Determination of loperamide in human plasma and saliva by liquid chromatography–tandem mass spectrometry

• LC/MS/MS method for analysis of loperamide in plasma and saliva has been developed and validated.
• Protein direct precipitation extraction method by methanol was used for loperamide determination.
• 20 pg/ml is new and the lowest validated LLOQ.
• The determinant method was validated upon European and US FDA guidelines.

A simple and sensitive liquid chromatography–tandem mass spectrometric method for quantification of loperamide in human plasma and saliva was developed and validated, and then successfully applied in pharmacokinetic clinical study to investigate and correlate bioavailability of Imodium® 2 mg quartet tablet dose in both human plasma and saliva. Loperamide with labeled internal standard was extracted from its biological matrix by methanol as protein direct precipitant in single extraction step. Adequate chromatographic separation for analytes from plasma and saliva matrices was achieved using ACE C18 (50 mm × 2.1 mm, 5 μm) column, eluted by water/methanol/formic acid (30:70:0.1%, v/v), delivered isocratically at constant flow rate of 0.75 ml/min. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to European guideline, and partial validation was applied on saliva, specificity, matrix effect, recovery, sensitivity, within and between day precision and accuracy. The calibration curve was linear through the range of 20–3000 pg/ml in both plasma and saliva using a 50 μl sample volume. The partial validation sections outcome in saliva was so close to those in plasma. The within- and between-day precisions were all below 8.7% for plasma and below 11.4% for saliva. Accuracies ranged from 94 to 105% for both matrices. In this study, 26 healthy volunteers participated in the clinical study, and 6 of gave their saliva samples in addition to plasma at the same time schedule. The pharmacokinetic parameters of Cmax, AUC0–t and AUC0–∞, Tmax and T1/2 in both plasma and saliva were calculated and correlated.

Illustrated mean concentration–time profile of 26 subjects for loperamide in plasma, correlated to mean concentration–time profile of 6 subjects for loperamide in saliva over 72 h following oral administration of 8 mg loperamide.Figure optionsDownload as PowerPoint slide