Journal of Analytical Toxicology,Vol.
28, April 2004
[CaseReport
The Analysisof Methyl Salicylateand SalicylicAcid
from Chinese Herbal Medicine Ingestion
Dawn Parker, Christina Martinez, Christina Stanley, Jerry Simmons,and lain M. Mclntyre*
County of San Diego, Medical Examiner'sOffice, 5555 OverlandAvenue, Bldg.14, San Diego, California 92123-1270
Abstract I
This paper presents a multi-drug fatality in which methyl salicylate
was ingested. It is presented to inform the toxicological
community that a particularly expeditious method of detection for
methyl salicylate exists. Previously published methods for the
analysis of methyl salicylate include a gas chromatographic-mass
spectrometric method and an alkaline/acidic extraction followed
by high-performance liquid chromatographic (HPLC) analysis. This
article describes a method for analyzing methyl salicylate using
HPLC, in which a simple, rapid extraction procedure is used. Using
a previously published HPLC method, methyl salicylate and
salicylic acid were easily identified in biological specimens. Methyl
salicylate and salicylic acid were detected using an extraction
solution of acetonitrUe coupled with internal standard and then
analyzed by HPLC-diode-array detection. Because of its
concentrated liquid form, methyl salicylate ingestion can cause
rapid onset salicylate toxicity. As the potentially fatal methyl
salicylate forms are readily available and easily found on drugstore
shelves, the need to rapidly detect and quantitate salicylic acid
concentrations that are due to methyl salicylate ingestion may
arise. In the case presented, the peripheral blood concentration of
salicylic acid from methyl salicylate ingestion was 320 rag/L, and
the concentration in gastric contents was 820 rag. It alone was not
the cause of death, however. The discovery of the ability to detect
and quantitate methyl salicylate was due to its suspected ingestion.
Introduction
Methyl salicylate is a liquid methyl ester of salicylic acid with
a distinct characteristic odor. It is colorless or light pale in
color, and commonly known as synthetic oil of wintergreen
(made from the distillation of wintergreen leaves). Over-the-
counter methyl salicylate preparations can be found in cos-
metics, flavorings, and perfumes. The most familiar medicinal
use of methyl salicylate is as a counter-irritant, analgesic, and
local anesthetic for relief of aches, pain, and stiffness in muscles,
joints, and tendons.
In China, methyl salicylate self-poisoning is commonly seen as
a problem among the Vietnamese population (1). A common
Chinese herbal medicinal, Koong Yick Hung Far oil, has a
* Author to whom correspondence and reprint requests should be addressed.
214 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
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methyl salicylate concentration of 67% (2). An article published
in the Journalof Family Practicestates that 5 mL of oil of win-
tergreen is equal to 7000 mg of salicylate (3). The minimal
lethal ingestion can be as little as 4 mL in a child. Symptoms of
severe methyl salicylate overdose include hyperventilation, con-
vulsion, hypotension, hallucinations, metabolic acidosis, severe
cerebral edema, and coma (3).
Therapeutic concentrations of methyl salicylate, averaged 4.9
mg/L at 15 rain after a 0.42-mL oral dose (4,5). Methyl salicylate
is thought to be converted to salicylic acid, predominately by the
liver. Davison et al. (5) note that very little hydrolysis of methyl
salicylate occurs in human blood. Baselt (4) cites two cases of
adult postmortem salicylic acid concentrations of 615 mg/L and
1050 mg/L following methyl salicylate ingestion.
Case History
The decedent was a 58-year-old Vietnamese woman who lived
alone in an apartment. A brother of the decedent discovered the
body, and emergency services was called. The San Diego Police
and Fire departments responded. Paramedics confirmed death
at the scene. Police officers observed a strong "menthol-like"
odor, coffee cups containing clear liquid with a similar odor, and
several empty medication containers, including Chinese herbal-
medicine bottles, on a bedside table. All were submitted to the
Medical Examiner's Office for further examination.
The autopsy revealed fine, tan precipitate in the stomach.
Abundant dried, chalky, tan to focally mint green emesis mate-
rial was also found on the face and clothing. The precipitate in
the stomach and the emesis material both had a similar men-
thol-like odor, as did the Chinese herbal-medicine bottles. Sev-
eral pill fragments were also noted in the gastric contents.
There was evidence of a remote mastectomy, but no evidence of
recurrent, residual, or metastatic tumor. No significant nat-
ural disease or trauma was discovered to account for the death.
Materials and Methods
Chemicalsand reagents
Potassium phosphate, dibasic, was obtained from Spectrum
Journal of Analytical Toxicology,Vol. 28, April 2004

Chemical Manufacturing Corp (Gardena, CA).A 20raM phos-
phate buffer was prepared, weighing 2.72 g of potassium phos-
phate (dibasic)per liter of HPLC grade DI water. The pH, was ad-
justed to 2.9 using concentrated phosphoric acid, obtained from
MallinckrodtChemicals (Phillipsburg, NJ), solution was filtered
before use. The internal standard, 5-(p-methylphenyl)-5-phenyl-
hydantoin (MPPH)was obtained from Sigma-Aldrich(St. Louis,
Table I. Instrumentation Parameters for the Agilent 1100
Series HPLC System
Zorbax.EclipseXDB-C18,5mm,microbore2.1
X150 mm (RocklandTechnologies,Inc.,
Column Newport, DE) with (HP) guard column
MO), and prepared as a 1.0 mg/mL methanolic solution.
Methanol and acetonitrile were of EMScience Optima grade
(Darmstadt, Germany). The methyl salicylate standard was ob-
tained from Sigma-AldrichCo. (St. Louis, MO).
Extraction
Followinga previouslypublished method (6), methyl salicylate
was extracted from toxicological samples. The extraction solu-
tion was prepared by adding 1.0 mL of MPPH (1 mg/mL in
methanol) to 30 mL of acetonitrile. A 250-1JLaliquot of sample
was added to a 2-mL Eppendorf microtube (VWR Scientific
Products, Willard, OH), followedby a 500-mL aliquot of extrac-
tion solution. Samples were vortex mixed, allowed to stand for
10 rain, and then centrifuged at 3200 rpm for 10 rain. The top

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solution was transferred to autosampler vials, capped, and placed
Injection Size 12 pL into the autosampler for analysis.
Temperature 26~
DAD-Monitoring 200-350, 4 nm Instrumentation
UV Wavelength 230 nm and 208 nm High-performance liquid chromatographic (HPLC) analysis
Flow Rate 0.45 mL/min
was accomplished using an Agilent 1100 series HPLC system.
Mobile PhaseA O.02M Phosphatebuffer
Mobile PhaseB Acetonitrile The system consisted of a quaternary pump with degasser, au-
Gradient 5% B Initial tosampler, thermostatted column compartment, and a UV-vis
35% B 9 min DADdetector. Data were recorded and analyzedusing HP Chem-
40% B 18 min station software. Instrumentation parameters are shown in
45% B 20 rain Table I.
55% B 24 min
5% B 27 min Identification and calculation
Run Time 30 rain with 5 min posttime Compounds were identified and confirmed by retention time;
retention time relating to the internal standard (• 0.03 rain);
and a UV spectra library match no less than
Table II. Toxicological Screening Results 95%. A calibration curve was calculated using
the relative peak areas of the methyl salicylate
Analysis Specimen Instrumentation Results and salicylic acid calibrators, compared to that
of the internal standard. The resulting linear
Alcohols Peripheral blood GC Negative regression utilized all data points and included
Drugs of abuse Central blood Gamma counter Negative zero. The relative retention times of methyl sal-
icylate (under these conditions) was 14.9 rain;
Basic drugscreen Peripheralblood GC-MS Carbamazepine, the retention time of the internal standard
Trazodone,Quetiapine (MPPH) was 13.5 rain.
metabolite,Trihexyphenidyl
Basic drugscreen Gastric contents GC-MS Carbamazepine,
Trazadone,Quetiapine
metabolite,Trihexyphenidyl, Results and Discussion
Ibuprofen, Salicylic acid
Acidic/neutraldrugs Peripheralblood HPLC Carbamazepine (25 mg/L),
Toxicological screening analyses were con-
Salicylic acid (320 mg/L) ducted on postmortem peripheral (pb), central
blood (cb) and urine collected at autopsy, in-
Acidic/neutraldrugs Gastriccontents HPLC Methyl salicylatedetected, cluding radioimmunoassay (RIA)screening for
Quetiapine (270 mg), drugs of abuse; gas chromatography (GC) for
Trazodone(770 rag), the analysis of alcohol; urine drug screen (Toxi-
Carbamazepine(130 mg),
lab); acid/neutral analysisby HPLCand capillary
Salicylic acid (820 mg)
GC with mass spectral confirmation or basic
Urine drugscreen Urine Toxi-Lab Carbamazepine, substances. The salicylic acid concentration
Trazodone,Quetiapine quantified in the gastric contents was deter-
metabolite,Trihexyphenidyl mined to be 820 rag. The concentration of sali-
Quantitations Peripheral blood HPLC Quetiapine (0.88 mg/L), cylicacid in the peripheral blood was 320 mg/L.
Trazodone(2.38 mg/L) Other significanttoxicologicalfindingsare sum-
marized in Table II.

215

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Time (rain)
Figure 1. Chromatogram A: methyl salicylate in gastric sample. Chro-
matogram B: methyl salicylatefrom (KYHF)evidence.ChromatogramC:
methyl salicylatestock (Sigma-Aldrich).
Chinese herbal medicine bottles were identified by labeling as
Koong Hung Yick Far oil (KHYF) containing 67% wintergreen
oil and Po Sum On oil (PSO) containing 15% menthol.
The gastric contents and KHYF evidence were analyzed for
verification of methyl salicylate content. A comparison was
made between a methyl salicylate stock standard with that of
the gastric contents and KHYF evidence (Figure 1). The KHYF
evidence was analyzed for verification of methyl salicylate con-
tent and found to contain a very high concentration of methyl
salicylate. Because of its concentration, a 1:100,000 dilution
was necessary to bring the methyl salicylate peak to scale with
the internal standard; no concentration was determined from
this analysis. The other bottle (PSO) was found to contain no
methyl salicylate.
216
Journal of Analytical Toxicology, Vol. 28, April 2004
Because methyl salicylate is potentially more toxic than sal-
icylic acid, it was necessary to determine its presence. In this
A
case, the presence of methyl salicylate in the gastric contents
confirmed that a potentially fatal amount of methyl salicylate
was ingested in conjunction with other medications. The po-
tential for early discovery of methyl salicylate exists in cases in
which detection can prevent possible future fatalities. The pur-
pose of this abstract is to provide the toxicological community
an alternate method for the expeditious detection of methyl sal-
~ . . . . ~ . . . .
icylate with the simultaneous quantitation of salicylic acid. The
cause of death in this case was an acute multi-drug intoxication,
and the manner of death was suicide. Obviously, the rapid de-
tection of methyl salicylate in this case was relatively non-im-
portant; however, its discovery played a role in determining
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the manner of death.
"
.
C
Acknowledgment
We wish to acknowledge Brian D. Blackbourne, M.D., Chief
Medical Examiner, County of San Diego, for his assistance in
making the biological and evidential specimens available for
analysis.
~ " " ~ ....
References
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to Chinese proprietary medicines. Hum. Exp. Toxicol. 14(5):
434-436 (1995).
2. T.Y. Chan. Medicated oils and severe salicylate poisoning: quan-
tifying the risk based on methyl salicylate content and bottle size.
Vet. Hum. Toxicol. 38:133-134 (1996).
3. W.L. Cauthen and W.H. Hester. Accidental ingestion of oil of win-
tergreen. J. Fam. Pract. 29:680-681 (1989).
4. R.C. Baselt. Disposition of Toxic Drugs and Chemicals in Man, 6th
ed. Biomedical Publications, Foster City, CA, 2002, pp 694-695.
5. C. Davison, E.F.Zimmerman, P.K. Smith. On the metabolism and
toxicity of methyl salicylate. J. Pharm. Exp. Ther. 132:207-211
(1961).
6. O.H. Drummer, A. Kotsos, and I.M. Mclntyre. A class independent
drug screen in forensic toxicology using a photodiode array
detector. J. Anal. Toxicol. 17:225-229 (1993).
Manuscript received February 3, 2003;
revision received June 25, 2003.