FORENSIC TOXICOLOGY - Bio Medical Forensics

of the plant are typically dried, crushed, and smoked for their
dissociative hallucinogenic effect. Plant concentrate or extract is also
commercially available. Salvia is a potent hallucinogen with effects
distinct from LSD, mescaline, and other hallucinogens. An effective
dose in humans is reportedly in the 200 to 1,000 microgram range when
smoked. Salvinorin A and Salvinorin B have both been identified in the
leaf and leaf extract; however, Salvinorin B is present in much smaller
amounts. The Salvinorin A and Salvinorin B contents have been
determined to be in the range of 3.2–5.0/0.10–0.17 mcg/mg in the dried
leaf products, and 4.1–38.9/0.26–2.42 in the “concentrated
extract” products.
After smoking Salvia, subjects experience rapid onset of an intense
hallucinatory dissociative effect, during which they cannot speak or
recognize their surroundings, lose psychomotor coordination and are
highly impaired. Acute symptoms resolve within 8 to 12 minutes;
however, longer term and residual effects have not been studied.
A validated a liquid chromatography/tandem mass spectrometry
(LC/MS/MS) method was developed for the identification and
quantitation of Salvinorin A and B in human blood, plasma, and urine.
Salvinorin A and B were extracted from biological matrices treated
and preserved with sodium fluoride by a single step liquid/liquid
extraction. Salvinorin A was analyzed under positive mode ESI-
LC/MS/MS and Salvinorin B was analyzed under negative mode ESI
LC/MS/MS (ABI 5000 Tandem Mass Spectrometer, Shimadzu SIL 20A,
HPLC). Ions monitored for Salvinorin A and its internal standard
Salvinorin A-d3 are: m/z 433/373; 436/373. Ions monitored for
Salvinorin B and its internal standard are: m/z 389/313; 391/359. HPLC
conditions included 2% methanol in water gradient, vs water, at
1mL/min, on a Phenomenex Luna C8(2) 150cm column.
The linear range for this assay was established as 1–40 ng/mL for
whole blood, plasma and urine. Response was linear, and the LLOQ was
established at 1 ng/mL for both analytes. LLOD was approximately
0.25ng/mL. Within-run precision at the LLOQ was 3.2 % for Salvinorin
A and 2.5% for Salvinorin B. The within-run accuracy was determined
as 100±5% for both Salvinorin A and B.
Following development, the assay was validated according to
laboratory procedure including assessment of inter- and intra- batch
precision and accuracy, storage, extraction and autosampler stability,
freeze thaw stability, dilution integrity, and recovery.
The stability experiments indicated that Salvinorin A and B in
unpreserved urine were stable for 28 days refrigerated and frozen,
Salvinorin A was stable for less than nine days at room temperature.
Both compounds were unstable in sodium fluoride/potassium oxalate
preserved whole blood at room temperature and refrigerated, being
undetectable after one day. Samples that were preserved with sodium
fluoride and EDTA and frozen, were stable for at least 28 days.
Challenges resulting from limited stability and likely low
concentrations in human subjects make this a challenging assay for
medico-legal applications and require the use of LC/MS/MS techniques.
Salvia Divinorum, Salvinorin A, LC/MS/MS
K32 Quantitative Analysis of Salvinorin A:
(Salvia) in Blood
Lyndsi J. Ayers, MS*, Sam Houston State University, 1003 Bowers
Boulevard, Huntsville, TX 77341; and Sarah Kerrigan, PhD, Sam
Houston State University Regional Crime Laboratory, 8301 New Trails
Drive, Suite 125, The Woodlands, TX 77341
After attending this presentation, attendees will be familiar with a
technique for the extraction and quantification of Salvinorin A from
blood specimens using solid phase extraction (SPE) and gas
chromatography/mass spectrometry (GC/MS).
This presentation will impact the forensic science community by
providing a new procedure for both qualitative and quantitative
determination of the potent hallucinogen, Salvinorin A, from whole
blood samples using GC/MS.
Salvia divinorum is a naturally occurring herb found within the
Lamiaceae (mint) family. Salvinorin A is the trans-neoclerodane
diterpene contained within its leaves that produces the plant’s
psychotropic properties. The drug is a potent naturally occurring
hallucinogen. The availability and psychotropic effects associated with
Salvinorin A have led to an increase in its use within the past decade.
Studies have shown a growing trend in the Salvia divinorum-related
media and internet traffic, as well as the use of the drug in persons age
12 or older. Salvinorin A is listed on the United States Drug
Enforcement Administration’s Drugs and Chemicals of Concern List but
is not currently scheduled under the Federal Controlled Substances Act.
Many states and other countries have already scheduled the drug and
some are currently in the process.
Despite growing concerns regarding the recreational use of Salvia
divinorum, published scientific literature describing Salvinorin A
identification in toxicological specimens is very limited. Liquid
chromatography/mass spectrometry (LC/MS) and related techniques
have been reported. The objective for this study was to develop and
validate a method for qualitative and quantitative identification of
Salvinorin A in whole blood using a technique that was universally
available in forensic toxicology laboratories (i.e. GC/MS). The
development of the procedure evaluated potential protein precipitants,
wash solvents, and elution solvents. The assay involves protein
precipitation with 0.2 M zinc sulfate/methanol (20/80, v/v), mixed-mode
solid phase extraction, a double wash step using hexane followed by
hexane/dichloromethane (90/10, (v/v) and dichloromethane/ethyl acetate
(80/20, v/v) as the elution solvent. Testosterone-d3 was used as the
internal standard, and quantification was performed in selective ion
monitoring mode. The ions selected were m/z 432, 273, and 94 for
Salvinorin A and m/z 291, 249, and 124 for testosterone-d3 (quantitation
ions underlined). The total run time was 23 minutes and the retention
times for Salvinorin A and testosterone-d3 were 10.655 and
5.479, respectively.
The optimized GC/MS assay was evaluated in terms of limit of
detection (LOD), limit of quantification (LOQ), precision, accuracy,
analytical recovery, linearity, interference, and carryover. The LOD and
LOQ for the assay were determined to be 2 ng/mL. Precision and
accuracy were evaluated at 15 and 150 ng/mL in blood. Both intra- and
inter-assay CVs were in the range 4.5 - 7.7%. The 95% confidence
intervals (95% CI) at 50 and 1000 ng/mL were 55.8 ± 5.3 and 1059.2 ±
43.5 ng/mL, respectively. Accuracy determined over a range of
concentrations was 87-104% and analytical recovery of Salvinorin A was
88%. Calibrations were linear at concentrations as high as 5,000 ng/mL
(the highest concentration tested). Carryover was evident at 2,000
ng/mL but this greatly exceeds concentrations expected in blood samples
of forensic interest, which are typically one hundred-fold lower. The
interference study included 27 commonly encountered drugs of abuse in
addition to other her structurally related Salvinorins and divinatorins.
No interferences were present, either qualitatively or quantitatively. This
presentation provides a reliable and effective method for the detection
and analysis of salvinorin A in whole blood by GC/MS at low
concentrations of forensic interest.
Salvinorin A, Gas Chromatography/Mass Spectrometry (GC/MS), Blood
17 * Presenting Author