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Method based on two-dimensional liquid chromatography to detect gelsemine, koumine and gelsenicine in biological specimen at same time

A technology of two-dimensional liquid chromatography and mesocarcinoma, which is applied in the field of analysis, can solve the problems of complicated purification of hookosin alkaloids, cumbersome processing operations, etc., and achieves the effects of reducing tailing phenomenon, simple pretreatment and low cost.

Active Publication Date: 2018-10-02
HUNAN AGRICULTURAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When Chen Weilin et al. used HPLC to measure the biological content of kinsine A, kinesin and cucurine A in the roots, stems and leaves of G. chinensis produced in Fujian, the limit of quantitation was only 0.012 mg / mL, and the analysis time required 30-50 minutes. At present, Qiu Hongqiang et al. established an HPLC method to determine the concentration of kelsin A and kelsin in human plasma, Lin Wang used UPLC-MS / MS to simultaneously determine the content of kelsin A and kelsin in rat plasma, and Yang Kun et al. Utilize LC-MS / MS to simultaneously measure kinesin A and kinesin in plasma, kinesin has been studied, but due to the complicated purification of kinesin alkaloids in plasma and cumbersome pretreatment operations, it is necessary to select an internal standard for calibration
And at present, there is no research and application about the simultaneous determination of three kinds of Gelkins alkaloids in biological samples (blood, urine, tissue) by two-dimensional liquid chromatography

Method used

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  • Method based on two-dimensional liquid chromatography to detect gelsemine, koumine and gelsenicine in biological specimen at same time
  • Method based on two-dimensional liquid chromatography to detect gelsemine, koumine and gelsenicine in biological specimen at same time
  • Method based on two-dimensional liquid chromatography to detect gelsemine, koumine and gelsenicine in biological specimen at same time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] qualitative analysis

[0065] 1. Reagents

[0066] a) water is ultrapure water;

[0067] b) ammonium dihydrogen phosphate is chromatographically pure;

[0068] c) Methanol, acetonitrile, phosphoric acid, and aqueous ammonia are all chromatographically pure;

[0069] d) 10mM ammonium dihydrogen phosphate (pH=3.0): Weigh 1.1503g of ammonium dihydrogen phosphate, put it into a 1000mL volumetric flask, add grade 1 water to dissolve, dilute to the mark, and adjust the pH to 3.0 with phosphoric acid;

[0070] e) 10mM ammonium dihydrogen phosphate (pH=7.5): Weigh 1.1503g of ammonium dihydrogen phosphate, put it in a 1000mL volumetric flask, add grade 1 water to dissolve, dilute to the mark, and adjust the pH to 7.5 with ammonia water;

[0071] 2. Standard solution:

[0072] 1) Standard stock solution of kelezin A, kelezin, and kelezin: Accurately weigh the standard products of kelezin A, kelezin, and kelezin, dissolve them in methanol, dilute them in a volumetric flask, an...

Embodiment 2

[0125] quantitative analysis

[0126] 1. The reagent is the same as in Example 1

[0127] 2. Apparatus and materials are the same as in Example 1

[0128] 3. Sample Extraction

[0129] 4. Accurately measure 200-400μL or 1.0g~2.0g of the case sample, crush it and put it in 2 parallel parts in the test tube. Take another blank sample of the same matrix, add the mixed standard solution of kelezin A, kelezin, and kelezin to prepare the added sample, parallel 6 copies, and mix well. Others are the same as embodiment 1.

[0130] 5. Record and calculate

[0131] Record the relative standard deviation, expressed by the following formula;

[0132]

[0133] In the formula:

[0134] P tr -The relative standard deviation;

[0135] - add peak area mean;

[0136] A i Indicates the peak area obtained for the nth time (i=1,2,3,...,6);

[0137] 6. Quantitative result evaluation

[0138] If the RSD of the target substance content in the spiked sample is >5%, the quantitative da...

Embodiment 3

[0140] Evaluation of the method of the invention using blood

[0141] 1. Reagent is with embodiment 1;

[0142] 2. Instrument and material are the same as in Example 1;

[0143] 3. Sample Extraction

[0144] Take 400mL of blank non-fresh blood in a test tube, add kelezin A, kelezin, kelezin

[0145] Prepare your own mixed standard to add the sample and mix well.

[0146] Add 1000 μL methanol-acetonitrile (80:20) treatment solution to the above sample, shake and vortex, 13000r / min

[0147] Centrifuge for 10min, take 500μL of supernatant from pig blood sample (100μL from rat and sheep) and inject into 2D-LC-UV

[0148] analyze;

[0149] 4. Instrument testing

[0150] New two-dimensional liquid chromatography conditions

[0151] The following are reference conditions, which can be adjusted according to the actual situation of different brands of instruments and different samples;

[0152] Chromatographic column: the extraction column (one-dimensional column) is ASTON SXI ...

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Abstract

The invention discloses a method based on two-dimensional liquid chromatography to detect gelsemine, koumine and gelsenicine in a biological specimen at same time. The method comprises the steps of 1)pretreating the biological specimen to obtain a solution under test; 2) injecting the solution under test into a 2D-LC-UV system for detection; 3) subjecting detection results to quantitative analysis. The method has the advantages that, for instance, specimen pretreatment is simple, detection sensitivity is higher than that of common chromatography, false positives can be effectively eliminated,instrument stability is high, and the method is applicable to the qualitative and quantitative detection of gelsemine, koumine and gelsenicine in various biological specimens (blood, urine and muscle) and also applicable to the inspection of in-vitro specimens and suspicious material evidences.

Description

technical field [0001] The invention relates to a detection method, in particular to a method for simultaneously detecting kelezin A, kelezin and kelezin in biological samples by two-dimensional liquid chromatography, and belongs to the technical field of analysis. Background technique [0002] Glycyrrhizae is a plant of the genus Echinacea, also known as Echinacea vine, Severus chinensis, Pueraria lobata, etc. Gelkinia genus is a woody vine, with ovate oblong leaves, yellow flowers, arranged in axillary bouquets or terminal ones, The seeds have wings, and their rhizomes and leaves are used for medicine. Warm in nature, pungent in taste, bitter, and highly poisonous. There are two kinds of medicinal Gelkins, one is North American Gelkins (G. sempervirens Ait), which is produced in America; the other is Chinese Gelkins (Gelsemiumelegans Benth.), which is produced in Asia, and is mainly distributed in Zhejiang, Fujian, and Guangdong in my country. , Guangxi, Hunan, Guizhou, Y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/86
CPCG01N30/02G01N30/06G01N30/8679
Inventor 刘兆颖刘莎莎孙志良杨昆
Owner HUNAN AGRICULTURAL UNIV
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