Analytical and Bioanalytical Chemistry (2023) 415:5323–5335

https://doi.org/10.1007/s00216-023-04807-3

RESEARCH PAPER

Conductive vial electromembrane extraction of opioids from oral fluid

Tonje Gottenberg Skaalvik1,2 · Chen Zhou2,3 · Elisabeth Leere Øiestad2,4 · Solfrid Hegstad1 · Roger Trones5 ·
Stig Pedersen‑Bjergaard2,6

Received: 28 April 2023 / Revised: 1 June 2023 / Accepted: 5 June 2023 / Published online: 29 June 2023
© The Author(s) 2023

Abstract
The use of oral fluid as sample matrix has gained significance in the analysis of drugs of abuse due to its non-invasive nature.
In this study, the 13 opioids morphine, oxycodone, codeine, O-desmethyl tramadol, ethylmorphine, tramadol, pethidine,
ketobemidone, buprenorphine, fentanyl, cyclopropylfentanyl, etonitazepyne, and methadone were extracted from oral fluid
using electromembrane extraction based on conductive vials prior to analysis with ultra-high performance liquid chroma-
tography–tandem mass spectrometry. Oral fluid was collected using Quantisal collection kits. By applying voltage, target
analytes were extracted from oral fluid samples diluted with 0.1% formic acid, across a liquid membrane and into a 300 μL
0.1% (v/v) formic acid solution. The liquid membrane comprised 8 μL membrane solvent immobilized in the pores of a flat
porous polypropylene membrane. The membrane solvent was a mixture of 6-methylcoumarin, thymol, and 2-nitrophenyloc-
tyl ether. The composition of the membrane solvent was found to be the most important parameter to achieve simultaneous
extraction of all target opioids, which had predicted log P values in the range from 0.7 to 5.0. The method was validated in
accordance to the guidelines by the European Medical Agency with satisfactory results. Intra- and inter-day precision and
bias were within guideline limits of ± 15% for 12 of 13 compounds. Extraction recoveries ranged from 39 to 104% (CV ≤
23%). Internal standard normalized matrix effects were in the range from 88 to 103% (CV ≤ 5%). Quantitative results of
authentic oral fluid samples were in accordance with a routine screening method, and external quality control samples for
both hydrophilic and lipophilic compounds were within acceptable limits.

Keywords Electromembrane extraction · Opioids · Oral fluid · Drug analysis · Liquid membrane

Introduction

Opioids are common analytes included in drug screening

methodologies, as they are frequently misused or abused
* Stig Pedersen‑Bjergaard analgesics. The overarching group of drugs includes nat-
stig.pedersen-bjergaard@farmasi.uio.no urally occurring opiates, such as morphine and codeine,
1
semi-synthetic opioids such as buprenorphine and synthetic
Department of Clinical Pharmacology, St. Olav University
Hospital, Professor Brochs Gate 6, 7030 Trondheim, Norway opioids like methadone and fentanyl. The latest class of syn-
2
thetic opioids to emerge on the illicit drug market are 2-Ben-
Department of Pharmacy, University of Oslo, P.O. Box 1068
Blindern, 0316 Oslo, Norway zyl benzimidazole analogues, also known as nitazenes [1, 2].
3
Oral fluid (OF) has gained popularity as an alternative
West China School of Public Health and West China Fourth
Hospital, Sichuan University, Chengdu 610041, China
biological matrix for the detection of drugs in forensic and
4
clinical settings, allowing simple and non-invasive sampling.
Division of Laboratory Medicine, Department of Forensic
Sciences, Oslo University Hospital, P.O. Box 4459 Nydalen,
The term OF refers to the mixture of saliva and other fluids
0424 Oslo, Norway and particles present in the oral cavity [3]. OF has been used
5
Extraction Technologies Norway, Verkstedveien 29,
for the analysis of a variety of drugs previously, including
1424 Ski, Norway opioids [4–6]. Several reviews on the drug disposition, col-
6
Department of Pharmacy, Faculty of Health and Medical
lection, interpretation and analysis of OF are available [7–9].
Sciences, University of Copenhagen, Universitetsparken 2, Sampling is often carried out using commercial collection
2100 Copenhagen, Denmark

13
Vol.:(0123456789)
5324
devices that ensure adequate volume collection to a collec-
tor pad. The OF sampling kits contain surfactants, buffers,
dyes and preservatives to improve analyte stability and elute
analytes off the collector pad. Analysis of such samples thus
usually includes an extraction step for sample clean-up prior
to LC-MS analysis [7, 8].
Microextraction techniques have emerged as alterna-
tive sample preparation methods as part of the develop-
ment towards smaller systems that consume less solvents
and reagents. Electromembrane extraction (EME) is a form
of membrane based liquid-phase microextraction (LPME)
that incorporates an electric field to enhance mass transfer.
Advantages are exhaustive extractions, sample clean-up and
enrichment using only few microlitres (< 10 μL) of organic
solvent, with faster kinetics compared to membrane based
LPME [10, 11]. Since the introduction of EME in 2006 [12],
extraction of organic and inorganic analytes from biological
[13–16], environmental [17–19] and food samples [20–22]
has been reported using a variety of technical formats.
Operational modes and configurations are based on hollow
fibres, 96-wells and microchip technology, among others,
and have been summarized in multiple reviews [23–26]. A
commercial device based on conductive vials was recently
launched, which is considered a step closer towards routine
implementation [27].
The typical EME system is a three-phase system con-
sisting of an aqueous sample separated from an aqueous
acceptor by a thin organic liquid membrane. Application
of voltage (i.e. extraction potential) across the liquid mem-
brane facilitates transfer of ionized compounds from the
sample, across the liquid membrane and into the acceptor,
which is collected for further analysis. Selectivity is tuned
by altering the electric field (polarity and magnitude) and the
physiochemical properties of the membrane. Highly selec-
tive extractions are thus possible with EME. Conditions
favouring high selectivity are low-voltage [28] and liquid
membranes with properties tuned towards analyte-specific
interactions [29–31]. On the other end of the scale, low
selectivity extractions that isolate a broad range of analytes
from the sample matrix are relevant for applications such
as multi-component drug screening. Finding robust EME
conditions for extraction of compounds with a wide range
of properties is not straightforward.
In theory, EME is applicable for all ionizable compounds;
however, the application range is currently limited by suit-
able liquid membranes. In general, the membrane solvent
should have minimal leakage to aqueous solution as this
may lead to excessive current, which further negatively
affects reproducibility due to electrolysis [32]. This is highly
important, as robust systems are essential for routine clini-
cal or forensic applications. In addition, the physiochemical
properties of the liquid membrane must favour some degree
of analyte partitioning for efficient extraction. Fundamental
13
Skaalvik T. G. et al.
studies and reviews on the molecular interactions and appli-
cation ranges of different liquid membranes can be found in
the literature [33–36]. However, the selection of the liquid
membrane remains to be by trial and error in most applica-
tions. To guide method development, recent work reported
on generalized EME conditions aimed to characterize sys-
tematically the application range of membrane solvents
using a large collection of model analytes [37].
The aim of the present study was to develop EME condi-
tions for extraction of target opioids from OF and evaluate
whether data quality was within recommended guidelines for
bioanalytical methods. Target opioids were morphine, oxyco-
done, codeine, O-desmethyl tramadol (metabolite), ethylmor-
phine, tramadol, pethidine, ketobemidone, buprenorphine,
fentanyl, cyclopropylfentanyl, etonitazepyne (N-pyrrolidino
etonitazene) and methadone. The opioids all have basic
functionality, which makes them applicable for simultane-
ous extraction with EME. However, lipophilicity varies, with
predicted n-octanol-water partition coefficients (log P) rang-
ing from 0.7 to 5.0 (Table S1), which complicates method
development. EME was carried out using prototype equip-
ment for a commercial EME device, employing conductive
vials to house the sample and acceptor. The study aimed to
find a suitable, robust liquid membrane for opioids in a wide
log P range. Applicability of the final EME-UHPLC-MS/MS
method was demonstrated through validation and comparison
of authentic samples with a pre-established routine method.
Materials and methods
Chemicals and reagents
Reference substances were purchased from two differ-
ent vendors for the majority of analytes to prepare quality
control samples independently from calibrators. Reference
substances were purchased from the following companies:
buprenorphine, codeine, codeine-d3, ethylmorphine, fenta-
nyl, morphine, morphine-d3, O-desmethyl tramadol (O-DM-
tramadol) and pethidine were from Lipomed (Arlesheim,
Switzerland). Fentanyl, oxycodone, pethidine and tramadol
were purchased from Sigma Aldrich (St. Louis, MA, USA).
Codeine, cyclopropylfentanyl, ethylmorphine-d5, fentanyl-
d5 methadone-d 3, morphine, N-pyrrolidino etonitazene
(etonitazepyne), methadone, oxycodone, oxycodone-d6
and tramadol-d6 were from Chiron (Trondheim, Norway).
Buprenorphine-d 3, ethylmorphine, ketobemidone and
O-DM-tramadol were from Toronto Research Chemicals
Inc. (TRC, Toronto, ON, Canada). Buprenorphine and meth-
adone were purchased from Reckitt Benckiser (Hull, UK)
and St. Olav University Hospital Pharmacy (Trondheim,
Conductive vial electromembrane extraction of opioids from oral fluid 5325
Norway), respectively. Ketobemidone-d3 was from Alsachim
(Strasbourg, France).
Bis(2-Ethylhexyl) phosphate (DEHP), bis(2-ethylhexyl)
phosphite (DEHPi), 2-nitrophenyl octyl ether (NPOE) and
ammonium formate (≥ 99.995% N ­ H4HCO2, trace metal basis)
were purchased from Sigma Aldrich. Thymol (Thy), 6-methyl-
coumarin (6MC), methanol (MeOH, LC-MS grade) and nitric
acid (69% H­ NO3) were from Merck (Darmstadt, Germany).
Ethyl acetate (EtOAc, HiPerSolv Chromanorm), n-heptane
(HiPerSolv, Chromanorm) and sodium carbonate ­(Na2CO3)
were purchased from VWR Chemicals (Leuven, Belgium).
Formic acid (99.0% HCOOH, Optima LC-MS grade) was
from Fischer Scientific (Leicestershire, UK). Type I water was
obtained in-house using a Milli-Q purification system from
Millipore (Molsheim, France). Quantisal extraction buffer was
obtained from Immunalysis Corporation (Pomona, CA, USA).
Preparation of solutions
Oral fluid samples
Oral fluid (OF) was sampled using Quantisal Oral Fluid Col-
lection device from Immunalysis. A collector pad collected 1
mL (± 10%) OF specimen confirmed by the use of a colour
indicator. The collector pad was subsequently submerged in
a tube containing 3 mL Quantisal extraction buffer (Immu-
nalysis). OF samples that arrive at the laboratory thus com-
prised OF diluted 1:3 (v/v) in the Quantisal buffer. Samples
consisting of OF diluted in the Quantisal extraction buffer
are henceforth referred to as “OF samples”. OF samples
used in method development and validation were prepared
by diluting drug-free OF with Quantisal buffer (1:3, v/v).
Drug-free OF was donated anonymously by healthy volun-
teers employed at the Department of Clinical Pharmacology
at St. Olav University Hospital (Trondheim).
External control samples of buprenorphine (n = 1),
codeine (n = 3), methadone (n = 2) and morphine (n = 3)
were obtained from LGC Proficiency Testing (Bury, UK).
The samples consisted of OF spiked by the manufacturer
and were diluted 1:3 (v/v) with Quantisal Extraction buffer
prior to analysis.
Working solutions, calibrators, and quality controls
Stock solutions of target analytes were combined and diluted
to working solutions in MeOH for each calibrator and qual-
ity control (QC) level. Calibrator and QC samples were
prepared by spiking 2% working solutions in drug-free OF
samples. Analytes were grouped in three calibration ranges,
0.1–5 μg/L, 1–50 μg/L and 3–50 μg/L, with four calibration
concentrations per range. The calibration range corresponds
to concentrations in pure OF. Calibrators and QC samples
were prepared to account for dilution in the collection
device. The internal standard (IS) working solution was
prepared in MeOH:H2O (1:1, v/v). Working solutions, cali-
brators, QC samples and IS solution were stored at −20°C.
Concentrations of target analytes and internal standards are
further detailed in Table S2-3.
Deep eutectic solvent
The deep eutectic solvent was prepared by heating appropri-
ate amounts of 6-methylcoumarin and thymol (1:2 molar
ratio) at 70°C for 12 min. The solvent was vortex-mixed
prior to every use.
Electromembrane extraction
EME was performed with a prototype device from Extrac-
tion Technologies Norway (Ski, Norway), shown in Fig. 1.
The prototype utilized vials of a conductive polymer and has
been described previously [27, 38]. Ten EME cells could
operate simultaneously. Each cell comprised a sample vial,
an acceptor vial and a flat polypropylene support membrane
contained in a leak-tight union. The sample vial held the
biological sample and sample diluent. The latter served to
control pH in the sample. The acceptor vial contained the
acceptor solution. The membrane was sandwiched between
the union and one vial to prepare the liquid membrane. The
liquid membrane was prepared by pipetting an organic sol-
vent onto the PP support, while the union was mounted on
a conductive vial. EME cells were assembled and placed on
the device where they were subjected to horizontal agita-
tion, and the electric field was coupled through the vials via
electrodes in the device lid and an external power supply
(ES 0300-0.45, Delta Elektronika BV, Zierikzee). Target
analytes migrated from the sample (anode) through the liq-
uid membrane contained in the union and into the acceptor
(cathode). The acceptor vial was re-capped after extraction
and transferred directly for UHPLC-MS/MS analysis of the
acceptor solution.
The final extraction protocol was as follows. The
sample vial held OF samples (100 μL), IS working solu-
tion (15 μL) and 0.1% (v/v) HCOOH (sample diluent,
175 μL). The liquid membrane was prepared by pipet-
ting 8-μL membrane solvent (2:1 (v/v) mixture of NPOE
and a deep eutectic solvent consisting of 6MC and Thy
(1:2, molar ratio)) onto the support membrane (Accurel
PP2E, 3M Deutschland GmbH, Wuppertal, Germany). A
punched out silicone disk (2 mm thick, D 9 mm, I.D. 6
mm) was sandwiched between the union and membrane
to provide electric insulation. The acceptor vial contained
13
5326
Fig. 1  a Prototype conduc-
tive vial EME setup with ten
EME cells (left). An EME
cell consisted of conductive
vials, a union, and PP support
membrane (right). b EME of
protonated bases (­ BH+). ­A− is
an anionic substance and N is a
neutral molecule
300 μL 0.1% (v/v) HCOOH. EME cells were assembled,
and extraction was carried out by applying 20 V for 20
min with agitation (875 rpm). Conditions during method
development were the same as described above when not
stated otherwise. Method development experiments were
carried out using OF samples spiked to the middle of the
calibration range of each analyte.
With the current prototype and liquid membrane, it
was found that the membrane should be mounted on the
donor vial, opposed to the acceptor vial, as recovery and
precision of the most lipophilic compounds seemed to be
affected by this detail. This has not been observed previ-
ously using other membrane solvents (e.g. NPOE) with
conductive vial EME. The remaining target opioids were
unaffected by the cell configuration. All reported recov-
ery data (method development and validation) of opioids
were obtained by mounting the membrane on the donor
vial. However, some validation experiments were carried
out with the opposite configuration.
Due to limited amount of prototype equipment, vials
were washed and re-used. To avoid carry-over, vials were
washed using the following procedure: (1) empty vials
were filled with 1% HCOOH in MeOH (t > 12 h), and (2)
vials were thoroughly rinsed with type I water followed
by (3) MeOH.
13
Skaalvik T. G. et al.
Liquid–liquid extraction — routine sample
preparation method
The final EME method was compared to a routine method
at St. Olav University Hospital (Trondheim, Norway) used
to screen multiple drugs of abuse in OF, including ten of the
target opioids (morphine, oxycodone, codeine, O-DM-tram-
adol, ethylmorphine, tramadol, ketobemidone, buprenor-
phine, fentanyl and methadone). The sample preparation
was as follows. Automated liquid handling was performed
by a Hamilton Microlab Star pipetting robot (Hamilton
Company, Bonaduz, Switzerland). Calibrators and quality
controls were prepared by the robot by spiking a mixture
of Quantisal extraction buffer and type I water (1:3, v/v)
with working solutions of analytes. Liquid–liquid extrac-
tion (LLE) was performed in a 2.2-mL 96-well plate (Irish
Life Sciences, Longford, Ireland) by mixing samples (200
μL), IS working solution (25 μL), 0.2 M ­Na2CO3 (200 μL)
and EtOAc/heptane (80:20, v/v, 1000 μL) for 1 min at 2100
rpm (Multi-format Plates & Tubes, Porvoir Sciences, Nor-
folk UK). The content was centrifuged for 5 min at 4235
rcf (Rotana 460, Hettich Zentrifugen, Tuttlingen, Germany).
Aliquots of supernatant (800 μL) were transferred to a new
96-well collection plate. The supernatant was acidified with
0.1% ­HNO3 in MeOH (10 μL) and then evaporated under
Conductive vial electromembrane extraction of opioids from oral fluid 5327
ambient air at 40 °C for 20 min using Ultravap sample con-
centrator (Porvair Sciences). Finally, samples were recon-
stituted in 60% MeOH in ­H2O (75 μL) and mixed (2100
rpm, 1 min). Chromatographic separation and detection were
achieved using the UHPLC-MS/MS conditions described in
the “UHPLC-MS/MS” section.
The method was validated prior to its implementation,
and the following validation data were achieved for opi-
oids: precision and accuracy at LLOQ CV ≤ 12% and bias
± 9%, intra-day CV ≤ 7%, inter-day CV ≤ 14%, bias ± 11%,
ME (uncorrected) 55–102% (CV ≤ 10%), ME (corrected)
95–110 (CV ≤ 5%) and recoveries 13–86% (CV ≤ 15%).
UHPLC‑MS/MS
Target analytes were separated and detected by ultra-high-
performance liquid chromatography–tandem mass spec-
trometry (UHPLC-MS/MS). Chromatographic conditions
were similar for EME and LLE samples. Gradient elution
was carried out at 60 °C on an Acquity UPLC BEH C ­ 18
(2.1 × 50 mm, 1.7 μm particles) analytical column with an
Acquity UPLC BEH ­C18 (2.1 × 5 mm, 1.7 μm particles)
pre-column on an Acquity UPLC I-Class instrument from
Waters (Milford, MA, USA). Mobile phase A consisted of
5 mM ­NH4HCO2 (pH 10.1) and B consisted of MeOH. The
flow rate was 0.5 mL/min and total run time 5.3 min. The
gradient started with 5% B and continued to 30% B after 0.3
min, 50% at 2.7 min, 90% at 3.8 min and 98% at 4.8 min.
The injection volume was 3 μL.
Analytes were detected using a Xevo TQ-S tandem mass
spectrometer (Waters, Manchester, UK) equipped with a
Z-spray electrospray interface. Positive ionization (ESI+)
was performed in multiple reaction monitoring (MRM). The
capillary voltage was 1 kV and ion source temperature 120
°C. The desolvation gas (nitrogen) was heated to 650 °C
and delivered with a flow rate of 1000 L/h. Cone gas flow
was 150 L/h. MRM transitions of analytes included in the
EME-UHPLC-MS/MS protocol are provided in Table S4.
Calibration and identification
Target opioids were identified by relative retention times and
ion ratios between MRM transitions compared to calibra-
tors. Weighted (1/×) quadratic calibration curves with four
calibration levels, excluding the origin, were used based on
analyte peak area normalized to IS peak area. Calculated
analyte concentrations related to the concentration in OF,
accounting for dilution in the collection buffer.
Validation
The method was validated based on guidelines given
by Peters et al. [39] and the European Medical Agency
(EMA) [40] and included linearity, precision, accuracy,
extraction recovery, matrix effects, post-extraction stabil-
ity and carry-over.
Linearity was evaluated by assessing the correlation
coefficient (R) when applying a 1/× weighted linear cali-
bration model with three replicates per calibrator. The
lower limit of quantification (LLOQ) was set as the lowest
calibrator concentration. Requirements of LLOQ was a
signal-to-noise (S/N) above ten and three for the quanti-
fying and qualifying ion, respectively, and inter-day CV
and accuracy within ± 20%. Precision and accuracy at
LLOQ were evaluated by analysing one spiked QC sample
on ten days.
The method precision and accuracy were assessed at
three QC levels. The intra- and inter-day precision at each
QC level was evaluated by analysis of six parallels on 1
day and one sample at three QC concentrations on 10 dif-
ferent days, respectively. Accuracy was determined using
inter-day precision data and by analysis of external control
samples of selected analytes (morphine, buprenorphine,
codeine and methadone). Z-scores were calculated as the
difference between our measured value and assigned value
(mean of laboratories included in the proficiency testing
programme), divided by the standard deviation for profi-
ciency assessment (SDPA).
Extraction recovery (RE) and matrix effects (ME) were
calculated by the following equations:
Rpre
RE = × 100% (1)
Rpost
Apost
ME = × 100% (2)
Aref
where Rpre and Rpost correspond to the IS-normalized
analyte peak area in OF samples spiked with analytes pre-
and post-extraction, respectively, with the internal stand-
ard added post-extraction in both cases. Apost and Aref cor-
respond to the analyte peak areas in spiked blank extracts
and a neat acceptor, respectively. The recovery was evalu-
ated at the high and low QC level with six parallels per
concentration. Recovery was related to the EME step and
did not include recovery from the OF collector pad. Matrix
effects were assessed with six OF samples from different
individuals at the high and low QC level.
Carry-over was evaluated by inspecting chromatograms
of analyte-free OF samples after injection of a sample five
times the concentration of the highest calibrator. Post-
extraction stability was assessed by reinjection of QC
samples (n = 5 per level) and calibrators left on the auto-
sampler (10°C) for 1, 3 and 7 days.
13
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Method comparison with reference method
To demonstrate the applicability of the method, authentic
anonymized OF samples were analysed, and results were
compared to a routine screening method at the Depart-
ment of Clinical Pharmacology at St. Olavs hospital
(Trondheim, Norway). Samples (n = 30) that had been
subjected to drug testing were anonymized and stored for
4 weeks at 4°C and up to 1 week at −20°C. The samples
were reanalysed with EME-UHPLC-MS/MS and a rou-
tine method that employed LLE-UHPLC-MS/MS. The
collected samples of methadone and buprenorphine were
above the highest calibrator level and were diluted with a
water/Quantisal buffer mixture (1:3 v/v) prior to analysis
with EME and the routine method. The routine screening
method targeted a total of 29 drugs of abuse, including
amphetamine-type and other stimulants, benzodiazepines,
hallucinogens, Z-hypnotics and opioids. Ten of the target
opioids (morphine, oxycodone, codeine, O-DM-tramadol,
ethylmorphine, tramadol, ketobemidone, buprenorphine,
fentanyl and methadone) were included in both methods.
Results were compared using Passing and Bablok regres-
sion [41] with MedCalc Statistical Software version 20
(MedCalc Software Ltd., Ostend, Belgium). According
to the Regional Committee of Research Ethics, no for-
mal approval is needed for a brief presentation of routine
results as part of a methodological article.
Characterization of the liquid membrane
The final membrane solvent was subjected to a characteri-
zation aiming to define the application range in terms of
analyte log P values. Experimental details and interpreta-
tions were the same as in previous work [37]. In short, based
on extraction of 90 basic model substances, the extraction
Fig. 2  Recoveries of target
opioids with six membrane
solvents: DEHPi, NPOE, and
NPOE with 1% and 5% (w/w)
DEHP, 6MC:Thy (1:2, molar
ratio), and a 1:2 (v/v) mixture of
6MC:Thy (1:2, molar ratio) and
NPOE. Applied voltage was 50
V for all membranes except for
6MC:Thy (1:2, molar ratio) (10
V). t = 20 min
13
Skaalvik T. G. et al.
window of a liquid membrane was expressed through analyte
log P values, from log Plow to log Phigh. Herein, log Plow is
the lowest log P value where RE ≥ 40% is achieved for ≥
50% of analytes in the range from log Plow to log Plow +0.5.
Similarly, ≥ 50% of model analytes in the log P range from
log Phigh – 0.5 to log Phigh are extracted with RE ≥ 40%.
Results and discussion
Method development
Selection of the liquid membrane
Selecting an appropriate membrane solvent is a key step for
efficient and robust EME. Attention should be on the extrac-
tion recoveries, repeatability and system stability. The latter
is assessed by monitoring the extraction current. Although
usually found by trial and error, efforts have been put for-
ward to study the application range of known membrane
solvents to guide method development. Here, the extraction
window of a liquid membrane is defined as the range of ana-
lyte log P values where high recoveries are expected [37].
For basic analytes, NPOE has been established as the first
choice based on stability, low current and high reproducibil-
ity. The extraction window of NPOE was recently defined
as 2 < log P < 6 [37]. Compounds with log P < 2 are likely
too hydrophilic for efficient transfer into NPOE. On the other
side, very lipophilic compounds have unfavourable partition-
ing coefficient between the liquid membrane and acceptor
and are trapped in the liquid membrane.
The target opioids were extracted from OF samples for
20 min with six membrane solvents. Recoveries are pre-
sented in Fig. 2. Analytes are ordered by their predicted
log P values, which ranged from 0.7 (morphine) to 5.0
Conductive vial electromembrane extraction of opioids from oral fluid 5329

(methadone) (Table S1). As expected from the extraction buprenorphine, fentanyl, cyclopropylfentanyl, etonitazepyne
window of NPOE (2 < log P < 6), opioids in the lower log and methadone the present experiment.
P range were not efficiently extracted with this membrane In a final set of experiments, the deep eutectic solvent was
solvent. For the other membrane solvents, extraction win- mixed with NPOE. The solvents were mixed in a 1:2 (v/v)
dows were not clearly defined at this point. ratio of 6MC:Thy (1:2, molar ratio) and NPOE. The mem-
DEHPi has been reported for extraction of hydrophilic brane solvent consisting of 6MC, Thy and NPOE covered
analytes [42], and this solvent was tested as an alterna- all target opioids, with high recoveries and acceptable preci-
tive to NPOE. In the present experiments, extractions with sion (CV ≤ 18%) for all analytes. In addition, stability was
DEHPi was generally only efficient for the most lipophilic improved compared to pure 6MC:Thy (1:2, molar ratio), and
opioids (log P > 2.5), similarly to NPOE, with the excep- stable extractions could be carried out at higher voltages.
tion of O-DM-tramadol (log P = 1.6), which was extracted
with medium efficiency (41%). Extraction window of the liquid membrane
Addition of the ionic carrier DEHP to NPOE is a well-
established measure to increase extraction efficiency of The selected liquid membrane was further characterized
hydrophilic analytes [43, 44]. As seen from Fig. 2, adding by studying its extraction window. The extraction window
1% DEHP to NPOE allowed exhaustive extraction of 11 of the membrane was determined by the same procedure
out of 13 target opioids and improved mass transfer of described by Zhou et al. [37], where 90 basic model ana-
all compounds in the lower end of the log P range. How- lytes were extracted from pseudo samples using conductive
ever, morphine recoveries were unsatisfactory (RE 19%, vial EME. The extraction window is here expressed through
CV 27%). Increasing the amount of carrier to 5% did not analyte log P values. Figure 3 shows the recoveries of model
improve morphine recoveries and resulted in membrane analytes plotted against their predicted log P values. The
trapping of the most lipophilic compounds. Mass trans- extraction window of the 1:2 (v/v) mixture 6MC:Thy (1:2,
fer of morphine was not improved upon increasing the molar ratio) and NPOE was found to be 1 < log P < 5.5.
extraction potential or time or upon altering the pH (data By comparison to pure NPOE (2 < log P < 6), the extrac-
not shown). tion window was shifted slightly towards more polar com-
Deep eutectic solvents have emerged as alternative extrac- pounds, and this allowed simultaneous extraction of all tar-
tion solvents due to their greenness, low-cost and applicabil- get opioids.
ity for a wide range of solutes [45]. A eutectic mixture is a
liquid comprising of two or more components that interact Sample diluent, acceptor, extraction potential, and time
through hydrogen bonds resulting in melting point depres-
sion relative to individual components. Hydrophobic deep After selecting a mixture of 6MC, Thy and NPOE as the
eutectic solvents have been used as membrane solvents in liquid membrane, further method development included
EME previously [46–49]. In particular, eutectic mixtures selection of sample diluent and acceptor, extraction potential
of coumarin and thymol (Thy) have demonstrated efficient
extraction of compounds in a log P range similar to the tar-
get opioids [47, 49]. In the present experiments, analytes
were first extracted with a deep eutectic solvent consisting
of 6-methylcoumarin (6MC) and Thy in a molar ratio of
1:2, which was previously used in extraction of methotrex-
ate and metabolites (−2.2 < log P < 0.9) from plasma [48].
6MC was used opposed to coumarin due to lower water solu-
bility making the liquid membrane inherently more stable.
Despite this, the deep eutectic solvent was less stable than
the other membrane solvents and had to be operated at lower
voltage (10 V) to avoid excessive current and attain system
stability. Extraction with 6MC:Thy (1:2, molar ratio) cov-
ered the lower log P range of target opioids, with satisfac-
tory results for morphine (RE 67%, CV 10%). Recoveries
were however low for the most lipophilic opioids. Although
some lipophilic analytes have been extracted with coumarin
and Thy in literature, the higher intrinsic hydrophobicity
Fig. 3  Extraction recovery versus log P for 90 bases using a 1:2 (v/v)
of 6MC could explain the unexpectedly low recoveries of mixture of 6MC:Thy (1:2, molar ratio) and NPOE as the liquid mem-
brane. The extraction window is highlighted

13
5330
and time. The agitation rate was set to 875 rpm based on
previous experience.
Solutions of 0.1% and 0.5% (v/v) HCOOH were evalu-
ated as sample diluent and acceptor (Fig. 4a). Although not
always possible, choosing the same reagent as the sample
diluent and acceptor was considered advantageous in rela-
tion to operational simplicity. OF samples were diluted 1:2
(v/v) with the sample diluent, resulting in a sample pH of 5
and 3 for 0.1% and 0.5% HCOOH, respectively. The acceptor
pH was 2.7 and 2.5, respectively. After applying 50 V for 20
min, both conditions resulted in high performance with an
average recovery for all analytes of 96% and 88% for 0.1%
and 0.5% HCOOH, respectively. The 0.1% HCOOH solution
was selected as the final sample diluent and acceptor.
The extraction potential was selected by studying ana-
lyte recovery when applying 0, 5, 15, 30, 50 and 80 V for
Fig. 4  Operational parameters for EME of opioids from OF samples
using a mixture of 6MC, Thy, and NPOE as membrane solvent. a
Recovery using two concentrations of HCOOH as sample diluent and
13
Skaalvik T. G. et al.
20 min. Based on the recorded system current (Fig. S1),
the system was considered stable with all conditions. Fig-
ure 4b show the extraction recovery with varying extrac-
tion potential for selected opioids. The optimal extraction
potential was compound dependent, and 20 V was selected
as a compromise. Method development data for all opioids
are presented in Table S5-6.
The final extraction time was selected to be 20 min as
a compromise between recovery, precision and sample
throughput. Figure 4c shows recovery after extraction
for 2, 5, 10 and 20 min for selected opioids. All com-
pounds were exhaustively extracted after 20 min with the
exception of morphine and etonitazepyne, which reached
recoveries of 80% and 68% respectively. The precision was
acceptable with CV ≤ 15%.
acceptor (V = 50 V, t = 20 min). b Recovery with varying extraction
potential selected opioids (t = 20 min). c Recovery as a function of
extraction time for selected opioids (V = 20 V)
Conductive vial electromembrane extraction of opioids from oral fluid 5331

Table 1  Validation data of opioids: coefficient of correlation (R), (ME), and internal standard corrected matrix effects. The quantifica-
precision (CV%), and accuracy (bias%) at the lower limit of quanti- tion range was from LLOQ to 50 × LLOQ for all analytes
fication (LLOQ) and three QC levels, recoveries (RE), matrix effects

Analyte, QC concen- LLOQ (μg/L) Intra-day CV (%) Inter-day CV (%) Bias (%) RE (CV%) ME (CV%) IS corrected Linearity (R)
tration (μg/L) ME (CV%)

n=6 n = 10 n = 10 n=6 n=6 n=6

Morphine 3 3 −0.2 0.9996
4.5 2 2 −0.8 66 (14) 98 (3) 102 (2)
30 2 3 5
120 1 2 8 85 (4) 97 (3) 101 (1)
Oxycodone 1 4 −8 0.9998
1.5 2 3 −9 104 (3) 97 (3) 102 (2)
10 1 2 −6
40 1 1 −2 97 (3) 96 (3) 98 (2)
Codeine 1 6 −6 0.9996
1.5 5 5 −8 97 (2) 95 (3) 103 (1)
10 3 5 0.2
40 2 9 −2 102 (3) 94 (3) 103 (1)
O-DM-tramadol 1 5 −11 0.9996
1.5 2 8 −6 96 (4) 97 (3) 104 (2)
10 3 5 −4
40 2 2 −5 102 (2) 97 (3) 105 (2)
Ethylmorphine 1 3 −8 0.9998
1.5 2 2 −8 97 (2) 96 (3) 100 (2)
10 1 3 −3
40 1 3 −2 100 (2) 96 (3) 99 (2)
Tramadol 3 2 −12 0.9998
4.5 2 5 −11 99 (1) 93 (4) 97 (2)
30 2 4 −9
120 2 4 −6 99 (1) 99 (4) 103 (2)
Pethidine 1 8 −4 0.9982
1.5 3 4 −5 101 (1) 97 (3) 100 (2)
10 2 4 −2
40 3 4 0.8 98 (2) 99 (3) 102 (2)
Ketobemidone 1 4 −5 0.9998
1.5 3 4 −6 98 (3) 96 (4) 102 (2)
10 2 3 −3
40 2 4 −1 102 (4) 92 (2) 99 (1)
Buprenorphine 3 2 −2 0.9994
4.5 4 4 −2 79 (7) 212 (1) 100 (2)
30 2 3 −1
120 2 3 −0.4 80 (6) 180 (4) 97 (1)
Fentanyl 0.1 13 −9 0.9993
0.15 2 8 −11 62 (13) 105 (4) 98 (4)
1 2 5 −8
4 2 3 −9 69 (14) 104 (4) 98 (2)
Cyclopropylfentanyl 0.1 16 −8 0.9954
0.15 7 11 −5 54 (18) 113 (3) 88 (2)
1 11 9 −6
4 6 12 −2 60 (20) 108 (4) 89 (1)
*Etonitazepyne 0.1 45 −16 0.9663
0.15 10 27 −12 39 (23) 127 (4) 99 (5)

13
5332 Skaalvik T. G. et al.

Table 1  (continued)
Analyte, QC concen- LLOQ (μg/L) Intra-day CV (%) Inter-day CV (%) Bias (%) RE (CV%) ME (CV%) IS corrected Linearity (R)
tration (μg/L) ME (CV%)

1 28 25 −20
4 34 25 −17 45 (21) 116 (4) 96 (2)
Methadone 3 6 −2 0.9995
4.5 2 3 −4 66 (19) 133 (3) 104 (3)
30 1 2 −1
120 2 3 0.2 66 (18) 122 (3) 101 (2)

*Validation criteria not fulfilled

Validation Regarding the quantitative data, recovery from the collec-

The method was validated according to guidelines by Peters
et al. and the European Medical Agency [39, 40]. Linearity,
LLOQ, intra-day precision, inter-day precision and bias, recover-
ies, matrix effects, carry-over and post-extraction stability were
assessed. A summary of validation data is shown in Table 1.
Etonitazepyne could not be quantified with acceptable pre-
cision (CV 25–34%) and accuracy (−20%). This is further
discussed in the “Extraction recoveries and the effect on preci-
sion” section. Validation results of etonitazepyne are included in
Table 1 but are excluded from the result and discussion below.
Calibration curves were linear in the calibration range
with correlation coefficients R ≤ 0.9954. The precision
and accuracy at LLOQ were acceptable (CV ≤ 16%, bias
± 16%). The intra-day CV was ≤ 11%, inter-day CV ≤ 12%
and bias within ± 11%, which was also considered satisfac-
tory. External control samples of buprenorphine, codeine,
methadone and morphine were quantified with Z-scores in
the range −1.5 ≤ Z ≤ 1.7, which was within requirements
(|Z| ≤ 2.0) and indicated good accuracy.
Matrix effects ranged from 93 to 133% (CV ≤ 4%) for all
analytes except buprenorphine, which had matrix effects of
212% and 180% (CV ≤ 4%) at the low and high concentra-
tor swab in the Quantisal Collection Device was not evalu-
ated. Previous studies have found the recovery from the col-
lection device to be high for opioids [51, 52].
Extraction recoveries and the effect on precision
Efficient extraction of all target opioids was achieved with
extraction recoveries in the range from 39 to 104%. Corre-
sponding CVs were ≤ 20% for all analytes except for etoni-
tazepyne (CV = 23%) (Table 1). Analytes that were regarded
as moderately lipophilic (1 < log P < 2.5) had recoveries
above 96% with low variation (CV ≤ 4%), whereas mor-
phine (log P = 0.7) was extracted with recovery above 66%
with CV ≤ 14%. Recovery of lipophilic analytes (log P >
2.5) was in the range 39–80% with CVs 6–23%. In general,
140
120
100
EME (µg/L)

tion, respectively. The matrix effects were 88–105% (CV ≤ 80

4%) for all analytes when corrected with internal standards.
In an additional experiment using a reference solution (neat 60
acceptor, Eq. 2) spiked with a drop of membrane solvent, the
calculated matrix effects of buprenorphine were 100–103%
40
(CV ≤ 4%), which suggests that the apparent signal enhance-
ment found in validation was likely attributed to the presence
20
of liquid membrane solvent in the acceptor.
Analytes were stable in extracts on the auto-sampler (10
0
°C) for at least seven days. Carry-over after injection of a 0 20 40 60 80 100 120 140
sample five times the concentration of the highest calibrator LLE (µg/L)
was acceptable (≤ 20% of LLOQ). However, OF concentra-
tions of buprenorphine may occasionally be much higher if Fig. 5  Passing and Bablok regression between EME-UHPLC-MS/
MS and LLE-UHPLC-MS/MS. The Y-intercept was −1.00 (95% CI
sampling occurs shortly after intake [50], and this must be
from [−1.0000, 0.1389]), and the slope was 1.00 (95% CI [0.9722,
considered in a routine setting. 1.000]). The Spearman rank correlation coefficient was 0.998 (95%
CI [0.996, 0.999])

13
Conductive vial electromembrane extraction of opioids from oral fluid 5333
recoveries of lipophilic analytes (i.e. buprenorphine, fen-
tanyl, cyclopropylfentanyl, etonitazepyne and methadone)
were associated with slightly higher variation compared to
other analytes in the method and compared to similar ana-
lytes extracted with NPOE previously [27]. The lipophilic
analytes ultimately required more correction by internal
standards than the remaining target opioids. Validation data
were satisfactory for buprenorphine, fentanyl and metha-
done, which all had deuterated analogues to compensate
variability. For cyclopropylfentanyl and etonitazepyne, no
deuterated standards were available, and methadone-d3 was
used. Validation data complied with guideline requirements
for cyclopropylfentanyl, while etonitazepyne suffered in
terms of linearity and precision and would likely benefit
from having a dedicated internal standard.
Application and comparison with routine method
Anonymized positive OF samples for buprenorphine (n =
13), codeine (n = 4), ketobemidone (n = 3), morphine (n =
2) and methadone (n = 9) from a routine screening method
were subjected to EME to demonstrate method applicabil-
ity. The sample preparation of the routine screening method
consisted of LLE, evaporation and reconstitution.
The correspondence between methods was within ± 15%
for 32 of the 33 concentration pairs. One sample of codeine
deviated by 23%, which was considered acceptable. The
Passing and Bablok regression line (Fig. 5) between EME
and LLE was [EME] = 1[LLE] − 1. The 95% confidence
intervals of the slope ([0.97, 1.00]) and intercept ([−1.00,
0.14]) contained one and zero, respectively, which implied
no systematic or proportional differences between methods.
Compared to the LLE protocol, EME involved fewer steps
with a significant reduction in organic solvent consumption,
which made EME more favourable with respect to principles
of green analytical chemistry [53, 54]. We recently demon-
strated this in another work by comparing greenness scores
of conductive vial EME and an LLE method similar to the
routine OF screening method [38].
Conclusion
In the present contribution, selected opioids were success-
fully extracted from OF samples using conductive vial
EME. Simultaneous EME of analytes in a wide log P range
is a challenge, as narrow extraction windows of many liq-
uid membranes cause discrimination of either lipophilic or
hydrophilic analytes. By adding 6-methyl coumarin and
thymol to 2-nitrophenyl octyl ether, the extraction window
was shifted slightly towards more polar substances, and
this allowed simultaneous extraction of all target opioids.
When EME was combined with UHPLC-MS/MS, valida-
tion data were within recommended guidelines for 12 out
of 13 target opioids. In general, the most lipophilic analytes
were prone to higher variability than the remaining analytes;
however, quantitative results were satisfactory when apply-
ing appropriate internal standards. External control samples
containing both hydrophilic opioids (morphine, codeine) and
lipophilic opioids (buprenorphine, methadone) had satisfac-
tory z-scores (|Z| ≤ 2.0). In addition, quantitative data of
authentic OF samples were in accordance with a reference
routine method that used conventional sample preparation.
The presented data thus illustrate the applicability of con-
ductive vial EME, which can be a greener alternative to
conventional sample preparation in the future. Lastly, the
results further motivate research on the application ranges
and characterization of liquid membranes in EME, which
will be useful for future method developers.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00216-0​ 23-0​ 4807-3.
Authors contribution Tonje Gottenberg Skaalvik, conceptualization;
investigation; writing, original draft; writing, review and editing; and
visualization; Chen Zhou, investigation and writing, review and edit-
ing. Elisabeth Leere Øiestad, conceptualization and writing, review
and editing. Solfrid Hegstad, conceptualization and writing, review
and editing. Roger Trones, writing, review and editing. Stig Pedersen-
Bjergaard, conceptualization and writing, review and editing.
Funding Open access funding provided by Royal Library, Copenhagen
University Library This work was supported by the Research Council
of Norway (NFR 310086).
Declarations
Ethics approval The healthy volunteers were employed at the Depart-
ment of Clinical Pharmacology at St. Olav University Hospital (Trond-
heim) and donated oral fluid for use in the study anonymously after
informed oral consent. Such studies are, in accordance with Norwegian
legislation, assessed by the Regional Committee of Research ethics not
to be subject to application.
Competing interests Co-author Roger Trones is working in the com-
pany Extraction Technologies Norway, and this company develops
EME technology towards commercialization. All other authors declare
that they have no known competing financial or non-financial interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.
13
5334 Skaalvik T. G. et al.

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