Method for measuring derivatization in valproic acid body fluid concentration process through high performance liquid chromatography

A technology of high performance liquid chromatography and valproic acid, which is applied in the field of derivatization in the process of determining the concentration of valproic acid in body fluids by high performance liquid chromatography, and can solve problems such as shortening the life of the chromatographic column, poor repeatability, and incomplete reaction , to achieve the effect of no endogenous impurity interference, accurate measurement results and prolonging the service life

Inactive Publication Date: 2014-11-12
LIUZHOU CITY HEALTHCARE HOSPITAL FOR WOMEN & CHILDREN
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AI Technical Summary

Problems solved by technology

[0009] Insufficient: When the serum valproic acid concentration is 182.2 μg / mL, 1.0 mL of the upper organic phase contains 0.063 μmol of valproic acid, 0.039 μmol of internal standard cyclohexanecarboxylic acid, and 0.52 μmol of derivatizing agent ω-bromoacetophenone μmol, that is, the molar ratio of the derivatizing agent to the reactant is 5.1 times, the reaction is not complete, the sensitivity of the method is low, and the repeatability is not good
[0014] Insufficient: When the concentration of serum valproic acid is 200 μg / mL, the upper organic phase contains 0.19 μmol of valproic acid, 0.14 μmol of internal standard caprylic acid, and 2.16 μmol of derivatizing agent 2,4′-dibromoacetophenone added. The molar ratio of the derivatizing agent to the reactant is 6.5 times, the reaction is incomplete, the sensitivity of the method is low, and the repeatability is not good
Excessive dosage of derivatizing agent 2,4′-dibromoacetophenone (>400μg) reduces the detection sensitivity of the methodology. Repeated use for a long time will easily increase the pressure of the chromatographic column, reduce the column efficiency and shorten the chromatographic column life
[0015] To sum up, the efficiency of triethylamine in catalyzing the derivatization reaction of valproic acid is not high, usually a derivatizing agent with a molar ratio of 9-11 times of reactants is required to completely derivatize valproic acid
However, excessive derivatization agent can easily cause uneven baseline of HPLC analysis, which reduces the sensitivity of the methodology; long-term repeated use of excessive derivatization agent (>400μg) can easily cause excessive column pressure, decrease column efficiency, and shorten column life

Method used

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  • Method for measuring derivatization in valproic acid body fluid concentration process through high performance liquid chromatography
  • Method for measuring derivatization in valproic acid body fluid concentration process through high performance liquid chromatography
  • Method for measuring derivatization in valproic acid body fluid concentration process through high performance liquid chromatography

Examples

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Effect test

Embodiment 1

[0034] Example 1: A derivatization method in the process of measuring the body fluid concentration of valproic acid by high performance liquid chromatography, using tetramethylammonium hydroxide as a catalyst and 2,4'-dibromoacetophenone as a derivatizing agent.

[0035] The reaction formula is as follows:

[0036]

[0037] Specific operation steps: Accurately take 50 μL of quality control or patient serum, add 60 μL of 0.10 μg / μL internal standard pelargonic acid solution, add 50 μL of 3mol / L sulfuric acid solution, and mix well; add 2 mL of diethyl ether, vortex extract for 1 min, and centrifuge for 5 min (3500r· min -1 ), draw the supernatant into a centrifuge tube, add 100 μL of 10 mmol / L tetramethylammonium hydroxide acetonitrile solution and vortex to mix, then add 63 μL of 2 mg / mL 2,4′-dibromoacetophenone acetonitrile solution (that is, the derivatizer is 126μg, 0.45μmol, which is equivalent to 5 times the molar multiple of the reactant, and the concentration of valpr...

Embodiment 2

[0046] Example 2: A derivatization method in the process of measuring the body fluid concentration of valproic acid by high performance liquid chromatography, using tetramethylammonium hydroxide as a catalyst and α-bromoacetophenone as a derivatizing agent.

[0047] The reaction formula is as follows:

[0048]

[0049] Specific operation steps: Accurately take 50 μL of quality control or patient serum, add 50 μL of 0.16 μg / μL internal standard cyclohexyl propionic acid solution, add 50 μL of 3mol / L sulfuric acid, and mix well; add 2 mL of ether, vortex extract for 1 min, centrifuge for 5 min (3500r / min), draw the supernatant into a centrifuge tube, add 100 μL of 10 mmol / L tetramethylammonium hydroxide acetonitrile solution, vortex and mix well, then add 55 μL of 2 mg / mL α-bromoacetophenone acetonitrile solution (that is, the derivatizer is 110 μg, 0.55μmol, which is equivalent to 5.3 times the molar multiple of the reactant, and the concentration of valproic acid in the re...

Embodiment 3

[0058] Example 3: A derivatization method in the process of measuring the body fluid concentration of valproic acid by high performance liquid chromatography, using tetrabutylammonium hydroxide as a catalyst and α-bromoacetophenone as a derivatizing agent.

[0059] The reaction formula is as follows:

[0060]

[0061] Specific operation steps: Accurately take 50 μL of quality control or patient serum, add 50 μL of 0.16 μg / μL internal standard cyclohexyl propionic acid solution, add 50 μL of 3mol / L sulfuric acid, and mix well; add 2 mL of ether, vortex extract for 1 min, centrifuge for 5 min (3500r / min), draw the supernatant into a centrifuge tube, add 40 μL of 10 mmol / L tetrabutylammonium hydroxide acetonitrile solution, vortex mix for 30 s, then add 55 μL of 2 mg / mL α-bromoacetophenone acetonitrile solution (that is, the derivatizer is 110 μg, 0.55 μmol, equivalent to 5.3 times the molar multiple of the reactant, the concentration of valproic acid in the reactant serum is...

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Abstract

The invention relates to a method for measuring derivatization in a valproic acid body fluid concentration process through a high performance liquid chromatography. According to the method, triethylamine is replaced by quaternary ammonium hydroxide acetonitrile solution (tetramethylammonium hydroxide acetonitrile solution or tetrabutyl ammonium hydroxide acetonitrile solution or tetraethyl ammonium hydroxide acetonitrile solution) to catalyze valproic acid and is derived into phenyl ester which is ultraviolet detectable with a derivating agent (2,4'-dibromo acetophenone acetonitrile solution, alpha-bromoacetophenone acetonitrile solution, 2-bromo-p-nitroacetophenone acetonitrile solution or 4-bromo-7-herniarin acetonitrile solution), the derivating agent which is only 3-5 molar times that of amount of the reactants is required, and the derivatization yield above 95 percent can be achieved. The method has the advantages that the derivatization reaction is complete, the chromatogram base line is stable, endogenous impurity interference is avoided, the measurement result is accurate, the precision is high, the sensitivity is high, the using amount of the derivating agent is small, the pressure of the chromatographic column is hardly increased, and the service life of the chromatographic column is prolonged.

Description

technical field [0001] The invention relates to a derivatization method in the process of measuring the body fluid concentration of valproic acid by high performance liquid chromatography. Background technique [0002] Valproic acid is the most commonly used broad-spectrum antiepileptic drug. Its therapeutic concentration range is narrow and individual differences are large. During the treatment, the blood concentration must be monitored. Through individualized medication, the control rate of epilepsy can be improved and adverse reactions can be reduced. [0003] HPLC (high performance liquid chromatography) is often used to monitor the plasma concentration of valproic acid, and the HPLC method often needs to derivatize valproic acid into esters with ultraviolet absorption or fluorescence. The commonly used derivatization method is to use triethylamine as a catalyst to catalyze valproic acid and α-bromoacetophenone, 2,4'-dibromoacetophenone, 2-bromo-p-nitroacetophenone or 4-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06
Inventor 毛桂福
Owner LIUZHOU CITY HEALTHCARE HOSPITAL FOR WOMEN & CHILDREN
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