Chemists carefully compare chromatography with immunoassay for VPA

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Ezine

  • Published: Apr 2, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Chemists carefully compare chromatography with immunoassay for VPA

Monitoring of valproic acid plasma levels required

Valproic acid (VPA, or 2-propylpentanoic acid) is an anticonvulsant often used in the treatment of epilepsy. It is also sometimes used to treat bipolar disorder and migraine. Too high a concentration of the drug in the body can cause liver damage and symptoms such as tremor, respiratory depression and coma. The liver toxicity has been linked with VPA’s main metabolites: 3-OH VPA, 5-OH VPA and 4-ene VPA. The drug is also known to cause foetal abnormalities, although it is still given to pregnant woman in some countries if no safer medication is effective in controlling their epilepsy. Monitoring of the levels of VPA in plasma or serum is thus an important safety measure, particularly in patients with impaired liver function or those on a particularly high dose of the drug. Immunoassay techniques are often used, although LC-MS and GC-MS have also been employed. The Shanghai researchers compared their own LC-MS/MS technique with a chemiluminescent microparticle immunoassay (CMIA) technique in routine use in a local hospital.

LC-MS/MS compared with CMIA for valproic acid plasma assay

The Shanghai researchers took 395 plasma samples from 271 patients being treated with the sodium salt of valproic acid (depakine). Each sample was extracted with an Oasis® HLB SPE cartridge (Waters). The HPLC was described in a previous paper (Gao et al.). An Agilent 1200 HPLC with a Zorbax® SB-C8 column and an isocratic mobile phase of 80:20 v/v methanol:10 mmol/L aqueous ammonium acetate, containing 0.1% formic acid, were used. The flow rate was 0.3 ml/min and the total elution time was only 2 minutes. An Agilent 6410 mass spectrometer with an electrospray source in negative mode was used. Distinctive precursor and product ions were found for valproic acid and its three metabolites.

CMIA assays were carried out on the samples using an Abbott ARCHITECT i1000 immunology analyser. The results were compared to those obtained by LC-MS/MS. The two sets of results showed good correlation, with the equation linking the two having a concordance correlation coefficient of 0.97, with no significant deviation from linearity. A further comparison was carried out using a Bland–Altman plot, which also found good agreement between the two techniques. A slight positive bias (overestimation) was found in the CMIA results, possibly due to the assay being affected by the presence of metabolites of VPA. It is not believed that the small difference in results is likely to significantly affect the treatment given to patients.

In a separate experiment, the effects of VPA and its metabolites on LO2 liver cells were examined. It was found that the 3-OH and 5-OH metabolites caused cell damage, while 4-ene VPA and the parent compound did not. The LC-MS/MS system has the advantage that it can identify and quantify these metabolites, while the CMIA assay cannot.

Advantages of LC-MS/MS over immunoassay demonstrated for VPA

The work has shown that LC-MS/MS gives results in line with CMIA. However, the former technique has the great advantage that it allows the monitoring of the VPA metabolites, which must be taken into account in any clinical measurement. The very short run time of 2 minutes means that quick results can be generated when needed. The authors point out that the method could be adapted to detect other drugs in addition to VPA in cases where patients are taking more than one medication. It would be interesting to compare the methods examined here with GC-MS to see which technique is the most useful.

Related Links

Journal of Clinical Laboratory Analysis, 2017, Early View paper. Wang et al. Comparison of LC-MS/MS vs chemiluminescent microparticle immunoassay in measuring the valproic acid concentration in plasma of epilepsy patients in a new perspective.

Journal of Chromatography B, 2011, 879, 1939-1944. Gao et al. LC–MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma.

Wikipedia, Valproate

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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