Journal of Analytical Toxicology, 2021;00:1–6

doi:10.1093/jat/bkaa199
Advance Access Publication Date: 4 January 2021
Article

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Article

Investigation and Resolution of Interference in

the LC–QTOF–MS Detection of 4-MePPP
Darren R. Allen* , Christopher Warnholtz and Brett C. McWhinney
Department of Chemical Pathology, Pathology Queensland Central Laboratory, Herston Hospital Campus, Herston
Road, Herston, QLD 4029, Australia

*Author to whom correspondence should be addressed. Email: darren.allen@health.qld.gov.au

Abstract
An interference resulting in the false-positive detection of the synthetic cathinone 4′ -methyl-α-
pyrrolidinopropiophenone (4-MePPP) in urine was suspected following the recent addition of
4-MePPP spectral data to a liquid chromatography–quadrupole time-of-flight–mass spectrome-
try drug library. Although positive detection criteria were achieved, it was noted that all urine
samples suspected of containing 4-MePPP also concurrently contained high levels of tramadol
and its associated metabolites. Using quadrupole time-of-flight–mass spectrometry software elu-
cidation tools, candidate compounds for the suspected interference were proposed. To provide
further confidence in the identity of the interference, in silico fragmentation tools were used to
match product ions generated in the analysis with product ions predicted from the theoretical
fragmentation of candidate compounds. The ability of the suspected interference to subsequently
produce the required product ions for spectral library identification of 4-MePPP was also tested.
This information was used to provide a high preliminary confidence in the compound identity prior
to purchase and subsequent confirmation with certified reference material. A co-eluting isobaric
interference was identified and confirmed as an in-source fragment of the tramadol metabo-
lite, N,N-bisdesmethyltramadol. Proposed resolutions for this interference are also described and
subsequently validated by retrospective interrogation of previous cases of suspected interference.

Introduction can assist further by theoretically predicting the product ions pro-
duced from the subsequent fragmentation of a compound (2). These
The utilization of liquid chromatography–quadrupole time-of-
theoretically predicted product ions can then be compared with
flight–mass spectrometry (LC–QTOF–MS) in toxicological analysis
fragmentation data acquired in the analysis to support compound
provides a powerful analytical technique for the detection of illicit
identification in the absence of certified reference material (CRM).
substances. With extensive spectral libraries containing detection
With this information, a high preliminary confidence in compound
information for potentially thousands of compounds, LC–QTOF–
identity can be achieved before the final confirmation is undertaken
MS provides comprehensive drug detection with the ability to rapidly
with CRM.
adapt methods for the detection of emerging substances. The use of
The Waters® Forensic Toxicology Screening Application with
untargeted data acquisition also allows for the retrospective analy-
UNIFI provides comprehensive drug screening by utilizing a sci-
sis of previously acquired data for the detection of newly identified
entific library containing accurate mass, fragmentation and chro-
drug targets (1). The analysis of untargeted accurate mass data
matographic retention time (RT) data for over 1,700 compounds.
can also provide information on the elemental composition of an
The requirement for all users of the application to maintain stan-
unknown compound, which can be further compared with external
dard chromatographic and mass spectrometer conditions allows
compound databases to provide tentative identification. With free
for externally validated compound information to be regularly
online compound databases providing additional structural informa-
added to the library. This beneficially allows all application users
tion for thousands of compounds, in silico fragmentation algorithms

© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Forensic Toxicologists, Inc. All rights reserved.
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2 Allen et al.
Figure 1. Tramadol metabolism pathway.
to keep their drug libraries up to date with new drugs and
emerging illicit substances. A recent library update included the
detection criteria for the amphetamine-type substance 4′ -methyl-α-
pyrrolidinopropiophenone (4-MePPP). 4-MePPP is a synthetic cathi-
none of the pyrrolidinophenone type, which gained popularity as a
recreational stimulant due to the euphoric effects it produces (3).
Considered a second-generation mephedrone analogue, 4-MePPP,
like other designer analogues, appeared in the illicit drug markets fol-
lowing legislative bans on mephedrone (4). Tramadol is a centrally
acting analgesic opioid that inhibits the reuptake of both serotonin
and norepinephrine. It is a commonly used drug with a low abuse
potential that is well tolerated in the treatment of acute and chronic
pain conditions (5). Tramadol’s main route of elimination is via the
kidneys, with numerous urinary metabolites arising from phase I
and II reactions. The major phase I metabolites of tramadol arise
from N- and O-demethylation, followed by phase II reactions of
O-desmethyl metabolites with glucuronide and sulfate conjugation
(see Figure 1) (5, 6).
A 22-year-old male presented to an emergency department feel-
ing unwell, and a comprehensive urine drug screen was performed
via LC–QTOF–MS using the Waters® Forensic Toxicology Screening
Application with UNIFI. 4-MePPP was detected in the analysis, hav-
ing fulfilled the required qualifying criteria (see Table I) with the addi-
tional compounds tramadol, tapentadol, atenolol and cannabinoid
metabolites concurrently detected in the analysis. Despite having ful-
filled the qualifying criteria, a small RT difference of 0.05 min from
the 4-MePPP RT library entry was noted. Having not previously
detected 4-MePPP, and not having immediate access to CRM to con-
firm the compound, the sample was referred to another laboratory
for confirmatory testing. A subsequent comparison by the refer-
ral laboratory using the identical LC–QTOF–MS application with
4-MePPP CRM confirmed a RT difference of 0.2 min, suggesting the
presence of an interfering substance. It was also noted in subsequent
screening results for additional patients that 4-MePPP was detected
in the presence of tramadol and the O- and N-desmethyltramadol
metabolites.
Suspecting the presence of an interfering substance, identifica-
tion was undertaken using the suite of elucidation tools available in
the UNIFI software to interrogate the sample data. Following the
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identification of possible candidate compounds, CRM could be pur-
chased to provide the final confirmation. We also describe in-house
resolutions for the false-positive detection of 4-MePPP in future
analyses using this application.
Methods
Rac N-bisdesmethyltramadol hydrochloride (CAS 541505-91-3)
and O,N-didesmethyltramadol (CAS 138853-73-3) were pur-
chased from Toronto Research Chemicals Inc. (Toronto, Canada).
4′ -Methyl-α-pyrrolidinopropiophenone hydrochloride (CAS 131
3393-58-6) was purchased from LGC Standards (Teddington,
United Kingdom). Molecular structure files (Molfiles) for the tra-
madol metabolites were obtained from Chemspider.com. Analysis
was performed using an iClass Acquity Ultra-High-Performance
Liquid Chromatography system coupled with a Xevo G2-XS
Quadrupole Time of Flight mass spectrometer (Waters Corporation,
Milford, MA, USA). Chromatographic separation was performed
at 50◦ C on a Waters Acquity HSS C18 Column (150 × 2.1 mm,
1.8 µm) using a 15-min gradient elution. Mobile phase A contained
5 mmol/L of ammonium formate (pH 3.0) (Sigma Aldrich) and
mobile phase B contained acetonitrile with 0.1% formic acid (Fisher
Chemical). Analysis was performed in positive electrospray ioniza-
tion mode using MSE acquisition mode to acquire data at both
low collision energy (6 eV CE) and high collision energy (10–40
eV CE ramp) for obtaining both the accurate mass of parent com-
pounds and associated high-energy fragments. Data were acquired
and processed using UNIFI (version 1.9.3) utilizing the UNIFI Sci-
entific Library (version 1.9). Criteria used for the identification of
4-MePPP include acceptable tolerances for mass error, RT error and
the number of expected fragments found following the collision-
induced dissociation of the precursor ion (see Table I). General
criterion for the acceptable number of expected fragments is ≥50%
(found vs expected), though the number of expected fragments listed
in the library may vary between analytes. For 4-MePPP, three frag-
ments are included in the library entry (see Table II), indicating that
two out of three fragments would be required for identification in
this application.
Investigation and Resolution of Interference in the LC–QTOF–MS Detection of 4-MePPP
Table I. Detection Results for 4′ -Methyl-α-pyrrolidinopropiophenone (4-MePPP)
Mass error (ppm) Retention time error (min)
Patient’s result −2.8 0.05
Positive criteria <±5.0 <±0.2
Table II. 4′ -Methyl-α-pyrrolidinopropiophenone (4-MePPP) Library
Detection Results
Precursor ion Retention time Product ions
[M + H] (m/z) (min) (m/z)
218.1539 3.70 147.0804
91.0542
98.0964
Results and Discussion
LC–QTOF–MS techniques can provide highly specific and com-
prehensive drug detection capability utilizing a combination of
accurate mass, fragmentation and RT data incorporated into large
compound libraries. Despite the inherent advantages offered with
these techniques including the convenience of rapidly updating com-
pound libraries for newly emerging substances, caution is still
warranted in the interpretation and reporting of new and rarely
encountered drugs. In the investigation by Petrie et al., the impor-
tance of caution in identifying the illicit use of the designer drug
m-chlorophenylpiperazine (mCPP) was highlighted following the
identification that mCPP was an active metabolite of the prescribed
medication trazodone (7). Gaining information on the patient’s con-
current polypharmacy either through a review of listed medications
or via untargeted drug analysis by LC–QTOF–MS can provide valu-
able information in interpreting the patient’s results. The potential
for isobaric interference from other drugs and/or metabolites is also
an important consideration when identifying the presence of newly
encountered drugs, even in high-resolution MS.
The unexpected detection of 4-MePPP across several patients
following the recent addition of 4-MePPP to our LC–QTOF–MS
compound library prompted an investigation into the validity of the
sample results. The additional findings that these patients exhibited
concurrently high levels of tramadol and tramadol metabolites fur-
ther suggested the potential presence of interference in our routine
LC–QTOF–MS application. The initial approach taken to identify
the interference was to use the suite of elucidation tools available in
the UNIFI software to propose an elemental formula for the inter-
ference. The ChemSpider database (Chemspider.com) could then be
searched for metabolites of tramadol matching the proposed elemen-
tal formula. The ChemSpider database provides the user with free
access to millions of chemical structures and associated chemical
properties. Using flexible search options, potential candidate com-
pounds can be matched to proposed elemental formula provided by
elucidation software. Data files containing the chemical structures of
candidate compounds can also be easily accessed and interrogated
with in silico fragmentation tools to provide further confidence in
the identity of unknown compounds.
Initial analysis of the suspected interference at m/z 218.15333
was conducted using the Elemental Composition Tool available
in UNIFI. A protonated molecular ion of a compound with the
elemental formula of C14 H19 NO was proposed (i-Fit confidence:
88.57%) based on additional isotopic information concurrently
3
Fragments: found vs expected (%) Counts (counts per second)
100 118,792
≥50 >10,000
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collected in the analysis. No compound matches corresponding to
known metabolites of tramadol were obtained for this proposed ele-
mental formula in the ChemSpider database, so the sample data
were further interrogated for the presence of additional compounds
acquired at the identical RT of the suspected interference. Iden-
tified in the low-energy scan was a protonated molecular ion at
m/z 236.16406, with a proposed elemental formula of C14 H21 NO2
(i-Fit confidence: 93.81%). A repeated search of this formula in the
ChemSpider database was able to identify two tramadol metabo-
lites, N,N-bisdesmethyltramadol and O,N-didesmethyltramadol.
The optimal approach for identifying candidate compounds would
be to allow the UNIFI software to interrogate the ChemSpider
database automatically, allowing more rapid candidate predic-
tion and automatic matching of product ions in the analysis by
in silico fragmentation of candidate structures. Due to cyberse-
curity settings in our laboratory at the time of this investigation,
this functionality was not possible and the ChemSpider database
was searched manually. Using Chemspider.com, molecular struc-
ture files (Molfiles) for N,N-bisdesmethyltramadol (CSID:2317896)
and O,N-didesmethyltramadol (CSID:7991856) were obtained and
added to the method component table.
Using the UNIFI Fragment Match tool, the candidate compound
at m/z 236.16406 was analyzed by in silico fragmentation using
both the molecular structures of N,N-bisdesmethyltramadol and
O,N-didesmethyltramadol. In this analysis, product ions acquired
in the high-energy scan of the sample are paired to fragments gener-
ated by UNIFI following theoretical fragmentation of the compound
structure. The default in silico fragmentation settings are configured
to theoretically provide the most plausible product ions produced
in the current method conditions, though it also allows for mod-
ification of these rules including the number of allowable bond
breakages. For N,N-bisdesmethyltramadol, 20 theoretically pre-
dicted fragments were matched to product ions acquired in the high-
energy scan of the patient data, and for O,N-didesmethyltramadol,
15 theoretical fragments were matched. The suspected interference
at m/z 218.15333 was proposed as a potential water loss fragment
of the parent molecule for both N,N-bisdesmethyltramadol and
O,N-didesmethyltramadol indicating two potential fragments that
were isobaric with 4-MePPP (see Figure 2).
To further support the identification of these metabolites as
sources of the interference, in silico fragmentation analysis was
undertaken to identify if the required product ions for the library
identification of 4-MePPP could be produced from these structures
(see Table II). Initial analysis using the default in silico fragmenta-
tion settings did not yield any of the 4-MePPP diagnostic product
ions from these structures. Reanalysis following an increase in the
allowable number of subsequent bond breaks was able to pre-
dict the generation of the m/z 147.0804 product ion from both
N,N-bisdesmethyltramadol and O,N-didesmethyltramadol.
Further review of the low-energy scan data indicated the pres-
ence of prominent in-source fragments proposed by UNIFI to
originate from the structures of either O,N-didesmethyltramadol or
N,N-bisdesmethyltramadol (see Figure 3). It was theorized that
the diagnostic product ions required for 4-MePPP identification
4 Allen et al.

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Figure 2. Isobaric structures of the protonated molecular ion C14 H19 NO (m/z 218.15333) for (A) 4-MePPP, (B) N,N-bisdesmethyltramadol (water loss), and
(C) O,N-didesmethyltramadol (water loss).

Figure 3. In-source fragmentation of N,N-bisdesmethyltramadol in low-energy scan.

may be produced from further fragmentation of the smaller in-
source fragments for both candidate compounds during the high-
energy scan component of the MSE analysis. To test this, individual
Molfiles were created for the most abundant in-source frag-
ments using the free PubChem Sketcher V.2.4 program (https://
pubchem.ncbi.nlm.nih.gov/edit3/index.html) and manually assigned
to the in-source fragments in UNIFI. Subsequent in silico frag-
mentation analysis using the newly assigned structures was able to
predict the formation of the isobaric product ions m/z 147.0804
(C10 H11 O) and m/z 91.0542 (C7 H7 ) from multiple in-source frag-
ments. Generation of the m/z 147.0804 product ion was predicted in
silico from both the m/z 189.12674 (C13 H17 O) and m/z 201.12697
(C14 H17 O) in-source fragments of N,N-bisdesmethyltramadol. Gen-
eration of the m/z 91.0542 product ion was predicted only
from the m/z 201.12697 (C14 H17 O) in-source fragment of
N,N-bisdesmethyltramadol. For O,N-didesmethyltramadol, the m/z
147.0804 product ion was predicted from the m/z 218.15333
in-source fragment, with both the m/z 147.0804 and m/z 91.0542
product ions predicted from the m/z 189.12674 in-source fragment.
It was also thought that additional in-source fragments from both
candidate compounds may also generate the required isobaric prod-
uct ions, despite not being predicted in silico due to the conservative
in silico fragmentation settings used in the analysis. Generation of
the m/z 98.0964 product ion could not be predicted from any of
the proposed structures. Review of all additional suspected false-
positive 4-MePPP patients also showed that the m/z 98.0964 product
ion was only matched once in the original patient results (low rela-
tive abundance), with all other results exhibiting the m/z 147.0804
and m/z 91.0542 product ions only. It was concluded that the
m/z 98.0964 product ion matched in the initial analysis was likely
produced from a co-eluting substance in the urine. The detection of
unrelated product ions in the analysis is considered possible in MSE
data acquisition as there is no specific precursor ion selection prior
to collision-induced dissociation.
With both N,N-bisdesmethyltramadol and O,N-didesmethyl-
tramadol qualifying as potential sources of both the isobaric pre-
cursor ion and diagnostic product ions, CRM was obtained for
both compounds to confirm a match based on the chromatographic
RT. Subsequent analysis with CRM was able to confirm that the
interfering compound corresponded to N,N-bisdesmethyltramadol
Investigation and Resolution of Interference in the LC–QTOF–MS Detection of 4-MePPP
Table III. Expected Ion Ratios for 4′ -Methyl-α-pyrrolidinopropiophenone (4-MePPP)
Expected fragment (m/z) Expected ion ratioa
147.08044 0.0210
91.05423 0.0121
98.09643 0.0277
a
Ion ratio of diagnostic product ion to the protonated precursor ion [M + H] (m/z 218.1539).
b
Counts per second <200.
(RT = 3.75 min), with O,N-didesmethyltramadol eluting earlier
(RT = 2.36 min). A false-positive match for 4-MePPP was also gen-
erated in the analysis from N,N-bisdesmethyltramadol reference
material (1,000 µg/L), confirming the source of the interference.
Resolution of this interference would ideally be achieved by
modification of the chromatography to improve the separation of
N,N-bisdesmethyltramadol and 4-MePPP. Due to the fixed chro-
matographic parameters required in the method, two alternative
solutions were demonstrated to differentiate the respective com-
pounds. Firstly, investigation of the expected ion ratios of each
4-MePPP diagnostic product ion to the 4-MePPP precursor ion was
established using CRM over a concentration range (60–500 µg/L)
(see Table III). The expected ion ratios for 4-MePPP were then
added to the UNIFI scientific library. Using these expected ion ratios
(ion ratio tolerance ±20%), the false-positive detection of 4-MePPP
due to N,N-bisdesmethyltramadol could be identified in the original
patient by the subsequent ion ratio failures. Retrospective interroga-
tion using this approach for all suspected false-positive results (n = 7)
indicated corresponding ion ratio failures for 4-MePPP.
As an alternative approach, a library entry was created for
the N,N-bisdesmethyltramadol in-source fragment (water loss) m/z
218.15333, with an associated chemical structure (Molfile) created
in PubChem Sketcher V.2.4. This approach was applied to test
if the UNIFI software would preferentially select the interference
over 4-MePPP in the analysis. High-energy product ions generated
from N,N-bisdesmethyltramadol using CRM were also added to the
corresponding library entry. The expected mass of both precursor
and product ions were obtained from their associated chemical for-
mulae. Following the retrospective interrogation of all suspected
false-positive patients (n = 7), the interference was only preferen-
tially selected for four patients. Review of the remaining three
patients showed that the N,N-bisdesmethyltramadol in-source frag-
ment was only offered as an alternative assignment in one instance.
This was likely due to the RT tolerance setting of ±0.05 min for the
N,N-bisdesmethyltramadol interference library entry. Expansion of
this setting to ±0.1 min improved the preferential selection to include
five patients. Analysis of results for the remaining two patients now
demonstrated a library match for the interference which was pro-
vided as an alternative assignment. This was believed to be due to the
subsequent RT of the interference appearing closer to the 4-MePPP
library RT of 3.70 min versus the interference entry at 3.75 min.
Variations in the observed RT of the interfering compound across all
patients were likely due to the performance of the analytical column
at different times of analysis.
The ability of the method to concurrently detect 4-MePPP
in the presence of the interference was also tested by spiking
4-MePPP (50 µg/L) into a urine sample containing tramadol and
its associated metabolites. At this spiked concentration, the sys-
tem was able to provide identification for both 4-MePPP and the
N,N-bisdesmethyltramadol in-source fragment despite the com-
pounds not exhibiting baseline separation in the chromatography.
5
Ion ratio tolerance (±20%) Interference ion ratio
0.0168–0.0252 0.0562 (Fail)
0.0097–0.0145 0.0816 (Fail)
0.0022b (Fail)
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0.0222–0.0332
Although identification was achieved, it was noted that the expected
ion ratios for 4-MePPP were also affected due to contribution from
isobaric product ions from the close eluting interference. It was
also suspected that greater concentrations of the interference may
also affect the ability to concurrently detect 4-MePPP, and there-
fore caution in the identification of 4-MePPP in the presence of
N,N-bisdesmethyltramadol needs to be taken.
Conclusion
The use of LC–QTOF–MS with comprehensive spectral libraries pro-
vides a powerful analytical technique in the identification of drugs
in biological samples. The analysis of untargeted data can also
provide valuable information regarding the chemical composition
of samples and assist in the identification of unknown substances.
QTOF–MS elucidation tools such as in silico fragmentation can
also provide the analyst with further diagnostic confidence in com-
pound identification in the absence of CRM. Although accurate
mass spectral libraries accompanied by fragmentation and RT data
generated from CRM provide a very high degree of specificity, cau-
tion is always warranted in identifying newly encountered drugs.
The potential of interference from isobaric substances is always
an important consideration, and the use of orthogonal techniques
using CRM when interferences are suspected is highly recommended.
Using the UNIFI elucidation toolset accompanied by information
from the untargeted analysis of samples, N,N-bisdesmethyltramadol
was proposed and subsequently identified as a source of false-
positive interference for 4-MePPP in this method. Resolutions for
this interference were also proposed and tested via retrospective
interrogation of sample data. The use of ion ratio tolerances for
4-MePPP in the library entry for this analyte was the most effective
approach in detecting false-positive 4-MePPP due to the presence of
N,N-bisdesmethyltramadol.
Acknowledgments
The authors would like to thank Michelle Johnston, Forensic and
Scientific Services, Brisbane, Australia, for assistance with this
investigation.
Conflict of Interest
The authors declare that they have no conflict of interest.
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