LC-MS/MS quantification of salvinorin A from biological fluids

Abstract

A facile method for quantifying the concentration of the powerful and widely available hallucinogen salvinorin A (a selective kappa opioid agonist) from non-human primate cerebrospinal fluid (CSF) and human plasma has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive electrospray ionization (ESI) mode. With CSF solid phase extraction can be avoided completely by simply diluting each sample to 10% (v/v) acetonitrile, 1% (v/v) formic acid and injecting under high aqueous conditions for analyte focusing. Extensive plasma sample preparation was investigated including protein precipitation, SPE column selection, and plasma particulate removal. Human plasma samples were centrifuged at 21 000 × g for 4 minutes to obtain clear particulate-free plasma, from which 300 μL was spiked with internal standard and loaded onto a C₁₈ SPE column with a 100 mg mL⁻¹ loading capacity. Guard columns (C₁₈, hand packed 1 mm × 20 mm) were exchanged after backpressure increased above 4600 psi, about 250 injections. A shallow acetonitrile/water gradient was used, 29–33% CH₃CN over 8 minutes to elute salvinorin A. Reduction of chemical noise was achieved using tandem mass spectrometry with multiple reaction monitoring while sensitivity increases were observed using a 50 μL injection volume onto a small bore analytical column (C₁₈, 1 mm ID × 50 mm) thus increasing peak concentration. Limits of quantification were found to be 0.0125 ng mL⁻¹ (CSF) and 0.05 ng mL⁻¹ (plasma) with interday precision and accuracy below 1.7% and 9.42% (CSF) and 3.47% and 12.37% (plasma) respectively. This method was used to determine the concentration of salvinorin A from an in vivo Rhesus monkey study and a trial of healthy human research participants, using behaviorally active doses.