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Sensitive method for determining ethambutol in blood plasma through liquid chromatography-tandem mass spectrometry

A tandem mass spectrometry and ethambutol technology, applied in the field of drug analysis, can solve the problems of time-consuming concentration steps, long analysis time, complicated operation, etc., and achieve the effect of increasing analysis speed, improving safety, and shortening analysis time

Inactive Publication Date: 2020-08-28
苏州必宜生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

An increase in the dose of the drug is likely to cause adverse effects on the body of the subject
In addition, the existing methods also have the characteristics of complex operation, time-consuming concentration steps, long analysis time, and poor signal-to-noise ratio.

Method used

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  • Sensitive method for determining ethambutol in blood plasma through liquid chromatography-tandem mass spectrometry
  • Sensitive method for determining ethambutol in blood plasma through liquid chromatography-tandem mass spectrometry
  • Sensitive method for determining ethambutol in blood plasma through liquid chromatography-tandem mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Determination of ethambutol in plasma by liquid chromatography-tandem mass spectrometry

[0027] 1. Instrument

[0028] Triple QuadTM 6500+ triple quadrupole tandem mass spectrometer and Analyst 1.6.3 data processing software from Sciex, USA; LC-30 high performance liquid chromatography system from Shimadzu, Japan, including DGU-20A5R degasser, LC-30AD infusion Pump, SIL-30AC autosampler, CTO-20A column thermostat.

[0029] 2. Pretreatment of plasma samples:

[0030] Add 50 µL of ethambutol-d4 internal standard solution (concentration is 100 ng / mL, solvent is methanol:water mixture with a volume ratio of 50:50) to 50 µL plasma sample, and then add 400 µL volume ratio of 90 :10 acetonitrile-water mixture, vortexed for 10 min, centrifuged at 3900 rpm for 10 min at 4°C, and transferred the centrifuged supernatant to another 96-well plate. Take 2.0 µL of the supernatant for LC-MS / MS analysis.

[0031] 3. Preparation of standard series samples and quality c...

Embodiment 2

[0038] Example 2: Methodological Validation

[0039] Carry out methodology verification to the assay method of embodiment 1, specifically as follows:

[0040] 1. Optional:

[0041] The selectivity of the method was evaluated by analyzing the lower limit of quantification samples prepared from 6 different sources of individual blank plasma, hemolyzed plasma, hyperlipidemia plasma and the above blank plasma.

[0042] The results showed that endogenous substances did not interfere with the determination of ethambutol and ethambutol-d4. For a typical chromatogram see image 3 and Figure 4 .

[0043] 2. Standard curve:

[0044] Take the theoretical concentration of ethambutol as the abscissa (x), and the peak area ratio of ethambutol and ethambutol-d4 as the ordinate (y), perform regression analysis to calculate the linear regression equation, weight factor W=1 / x 2 , ethambutol plasma concentration range 4.00 ~ 1600ng . mL −1 Internal linear relationship is good. A typi...

Embodiment 3

[0060] Embodiment 3: clinical sample detection

[0061] The human bioequivalence test of ethambutol hydrochloride tablets was approved by the ethics committee and included 8 healthy subjects. Subjects took one tablet of ethambutol hydrochloride test preparation (T) or reference preparation (R) 250 mg orally on an empty stomach in the two periods respectively, and blood samples were collected at 0 h (within 1 h before taking the medicine) and at different times after taking the medicine. After plasma was isolated, ethambutol concentrations in plasma were determined using established methods. The plasma concentration-time curve of 1 subject is shown in Figure 5 .

[0062] Based on the above examples, it can be seen that the present invention establishes a sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of ethambutol in human plasma. Plasma samples were processed by the precipitated protein method and then directly injected for ...

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Abstract

The invention discloses a sensitive method for determining ethambutol in blood plasma through liquid chromatography-tandem mass spectrometry, and belongs to the technical field of drug analysis. According to the invention, a method for determining ethambutol in human plasma by liquid chromatography-tandem mass spectrometry is established, a plasma sample is treated by a protein precipitation method, quick gradient elution chromatographic separation is carried out by using an Atantis-dC18 chromatographic column, and detection is carried out by using a tandem quadrupole mass spectrometer. The ion reaction is monitored to be m / z 205.2 to 116.2 (ethambutol) and m / z 209.2 to 120.2 (internal standard ethambutol-d4) by mass spectrometry. The method provided by the invention can realize quick andsensitive detection of ethambutol in blood plasma, and can be applied to bioequivalence research of ethambutol hydrochloride tablets with a specification of 250 mg.

Description

technical field [0001] The invention relates to the technical field of drug analysis, in particular to a sensitive liquid chromatography-tandem mass spectrometry method for determining ethambutol in blood plasma. Background technique [0002] Ethambutol hydrochloride is a first-line anti-tuberculosis drug. During the research and development of ethambutol hydrochloride generic drugs, clinical bioequivalence studies are required to compare the exposure and absorption of generic drugs and original drugs in the human body. Therefore, it is necessary to determine the concentration of ethambutol in the blood. At present, the main method for determining the concentration of ethambutol in plasma is liquid chromatography-tandem mass spectrometry. However, the sensitivity of the existing detection methods is poor, and the lower limit of quantitation can only reach 10 ng / mL. At present, most domestic ethambutol specifications are 0.25g. Limited by the sensitivity of the detection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/34G01N30/724G01N30/8675
Inventor 黄立峰孙叶顾琴
Owner 苏州必宜生物科技有限公司
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