Enzymatic cycling assay for D-carnitine determination

Area of Science:

• Biochemistry
• Analytical Chemistry
• Enzymology

Background:

• Carnitine plays a vital role in cellular energy metabolism.
• Accurate determination of d-carnitine is crucial for various biological and pharmaceutical applications.
• Existing methods for d-carnitine determination may lack sensitivity or automation capabilities.

Purpose of the Study:

• To develop and validate a novel enzymatic assay for the sensitive and accurate determination of d-carnitine.
• To optimize assay conditions using different tetrazolium salt pairs and electron carriers.
• To assess the applicability of the developed method in real-world scenarios, including microbial transformations and pharmaceutical analysis.

Main Methods:

• Enzymatic assay utilizing d-carnitine dehydrogenase.
• NAD(+) cycling coupled with a tetrazolium salt (MTT or INT) and phenazine methosulfate.
• Optimization of reaction parameters and validation of linearity, quantification limit, and precision.
• Application of the assay to monitor microbial d-carnitine to l-carnitine conversion and quantify d-carnitine in pharmaceutical preparations.

Main Results:

• The assay demonstrated high sensitivity, with optimal performance using the MTT/MTT-formazan system.
• A linear calibration curve was established for d-carnitine concentrations ranging from 0.1 to 5 mM.
• The method exhibited a low quantification limit (0.1 mM) and high precision (RSD of 1.51%).
• The assay was successfully applied to track d-carnitine levels during microbial transformations and to determine d-carnitine content in pharmaceutical samples.

Conclusions:

• A simple, rapid, accurate, and automatable enzymatic method for d-carnitine determination has been established.
• The developed assay offers superior sensitivity and reliability compared to existing methods.
• This method provides a valuable tool for research and quality control in fields involving d-carnitine analysis.