Development and validation of a LC–MS/MS method for homocysteine thiolactone in plasma and evaluation of its stability in plasma samples

• We developed LC–MS/MS method for homocysteine thiolactone (HCTL) in human plasma.
• Validated according to US FDA Bioanalytical Method Validation Guidelines.
• HCTL levels in plasma found to be highly unstable.
• Immediate extraction and storage at −86 °C is found to stabilize HCTL up to 1 month.
• Applied for quantifying HCTL levels in plasma of healthy human volunteers.

The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC–MS/MS. The gradient elution of HCTL was achieved within 5 min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5–32.5 nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at −86 °C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers.