CN104569215A - Method for determining concentration of sclareol in blood plasma - Google Patents
Method for determining concentration of sclareol in blood plasma Download PDFInfo
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- CN104569215A CN104569215A CN201510044234.0A CN201510044234A CN104569215A CN 104569215 A CN104569215 A CN 104569215A CN 201510044234 A CN201510044234 A CN 201510044234A CN 104569215 A CN104569215 A CN 104569215A
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Abstract
The invention relates to a method for determining concentration of sclareol in blood plasma and belongs to the technical field of biological detection. Nitrazepam is taken as an internal standard, a protein precipitation method is adopted, and the concentration of sclareol in the determined blood plasma is calculated by virtue of blood plasma sample pre-treatment, chromatograph, mass spectrometry and an internal standard method. The method for determining the concentration of sclareol in the blood plasma is rapid, accurate, high in sensitivity and easy to operate, and a methodological foundation is laid for drug metabolism dynamic research of sclareol in an animal body.
Description
Technical field
The present invention relates to a kind of method measuring sclareol concentration in blood plasma, belong to technical field of biological.
Background technology
Sclareol (sclareol) has another name called sclareol, is a kind of Ladanum alkanes diterpene two tert-alcohols of separation and Extraction from fragrant purple perilla, tobacco and other Salvia labiates.The inside and outside research of body confirms, it has anti-inflammatory, antibacterial, bactericidal action, all having lethal effect in various degree for malignant cells such as human leukemia cell, human colon cancer cell, human breast cancer cells, is a kind of source new drugs compound with good DEVELOPMENT PROSPECT.Its structural formula is as follows:
Blood concentration detection method at present about sclareol has no bibliographical information, the present invention establishes the assay method of sclareol in rat plasma first, and carry out pilot study by the drug metabolism situation of the method to sclareol, for its Preclinical metabolism and pharmacokinetics study has established solid methodology basis.
Summary of the invention
The object of the invention is to overcome above-mentioned weak point, a kind of method that can detect sclareol concentration in blood plasma fast, is accurately and efficiently provided.
According to technical scheme provided by the invention, a kind of method measuring sclareol concentration in blood plasma, step is:
(1) plasma sample pre-service: get plasma sample, adds interior mark intrazepam, adds methanol extraction albumen, centrifuging and taking supernatant;
(2) chromatogram: step (1) pretreated plasma sample is carried out liquid chromatography separation: chromatographic column in chromatographic condition: Phenomenex kinetex XB-C18 post; Mobile phase: methyl alcohol: water volume ratio is 80:20, also containing 10mM ammonium acetate in mobile phase; Flow velocity: 200 ~ 300 μ L/min; Column temperature: 30 ~ 35 DEG C; Sample size: 10 μ L;
(3) mass spectrum: ion gun: electro-spray ionization ESI ion gun, mode scans: select reaction detection scanning (SRM), spray voltage: 3500 ~ 4000V, sheath gas: 20 ~ 50psi, assisted gas: 10 ~ 20arb, capillary temperature: 350 DEG C, ion source temperature: 300 DEG C; Detect ion: sclareol selective reaction detects ion [M+H]
+m/z 309.0 → m/z 230.9 CE:11eV; Interior mark intrazepam selective reaction detects ion [M+H]
+m/z 282.3 → m/z 236.2 CE:23eV;
(4) calculate: adopt internal standard method, substitute into typical curve equation with the peak area ratio of sclareol and interior mark intrazepam, calculate the blood concentration of sclareol.
Step (1) is specially: get plasma sample 100 μ L, and add mark intrazepam solution 10 μ L in 500ng/mL, then add methyl alcohol 290 μ L, the centrifugal 10 ~ 15min of vortex mixing 5 ~ 10min, 13000rpm, gets supernatant after centrifugal.
In step (2), chromatogram column length is 100mm, and internal diameter is 2.1mm, and packing material size is 2.6 μm.
Described plasma sample is the blood plasma containing sclareol.
Beneficial effect of the present invention:
(1) preprocess method is easy: adopt a step precipitation of protein, be applicable to conventional sense.
(2) specificity is strong, Phenomenex kinetex XB-C18 chromatographic column is adopted to be separated, methyl alcohol: the mixed liquor of water (10mM ammonium acetate) is as mobile phase, isocratic elution, the retention time of sclareol is about 1.4min, the retention time of interior mark intrazepam is about 1.7min, and both have good degree of separation, can complete mensuration in 3min.In addition, endogenous material does not disturb both mensuration.
(3) highly sensitive: in blood plasma, sclareol is minimum is quantitatively limited to 10ng/mL.
(4) detect fast: 3min completes the once mensuration of sample, and the time is short, be therefore applicable to the detection of large batch of biological sample.
(5) amount of samples is little: only need 100 μ L plasma samples, can determine actual concentrations.
(6) the inventive method is quick, accurate, highly sensitive, easy and simple to handle, for the determination of plasma concentration of sclareol provides foundation.The sclareol plasma standard curve linear scope of this method is 10 ~ 1000ng/mL, and in a few days, day to day precision RSD is all less than 10%.
Accompanying drawing explanation
Fig. 1 is blank rat plasma mass spectrogram.
Fig. 2 adds sclareol and interior target mass spectrogram in blank rat plasma.
Fig. 3 is that the plasma sample after rat gives sclareol adds interior target mass spectrogram.
In figure, passage b is sclareol, and retention time is 1.4min; Passage a is interior mark intrazepam, and retention time is 1.7min.
Embodiment
According to following embodiment, the present invention may be better understood, but those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment: the mensuration of sclareol concentration in rat plasma
Experiment material and instrument
Sclareol: purity >98%, lot number: MUST-14072810, is purchased from Man Site bio tech ltd, Chengdu; Interior mark: intrazepam, lot number: 9201, is purchased from national arcotic laboratory; Methyl alcohol (CNW company), lot number: HX080973; Ammonium acetate (Chemical Reagent Co., Ltd., Sinopharm Group), lot number: F20090520; Water is ultrapure water (Milli-Q water purification machine) self-control.
Liquid chromatographic system: ACCELA autosampler, ACCELA 1250pump, Thermo Fisher company of the U.S.; MS/MS system: the triple quadrupole rods tandem mass spectrometry instrument of TSQ Quantum Access type, is equipped with electro-spray ionization ESI ion gun, Thermo Fisher company of the U.S.; Data acquisition: LC QUAN
tM2.6 softwares, Thermo Fisher company of the U.S.; The miniature vortex mixed instrument of XW-80A, Instrument Factory of Shanghai Medical Univ.; Z233MK-2 supercentrifuge, German HERMLE company; BT25S type analysis balance, German Sartorius company; HGC-12A Nitrogen evaporator, Tianjin Heng Ao company.
Liquid matter condition
1, liquid phase chromatogram condition:
Chromatographic column: Phenomenex kinetex XB-C18(2.6 μm, 2.1 × 100mm); Mobile phase: methyl alcohol: water (10mM ammonium acetate)=80:20 (
v/V); Flow velocity: 200 ~ 300 μ L/min; Column temperature: 30 ~ 35 DEG C; Sample size: 10 μ L.
2, Mass Spectrometry Conditions:
Ion gun: electro-spray ionization ESI ion gun, mode scans: select reaction detection scanning (SRM), spray voltage: 3500 ~ 4000V, sheath gas: 20 ~ 50psi, assisted gas: 10 ~ 20arb, capillary temperature: 350 DEG C, ion source temperature: 300 DEG C.Detect ion: sclareol selective reaction detects ion [M+H]
+m/z 309.0 → m/z 230.9 CE:11eV; Interior mark intrazepam selective reaction detects ion [M+H]
+m/z 282.3 → m/z 236.2 CE:23eV.
Experimentation
1, sclareol and interior mark intrazepam standard solution preparation
Precision takes sclareol standard items 10.21mg, is placed in 100mL volumetric flask, adds dissolve with methanol solution and is settled to scale, obtains 100 μ g/mL sclareol storing solutions.Precision measures appropriate storing solution methyl alcohol and dilutes successively, be made into concentration be respectively 100,250,500,1000,2500,5000, the sclareol standard solution of 10000ng/mL.
Precision takes interior mark intrazepam standard items 10.02mg, is placed in 100mL volumetric flask, adds dissolve with methanol solution and be settled to scale, obtains 100 μ g/mL intrazepam storing solutions.Precision measures appropriate storing solution methanol dilution, and being configured to concentration is 500ng/mL intrazepam standard solution.
2, the Acquire and process of rat plasma sample
After rat oral gavage gives 100 mg/kg sclareols, to take a blood sample 400 μ L respectively at 5min, 10 min, 20min, 30min, 45min, 1h, 2h, 4h, 8h, 12h, 24h time point intraocular corners of the eyes before administration and after administration, in injection calparine pipe, the centrifugal 5min of 3500rpm, prepares blood plasma.
Get rat plasma 100 μ L, add mark intrazepam solution 10 μ L in 500ng/mL, then add methyl alcohol 290 μ L, the centrifugal 10 ~ 15min of vortex mixing 5 ~ 10min, 13000rpm, gets supernatant after centrifugal, gets supernatant 10 μ L and carry out LC-MS/MS analysis.
3, specificity
Get 100 μ L blank rat plasma, add 300 μ L methanol extraction albumen, the centrifugal 10 ~ 15min of vortex mixing 5 ~ 10min, 13000rpm, gets supernatant 10 μ L and carries out LC-MS/MS analysis.
Get 1.5mL EP pipe number to prop up, add sclareol standard solution 10 μ L respectively, after nitrogen dries up, add 100 μ L blank rat plasma, vortex mixing 30s, adds 10 μ L inner mark solutions and 290 μ L methanol extraction albumen, vortex mixing 5 ~ 10min, centrifugal 10 ~ the 15min of 13000rpm, gets supernatant 10 μ L and carries out LC-MS/MS analysis.
Result shows, and under the LC-MS/MS condition that this experiment adopts, cause interference without assorted peak to detection material in blood plasma, the retention time of sclareol is about 1.4min, and the retention time of interior mark intrazepam is about 1.7min, completes mensuration in 3min.Sclareol and interior mark do not interfere with each other, and peak shape is good, and baseline is steady.
If Fig. 1 is blank rat plasma mass spectrogram; Fig. 2 adds sclareol and interior target mass spectrogram in blank rat plasma.Fig. 3 is that the plasma sample after rat gives sclareol adds interior target mass spectrogram.
In figure, passage b is sclareol, [M+H]
+m/z 309.0 → m/z 230.9, retention time is 1.4min; Passage a is interior mark intrazepam, [M+H]
+m/z 282.3 → m/z 236.2, retention time is 1.7min.
4, typical curve
Get 1.5mL EP pipe number to prop up, precision adds 10 μ L sclareol series standard solution, and nitrogen dries up, and adds 100 μ L blank rat plasma, vortex mixes, be mixed with sclareol concentration be respectively 10,25,50,100,250,500, the standard plasma containing drug of 1000ng/mL.Add 10 μ L inner mark solutions and 290 μ L methanol extraction albumen, the centrifugal 10 ~ 15min of vortex mixing 5 ~ 10min, 13000rpm, gets supernatant 10 μ L and carries out LC-MS/MS analysis.Calculate the peak area As of sclareol and the ratio (=As/Ai) of interior mark peak area Ai respectively, with testing concentration c for horizontal ordinate, to be worth for ordinate, with weighting (weight coefficient: 1/x
2) least square method carries out linear regression operation and obtain regression equation=-0.0157+1.4362*c,
r=0.997.Sclareol is good in 10 ~ 1000ng/mL scope internal linear relation, is minimumly quantitatively limited to 10ng/mL.
5, accuracy and precision
Be the basic, normal, high containing sclareol plasma sample of 25,250,800 ng/mL according to the preparation of rat plasma typical curve method containing sclareol concentration, process by " process of rat plasma sample " disposal route.For three days on end, each concentration every day 5 Duplicate Samples, calculate the peak area As of sclareol and the ratio of interior mark peak area Ai, substitute in the plasma standard curve on the same day measured concentration of trying to achieve containing sclareol, calculated in a few days by measured concentration, day to day precision and relative standard deviation (RSD), the ratio of measured concentration and theoretical concentration is accuracy.Result shows, and in a few days day to day precision RSD is all less than 10%, and accuracy meets the requirements.
6, measurement result
Above-mentioned 6 gavages give sclareol concentration (after administration 2h) in the rat plasma of sclareol be respectively 327.5,326.5,298.5,387.4,306.1,316.6ng/mL.
Claims (4)
1. measure a method for sclareol concentration in blood plasma, it is characterized in that step is:
(1) plasma sample pre-service: get plasma sample, adds interior mark intrazepam, adds methanol extraction albumen, centrifuging and taking supernatant;
(2) chromatogram: step (1) pretreated plasma sample is carried out liquid chromatography separation: chromatographic column in chromatographic condition: Phenomenex kinetex XB-C18 post; Mobile phase: methyl alcohol: water volume ratio is 80:20, also containing 10mM ammonium acetate in mobile phase; Flow velocity: 200 ~ 300 μ L/min; Column temperature: 30 ~ 35 DEG C; Sample size: 10 μ L;
(3) mass spectrum: ion gun: electro-spray ionization ESI ion gun, mode scans: select reaction detection scanning (SRM), spray voltage: 3500 ~ 4000V, sheath gas: 20 ~ 50psi, assisted gas: 10 ~ 20arb, capillary temperature: 350 DEG C, ion source temperature: 300 DEG C; Detect ion: sclareol selective reaction detects ion [M+H]
+m/z 309.0 → m/z 230.9 CE:11eV; Interior mark intrazepam selective reaction detects ion [M+H]
+m/z 282.3 → m/z 236.2 CE:23eV;
(4) calculate: adopt internal standard method, substitute into typical curve equation with the peak area ratio of sclareol and interior mark intrazepam, calculate the blood concentration of sclareol.
2. measure the method for sclareol concentration in blood plasma as claimed in claim 1, it is characterized in that: step (1) is specially: get plasma sample 100 μ L, add mark intrazepam solution 10 μ L in 500ng/mL, add methyl alcohol 290 μ L again, vortex mixing 5 ~ 10min, centrifugal 10 ~ the 15min of 13000rpm, gets supernatant after centrifugal.
3. measure the method for sclareol concentration in blood plasma as claimed in claim 1, it is characterized in that: in step (2), chromatogram column length is 100mm, and internal diameter is 2.1mm, and packing material size is 2.6 μm.
4. measure the method for sclareol concentration in blood plasma as claimed in claim 2, it is characterized in that: step (1) described plasma sample is the blood plasma containing sclareol.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116559307A (en) * | 2023-01-10 | 2023-08-08 | 江南大学 | High performance liquid chromatography detection method for perillyl alcohol in bacterial strain fermentation liquor |
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| MD2823F1 (en) * | 2004-02-12 | 2005-07-30 | Institutul De Chimie Al Academiei De Stiinte A Republicii Moldova | Process for quantitatively determining the sclareol in the obtained from clary sage |
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| CN104101662A (en) * | 2014-08-01 | 2014-10-15 | 中国烟草总公司郑州烟草研究院 | Metabonomics method for testing terpenoids in fresh tobacco leaves |
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| MD2823F1 (en) * | 2004-02-12 | 2005-07-30 | Institutul De Chimie Al Academiei De Stiinte A Republicii Moldova | Process for quantitatively determining the sclareol in the obtained from clary sage |
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| CN104101662A (en) * | 2014-08-01 | 2014-10-15 | 中国烟草总公司郑州烟草研究院 | Metabonomics method for testing terpenoids in fresh tobacco leaves |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116559307A (en) * | 2023-01-10 | 2023-08-08 | 江南大学 | High performance liquid chromatography detection method for perillyl alcohol in bacterial strain fermentation liquor |
| CN116559307B (en) * | 2023-01-10 | 2024-03-15 | 江南大学 | High performance liquid chromatography detection method for perillyl alcohol in bacterial strain fermentation liquor |
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