Psychopharmacology (2011) 214:933–939 In conclusion, in this first controlled study of the effects of SA in human subjects, sublingual doses up to 4,000 μg in DMSO/PEG-400 were not psychoactive. These results suggest that the sublingual bioavailability of pure SA is low and that oral absorption of SA from SD depends on other components of SD or the local effects of SD on conditions in the mouth. Acknowledgements The authors wish to thank Margie Jang for her assistance with data collection. Contributors Baggott, Cohen, Galloway, Mendelson, and Munro designed the protocol. Coyle, Everhart, Flower, Galloway, Lopez, and Mendelson implemented the study procedures. Munro isolated the salvinorin A and wrote the discussion of solvent and formulation effects. Coyle conducted the statistical analyses and wrote the first draft of the manuscript. All authors contributed to and approved the final manuscript. Role of funding source This study was supported by a grant from an anonymous donor. Baggott received support from the Beckley Foundation. These sources had no further role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. Conflicts of interest We have no conflicts of interest to declare. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Appendix Method for determination of salvinorins A and B in DMSO/PEG-400 by APCI LC/MS/MS Sample preparation Two solutions of salvinorin A were prepared at a concen- tration of 4.00 mg/mL in DMSO–PEG-400 (25:75, v/v). One was prepared and allowed to sit on the laboratory benchtop at room temperature for 6 days, while the other was prepared just before sample analysis. Just before analysis, 100 μL of each solution was dissolved in 9.90 mL of ethyl acetate to provide two 10-mL solutions at concentrations of 40 μg/mL. Calibration standards were prepared by spiking mixtures of salvinorin A and B in the following amounts in 16 × 100 mm silanized screw-top culture tubes: 0, 10, 50, 100, 250, 500, 750, 1,000, and 2,000 ng/mL. Quality control 937 (QC) samples were prepared in duplicate at the following concentrations: 50, 250, and 1,000 ng/mL. Each tube also contained a mixture of the internal standards, salvinorin A 4-CO2CD3 and salvinorin B 4-CO2CD3, in the amount of 2,000 ng each. Twenty-five microliters (1,000 μg) of the ethyl acetate dilutions of the DMSO–PEG400 solutions were trans- ferred to 16 × 100 mm silanized screw-top culture tubes, which had been supplemented with 2,000 ng of each of the internal standards. These samples were prepared in duplicate. Extraction and derivatization To each tube was added 1 mL of water and 4 mL of an ethyl acetate-heptane (1:3, v/v) mixture. The tubes were vortex-mixed for 5 min and centrifuged at 3,000 rpm for 5 min. The aqueous layer was frozen in a dry ice–acetone bath, the organic layer decanted into a clean, silanized tube, and the solvent evaporated in the centrifugal evaporator. To each tube was added 100 μL of a 5% (v/v) solution of acetic anhydride-d6 in methylene chloride (CH2Cl2) and 100 μL of 0.5% (w/v) N,N-dimethylaminopyridine in CH2Cl2. The tubes were capped and vortex-mixed for 2 h, and the reaction mixtures quenched by the addition of 1 mL of methanol and vortex mixing for another 5 min. After evaporation of the solvent the residue was partitioned between 4 mL of an ethyl acetate-heptane (4:1, v/v) mixture and 0.5 mL of 0.25 M sulfuric acid. Vortex-mixing for 5 min was followed by centrifuging for 5 min, freezing of the aqueous layer, decanting of the organic layer into a clean tube, and evaporation to dryness in the centrifugal evaporator. The residues were reconstituted in 200 μL of MeOH, filtered through 0.45 μm filters, and transferred to 200 μL autosampler vial inserts. The inserts were placed in vials, capped, and loaded onto the autosampler tray. Liquid chromatography LC analyses were performed on a Thermo Separation Products P4000 gradient pump. Separations were achieved on a Phenomenex Synergi Polar (4.6 × 150 mm, 4 μm particle size) reversed-phase HPLC column employing 0.1% formic acid and methanol as the mobile phase components. The solvents were degassed before use, employing a Thermo Separation Products SCM1000 vacu- um degasser equipped with the optional helium sparging module. The column was protected by the use of two Phenomenex Security Guard cartridges installed in tandem with the analytical column. The flow rate was 1 mL/min. Analyses were carried out with a linear gradient of 0.1% formic acid-methanol (50:50, v/v) to 100% methanol over a