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Method for quantitatively detecting content of leonurine in blood plasma

A technology for quantitative detection of leonurine, which is applied in the field of medical testing, can solve the problems of undetectable leonurine and low sensitivity, and achieve the effects of high sensitivity, strong specificity, and easy operation

Active Publication Date: 2012-09-19
ZHUHAI HENGQIN NEW DISTRICT ZHONGZHU ZHENGTAI MEDICAL MANAGEMENT CO LTD
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Problems solved by technology

However, what is reported in the existing literature is mainly the application of reverse high-performance liquid chromatography (HPLC) or ultra-high pressure liquid chromatography (UPLC) to determine the content of motherwort in the raw materials of motherwort or Chinese patent medicine preparations; The plasma concentration of leonurine in rat plasma was used to calculate the pharmacokinetic parameters of leonurine after intravenous injection, but the sensitivity of this method is very low, and the detection limit is about 0.25 μg / mL; After oral administration, leonurine cannot be detected by the above method, indicating that the blood concentration of leonurine after oral administration is lower than the detection limit of this method; therefore, a more sensitive, more rapid and agile method needs to be established

Method used

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  • Method for quantitatively detecting content of leonurine in blood plasma
  • Method for quantitatively detecting content of leonurine in blood plasma
  • Method for quantitatively detecting content of leonurine in blood plasma

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Detection conditions and method examples

[0041] 1. Reagent preparation:

[0042] (1) Standard curve: Precisely prepare the motherwortine stock solution with deionized double-distilled water, the concentration is 1mg / mL, and then dilute it with double-distilled water to form different concentrations of the motherwortine standard working solution; take each concentration of the motherwortine standard working solution 10 μL, add 90 μL of blank rat plasma, and prepare standard curve solutions with concentrations of 4, 8, 16, 32, 64, 128, and 256 ng / mL respectively, a total of 7 concentration points, prepare 1 mL for each concentration, and store in aliquots At -80°C, to be tested;

[0043] (2) Quality control product: Take 10 μL of the standard working solution of motherwortine at an appropriate concentration, add 90 μL of blank rat plasma, and prepare four concentrations of motherurine with concentrations of 4, 10, 50, and 200 ng / mL as quality controls. Prepare 2 mL of...

Embodiment 2

[0073] Embodiment 2. Methodological verification:

[0074] Using the detection conditions and methods listed in Example 1, the assay method was validated methodologically. In the concentration range of 4-256ng / mL motherwortine, the linear relationship is good (r>0.995), the lower limit of quantification is 4ng / mL, and the lowest detection limit can reach 2ng / mL. The lower limit of quantitation precision is less than 20%, the other quality control intra-assay and inter-assay precisions are less than 15%, the accuracy is 98.73-105.42%, the recovery rate of motherwort extraction is 66.2-69.98%, RDS<10%, internal standard extraction The recovery rate was 62.78%, the RSD was 6%, the matrix effect of leonurine was 79.3–88.49%, and the matrix effect of the internal standard was 88.32%. Good stability. The results of method validation show that the established LC-MS / MS can quickly, sensitively, accurately and reliably detect the blood concentration of leonurine in rats administered ...

Embodiment 3

[0099] Detection of plasma concentration and calculation of pharmacokinetic parameters after oral administration of leonurine in rats.

[0100] Take 6 healthy male SD rats, take the blank blood before administration, and then give 60mg / kg intragastric administration, and then at 0.083, 0.167, 0.333, 0.5, 0.75, 1, 1.5, 2, 4, 8, 12 h Blood was collected and centrifuged to obtain plasma, and then the concentration of leonurine in the blood sample was measured according to the method of Example 1, and its pharmacokinetic parameters were calculated using the statistical moment method using DAS2.0 software (as shown in Table 6).

[0101] Table 6

[0102] Pharmacokinetic parameters unit Mean±S.D. Cmax μg / L 126.32±63.69 Tmax h 0.95±0.11 AUC(0-t) μg / L*h 411.60±147.96 AUC(0-∞) μg / L*h 432.87±146.72 MRT(0-t) h 3.21±0.44 MRT(0-∞) h 4.07±0.91 t 1 / 2 z h 2.77±0.61 CLz / F L / h / kg 151.54±49.05 Vz / F L / kg 622.83±289....

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Abstract

The invention belongs to the technical field of medicine detection and relates to a method for quantitatively detecting the content of leonurine in blood plasma. The method is characterized by adopting the high performance liquid chromatography-mass spectrometry technology, using benzoyl arginine ethyl ester as an internal standard of a detected substance, using a method of precipitating the proteins with perchloric acid to pretreat the blood plasma sample and calculating the blood concentration of leonurine in the blood plasma by using the peak area according to an internal standard method, wherein liquid chromatography adopts Agilent ZorbaxSB-C18 column as the stationary phase, the acetonitrile / ammonium acetate buffer solution mixed solution is used as the mobile phase and gradient elution is carried out; mass spectrometry adopts an electrospray ionization source; leonurine is quantified by adopting multiple reaction monitoring; and the lowest limit of quantification of leonurine in the method reaches 4ng / mL. The method has the following beneficial effects: the method has strong sample detection specificity and high sensitivity and is simple and convenient to operate; all the textual research indexes of methodology can meet relevant technical requirements of Chinese pharmacopoeia; and the method can be used for pharmacokinetics research after oral administration of leonurine.

Description

technical field [0001] The invention belongs to the technical field of medical detection, and relates to a method for detecting the content of motherurine in plasma, in particular to a method for quantitatively detecting the content of motherurine in plasma by liquid chromatography-mass spectrometry (high performance liquid chromatography tandem mass spectrometry technology). Background technique [0002] The traditional Chinese medicine Motherwort is the dried whole herb of the Lamiaceae plant Leonurus helerophyllus sweet. It has the functions of promoting blood circulation, removing scars, regulating menstruation, eliminating water, clearing away heat and detoxifying. It is often used to treat irregular menstruation, amenorrhea, postpartum abdominal pain, bruises, edema wait. Recent studies have shown that motherwort has a variety of biological activities. For example, it has been reported that the extract of motherurine can improve myocardial ischemia, increase coronary b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/72G01N30/06G01N30/36
Inventor 朱依谆朱清
Owner ZHUHAI HENGQIN NEW DISTRICT ZHONGZHU ZHENGTAI MEDICAL MANAGEMENT CO LTD
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