Determination of Salvinorin A in body fluids by high performance liquid chromatography–atmospheric pressure chemical ionization

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Abstract

Salvinorin A was quantitated in human and rhesus monkey plasma, rhesus monkey cerebrospinal fluid, and human urine by negative ion LC–MS/APCI. The method for Salvinorin A has been fully validated, the LLOQ using FDA guidelines is 2 ng/mL for 0.5 mL plasma samples. The linear range was from 2 to 1000 ng/mL. Several derivatives in the Salvinorin family can also be analyzed by this method; d3-Salvinorin A was prepared and used as internal standard. The metabolite Salvinorin B can be semi quantitatively determined. The method has been used to establish that Salvinorin B is the principal metabolite of Salvinorin A ex vivo and to establish the analytical method to study in vivo samples.

Introduction

The Mexican mint, Salvia divinorum, is a hallucinogenic plant whose availability has recently increased rapidly, partially due to internet trading [1]. Among the structurally related compounds found in the plant are Salvinorin A, 1, and Salvinorin B, 2 (Fig. 1). At present, only 1 appears to be pharmacologically active. Currently the plant is unregulated and legally available in all countries except Denmark, Italy, and Australia. The leaves of the plant and various extracts and tinctures are readily available over the internet [2]. The active ingredient, Salvinorin A, is typically self-administered in humans either by smoking or buccal absorption [3], [4]. Salvinorin A based products can produce profound hallucinations lasting up to 1 hour [3], [5]. It is expected that misuse of Salvia divinorum based products will increase rapidly [1].

Rather than affecting the serotonin 5-HT2A receptors as do classical hallucinogens, 1 is a potent, efficacious, and selective κ opioid receptor agonist [6], [7], [8]. Selective synthetic κ opioid receptor agonists have been explored as analgesics with potential for reduced dependence and tolerance. However, dysphoria, diuresis, and psychotomimesis are associated with their administration in clinical studies in humans [9], [10]. A growing body of evidence indicates that κ receptors may be involved in the modulation of some abuse related effects of CNS stimulants [11], [12], [13], [14].

κ Agonists are able to modulate the behavioral and neurochemical effects of cocaine [11]. They appear to attenuate cocaine's discriminative stimulus properties, its conditioned reinforcing effects, and its self-administration. κ Agonists also lowered the reinstatement of cocaine-reinforced responding. The findings indicate the endogenous κ opioid receptor/dynorphin system may be involved in the physiological modulation of some abuse related effects of cocaine, offering a valuable pharmacological target to treat cocaine abuse or its relapse. However, while κ opioid receptor agonists are effective in reducing cocaine self-administration in monkeys, they produce side effects including sedation and vomiting [11]. It has been speculated that the addition of μ agonist/antagonist activity to the κ agonist might lessen the incidence of side effects and encompass a useful treatment for cocaine abuse [11].

A method for determining 1 in plant tissues by HPLC with UV detection exists [15]. The presently reported method is 3 orders of magnitude more sensitive than the previously reported method. At present, there are no methods available for the detection of 1 and related analogues in biological fluids. In addition, the identities of metabolites of 1 are unclear. In order to support the study of the disposition of 1 in vivo, we set out to develop a method to determine the concentration of 1 in biological fluids.

Section snippets

Chemicals and solutions

Salvinorin A and B were extracted from Salvia divinorum as previously described with modifications [16], [17], [18], [19]. d3-Salvinorin A, 1A, was prepared from Salvinorin A [19]. Purities of the above were >98%. Salvinorin A for use in monkey studies was commercially obtained from Biosearch (Novato, CA, USA) and was approximately 92% pure, the residual was mostly Salvinorin B. ACS grade ammonium hydroxide, dimethylsulfoxide, dichloromethane, and Optima HPLC grade acetonitrile were purchased

Results and discussion

The Salvinorin concentrations were calculated from the peak-area ratio of 1 or 2 to 1A for standards and samples. Peak areas of standards taken through solid phase extraction were approximately 102% those of standards evaporated and reconstituted. The linear least squares equations were calculated with 1/C weighting. For 1, the mean equation of the linear portion of the curve derived from human plasma was 0.0145x + 0.0264 (R.S.D. slope 5.4%, R.S.D. intercept 9.1%), the correlation coefficient was

Conclusion

A sensitive method for the determination of Salvinorin compounds in body fluids has been developed and its utility demonstrated in an ex vivo pharmacokinetic study supporting the feasibility of future in vivo investigations. This is the first study to demonstrate the formation of Salvinorin B as a metabolite of Salvinorin A in biological fluids. The ex vivo data suggests that Salvinorin A is metabolized by blood esterases primarily to Salvinorin B.

Acknowledgements

Studies were supported in part by NIH-NIDA grants DA11113 (ERB) and DA05130 and DA00049 (MJK).

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