CN105738523B - The method of epicatechin and table catechu mercapto for thing content in LC-MS detection blood plasma - Google Patents
The method of epicatechin and table catechu mercapto for thing content in LC-MS detection blood plasma Download PDFInfo
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Abstract
Epicatechin and table catechu mercapto in blood plasma are detected the invention discloses a kind of Liquid Chromatography/Mass Spectrometry, for the method for thing content, to include the following steps：(1) plasma containing drug sample preparation；(2) preparation of working curve sample；(3) LC determination conditions；(4) MS/MS conditions：(5) inner mark method ration is used, using serial epicatechin and epicatechin mercapto for thing blank plasma drug concentration as abscissa, epicatechin and epicatechin mercapto are ordinate for the peak area ratio of thing and internal standard compound Propranolol, regressing calculation, weight coefficient 1/x are carried out with weighted least-squares method2, the linear regression equation tried to achieve, is working curve, using working curve, the epicatechin in plasma containing drug sample and epicatechin mercapto is calculated for the drug concentration of thing.Method of the invention is quick, exclusive, accurate, high sensitivity.
Description
Technical field
The invention belongs to medical detection technique field, is related to epicatechin and table catechu mercapto in a kind of detection blood plasma and contains for thing
The method of amount.
Background technology
Condensed tannin has many medicines such as anti-oxidant, resisting pathogenic microbes, anti-inflammatory, blood pressure lowering, Adjust-blood lipid, hypoglycemic
Reason activity, in the past 10 years research find that condensed tannin has good application prospect in antitumor, AntiHIV1 RT activity, antirheumatic etc., gold
Wei Maining made of buckwheat condensed tannin is used as antineoplastic in clinic, and oligomerization condensed tannin is that anti-HI V-1 are whole in root of three-nerved spicebush stem
The main active of synthase, and the main matter basis of its anti-inflammatory, antirheumatic activity.The condensation of condensed tannin active ingredient is single
Peaceful class compound, it is not easy to be detected in blood plasma, there is no a kind of accurate accurate method at present measure the contracting in blood plasma and
The content of tannins compound.
The content of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide in a kind of LC-MS detection blood plasma epicatechin and
Method of the table catechu mercapto for thing content.
Technical scheme is summarized as follows：
Epicatechin and table catechu mercapto are for the method for thing content, including following step in a kind of Liquid Chromatography/Mass Spectrometry detection blood plasma
Suddenly：
(1) plasma containing drug sample preparation
Take and take the 50 μ L of animal blood plasma containing after condensed tannin medicine, add 10 μ L methanol, the 20 general naphthalenes of μ L 100ng/mL
Luo Er solution, 50 μ L 50mg/mL mercamine hydrochloride solution, after vortex mixes, 60 DEG C of water-bath 20min, add 50 μ L methanol,
After vortex mixes, centrifugation, takes 50 μ L of supernatant to add the methanol aqueous solution that 50 μ L volumetric concentrations are 50%, is measured for LC/MS/MS；
The solvent of the Propranolol solution is the acetonitrile-methanol solution that volumetric concentration is 50%；The mercamine hydrochloride solution it is molten
Agent is that concentrated hydrochloric acid and methanol are 1 by volume：9 are formulated.
(2) preparation of working curve sample
1. the standard items storing solution mother liquor of epicatechin and epicatechin mercapto for thing is produced in matching somebody with somebody for standard serial solution, first is used
Alcohol is configured to epicatechin and epicatechin mercapto and dilutes, obtain respectively for the storing solution that the concentration of thing is 1mg/mL, again with methanol
For the concentration of thing it is 50,100,250,500,1000,2500 to catechin and epicatechin mercapto, 5000 and 10000ng/mL；
2. take 8 parts of 50 μ L of blank plasma, be separately added into step 1. with each epicatechin and epicatechin mercapto for thing
10 μ L of methanol solution, the concentration being configured to equivalent to epicatechin and epicatechin mercapto for thing in blood plasma is 10,20,50,
100th, 200,500, the sample of 1000 and 2000ng/mL, then it is separately added into 20 μ L 100ng/mL Propranolols solution, 50 μ
L50mg/mL mercamine hydrochloride solution, after vortex mixes, 60 DEG C of water-bath 20min, add 50 μ L methanol, after vortex mixes, from
The heart, takes 50 μ L of supernatant to add the methanol aqueous solution that 50 μ L volumetric concentrations are 50%, is measured for LC/MS/MS；The Propranolol is molten
The solvent of liquid is the acetonitrile-methanol solution that volumetric concentration is 50%；The solvent of the mercamine hydrochloride solution is concentrated hydrochloric acid and first
Alcohol is 1 by volume：9 are formulated.
(3) LC determination conditions
Liquid-phase chromatographic column：Waters BEH Shield RP18 columns, 2.1mm × 100mm, 1.7um
Mobile phase：A：The aqueous formic acid of volumetric concentration 0.1%, B：The formic acid acetonitrile solution of volumetric concentration 0.1%, gradient
It is as follows：
0-1 minutes, mobile phase A was from 95% to 90%, and Mobile phase B is from 5% to 10%；
1-4.8 minutes, mobile phase A was from 90% to 84%, and Mobile phase B is from 10% to 16%；
4.8-6.3 minutes, mobile phase A was from 84% to 60%, and Mobile phase B is from 16% to 40%；
6.3-6.5 minutes, mobile phase A was from 60% to 5%, and Mobile phase B is from 40% to 95%；
6.5-7.5 minutes, mobile phase A kept 5%, and Mobile phase B keeps 95%
7.5-7.51 minutes, mobile phase A was from 5% to 95%, and Mobile phase B is from 95% to 5%；
7.51-10.0 minutes, mobile phase A kept 95%, and Mobile phase B keeps 5%.
Flow velocity：0.3mL/min；
Column temperature：35℃；
Sample size：5μL.
(4) MS/MS conditions：
Ion gun：Electric spray ion source；
Capillary voltage：-3.2KV；
Ion source temperature：120℃；Desolvation temperature：350℃；
Desolvention gas velocity：800L/Hr；
Taper hole gas velocity：50L/Hr；
Detection mode：Cation；
Scan mode：Multiple reaction monitors (MRM)；
Quota ion pair：Epicatechin：m/z 291.15→139；Epicatechin mercapto is for thing：m/z 366.25→
289.15；Internal standard Propranolol：m/z260.15→116.19
(5) use inner mark method ration, with serial epicatechin and epicatechin mercapto for thing blank plasma drug concentration
For abscissa, epicatechin and epicatechin mercapto are ordinate for the peak area ratio of thing and internal standard compound Propranolol, with weighting
Least square method carries out regressing calculation, weight coefficient 1/x2, the linear regression equation tried to achieve, is working curve, uses work
Make curve, the epicatechin in plasma containing drug sample and epicatechin mercapto are calculated for the drug concentration of thing.
Advantage：
Then the mercapto that the present invention obtains main component epicatechin by the method for thiolysis measures table in plasma sample for thing
Catechin and epicatechin mercapto have quick, exclusive, accurate, clever for the analysis method of the liquid chromatograph mass spectrography of thing concentration
The characteristics of sensitivity is high.
Brief description of the drawings
Fig. 1 is specific chromatogram.
Fig. 2 is quantitative limit 10ng/mL chromatograms.
Fig. 3 is standard curve 200ng/mL chromatograms.
Fig. 4 be beasle dog take orally epicatechin after Wei Mai Ning Capsule, epicatechin mercapto for thing 2 when small after plasma sample color
Spectrogram.
It is bent for thing blood concentration-time that Fig. 5 a for beasle dog take orally epicatechin and epicatechin mercapto after Wei Mai Ning Capsule
Line.
(epicatechin and epicatechin mercapto are for table in thing for total epicatechin after Fig. 5 b take orally Wei Mai Ning Capsule for beasle dog
The summation of theine) pharmaceutical concentration-time curve.
Embodiment
The present invention is further elaborated with reference to specific embodiment and with reference to attached drawing, but does not limit the present invention.
1 detection method of embodiment determines
1.1 laboratory apparatus and reagent
Instrument：
Liquid chromatograph is Waters ACQUITY UPLC (Waters companies), equipped with Binary Solvent
Manager, Column, PDA detector；Chromatographic column：Waters BEH Shield RP18 (2.1 × 100mm, 1.7mm)；
Mass spectrograph is Quattro Premier XE (Waters), equipped with electron spray ionisation ion gun (ESI) and atmospheric pressure
Chemical ionization source (APCI), Masslynx V4.1 work stations, Ultra Sonic Cleaner USK Type ultrasonic cleanings
Device；
FA604A electronic balances (Shanghai Ke Tian Electron equipment Co., Ltd)；
Vortex mixed instrument XW-80A (its woods Bell's instrument manufacturing Co., Ltd of Haimen City)
TGL-16G high speed tabletop centrifuges (Anting Scientific Instrument Factory, Shanghai)
Liquid-transfering gun (BIOHT PROLINE) 20-200 μ l；10-1000μl
Reagent：
Epicatechin, is purchased from Shanghai Yuan Ye bio tech ltd, purity >=98%, lot number：YA0402SC13；
Epicatechin mercapto is for thing, and by Chinese medicine, company provides, former purity >=98%, and present HPLC purity is 93%；
Propranolol Hydrochloride：U.S.'s Sigma Aldriches, purity >=99%, lot number：16524AH；
Wei Mai Ning Capsule, by Chinese medicine, company provides, lot number 130401；
Mercamine hydrochloride：U.S.'s Sigma Aldriches, purity >=98%, lot number MKBQ8406V；
Methanol, acetonitrile and formic acid are chromatographically pure, and other reagents are pure to analyze；
Watson distilled water；
Preparation of reagents：
The preparation precision of standard items storing solution mother liquor weighs epicatechin and epicatechin mercapto for each 2mg of thing, with 1mL methanol
Dissolving is made into 2mg/mL standard items storing solution mother liquors.
The preparation precision of Wei Maining solution weighs Wei Maining extract 100mg, puts in 10mL volumetric flasks, adds methanol to dissolve,
And scale is diluted to, and the Wei Maining solution of 10mg/mL is configured to, it is now with the current.
Standard serial solution it is appropriate with standard items storing solution mother liquor is produced, with methanol dilution, obtain catechin and table
Theine mercapto is 50,100,250,500,1000,2500 for the concentration of thing, 5000 and 10000ng/mL.
Interior target prepares precision and weighs 10mg Propranolols (propranolol) standard items, puts in 10mL volumetric flasks, adds second
Nitrile-methanol (50:50, v/v) dissolve, and be diluted to scale, be configured to the storing solution of 1.0mg/mL, take appropriate Propranolol deposit
Liquid, with acetonitrile-methanol (50:50, v/v) it is 100ng/mL to be diluted to concentration.
The preparation precision of mercamine hydrochloride reagent weighs the mercamine hydrochloride of 100mg, adds 200mL concentrated hydrochloric acids and 1.8mL
Methanol fully dissolves, and is made into 50mg/mL solution, now with the current.
Experimental animal：Beagle dogs, weight 9-10kg, herds positive experimental animal purchased from Beijing gold and cultivates Co., Ltd.
Choose health Beagle dogs 8, weight (9 ± 1.0) kg, 1 day more than fasting 12h before experiment, can't help water, be administered after 4h uniformly into
Food.
1.2 plasma containing drug sample preparations (Plasma sample analysis method)
Take and take the 50 μ L of Dog Plasma containing after condensed tannin medicine (the present embodiment is by taking Wei Mai Ning Capsule as an example)
Put in 1.5mL plastics EP pipes, be separately added into 10 μ L methanol, 20 μ L inner mark solutions, 50 μ L mercamine hydrochloride solution, vortex mixes
Afterwards, 60 DEG C of water-bath 20min, after question response, add 50 μ L methanol, after vortex mixes, centrifugation 10min (15000rpm, 4
DEG C), take 50 μ L of supernatant and with 50 μ L methanol-waters (50:50, v/v) dilute after 5 μ L of sample introduction carry out LC/MS/MS by following condition into
Row analysis；
1.2.1 liquid-phase condition
Chromatographic column：Waters BEH Shield RP18 columns (2.1 × 100mm, 1.7 μm)；Mobile phase：A：Water (contains 0.1%
Formic acid, v/v), B：Acetonitrile (containing 0.1% formic acid, v/v) gradient elution, elution requirement are shown in Table 1；Flow velocity：0.3mL/min；Column temperature：
35℃；Sample size：5μL.
1 liquid phase gradient elution program of table
1.2.2 Mass Spectrometry Conditions
Ion gun：Electric spray ion source；Capillary voltage：-3.2K V；Ion source temperature：120℃；Desolvation temperature：
350℃；Desolvention gas velocity：800L/Hr；Taper hole gas velocity：50L/Hr；Positive ion mode detects；Scan mode is multiple anti-
It should monitor (MRM)；Epicatechin, epicatechin mercapto are respectively for the taper hole voltage of thing and internal standard Propranolol (Cone)：22V、
15V and 35V；Collision energy (Collision) is respectively：16V, 10V and 18V；Ionic reaction for quantitative analysis is respectively
M/z 291.15 → 139 (epicatechin), m/z 366.25 → 289.15 (epicatechin mercapto is for thing) and m/z 260.15 →
116.19 (internal standards：Propranolol).
2 methodology validation of embodiment is tested
The specificity of method, linear and related coefficient
The specificity of method takes the 50 μ L of blank plasma of 6 beasle dogs respectively, by being grasped under " Plasma sample analysis method " item
Make, 5 μ L of sample introduction, obtain specific chromatogram 1；Certain density epicatechin standard solution, epicatechin mercapto is molten for thing standard
Liquid and internal standard Propranolol solution are added in blank plasma, are operated according to same method, are obtained quantitative limit 10ng/mL chromatograms, see Fig. 2, Fig. 3
(for standard curve 200ng/mL chromatograms)；The plasma sample for taking tested dog to be collected after being administered, operates according to same method, obtains corresponding chromatography
Fig. 4 (see Fig. 4).The result shows that the endogenous material in blank plasma does not disturb epicatechin, epicatechin mercapto for thing and internal standard
The measure of Propranolol.
The sample preparation of working curve
1. the standard items storing solution mother liquor of epicatechin and epicatechin mercapto for thing is produced in matching somebody with somebody for standard serial solution, first is used
Alcohol is configured to catechin and epicatechin mercapto and dilutes, obtain respectively for the storing solution that the concentration of thing is 1mg/mL, again with methanol
Catechin and epicatechin mercapto are 50,100,250,500,1000,2500 for the concentration of thing, 5000 and 10000ng/mL；
2. take 8 parts of 50 μ L of blank plasma, be separately added into step 1. with each epicatechin and epicatechin mercapto for thing
10 μ L of methanol solution, the concentration being configured to equivalent to epicatechin and epicatechin mercapto for thing in blood plasma is 10,20,50,
100th, 200,500, the sample of 1000 and 2000ng/mL, then it is separately added into 20 μ L 100ng/mL Propranolols solution, 50 μ
L50mg/mL mercamine hydrochloride solution, after vortex mixes, 60 DEG C of water-bath 20min, add 50 μ L methanol, after vortex mixes,
15000rpm, centrifuges 10min under the conditions of 4 DEG C, takes 50 μ L of supernatant to add the methanol aqueous solution that 50 μ L volumetric concentrations are 50%, supplies
LC/MS/MS is measured；
The solvent of the Propranolol solution is the acetonitrile-methanol solution that volumetric concentration is 50%；The mercamine hydrochloride
The solvent of solution is formulated for 200uL concentrated hydrochloric acids and 1.8mL methanol；
5 μ L sample introductions are taken, carry out LC/MS/MS analyses, record chromatogram；With serial epicatechin and epicatechin mercapto for thing
It is the peak area of abscissa, epicatechin and epicatechin mercapto for thing and internal standard compound Propranolol in the drug concentration of blank plasma
Ratio is ordinate, with weighting (W=1/x2) least square method progress regressing calculation, the linear regression equation tried to achieve, is work
Make curve.
Epicatechin exemplary operation curve is y=0.00265x-0.00621, range of linearity 10- in plasma sample
2000ng/mL.Coefficient R 2=0.990.Epicatechin mercapto is y=0.00179x for thing exemplary operation curve in plasma sample
+ 0.00341, range of linearity 10-2000ng/mL.Coefficient R 2=0.997.Meet quantitative testing requirements.
The range of linearity according to standard curve, in blood plasma epicatechin and epicatechin mercapto for the range of linearity of thing be 10~
2000ng/mL, lower limit of quantitation 10ng/mL.
The accuracy of method, precision
Veracity and precision is tested, and by being operated under " preparation of working curve " item, prepares epicatechin and epicatechin
Mercapto is for quality control (QC) sample of basic, normal, high three concentration of thing, and each concentration carries out 6 sample analyses, METHOD FOR CONTINUOUS DETERMINATION three days,
According to the working curve on the same day, calculate QC samples measures concentration, and the accuracy and precision of this law are calculated according to QC sample results
Degree, the results are shown in Table 2 and table 3.
Epicatechin LC/MS/MS assay method veracity and precision experimental results in 2 plasma sample of table
Epicatechin mercapto is for thing LC/MS/MS assay method veracity and precision experimental results in 3 plasma sample of table
From table 2, table 3 is it can be seen that epicatechin and epicatechin mercapto are for thing concentration in LC/MS/MS measure plasma samples
In a few days relative standard deviation is respectively between 3.62-6.95% and 3.43-10.32% for method, and relative standard deviation is distinguished in the daytime
Between 12.13-14.82% and 2.53-3.64%, meet the requirement of analysis in beasle dog body.The accuracy of illustration method and
Precision is preferable, fully verifies the operability of institute's method for building up.
The method rate of recovery and matrix effect
The method rate of recovery and matrix effect experiment, take 50 μ L of blank plasma, by being operated under " preparation of working curve " item, make
The QC samples of standby basic, normal, high three concentration, each 3 sample of concentration, carries out LC-MS/MS analyses, obtains peak area A.Separately take sky
50 μ L of white blood plasma, add 50 μ L mercamine hydrochloride solution, are vortexed after mixing, 60 DEG C of water-bath 20min, after question response, centrifugation
10min (15000rpm, 4 DEG C), take 50 μ L of supernatant add epicatechin and the thio thing of epicatechin 5 μ L of mixed standard solution,
10 μ L of inner mark solution and 65 μ L methanol-waters (50:50, v/v) respective concentration, is configured to, vortex mixing, takes 5 μ L to carry out LC/MS/
MS is analyzed, and obtains respective peaks area B.50 μ L ultra-pure waters are separately taken again, add 10 μ L standard solution, 20 μ L internal standards and 50 μ L hydrochloric acid mercaptos
Ethylamine solution, after vortex mixes, prepares basic, normal, high three concentration without plasma matrix sample, take 5 μ L to carry out LC/MS/MS and divide
Analysis, obtains corresponding peak area C.With peak area A and peak face B be compared to evaluation epicatechin, epicatechin mercapto for thing with it is interior
The rate of recovery is marked, calculation formula is R=A/B × 100%；Evaluation epicatechin, table are compared to peak area B and peak face C
Theine mercapto is M=B/C × 100% for thing and internal standard matrix effect, calculation formula, the results are shown in Table 4 and 5.
4 epicatechin of table, epicatechin mercapto are for thing and internal standard extraction recovery
5 epicatechin of table, epicatechin mercapto are for thing and internal standard matrix effect
From table 4 to find out, the extraction recovery of basic, normal, high three concentration of epicatechin is respectively 97.5 ± 6.6%,
94.4 ± 8.3%, 89.5 ± 6.7%, the extraction recovery of basic, normal, high three concentration of the thio thing of epicatechin is respectively 83.8
± 7.3%, 82.5 ± 4.4%, 88.5 ± 2.4%, all meet the requirement analyzed in vivo.Epicatechin, epicatechin are known by table 5
Mercapto is all higher than 87.4% for thing and internal standard matrix effect ratio, shows that selected mass spectrum and chromatographic condition efficiently avoid matrix
Effect, meets the requirement analyzed in vivo, fully verifies the operability of institute's method for building up.
The measure of 3 actual sample of embodiment
The collection of plasma sample
Health Beagle dogs 8, weight (9 ± 1.0) kg, 1 day more than fasting 12h before experiment are chosen, can't help water, 4h is administered
Unify feed afterwards.Binary cycle intersects administration, cleans two weeks phases.8 (200mg/) are administered orally in Wei Mai Ning Capsule.Taken out before experiment
Blank blood is taken, respectively at 0,10,20,30min, 1,2,4,6,8,12,24,36,48,60,72 and 84h totally 16 times after administration
Point takes foreleg vein blood 1mL, is placed in the centrifuge tube containing heparin, is placed in ice bath, is centrifuged at 4 DEG C in 3000rpm in 0.5hr
10min collects upper plasma, and -80 DEG C of stored frozens are to be measured.
The measure of actual plasma sample
The plasma sample of above-mentioned gained is handled into plasma sample according to " plasma containing drug sample preparation ", according in embodiment 1
Liquid-phase condition and Mass Spectrometry Conditions carry out liquid matter analysis, each analysis batch (the same sample tested in one day) prepares a job
Curve, while the QC samples of basic, normal, high three concentration are prepared, per concentration double sample, and QC sample sizes are no less than each analysis
The 5% of sample total amount in batch.The concentration of QC samples and unknown sample is calculated according to the working curve of each analysis batch.Above-mentioned Quality Control
At most allow the concentration of two different samples in sample beyond 15% (low concentration point is 20%) of theoretical value, otherwise this batch data
Do not received.Acquired results are shown in Table 6.
6 beasle dog of table takes orally blood medicine mean concentration of the epicatechin medicine with epicatechin mercapto for thing after Wei Mai Ning Capsule
(ng/mL) (n=8)
Beasle dog vivo medicine concentration and the change curve of time are drawn after administration
According to the data of table 6, epicatechin is drawn out respectively, epicatechin mercapto compares after being administered for thing and total epicatechin
Lattice dog vivo medicine concentration and the change curve of time, are specifically shown in Fig. 5 a, shown in 5b, as can be seen from the figure epicatechin, table
Drug concentration change of the catechin mercapto for thing and total epicatechin in beasle dog body, therefore taken orally available for Wei Mai Ning Capsule
The research of pharmacokinetics after administration.
Above-mentioned LC-MS quantitatively detects in Dog Plasma epicatechin and epicatechin mercapto for the range of linearity of thing
It is 10~2000ng/mL, lower limit of quantitation 10ng/mL, linear relationship is good in the range of 10~2000ng/mL.Reviewing party
Method is 89.5~97.5% to the rate of recovery of epicatechin, and in a few days standard deviation is 3.62~6.95%, and standard deviation is in the daytime
12.13-14.82%；Inspection method is 82.5~88.5% for the rate of recovery of thing to epicatechin mercapto, and in a few days standard deviation is
3.43~10.32%, standard deviation is 2.53~3.64% in the daytime, meets epicatechin and epicatechin mercapto generation in beasle dog body
The requirement of thing detection of drug concentration analysis.
In conclusion the LC-MS of the present invention quantitatively detects in blood plasma epicatechin and epicatechin mercapto for thing content
Analysis method can sensitive, accurate, reliable detection blood sample drug concentration.
The above is only the citing of embodiments of the present invention, it is noted that for the ordinary skill of the art
For personnel, without departing from the technical principles of the invention, some improvement and modification can also be made, these improve and become
Type also should be regarded as protection scope of the present invention.
Claims (1)
1. in a kind of Liquid Chromatography/Mass Spectrometry detection blood plasma epicatechin and epicatechin mercapto for thing content method, it is characterized in that including
Following steps：
(1) plasma containing drug sample preparation
Take and take the 50 μ L of animal blood plasma containing after condensed tannin medicine, add 10 μ L methanol, 20 μ L 100ng/mL Propranolols
Solution, 50 μ L 50mg/mL mercamine hydrochloride solution, after vortex mixes, 60 DEG C of water-bath 20min, add 50 μ L methanol, vortex
After mixing, centrifugation, takes 50 μ L of supernatant to add the methanol aqueous solution that 50 μ L volumetric concentrations are 50%, is measured for LC/MS/MS；It is described
The solvent of Propranolol solution is the acetonitrile-methanol solution that volumetric concentration is 50%；The solvent of the mercamine hydrochloride solution is
Concentrated hydrochloric acid and methanol are 1 by volume：9 are formulated；
(2) preparation of working curve sample
1. the standard items storing solution mother liquor of epicatechin and epicatechin mercapto for thing is produced in matching somebody with somebody for standard serial solution, matched somebody with somebody with methanol
Epicatechin and epicatechin mercapto is set to dilute, obtain respectively for the storing solution that the concentration of thing is 1mg/mL, again with methanol
Theine and epicatechin mercapto are 50,100,250,500,1000,2500 for the concentration of thing, 5000 and 10000ng/mL；
2. take 8 parts of 50 μ L of blank plasma, be separately added into step 1. with each epicatechin and epicatechin mercapto for thing first
10 μ L of alcoholic solution, the concentration being configured to equivalent to epicatechin and epicatechin mercapto for thing in blood plasma is 10,20,50,100,
200th, 500, the sample of 1000 and 2000ng/mL, then it is separately added into 20 μ L 100ng/mL Propranolols solution, 50 μ L50mg/mL
Mercamine hydrochloride solution, after vortex mixes, 60 DEG C of water-bath 20min, add 50 μ L methanol, and after vortex mixes, centrifugation, takes supernatant
50 μ L add the methanol aqueous solution that 50 μ L volumetric concentrations are 50%, are measured for LC/MS/MS；The solvent of the Propranolol solution
The acetonitrile-methanol solution for being 50% for volumetric concentration；The solvent of the mercamine hydrochloride solution presses volume for concentrated hydrochloric acid and methanol
Than for 1：9 are formulated；
(3) LC determination conditions
Liquid-phase chromatographic column：Waters BEH Shield RP18 columns, 2.1mm × 100mm, 1.7um
Mobile phase：A：The aqueous formic acid of volumetric concentration 0.1%, B：The formic acid acetonitrile solution of volumetric concentration 0.1%, gradient is such as
Under：0-1 minutes, mobile phase A was from 95% to 90%, and Mobile phase B is from 5% to 10%；
1-4.8 minutes, mobile phase A was from 90% to 84%, and Mobile phase B is from 10% to 16%；
4.8-6.3 minutes, mobile phase A was from 84% to 60%, and Mobile phase B is from 16% to 40%；
6.3-6.5 minutes, mobile phase A was from 60% to 5%, and Mobile phase B is from 40% to 95%；
6.5-7.5 minutes, mobile phase A kept 5%, and Mobile phase B keeps 95%
7.5-7.51 minutes, mobile phase A was from 5% to 95%, and Mobile phase B is from 95% to 5%；
7.51-10.0 minutes, mobile phase A kept 95%, and Mobile phase B keeps 5%.
Flow velocity：0.3mL/min；
Column temperature：35℃；
Sample size：5μL；
(4) MS/MS conditions：
Ion gun：Electric spray ion source；
Capillary voltage：-3.2KV；
Ion source temperature：120℃；Desolvation temperature：350℃；
Desolvention gas velocity：800L/Hr；
Taper hole gas velocity：50L/Hr；
Detection mode：Cation；
Scan mode：Multiple reaction monitors；
Quota ion pair：Epicatechin：m/z291.15→139；Epicatechin mercapto is for thing：m/z 366.25→289.15；Internal standard
Propranolol：m/z 260.15→116.19
(5) use inner mark method ration, using serial epicatechin and epicatechin mercapto for thing blank plasma drug concentration as horizontal stroke
Coordinate, epicatechin and epicatechin mercapto are ordinate for the peak area ratio of thing and internal standard compound Propranolol, minimum with weighting
Square law carries out regressing calculation, weight coefficient 1/x2, the linear regression equation tried to achieve, is working curve, uses work bent
Line, the epicatechin in plasma containing drug sample and epicatechin mercapto are calculated for the drug concentration of thing.
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| CN105153092A (en) * | 2015-10-21 | 2015-12-16 | 中国人民解放军军事医学科学院毒物药物研究所 | Preparation method of mercaptamine substituted degraded product of condensed tannin |
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| CN105153092A (en) * | 2015-10-21 | 2015-12-16 | 中国人民解放军军事医学科学院毒物药物研究所 | Preparation method of mercaptamine substituted degraded product of condensed tannin |
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| Simultaneous determination of catechin, epicatechin and epicatechin gallate in rat plasma by LC–ESI-MS/MS for pharmacokinetic studies after oral administration of Cynomorium songaricum extract;Qiu-Hong Zhang;《Journal of Chromatography B》;20121231;第880卷;第168-171页 * |
| 儿茶素类化合物巯乙胺基取代物的制备;李姝靓 等;《国际药学研究杂志》;20151031;第42卷(第5期);第630-633页 * |
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