DETECTION OF BLOOD PLASMA AMYGDALIN OF DISSIPATING BLOOD STASIS BOTANICAL

Abstract

A detection method of blood plasma amygdalin of dissipating blood stasis botanical is disclosed. The method includes: (1) adding 2-5 mol/L phosphoric acid to plasma of mammalian administered dissipating blood stasis botanical and mixing the solution of plasma-phosphoric acid (1:2-3, v/v), applying the solution to small column of Waters Oasis HLB activated by methanol and water; after leaching by water and 80-100% methanol and eluting by 0.2-1% ammonia-methanol, drying and enriching the eluent 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 150-800. The method can be used for pharmacokinetics study of amygdalin of dissipating blood stasis botanical.

Description

TECHNICAL FIELD

This invention belongs to the field of pharmacokinetics, particularly involves the determination method of blood plasma Amygdalin 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 relevant pharmacokinetic research report on strengthening body resistance and dissipating blood stasis botanical has been found, and there is not any detection method of Amygdalin of the compound prescription in biological samples (including blood plasma sample).

CONTENTS OF THE INVENTION

The technical matters aim to be resolved by this invention is to provide a detection method of blood plasma Amygdalin 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 Amygdalin of the strengthening body resistance and dissipating blood stasis.

• • The technical matters solved by this invention are achieved through the following technical solutions:
  • The detection method of blood plasma Amygdalin of strengthening body resistance and dissipating blood stasis botanical (vegetable) includes the following steps:
  • (1) Pretreatment of Mammalian Blood Plasma Samples
• a. Collect mammal plasma containing drugs after being administered the strengthening body resistance and dissipating blood stasis botanical, misce bene after adding 2-5 mol/L phosphoric acid, and the volume ratio between the blood plasma and phosphoric acid is 1:2-3, applying the blood plasma with drugs to small column of Waters Oasis HLB activated by methanol and water; after leaching with water and 80-100% methanol and eluting with 0.2-1% ammonia-methanol, drying and enriching the eluent under the condition of 25-30° C., and redissolving the eluent with mobile phase.
• b. Detection with UPLC-MS method;
  • (2). UPLC/MS Detection Method

UPLC condition: chromatographic column: Acquity UPLC BEH C18, 2.1×100mm, mobile phase A: water-Acetonitrile-Formic acid 95:5:0.1 v/v/v, mobile phase B: Acetomtrile-Formic acid 100: The electrospray ionization (ESI) ion source of the described step (2), detects with 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 mass scanning at the range of m/z 150-800. In solid-phase extraction process of this invention, the adsorption capacity of the solid phase to the analytes is greater than the sample mother liquor, when the samples pass through the solid-phase extraction column, the analytes and a number of similar ingredients were adsorbed on the surface of solid, and other ingredients then pass through the column with the sample mother liquor, first leach columns with larger polar solvents, to rinse and remove a number of unrelated ingredients, and filially elute the analytes with appropriate solvent, drain and enrich the eluent; use UPLC system, separate the analytes with other ingredients in the elution, and finally detected with mass spectrometry detector.

The Amygdalin in this invention is water-soluble ingredients, using solid-phase extraction method, can fully extract the Amygdalin in the samples, combined with using a UPLC/MS system to detect, markedly improve the resolutions of Amygdalin among other ingredients in the samples, and the analysis method is more sensitive and faster, to facilitate the detection of the blood plasma concentration of the Amygdalin in pharmacokinetic research.

DESCRIPTION OF FIGURES

FIG. 1. UPLC-MS chromatogram of Amygdalin.

MODE OF CARRYING OUT THE INVENTION

Combining 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, after reading the contents taught in this invention, technicians in this field can make various changes or modification 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 Amygdalin of strengthening body resistance and dissipating blood stasis botanical (vegetable).

• 1. Method of pretreating mammalian blood plasma sample: Collect mammal plasma containing drugs after being administered the strengthening body resistance and dissipating blood stasis botanical, mince bene after adding 2-5 mol/L phosphoric acid, and the volume ratio between the biological sample and phosphoric acid is 1:2-3, applying the blood plasma with drugs to small column of Waters Oasis HLB activated by methanol and water; after leaching with water and 80-100% methanol and eluting with 0.2-1% ammonia-methanol, drying and enriching the eluent under the condition of 25 to 30° C., and redissolving the eluent with mobile phase.
• 2. UPLC/MS determination method: The analysis conditions of the applied UPLC/MS method in this invention, 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: Acetomtrile-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 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 mass scanning at the range of m/z 150 to 800.

Detection results: Amygdalin can be detected in the blood plasma of mammals after drenched with strengthening body resistance and dissipating blood stasis botanical (see FIG. 1).

Claims

1. The detection method of blood plasma Amygdalin of strengthening body resistance and dissipating blood stasis botanical includes the following steps: (1) Pretreatment of Mammalian Blood Plasma Samplesa. Collect mammal plasma containing drugs after being administered the strengthening body resistance and dissipating blood stasis botanical, misce bene after adding 2 to 5 mol/L phosphoric acid, and the volume ratio between the blood plasma and phosphoric acid is 1:2-3, applying the blood plasma with drugs to small column of Waters Oasis HLB activated by methanol and water; after leaching with water and 80-100% methanol and eluting with 0.2-1% ammonia-methanol, drying and enriching the eluent under the condition of 25 to 30° C., and redissolving the eluent with mobile phase;b. Detection with UPLC/MS Method;(2) UPLC/MS detection method.

2. The detection method of blood plasma Amygdalin of strengthening body resistance and dissipating blood stasis botanical described in accordance with claims 1, its features lie in: the described step (2) detects with 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 mass scanning at the range of m/z 150 to 800.