FORENSIC TOXICOLOGY - Bio Medical Forensics

including equilibration time. MS/MS analysis was conducted using a
tandem mass spectrometer equipped with ESI in negative ion mode for
THC-COOH/ D3-THC-COOH and was operated with multiple reaction
monitoring (MRM) under the following conditions: curtain gas 15,
collision gas medium, ion spray voltage -4500V, temperature 650 °C, ion
source gas(1) 50, ion source gas (2) 50. The following transitions were
monitored (quantification ions underlined): m/z 343.1 → 299.3 and
245.3 for THC-COOH, and m/z 346.1 → 302.3 and 248.3 for D3-THC-
COOH. Positive ion mode was employed for THC/ D3-THC under the
following conditions: curtain gas 15, collision gas medium, ion spray
voltage 5000V, temperature 650 °C, ion source gas(1) 50, ion source gas
(2) 50. The following transitions were monitored (quantification ions
underlined): m/z 315.2 → 193.2 and 123.1 for THC, and m/z 318.2 →
196.2 and 123.1 for D3-THC.
Linearity (r 2 >0.99) was achieved from 0.25 ng/mL to 50 ng/mL,
(THC/ THC-COOH) and the limits of detection were determined to be
0.1 ng/mL for THC and 0.25 ng/mL for THC-COOH, respectively. The
limits of quantification were 0.25 ng/mL for THC and 0.5 ng/mL for
THC-COOH, respectively. Recoveries were > 92% for THC and > 87%
for THC-COOH, respectively measured at a target value of 4.0 ng/mL.
Intra and inter-day precision was less than 7% and 11%, respectively for
THC and less than 8% and 12%, respectively for THC-COOH. Ion
suppression studies revealed that suppression of monitored ions was less
than 6%.
This SPE method coupled with and fast LC-MS/MS provides a
simple, sensitive, and reproducible quantitative method for the analysis
of THC and its primary metabolite in whole blood. This procedure
should be of great assistance to those analysts actively involved with the
LC-MS/MS analysis of these drugs in biological matrices.
THC and Metabolite, Solid Phase Extraction, LC-MS/MS
K3 Simultaneous Quantification of Twenty
Common Drugs of Abuse and Metabolites in
Human Meconium by LCMSMS
Teresa R. Gray, MS*, National Institute on Drug Abuse, Chemistry and
Drug Metabolism, IRP, NIH, 251 Bayview Boulevard, Suite 05A406,
Baltimore, MD 21224; Diaa M. Shakleya, PhD, National Institute on
Drug Abuse, 251 Bayview Boulevard, Suite 05A729.02, Baltimore, MD
21224; and Marilyn A. Huestis, PhD, National Institute on Drug Abuse,
Chemistry & Drug Metabolism, Intramural Research, National Institute
on Drug Abuse, NIH, 251 Bayview Boulevard, Suite 05A721A,
Baltimore, MD 21224
After attending this presentation attendees will be introduced to a
liquid chromatography tandem mass spectrometry (LCMSMS) method
for simultaneous quantification of common drugs of abuse in human
meconium.
This presentation will impact the forensic community by offering a
novel analytical method for sensitive and specific simultaneous
quantification of 20 analytes in a single extraction and small meconium
specimen, offering time and resource savings.
Drug abuse during pregnancy is associated with adverse obstetrical
and neonatal outcomes. Detection of in utero drug exposure is often
accomplished by meconium analysis due to ease and non-invasiveness of
specimen collection and a long window of drug detection. However, the
amount of meconium is often limited, prohibiting multiple assays for
different drugs of abuse. Attendees will be introduced to a liquid
chromatography tandem mass spectrometry (LCMSMS) method for
simultaneous quantification of common drugs of abuse in human
meconium.
An LCMSMS method for the simultaneous quantification of
amphetamine (AMP), methamphetamine (MAMP), p-
* Presenting Author
hydroxymethamphetamine (pOHMAMP), cocaine (COC),
benzoylecgonine (BE), cocaethylene (CE), m-hydroxybenzoylecgonine
(mOHBE), nicotine (NIC), cotinine (COT), 3’-trans-hydroxycotinine
(OHCOT), morphine (MOR), 6-acetylmorphine (6AM), codeine (COD),
hydromorphone (HYM), hydrocodone (HYC), oxycodone (OXY),
methadone (MTD), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine
(EDDP), buprenorphine (BUP), and norbuprenorphine (NBUP) in
meconium in only 0.25 g of meconium was developed and validated.
Meconium specimens (0.25 g) fortified with deuterated internal
standards were homogenized in acidic methanol. After centrifugation
and supernatant evaporation, analytes were isolated using mixed mode
solid phase extraction and analyzed by LCMSMS operating in positive
multiple reaction monitoring (MRM) mode. Two analytical runs
utilizing the same extract were required: a 5-μL injection, 18 minute run
with gradient elution that quantified all analytes except BUP and NBUP.
These two analytes were measured in a second 5 min isocratic run with
a 10-μL injection volume to enhance sensitivity. The analytical method
was validated over four days for limits of quantification, recovery,
imprecision, extraction efficiency, matrix effects, carryover, and
endogenous and exogenous interference.
Limits of quantification were 1 ng/g for COT, CE, BE, and COC,
2.5 ng/g for MAMP, EDDP, MTD, and pOHMAMP, 5 ng/g for AMP,
mOHBE, NIC, OHCOT, MOR, 6-AM, HYM, HYC, OXY, and 25 ng/g
for BUP and NBUP. The upper limit of quantification for all analytes
was 500 ng/g, except for pOHMAMP at 250 ng/g. Correlation
coefficients for each calibration curve were >0.996 with all calibrators
quantifying within ±20% of target when calculated against the
calibration curve. Validation parameters were tested at three
concentrations spanning the linear dynamic range. Intra- and inter-day
recovery ranged from 83.3 – 126.6% and 80.1 – 129.0%, respectively.
Inaccuracies of up to 30% were considered acceptable due to
meconium’s complexity. Intra- and inter-day imprecision ranged from
0.9 – 16.9% relative standard deviation (RSD) and 3.1 – 9.8% RSD,
respectively. Extraction efficiencies ranged from 46.7 – 96.0%. Matrix
effects ranged from -305.7 – 40.7%, depending on the analyte, with
negative values indicating ion enhancement. Matrix effects at each
quality control concentration were similar for native and corresponding
deuterated compounds, highlighting the importance of employing
matched deuterated internal standards in LCMSMS quantification
procedures, especially with complex matrices. Similar results were
observed for matrix effects determination in seven different blank
meconium sources fortified with low quality control concentrations;
while matrix effects varied between meconium specimens, matrix
enhancement or suppression of related native, and deuterated compounds
were similar and quantification was within acceptable limits. Analyte
stability was assessed under the following conditions: 24 h at room
temperature, 72 h at 4°C, three -20°C freeze-thaw cycles, and 48 h on the
15°C autosampler. Losses of less than 34.0% were observed for each
condition, except for 6AM and MOR. After room temperature, 4°C, and
three freeze-thaw cycles, up to 85.8% of 6AM was lost; however, MOR
concentrations under these conditions increased by up to 31.2%. In cases
of suspected heroin exposure, meconium should be immediately frozen
and repeated freeze thaw cycling should be avoided. No analyte
carryover was observed at two times the upper limit of quantification.
No interference by 57 illicit and therapeutic drugs or endogenous
meconium compounds was observed. Method applicability for all
analytes except 6AM, BUP, and NBUP was demonstrated by analysis of
meconium from drug-exposed neonates.
The most comprehensive chromatographic method for the
identification and quantification of drugs and metabolites in meconium
is described. This LCMSMS method will impact the clinical and
forensic community by offering a novel analytical method for sensitive
and specific simultaneous quantification of 20 analytes in a single
extraction and small meconium specimen, offering time and resource
savings. This method will be employed in prenatal drug exposure
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