be 80.9 eluting peak be pellitorine;
Described pepper is black pepper or white pepper;
In described Mass Spectrometer Method condition, the ionization voltage in electron spray ionisation source is+5000V, and ion source temperature is 500 DEG C-600 DEG C;
The pressure of atomization gas is 40-60psi;
The pressure of auxiliary heating gas is 40-60psi;
Gas curtain gas CUR pressure is 10-30psi;
The solution bunch voltage that often kind of alkaloid is corresponding, impact energy, collision cell exit potential are as follows:
Pipering: separating bunch voltage is 71eV, and impact energy is 27eV, and collision cell exit potential is 10V;
The bright peaceful alkali of the Bi roots of grass: separating bunch voltage is 56eV, and impact energy is 25eV, and collision cell exit potential is 10V;
The bright peaceful alkali of the dihydro Bi roots of grass: separating bunch voltage is 66eV, and impact energy is 29eV, and collision cell exit potential is 10V;
Pellitorine: separating bunch voltage is 96eV, and impact energy is 31eV, and collision cell exit potential is 4V;
Piperanine: separating bunch voltage is 111eV, and impact energy is 39eV, and collision cell exit potential is 10V.
2. method according to claim 1, is characterized in that: in described Mass Spectrometer Method condition, ion source temperature is 550 DEG C;
The pressure of atomization gas is 50psi;
The pressure of auxiliary heating gas is 50psi;
Gas curtain gas CUR pressure is 20psi.
3., according to the arbitrary described method of claim 1-2, it is characterized in that: described method also comprises the steps:
Before described liquid chromatography-tandem mass spectrometry detecting step, after testing sample and methyl alcohol are mixed with the ratio of 100mg:100mL, after filtering with microporous membrane, get filtrate, add 10 times wherein to the mixed liquor a of described filtrate volume, after vortex mixing, then add 19 times of mixed liquor vortexs be made up of isopyknic first alcohol and water to vortex gained liquor capacity wherein and mix;
Wherein, the aperture of described miillpore filter is 0.45 μm;
Described mixed liquor a is the solution of teldane;
In described mixed liquor a, solvent is methyl alcohol and acetonitrile; The concentration of described teldane is 50ng/mL;
Described mixed liquor a is obtained by following steps: teldane dissolved with methyl alcohol, and being mixed with concentration is after the methanol solution of the teldane of 1.00mg/mL, then to add isopyknic methyl alcohol and dilution in acetonitrile to the concentration of teldane be 50ng/mL, and obtains;
In described vortex mixing step, the time of vortex is 10s.
4. measure a method for alkaloid in the Bi roots of grass and/or pepper, comprise the steps:
1) potpourri of the described alkaloidal reference substance of concentration known is separated according to described method arbitrary in claim 1-3, and the peak area that record often kind of alkaloid is corresponding; With often kind of alkaloidal concentration value for independent variable, with the peak area of its correspondence for dependent variable, obtain unary linear regression equation;
2) testing sample is separated according to described method arbitrary in claim 1-3, and the peak area that record often kind of alkaloid is corresponding;
3) by the middle unary linear regression equation of peak area substitution step (1) corresponding for often kind of alkaloid, each alkaloidal concentration in described testing sample is obtained;
Described testing sample is the Bi roots of grass and/or pepper;
Simultaneously test five kinds of alkaloids are pipering, the bright peaceful alkali of the Bi roots of grass, the bright peaceful alkali of the dihydro Bi roots of grass, piperanine and pellitorine.
5. method according to claim 4, is characterized in that: described pepper is black pepper or white pepper;
The unary linear regression equation that various described alkaloid is corresponding is as follows:
Pipering: y=0.00353x-0.00046, independent variable concentration range is 10-10000ng/mL;
The bright peaceful alkali of the Bi roots of grass: y=0.00263x+0.00157, independent variable concentration range is 10-10000ng/mL;
The bright peaceful alkali of the dihydro Bi roots of grass: y=0.00362x+0.00670, independent variable concentration range is 10-10000ng/mL;
Piperanine: y=0.00327x-0.00184, independent variable concentration range is 10-10000ng/mL;
Pellitorine: y=0.00067x+0.00326, independent variable concentration range is 10-10000ng/mL.
6. the method according to claim 4 or 5, is characterized in that: described method also comprises the steps:
Before described liquid chromatography-tandem mass spectrometry detecting step, after testing sample and methyl alcohol are mixed with the ratio of 100mg:100mL, after filtering with microporous membrane, get filtrate, add 10 times wherein to the mixed liquor a of described filtrate volume, after vortex mixing, then add 19 times of mixed liquor vortexs be made up of isopyknic first alcohol and water to vortex gained liquor capacity wherein and mix;
Wherein, the aperture of described miillpore filter is 0.45 μm;
Described mixed liquor a is the solution of teldane;
In described mixed liquor a, solvent is methyl alcohol and acetonitrile; The concentration of described teldane is 50ng/mL;
Described mixed liquor a is obtained by following steps: teldane dissolved with methyl alcohol, and being mixed with concentration is after the methanol solution of the teldane of 1.00mg/mL, then to add isopyknic methyl alcohol and dilution in acetonitrile to the concentration of teldane be 50ng/mL, and obtains;
In described vortex mixing step, the time of vortex is 10s.
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| Simultaneous UFLC–ESI–MS/MS determination of piperine and piperlonguminine in rat plasma after oral administration of alkaloids from Piper longum L.: Application to pharmacokinetic studies in rats;Junhui Liu等;《Journal of Chromatography B》;20110824;第879卷;2885-2890 * |
| UFLC-ESI-MS/MS 法测定荜茇总生物碱中3种成分在大鼠组织中的分布;刘浩龙等;《第十一届全国青年药学工作者最新科研成果交流会论文集》;20120820;832-842 * |
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