# Patent

## Title
Detection of blood plasma schizadrin B of dissipating blood stasis botanical

## Abstract

A detection method of blood plasma schizadrin B of dissipating blood stasis botanical is disclosed. The method includes: (1) extracting schizadrin B from plasma of mammalian administered dissipating blood stasis botanical by ethyl acetate with the volume ratio 1:4, whirling 3-5 mins, centrifugating at 9600 rpm for 10 mins, drying and enriching the upper layer at 25-30° C., and redissolving with mobile phase; (2) UPLC/MS measuring: UPLC condition: chromatographic column: Acquity UPLC BEH C18, 2.1′100 mm, mobile phase A: water-acetonitrile-formic acid 95:5:0.1 v/v/v, mobile phase B: acetonitrile-formic acid 100:0.1 v/v; MS condition: electric spraying ion source (ESI), detecting with positive ion mode, scanning at the range of m/z 150-1200. The method can be used for pharmacokinetics study of schizadrin B in dissipating blood stasis botanical.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a 35 U.S.C. §371 national stage filing of International Patent Application No. PCT/CN2008/000865, filed Apr. 28, 2008, and through which priority is claimed to Chinese Patent Application No. 200710040140.1, filed Apr. 27, 2007, the disclosures of which are incorporated herein by reference in their entireties.
TECHNICAL FIELD
The invention belongs to the field of pharmacokinetics, particularly involves a detection method of blood plasma schizadrin B of strengthening body resistance and dissipating blood stasis botanical (vegetable).
BACKGROUND ART
Strengthening body resistance and dissipating blood stasis botanical are composed of compound prescriptions including salvia miltiorrhiza, peach kernel, Schisandra chinens etc., which have the effect of curing liver, lung and kidney fibrosis; however, due to lack of pharmacokinetics research has been carried out on the strengthening body resistance and dissipating blood stasis botanical, so it is not clear about the effective ingredients in vivo, and it is difficult to provide a basis for quality control and guiding clinical rational administration, thus hinder those drugs from entering the international market.
So far, no pharmacokinetics research report on the strengthening body resistance and dissipating blood stasis botanical has been found, and there has not any detection method of schizadrin B of the compound prescription in biological samples (including blood plasma).
CONTENTS OF THE INVENTION
The technical matters aim to be resolved by this invention is to provide a detection method of blood plasma schizadrin B of strengthening body resistance and dissipating blood stasis botanical (vegetable), and the method is used for pharmacokinetics research, and clarifying the pharmacokinetics rules of the blood plasma schizadrin B of strengthening body resistance and dissipating blood stasis botanical (vegetable).
The technical problems solved by the invention are achieved through the following technical solutions:
Detection method of blood plasma schizadrin B of strengthening body resistance and dissipating blood stasis botanical includes the following steps:

    
    
        (1) Pretreatment of mammalian plasma samples
        a. Mammal plasma containing drugs after being administered the strengthening body resistance and dissipating blood stasis botanical was extracted with ethyl acetate at the volume ratio of 1:4, whirling for 3 to 5 min, then centrifuging at 9600 rpm for 10 min; and drying and enriching the supernatant under the condition of 25 to 30° C.;
        b. Redissolve eluent with mobile phase and then analyze it with UPLC/MS;
        (2) UPLC/MS detection
    
    


UPLC condition: chromatographic column: ACQUITY UPLC BEH C18 (a type of chromatographic column), 2.1×100 mm, mobile phase A: Water-Acetonitrile-Formic acid 95:5:0.1 v/v/v, mobile phase B: Acetonitrile-Formic acid 100:0.1 v/v; MS condition: electrospray ionization (ESI) ion source, detection with positive ion mode, scanning at the range of m/z 150 to 1200.
The described step (2) detects with the positive ion mode; the desolvation gas flow is 440 L/h, the desolvation gas temperature is 300° C., the cone gas flow is 50 L/h, the ion source temperature is 100° C., the spray capillary voltage is 3800 V, the sampling cone voltage is 30V, the extracting cone voltage is 2.00 V, the lens voltage is 0.1 V, and the mass scanning at the range of m/z 150 to 1200.
In the process of the liquid-liquid extraction in this invention, the detected components are distributed in two solvents which are immiscible, select appropriate organic solvents and appropriate proportion, can extract the non-polar components dissolved in the blood plasma entering the organic phase, and then the draining and enriching the organic phase; a UPLC system is used to separate analyte with other components, and finally detected with a mass spectrometric detector.
The ethyl acetate liquid-liquid extraction method adopted by the invention can extract the schizadrin B in the blood plasma, combined with using a UPLC/MS system to detect, markedly improve the resolutions of schizadrin B among other ingredients in the samples, and the analysis method is more sensitive and faster, to facilitate detection of the plasma concentration of the schizadrin B in pharmacokinetic research.



DESCRIPTION OF FIGURES
FIG. 1. UPLA-MS Chromatogram of rat's blood plasma Schizandrin B 0.5 hours after being administered with Strengthening Body Resistance and Dissipating Blood Stasis Botanical.



MODE OF CARRYING OUT THE INVENTION
Combined with the specific embodiments, further elaboration of this invention is given below. It should be understood that these embodiments are only for description of the present invention but not for the use of limiting the scope of the present invention. It should also be understood that, after reading the contents taught in this invention, technicians in this field can make various changes or modifications to this invention, these equivalent forms are all included in the scope defined by the claims attached to this application.
Embodiment 1
A detection method of blood plasma schizadrin B of strengthening body resistance and dissipating blood stasis botanical includes:
1. Mammal plasma containing drugs after being administered the strengthening body resistance and dissipating blood stasis botanical was extracted with ethyl acetate with the volume ratio of 1:4, whirling for 3 min, then centrifugating at 9600 rpm for 10 min, and drying and enriching the supernatant under the condition of 25 to 30° C., redissolving the eluent with mobile phase and then analyzing it with UPLC/MS.
2. UPLC/MS detecting method: the analysis conditions of the UPLC/MS method adopted in this invention: chromatographic column: ACQUITY UPLC BEH C18 (a type of chromatographic column), 2.1×100 mm, mobile phase A: Water-Acetonitrile-Formic acid 95:5:0.1 v/v/v, mobile phase B: Acetonitrile-Formic acid 100:0.1 v/v, eluting in accordance with the following gradient:

















Flow Rate





Time (min)
(ml/min)
% A
% B





















0
0.300
100
0



5.00
0.300
85.0
15.0



10.00
0.300
70.0
30.0



20.00
0.300
40.0
60.0



30.00
0.300
20.0
80.0



35.00
0.300
20.0
80.0



35.01
0.300
100.0
0.0



38.00
0.300
100.0
0.0










MS conditions: electrospray ionization (ESI) ion source, detecting with positive ion mode; the desolvation gas flow is 440 L/h, the desolvation temperature is 300° C., the cone gas flow is 50 L/h, the ion source temperature is 100° C., the spray capillary voltage is 3800 V, the sampling cone voltage is 30V, the extracting cone voltage is 2.00 V, the lens voltage is 0.1 V, and the mass scanning at the range of m/z 150 to 1200.
Detection results: Schizadrin B can be detected in the plasma of mammals after drenched with strengthening body resistance and dissipating blood stasis botanical (see FIG. 1).

## Claims

1. A method for detecting the presence of schizandrin B in the blood plasma of an animal after administering the botanical extract composition Strengthening Body Resistance and Dissipating Blood Stasis Botanical to said animal, comprising the steps of:
a. collecting the blood plasma from said animal 0.5 hours after administration of Strengthening Body Resistance and Dissipating Blood Stasis Botanical to said animal;
b. extracting said blood plasma with ethyl acetate in a blood plasma to ethyl acetate ratio of 1:4 (v/v) for 3 minutes;
c. centrifugating the product resulting from step b at 9600 rpm for 10 minutes;
d. collecting the supernatant after said centrifugation and drying said supernatant at 25-30° C. to produce a dried supernatant;
e. redissolving said dried supernatant in a mixture of water-acetonitrile-formic acid at a ratio of water:acetonitrile:formic acid equals to 95:5:0.1 (v/v/v) to form a blood plasma solution;
f. applying said blood plasma solution to a liquid chromatography column having a size of 2.1×100 mm, wherein the chromatography is performed having conditions including a mobile phase A and a mobile phase B, said mobile phase A is the mixture of water-acetonitrile-formic acid in step e and said mobile phase B is a mixture of acetonitrile-formic acid in a ratio of acetonitrile:formic acid equals to 100:1 (v/v); and
g. detecting the presence of schizandrin B with mass scan at a range of m/z 150-1200, using an electrospray ionization (ESI) with a positive ion mode.

2. The method in claim 1, wherein said detection of the presence of schizandrin B in step g with a positive ion mode, wherein the desolvation gas flow being 440 L/h, the desolvation gas temperature being 300° C., the cone gas flow being 50 L/h, the ion source temperature being 100° C., the spray capillary voltage being 3800 V, the sampling cone voltage being 30V, the extracting cone voltage being 2.00 V, and the lens voltage being 0.1 V.