Quantitative measurement of acetyl fentanyl and acetyl norfentanyl in human urine by LC-MS/MS

Article abstract of DOI:10.1021/ac4036197

Opioid abuse involving emerging opioid compounds is a growing public health problem, which was highlighted recently by cases of human morbidity and mortality linked to acetyl fentanyl abuse. Unfortunately, the lack of information available on the toxicology and metabolism of acetyl fentanyl precludes its detection in human samples. The following study was conducted to test a new analytical procedure for the simultaneous quantification of acetyl fentanyl and its predicted metabolite, acetyl norfentanyl, in human urine. Metabolic reference standards and deuterium-labeled internal standards were synthesized for use in an assay that coupled solid-phase extraction (SPE) with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The accuracy (% Relative Error <5%) and inter- and intrarun precision (%CV <20%) of this new method resulted in low levels of quantification (～1 ng/mL). Similar results were obtained using liquid chromatography columns manufactured with phenyl-hexyl and biphenyl stationary phases (r² > 0.98). Preliminary human liver microsomal and in vivo rodent studies demonstrated that acetyl fentanyl is metabolized by cytochrome P450s to acetyl norfentanyl. Urine samples from rats treated with a toxic dose of acetyl fentanyl contained high concentrations of acetyl fentanyl and acetyl norfentanyl. Further toxicokinetic studies are required to fully elucidate the metabolic pathways responsible for acetyl fentanyl detoxification and excretion.

Full text of DOI:10.1021/ac4036197

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Article
Quantitative Measurement of Acetyl Fentanyl and
Acetyl Norfentanyl in Human Urine by LC-MS/MS
Amy Leah Patton, Kathryn A. Seely, Sharon Pulla, Nancy J. Rusch, Cindy
L. Moran, William E. Fantegrossi, Laura D. Knight, Jeanna M. Marraffa, Paul
Douglas Kennedy, Laura P. James, Gregory W. Endres, and Jeffery H. Moran
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Dec 2013
Downloaded from http://pubs.acs.org on December 22, 2013
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Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth
Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
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of their duties.

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Quantitative Measurement of Acetyl Fentanyl and Acetyl Norfentanyl in Human
Urine by LCꢀMS/MS
‡
‡
§
§
#
Amy L. Patton , Kathryn A. Seely , Sharon Pulla , Nancy J. Rusch , Cindy L. Moran ,
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¥
Ө
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William E. Fantegrossi , Laura D. Knight , Jeanna M. Marraffa , Paul D. Kennedy ,
^
||
‡,§
Laura P. James , Gregory W. Endres , and Jeffery H. Moran *
‡
Arkansas Department of Health, Public Health Laboratory, Little Rock, Arkansas,
§
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2205 United States; Department of Pharmacology & Toxicology, College of Medicine,
University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205 United
#
States; Arkansas State Crime Laboratory, Little Rock, Arkansas, 72205 United States;
¥
Onondaga County Medical Examiner's Office, Syracuse, New York, 13210 United
Ө
States; Upstate New York Poison Center, SUNY Upstate Medical University,
||
Syracuse, New York, 13210 United States; Cayman Chemical Co., Ann Arbor,
^
Michigan, 48108 United States; Section of Clinical Pharmacology and Toxicology,
Arkansas Children’s Hospital and Department of Pediatrics, College of Medicine,
University of Arkansas for Medical Sciences and Arkansas Children’s Hospital, Little
Rock, Arkansas, 72205
*
Corresponding Author: Jeffery H. Moran, PhD, Arkansas Department of Health,
Public Health Laboratory, 201 S. Monroe Street, Little Rock, AR, 72205, USA. Phone:
(501) 661ꢀ2826; Fax: (501) 661ꢀ2972; email: jeffery.moran@arkansas.gov.
Keywords: Acetyl fentanyl, emerging drug of abuse, human testing, human metabolism
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ABSTRACT
Opioid abuse involving emerging opioid compounds is a growing public health
problem, which was highlighted recently by cases of human morbidity and mortality
linked to acetyl fentanyl abuse. Unfortunately, the lack of information available on the
toxicology and metabolism of acetyl fentanyl precludes its detection in human samples.
The following study was conducted to test a new analytical procedure for the
simultaneous quantification of acetyl fentanyl and its predicted metabolite, acetyl
norfentanyl, in human urine. Metabolic reference standards and deuteriumꢀlabeled
internal standards were synthesized for use in an assay that coupled solidꢀphase
extraction (SPE) with liquid chromatographyꢀtandem mass spectrometry (LCꢀMS/MS).
The accuracy (% Relative Error <5%), interꢀ and intraꢀrun precision (%CV <20%) of this
new method resulted in low levels of quantification (~ 1 ng/ml). Similar results were
obtained using liquid chromatography columns manufactured with phenylꢀhexyl and
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biphenyl stationary phases (r > 0.98). Preliminary human liver microsomal and in vivo
rodent studies demonstrated that acetyl fentanyl is metabolized by cytochrome P450 to
acetyl norfentanyl. Urine samples from rats treated with a toxic dose of acetyl fentanyl
contained high concentrations of acetyl fentanyl and acetyl norfentanyl. Further
toxicokinetic studies are required to fully elucidate the metabolic pathways responsible
for acetyl fentanyl detoxification and excretion.
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INTRODUCTION
Abuse of prescription opioids is an ongoing epidemic that continues to burden
the public health system. Since 2001, opioid abuse has been of great concern and
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2.2% of high school seniors report use of prescription narcotics for nonꢀmedical
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reasons. In addition, unintentional poisoning deaths involving opioid analgesics have
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increased over the last decade. For example, deaths from opioid pain relievers
increased fiveꢀfold in women between 1999 and 2010, and since 1997, opioidꢀrelated
drug overdoses exceeded motor vehicle collisions as the cause of death in this
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population. The etiology of increased opioidꢀrelated deaths is multifactorial and
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includes readyꢀtoꢀuse heroin and fentanyl tablet formulations and synthetic opioid
derivatives that circumvent analog drug laws and detection in routine toxicological
assays. For example, United States’ analog laws were implemented in response to
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“China White” (4ꢀmethylfentanyl) as early as the 1980s , but now acetyl fentanyl is the
‘new emerging’ drug of abuse that escapes detection and is linked to over 50 human
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fatalities in Rhode Island and Pennsylvania.
Acetyl fentanyl has never been
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approved for human use and is considered a controlled substance analogue.
When faced with an unsuspected acetyl fentanyl death, forensic toxicology
laboratories may be perplexed when enzymeꢀlinked immunosorbent assay (ELISA)
screens are positive for fentanyl, yet confirmatory gas chromatographyꢀmass
spectrometry (GCꢀMS) and/or liquid chromatographyꢀmass spectrometry (LCꢀMS)
analyses yield no fentanyl result. Thus, development of a validated testing procedure to
quantify acetyl fentanyl and acetyl norfentanyl in human samples is critical to accurately
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diagnosing human morbidity and mortality from this deadly emerging drug of abuse.
This study reports a liquid chromatographyꢀtandem mass spectrometry (LCꢀMS/MS)
analytical method for acetyl fentanyl and the predicted human metabolite acetyl
norfentanyl.
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EXPERIMENTAL SECTION
Reagents and Chemicals
Acetyl fentanyl, acetyl norfentanyl, acetyl fentanylꢀd , and acetyl norfentanylꢀd5
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standards were provided by Cayman Chemical (Ann Arbor, MI). Sodium acetate
trihydrate, ammonium formate, and βꢀglucuronidase were purchased from Sigmaꢀ
Aldrich (St. Louis, MO). Solid phase extraction was performed using Strata XꢀDrugꢀB
polymeric strong cationꢀexchange cartridges provided by Phenomenex (Torrance, CA).
Pooled human urine void of synthetic opioids is maintained by the Arkansas Public
Health Laboratory and routinely used as a drugꢀfree blank sample for various clinical
analyses. Deionized water was purified to resistivity of 18.2 mꢁꢀcm using an Elga
Purelab Ultra laboratory water purification system (Woodridge, IL). Optima grade
acetonitrile and other analytical grade chemicals were obtained from Fisher Scientific
(Pittsburgh, PA).
Equipment
Aliquots of all specimens and standards were extracted using solid phase
extraction (SPE) on a Gilson Nebula 215 solidꢀphase extraction system (Middleton, WI)
controlled by Gilson 735 Sampler software. Extracted samples were analyzed using an
Agilent 1200 series quaternary liquid chromatography system (Santa Clara, CA)
interfaced with an APIꢀ4000 QꢀTrap tandem mass spectrometer (AB SCIEX,
Framingham, MA). The operation of the LCꢀMS/MS system was controlled by Analyst
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software (version 1.5.1, AB SCIEX, Framingham, MA).
Preparation of Analytical Standards and Quality Control Material
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Analytical calibration standards and quality control (QC) material were prepared
from a common aqueous stock solution containing 10 ꢂg/mL of acetyl fentanyl and
acetyl norfentanyl. An internal standard spiking solution (100 ng/ml final concentration of
each analyte) was prepared in deionized water from a common aqueous stock solution
containing 10 ꢂg/mL of acetyl fentanylꢀd and acetyl norfentanylꢀd . All analytical and
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internal standard solutions were stored at ꢀ40°C until needed.
All calibration standards and QCs were matrix matched by first preparing a 500
ng/mL intermediate working standard in pooled, blank human urine. Final working urine
standards (0 ng/mL to 100 ng/mL) were prepared daily by serially diluting the
intermediate working standard with blank urine. QCs were prepared in urine
independent of calibration standards and at three concentrations spanning the
calibration range (QCL, QCM, and QCH).
βꢀGlucuronidase Treatment & Solid Phase Extraction
Sample hydrolysis procedures were incorporated as part of this new analytical
method because several synthetic opioids like morphine are conjugated with glucuronic
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acid prior to urinary excretion. Thus, it is important to validate testing procedures to
ensure they are capable of using hydrolyzed urine samples. All standards, QCs, and
samples were subjected to βꢀglucuronidase treatment prior to solidꢀphase extraction
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(
SPE). Urine standards, QCs, and samples were prepared identically by diluting 80 ꢂL
aliquots with 310 ꢂL of 0.1 M sodium acetate buffer (pH 5.0) containing βꢀ
glucuronidase, spiking with 10 ꢂL of the internal standard solution, and incubating for 60
minutes at 37°C.
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The entire reaction mixture (400 ꢂL) was loaded onto the polymeric strongꢀ
cationic exchange solid phase extraction cartridge (Strata XꢀB 33 ꢂ, 30 mg/3mL),
washed with 1 mL of a 0.1% formic acid aqueous solution and 1 mL of a 70:30
water:methanol solution. Analytes were eluted twice with 0.5 mL of a basic 50:50
methanol:acetonitrile solution (2% ammonium hydroxide). The collected eluent was
evaporated at 60°C under a stream of nitrogen until dry, and analytes were
reconstituted in a 50:50 mixture of mobile phase (85% mobile phase A and 15% mobile
phase B) and acetonitrile. Mobile phase A (MPA) consisted of 10 mM ammonium
formate in water and mobile phase B (MPB) consisted of 0.1% formic acid in methanol.
Liquid Chromatography Tandem Mass Spectrometry
The primary LCꢀMS/MS method reported here utilizes a Phenomenex phenylꢀ
hexyl (2.6 ꢂm, 100 Å) analytical column (50 mm x 4.6 mm) heated to 30°C. Analytes are
resolved using a gradient starting at 98% MPA, ramping to 2% MPA over 4 min, holding
for 2 min, and returning to 98% MPA for an additional 2 min. The total run time was 8
min, including the column equilibration period between injections. Second column
confirmation studies were completed using a Restek Raptor biphenyl (2.7 ꢂm) analytical
column (50 mm x 3.0 mm). This column required a higher flow rate (800 uL/min), but
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used the same mobile phases. Analytes of interest were resolved over a 5 minute total
runꢀtime, with a gradient starting at 98% MPA, ramping to 2% MPA over 1.5 minutes,
holding for 1.5 minutes, and returning to 98% MPA for an additional 2 minutes.
Significant carryover was observed with both columns following the evaluation of highly
concentrated samples. Carryover was controlled by injecting a series of blanks after
each high standard, QC, and unknown sample.
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Electrospray ionization in positiveꢀion mode was used to collect mass spectral
data. Source temperature and turbo ion spray voltage were maintained at 650°C and
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,500 V, respectively. Nitrogen gas settings for the source gases GS1 and GS2, curtain
gas and collision gas were 50.0 cm/s, 50.0 cm/s, 20.0 cm/s, and “medium”, respectively.
Molecular specific parameters for specific reaction monitoring (SRM) experiments are
listed in Table 1. The SRMꢀinformation dependent acquisition (IDA) transition threshold
that triggered enhanced product ion (EPI) experiments was set to an intensity of 500
counts per second. Specific EPI parameters are as follows: 4000 Da/S; 800 – 600 Da;
declustering potential (DP) set at 60 V; collision energy spread (CES) set at 5 V; and
collision energy (CE) set at 35 V.
Acetyl Fentanyl Metabolic Studies
Pooled human liver microsomes (5 ꢂg) from a 50ꢀdonor pool were assayed for
cytochrome P450 (CYP) activity toward acetyl fentanyl. Substrate (acetyl fentanyl, 10
ꢂM final concentration) was added in ethanol and allowed to dry at ambient
temperature. When dry, protein was added in the presence of 0.5 M potassium
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phosphate buffer (pH 7.4) and 10% acetonitrile. Reactions were initiated by the addition
of NADPHꢀregenerating system (solution A: 20 mM NADP+, 60 mM glucoseꢀ6ꢀ
phosphate, and 60 mM MgCl ; solution B: 100 U/mL glucoseꢀ6ꢀphosphate
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dehydrogenase), incubated at 37°C for 60 min, and terminated by the addition of an
equal volume of ethanol. Before LCꢀMS/MS analysis, 10 ꢂL of internal standard spiking
mix was added, and proteins were precipitated via centrifugation at 20,800 g for 8
minutes. Controls omitting the substrate, protein, and NADPH were included with each
assay, and all incubations were performed in triplicate.
In vivo metabolic studies were performed in two adult, male Sprague Dawley rats
placed into new cages with grid floors to facilitate collection of urine. After collection of a
baseline urine sample (time 0), acetyl fentanyl dissolved in 0.9% physiological saline
was infused through an indwelling intraꢀjugular venous catheter (3 mg/kg), then flushed
with approximately 2.0 mL sterile saline. Urine samples were collected after 10, 180,
and 360 min.
Statistical Methods
Accuracy and interꢀrun precision were determined by evaluating QC results from
six independent experiments performed over several nonꢀconsecutive days. Intraꢀrun
precision was calculated by evaluating results from triplicate QCs prepared
independently on a single day. Accuracy was calculated as the absolute percent relative
error for each of the expected QC concentrations. Replicate measurements at the three
QC concentrations (3.75 ng/mL, 7.5 ng/mL, and 75 ng/mL) were used to determine the
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analytical precision, calculated as the percent coefficient of variation (%CV), for both
withinꢀrun and betweenꢀrun measurements. The limit of detection was defined at less
than the lowest calibrator (0.5 ng/mL for acetyl fentanyl and 1 ng/mL for acetyl
norfentanyl), and the lower limit of quantitation (LLQ) was calculated as three times the
standard deviation of six replicate analyses of the low QC standard. Correlation studies
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were evaluated using Pearson correlation coefficients (r ).
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RESULTS AND DISCUSSION
Abuse of synthetic opioids is a growing public health problem, and recent clinical
morbidity and mortality reports from Rhode Island and Pennsylvania involving acetyl
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fentanyl are the latest examples to highlight this dangerous trend.
Very little is
known about the toxicology of this emerging drug of abuse, and several research
barriers remain. For example, assays that simultaneously measure the parent drug and
primary human metabolites are required to facilitate future clinical studies designed to
understand the relationship between drug metabolism and clinical symptoms
documented after acetyl fentanyl use.
This report coupled solidꢀphase extraction with an LCꢀMS/MS approach that
incorporated deuterium labeled internal standards to provide adequate sensitivity and
1
precision for measuring acetyl fentanyl and acetyl norfentanyl. H NMR confirmation
studies showed chemical shifts and coupling constants that were in agreement with
chemical structures (Figure 1). Mass spectra showed the presence of molecular ions
[MH+] and diagnostic fragment ions (Figures 2 & 3).
The LCꢀMS/MS approach achieved baseline separation of acetyl fentanyl and
acetyl norfentanyl and resolved potential interfering substances detected in urine. The
isobaric interference (labeled as peak “1”) shown in Figure 4A coꢀeluted with acetyl
norfentanyl (peak “2”) in preliminary studies. A slower flow rate allowed baseline
resolution of the interference but extended the total run time to 8 min (Figure 4B). Initial
conditions established for the second column confirmation studies allowed for
equivalent resolution of the interference (Figure 4C). Chromatography of standards,
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QCs, and samples remained consistent throughout this study (Figure 4B&C) in which
deuterium labeled internal standards coꢀeluted with each analyte of interest and
retention times remained constant (± 0.1 minute). Accuracy, precision, and lower limits
of quantification (LLQ) for each analyte in urine are presented in Table 2. All calibration
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curves were linear over the tested analytical range, where r values were ≥0.99.
In some instances, second column confirmation studies may be required to meet
clinical or forensic method validation requirements. Therefore, the use of phenylꢀhexyl
and biphenyl stationary phase columns was examined and no significant differences
were detected between the two columns. Fifteen blank human urine samples spiked
with acetyl fentanyl and acetyl norfentanyl at concentrations spanning the calibration
2
range had comparable values for both compounds (r =0.98 and 0.99, respectively)
(Figure 5).
Acetyl fentanyl metabolism was also investigated as part of this study. When
incubated with pooled human liver microsomes, hepatic cytochrome P450s catalyzed
the production of acetyl norfentanyl (Figure 6A). These in vitro data are in agreement
14,15
with previous studies investigating fentanyl metabolism
and suggest the involvement
of similar metabolic pathways. Since authentic human urine samples were not available
for this research, rodent studies were included to further assess in vivo metabolic
pathways and the urinary excretion of acetyl fentanyl and the predicted metabolite
acetyl norfentanyl. Baseline urine collected at time zero did not contain either the
parent drug or the norfentanyl metabolite, and behavioral observations were entirely
speciesꢀtypical. However, immediately following the intravenous administration of acetyl
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fentanyl (i.v. 3 mg/kg), one rat became rigid, lost its righting reflex, and did not respond
to nociceptive mechanical pressure applied to the hindlimbs, while the second subject
exhibited a sedation consistent with that observed with other opioids. Within
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approximately 5 minutes, the most impaired rat began to exhibit labored breathing, and
manual chest compressions were administered to stimulate ventilation, but the rat
expired approximately 15 minutes after drug administration. In contrast, the other rat
survived the 6ꢀhour observation period, although it remained sedated the entire time.
Urine collected at various times points showed a timeꢀdependent excretion of both
acetyl fentanyl and acetyl norfentanyl (Table 3). A chromatograph from a urine sample
taken three hours postꢀadministration from the rat which survived is shown in Figure
6
B. Chromatographic and mass spectral data are nearly identical to results obtained
with human liver microsomes (Figure 6A).
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CONCLUSIONS
The LC−MS/MS method presented in this study provides low levels of
quantification and a high level of accuracy and reproducibility for acetyl fentanyl and
acetyl norfentanyl. The sensitivity, accuracy, and precision of this method are adequate
for clinical, public health, and forensic applications. This is the first method to study
acetyl fentanyl metabolism and to suggest that human cytochrome P450 metabolism
leads to the urinary excretion of acetyl norfentanyl. Future clinical investigations and
toxicokinetic studies are required to confirm these preliminary findings and to further
elucidate the metabolic pathways responsible for acetyl fentanyl detoxification and
excretion.
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ACKNOWLEDGEMENTS
This work was supported by the Association of Public Health Laboratories [Grant
Innovations in Quality Public health Laboratory Practice] (JHM) and by the Centers for
Disease Control (Contract No. 200ꢀ2007ꢀ21729) (JHM).
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AUTHOR CONTRIBUTION
All authors contributed to the writing of this manuscript and the design and/or execution
of the studies. All authors provided approval for the final version of this manuscript.
CONFLICT OF INTEREST DISCLOSURE
None of the authors have personal financial conflicts of interest. However Paul
Kennedy and Gregory Endres are employees of Cayman Chemical Company.
Commercial products from Cayman Chemical Co. were used in the study.
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FIGURES
Figure 1.
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Figure 2.
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Figure 4.
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Figure 6.
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FIGURE LEGENDS
1
Figure 1. H NMR confirmation data for (A) acetyl fentanyl, (B) acetyl norfentanyl, (C)
acetyl fentanylꢀd , and (D) acetyl norfentanylꢀd reference standards.
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Figure 2. Representative mass spectra for (A) acetyl fentanyl, (B) acetyl norfentanyl,
C) acetyl fentanylꢀd , and (D) acetyl norfentanylꢀd reference standards.
(
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Figure 3. Proposed MS/MS fragmentation pathways for (A) acetyl fentanyl and (B)
acetyl norfentanyl. The fragmentation products in the schematic correspond to the
fragments observed in the mass spectra in Figure 2.
Figure 4. Representative chromatograms from analytical columns manufactured with
phenylꢀhexyl (A & B) and biphenyl (C) stationary phases. The different color tracings
represent the specific reaction monitoring experiments used for each analyte. The
notation “1” illustrates an isobaric interference that coꢀeluted with acetyl norfentanyl that
is denoted as “2”. The chromatogram illustrated in panel A used the initial flow rate
conditions of 800 ꢂL/min, but as illustrated in panel B, a slower flow rate of 600 ꢂl/min
was required to resolve the isobaric interference. Data presented in panel C
demonstrate that resolution of the isobaric interference is maintained at higher flow
rates (800 ꢂL/min) when using the biphenyl stationary phase.
Figure 5. A comparison of quantitative results obtained with liquid chromatography
columns manufactured with phenylꢀhexyl and biphenyl stationary phases. Acetyl
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fentanyl (A) and acetyl norfentanyl (B) were simultaneously assayed as described in
materials and methods. Fifteen human urine samples were spiked with concentrations
2
spanning the calibration range of each analyte. Pearson correlation coefficients (r ) are
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inset within each panel.
Figure 6. Representative chromatograms demonstrating that cytochrome P450
metabolizing enzymes in (A) human liver microsomes produce the predicted acetyl
norfentanyl metabolite. The different color tracings represent combined extracted
specific reaction monitoring (SRM) experiments optimized for the acetyl fentanyl
substrate and acetyl norfentanyl metabolite. Specific conditions for each SRM are
provided in the Experimental Section. Each reaction condition noted in the figure legend
was run in triplicate and none of the negative controls generated measurable levels of
the acetyl norfentanyl metabolite. Results obtained from urine collected from rats
acutely exposed to acetyl fentanyl (3 mg/kg i.v.) produced similar results. The
representative chromatogram illustrated in panel B were the results obtained from urine
collected 3 hours postꢀadministration. Quantitative results obtained for each time point
are provided in Table 3.
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TABLES
Table 1. Specific Reaction Monitoring (SRM) Experimental Parameters
a
b
c
d
Analyte
Q1 (m/z) Q3 (m/z) DP (V) EP (V) CE (V) CXP (V)
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Acetyl Fentanyl
Acetyl Fentanylꢀd5
Acetyl Norfentanyl
328
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Acetyl Norfentanylꢀd5
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DP, declustering potential; EP, entrance potential; CE, collision energy;
CXP, collision cell exit potential
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Table 2. Summary of accuracy, precision, and quantification limits in human urine*
Quality Control High
(75 ng/ml)
Quality Control Medium
(7.5 ng/ml)
Quality Control Low
(3.75 ng/ml)
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Interꢀ Intraꢀ
Interꢀ Intraꢀ
Interꢀ Intraꢀ
Conc. ± SD
ng/ml)
b
Conc. ± SD
(ng/ml)
b
Conc. ± SD
(ng/ml)
b
Analyte
Daya
Day
%CV
%RE
Day
%CV
Day
%CV
% RE
Day
%CV
Day
%CV
%RE
LLQ
(
a
a
a
a
a
%
CV
Acetyl
Fentanyl
7
7.7 ± 7.32
6.8 ± 5.93
9.4
4.3
7.9
3.65
7.39 ± 0.59
7.59 ± 0.44
7.9
5.8
5.0
3.8
1.43
1.19
3.71 ± 0.35
9.5
7.5
3.8
0.96 1.06
2.27 1.62
Acetyl
Norfentanyl
7
7.7
2.34
3.84 ± 0.54 14.0
a
b
%
CV , Coefficient of Variation; %RE , Absolute Relative Error; LLQ, Lower Limit of Quantification
*
Data are based on 6 independent experiments conducted on multiple, nonconsecutive days
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Table 3. Quantitative measurement of acetyl fentanyl and acetyl norfentanyl detected in
rat urine collected after acute exposure to acetyl fentanyl (3 mg/kg i.v.)
Rat Subject #1
Rat Subject #2
Acetyl
Fentanyl
Acetyl
Norfentanyl
(ng/mL)
Acetyl
Fentanyl
(ng/mL)
Acetyl
Norfentanyl
(ng/mL)
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Time
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ND*
ND*
ND*
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ND*
ND*
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ND*
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ꢀꢀ
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#
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16500
#
#
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ꢀꢀ
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3810
*
ND: Not detected
Rat 1 expired 15 min postꢀdrug administration
ꢀꢀ
#
All samples were diluted within the range of the calibration curve
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REFERENCES
(
1) AAPCC. 2011.
2) Johnston, L.; O'Malley, P.; Bachman, J.; Schulenberg, J. University of Michigan
(
News Service 2011, www.monitoringthefuture.org.
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(
(
(
3) Manchikanti, L.; Singh, A. Pain physician 2008, 11. S63ꢀ88.
4) Okie, S. N Engl J Med 2010, 363. 1981ꢀ5.
5) CDC. Morbidity and Mortality Weekly Report 2013, 62. 537ꢀ542.
(6) Perger, L.; Rentsch, K. M.; KullakꢀUblick, G. A.; Verotta, D.; Fattinger, K. Eur J
Pharm Sc 2009, 36. 421ꢀ32.
(7) Reisinger, M. Heroin Addiction and Related Clinical Problems 2006, 8. 29ꢀ40.
(8) EMCDDA. 2012.
(
9) Kram, T. C.; Cooper, D. A.; Allen, A. C. Anal Chem 1981, 53. 1379Aꢀ1386A.
10) In CBC News: Montreal, 2013.
(
(11) CDC. CDC Health Alert Network 2013, 00350.
(
(
(
12) CDC. Morbidity and Mortality Weekly Report 2013, 62. 703ꢀ704.
13) DEA. 2013.
14) Labroo, R. B.; Paine, M. F.; Thummel, K. E.; Kharasch, E. D. Drug Metab Dispos
1
997, 25. 1072ꢀ80.
(15) Feierman, D. E.; Lasker, J. M. Drug Metab Dispos 1996, 24. 932ꢀ9.
(16) Smith, H. S. Mayo Clinic proceedings. Mayo Clinic 2009, 84. 613ꢀ24.
(17) PDoH. 2013.
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