Chapter 16
Analysis of Illicit Drugs in Wastewater Using High-
Performance Liquid Chromatography-Electrospray
Ionization-Tandem Mass Spectrometry (HPLC-ESI-MS/MS)
Katelyn S. Foppe and Bikram Subedi
Abstract
Consumption of illicit drugs is considered to be a global socioeconomic burden of adverse ecological and
public health. Proper identification and quantification of trace level residues of illicit drugs in wastewater
allow for an estimation of drug usage in a community, the amount of drug discharge into the environment,
and the overall fate of drugs in the environment. This chapter provides a procedural detail of the determina-
tion of select illicit drugs and their metabolites in wastewater, suspended particulate matter, and sewage
sludge using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.
Key words HPLC, Mass spectrometry, Illicit drugs, Wastewater, Sewage sludge, Analytical method
1 Introduction
Drug abuse is considered a national epidemic in many countries
including the USA [1]. The consumption of illicit drugs is directly
associated with impaired social indicators such as economic loss
and adverse public health. Consumed and/or disposed illicit drugs
are routinely flushed down the drain as residuals, bioconjugates,
and their metabolites [2]. The discharged drug residues from
wastewater treatment plants (WWTPs) into the environment con-
taminate source water and can cause unidentified effects on non-
target organisms [3]. Assessment of trace levels of drug residues in
wastewater provides opportunities for the estimation of drug usage
in a community, the amount released into the environment, and
the overall environmental fate of drug residues [4]. Despite several
reports available on identification and quantification of illicit drugs
in wastewater [4–7], there are no standardized protocols estab-
lished to identify and quantify the trace-level residues of illicit
drugs in environmental matrices including wastewater.
Rabi A. Musah (ed.), Analysis of Drugs of Abuse, Methods in Molecular Biology, vol. 1810,
https://doi.org/10.1007/978-1-4939-8579-1_16, © Springer Science+Business Media, LLC, part of Springer Nature 2018
183
184 Katelyn S. Foppe and Bikram Subedi
Drug metabolites can be at higher concentration levels than
parent drugs in wastewater. For example, the excretion profile of
cocaine from the human body is 1–9% unchanged cocaine, 35–54%
benzoylecgonine, 32–49% ecgonine methyl ester, 0.7% cocaethyl-
ene, and trace amounts of norcocaine [8]. Among all possible drug
residues, one having relatively higher solid-water partitioning coef-
ficient (such as EDDP) can preferentially adsorb onto suspended
particulate matter (SPM) compared to the dissolved phase.
Therefore, estimations based on the concentration of only parent
drugs in dissolved phase typically underestimate the actual drug
usage in a community and/or drug discharge into the environ-
ment [4]. This chapter provides stepwise procedures for the iden-
tification and quantification of select illicit drugs and their
metabolites (Fig. 1) in wastewater, SPM, and sludge using high-
performance liquid chromatography-electrospray ionization-
tandem mass spectrometry (HPLC-ESI-MS/MS) as summarized
elsewhere [4].
2 Materials
2.1 Samples 1. Wastewater influent (100 mL).
2. Wastewater effluent (100 mL).
3. Freeze-dried sludge (0.1 g).
4. Freeze-dried SPM (0.1 g).
2.2 Solvents 1. HPLC-grade methanol.
and Reagents 2. HPLC-grade or ultrapure water (18.2 MΩ cm).
3. HPLC-grade acetonitrile.
4. Formic acid (>98%).
2.3 Standards 1. Stock solutions of target illicit drugs: Commercially avail-
able typically at 1000 μg/mL or 100 μg/mL concentrations
(≥99%).
2. Individual internal standard for each target illicit drug:
Deuterated analogs of illicit drugs are commercially available
typically at 1000 μg/mL or 100 μg/mL concentrations (≥99%).
2.4 Instrument HPLC-ESI-MS/MS (API 2000 electrospray triple quadrupole
and Supplies mass spectrometer combined with an Agilent 1100 Series HPLC
system), an ultra-biphenyl column (5 μm × 100 mm × 2.1 mm),
centrifuge, freeze-dryer, ultrasonic bath, vortex, glass fiber filter
(pore size ≤1 μm), HLB 6 cm3 solid-phase extraction (SPE) car-
tridges, and silanized amber glass jars for sample collection with
Teflon™ screw caps.
Analysis of Illicit Drugs in Wastewater Using High-Performance Liquid… 185
Fig. 1 Chemical structures of select illicit drugs and their metabolites
3 Methods
3.1 Method 1. Prepare working solution [1000 ng/mL (1 ppm)] of each
Development standard in methanol. Store all standard stock solutions and
working solutions at −20 °C.
2. Identify the parent ion, two major MS/MS transitions
(Table 1), optimum compound-specific parameters (collision
energy, entrance potential, declustering potential, focusing
potential, and exit potential), and method-specific source
parameters (ion source temperature and ion spray voltage).
186 Katelyn S. Foppe and Bikram Subedi
Table 1
ESI-MS/MS parametersa for analytes and internal standards
Analytes Q1 Q3 DP, FP, EP, CP, EXP
Cocaine (CCN) 304 182;105 30;400;6;27;7
CCN-D3 307 185;85 35;390;8;27;7
Benzoylecgonine (BEG) 290 168;105 35;390;9;25;7
BEG-D8 298 171;110 30;390;10;27;7
Norcocaine (NCCN) 290 168;136 22;390;10;22;7
NCCN-D3 293 171;136 28;400;10;22;7
Cocaethylene (CCE) 318 196;82 25;380;10;27;9
CCE-D8 326 204;85 22;390;10;27;9
Amphetamine (APT) 136 119;91 35;400;12;30;5
APT-D8 144 97 20;400;11;22;5
Methamphetamine (MAPT) 150 91;119 20;400;12;25;5
MAPT-D8 158 93;124 25;400;12;27;5
Morphine (MPH) 286 165;153 40;400;10;55;7
MPH-D6 292 153;165 40;400;10;55;5
Morphine-3-β-d-glucuronide (M3G) 462 286 35;400;12;40;5
M3G-D3 465 289 32;400;12;42;6
Morphine-6-β-d-glucuronide (M6G) 462 286 50;400;12;45;5
M6G-D3 465 289 35;400;12;45;5
Methadone (MTD) 310 265;105 10;400;10;20;12
MTD-D9 319 268;105 15;375;10;22;5
EDDP 278 234;249 25;400;12;38;10
EDDP-D3 281 234;249 30;400;12;38;10
MDA 180 105;133 15;400;12;32;5
MDA-D5 185 110;138 17;400;10;30;5
MDMA 194 163;105 15;375;5;15;5
MDMA-D5 199 165;107 20;375;12;20;6
MDEA 208 163;105 25;400;11;20;6
MDEA-D5 213 163;105 22;400;10;20;7
Reprinted with permission [4]. Copyright © 2017 American Chemical Society
DP declustering potential, FP focusing potential, EP entrance potential, CP collision potential, EXP exit potential
a
Source parameters: ion spray voltage (4500 V) and temperature (450 °C)
Analysis of Illicit Drugs in Wastewater Using High-Performance Liquid… 187
3. Create a multiple reaction monitoring (MRM) method includ-
ing all optimized parameters and optimize the solvent program
for the baseline separation using chromatographic column
(Table 2 and Subheading 2.4).
4. Prepare seven to nine calibration solution mixtures in metha-
nol with each analyte’s concentration range from 0.1 to
1000 ng/mL and 10 ng/mL level of each internal standard
(see Notes 1 and 2).
3.2 Sample 1. Centrifuge 100 mL of wastewater samples at ~2500 × g for
Preparation 10 min to separate SPM. Weigh the glass fiber filter (pore size
3.2.1 Sample ≤1 μm), filter supernatant, combine the decanted SPM from
Pretreatment centrifugation and residue from filtration, weigh, and freeze-
dry the combined SPM.
Table 2
HPLC mobile-phase compositions and programming for illicit drugs
(flow rate = 400 μL/min)
% B (0.1% formic
Time (min) % A (methanol) acid in water)
0 5 95
2 10 90
2.1 30 70
4 30 70
6 50 50
7 50 50
7.1 65 35
10 65 35
10.1 75 25
12 75 25
12.1 90 10
13.5 90 10
14 10 90
19 10 90
20 100 0
25 100 0
26 5 95
30 5 95
Reprinted with permission [4]. Copyright © 2017 American Chemical Society
188 Katelyn S. Foppe and Bikram Subedi
2. Freeze-dry the ~100 g sludge.
3. Weigh the total freeze-dried SPM and sludge and calculate the
moisture content using an equation:
Wet mass − Dry mass
%Moisture = ×100.
Wet mass
3.2.2 Extraction 1. Spike a mixture of internal standards (25 ng each).
of Wastewater (See Note 3) 2. Mix well and equilibrate for ~30 min at room temperature.
3. Condition the SPE cartridge with 5 mL of methanol followed
by 5 mL of water.
4. Load 100 mL wastewater sample via conditioned cartridge and
allow to dry under vacuum for ~30 min (see Note 4).
5. Elute analytes with 6 mL of methanol, 3 mL of a mixture of
acetone, methanol, and ethyl acetate (2:2:1 v/v/v), and 3 mL
of methanol containing 5% ammonia into a single 15 mL
capacity polypropylene tube.
6. Concentrate eluate mixture to ~100 μL under a gentle stream
of nitrogen at 35 °C (see Note 5).
7. Transfer concentrated eluate into an LC vial using Pasteur
pipette considering multiple rinses with methanol to a final
volume of 1 mL.
3.2.3 Extraction of SPM 1. Spike 0.1 g of a freeze-dried sludge with a mixture of internal
and Sludge standards (10 ng each).
2. Equilibrate for ~30 min at room temperature, and add 6 mL
mixture of methanol and water (5:3 v/v).
3. Sonicate SPM and sludge samples using ultrasonic bath for
30 min at ambient temperature.
4. Centrifuge extract at 1924 × g for 5 min.
5. Transfer decanted extract to a new 15 mL capacity polypropyl-
ene tube.
6. Repeat extraction with 6 mL methanol, combine extracts, and
concentrate extract to ~1 mL under a gentle stream of nitro-
gen as explained previously.
7. Dilute the extract to 12 mL using ultrapure water.
8. Purify extract passing through the cartridge, concentrate, and
transfer to an LC vial as explained above.
3.3 Method 1. Calculate signal-to-noise ratio (S/N) at lowest 2–3 calibration
Validation levels to determine the limit of quantitation (LOQ: a concen-
tration of analyte that provides S/N ratio of 10) and limit of
detection (LOD: a concentration of analyte that provides S/N
ratio of 3) for each analyte (see Note 2).
Analysis of Illicit Drugs in Wastewater Using High-Performance Liquid… 189
2. Spike 100 mL of wastewater samples (n = 3) with 10 ng of
each analyte, extract, purify, and concentrate as explained
above. Spike 10 ng of each internal standard after concentra-
tion to ~100 μL and adjust final volume to 1 mL prior to
analysis. Include one wastewater sample as sample blank (no
analyte spiking). Repeat this experiment with 0.1 g of SPM
and 0.1 g of sludge. Calculate percentage recovery (see
Note 6) of analytes as
Concentration Determined − Concentration in Blank Samples
%Recovery = ×100.
Concentration Spiked
3.4 Sampling 1. Rinse silanized amber glass bottles (1 or 2 L capacity) and
(See Notes 7–9) Teflon™ screw caps with at least three aliquots of methanol
followed by three aliquots of ultrapure water (18.2 MΩ cm).
2. Collect an aliquot of wastewater samples at least once every
hour for 24 h, store at <4 °C until 24 h, and composite to get
at least 100 mL of sample. Collect 24-h composite samples
over a week to examine daily trends of drug usage over a week.
3. Transport samples in ice (or dry ice for long-distance transpor-
tation) to the laboratory as soon as possible to retain the sam-
ple integrity.
3.5 Quality 1. Analyze continuous calibration verification (CCV) solutions
Assurance and Quality containing analytes at midpoint calibration concentration lev-
Control els and 10 ng/mL internal standards before and after every ten
samples. Recovery of more than ±25% of analytes in CCV solu-
tions requires rerunning calibration solutions.
2. Prepare a method blank (spiked with 10 ng/mL internal stan-
dards) exactly as other samples and analyze before and after
each batch of sample analysis to correct for potential labora-
tory contamination.
3. Prepare a matrix spike sample in duplicates by spiking analytes
(midpoint calibration levels) and internal standards (10 ng/
mL) to a randomly selected sample of each batch of sample
preparation. Calculate the percentage recovery of analytes.
Prepare and analyze matrix-spike samples for wastewater, SPM,
and sludge samples.
3.6 Analysis Report any concentrations ≥LOQ. Correct instrumental read-
out with the corresponding concentration in a method blank.
The concentration readout from the instrument is the concen-
tration of drug in the final 1 mL purified extract. The influent
extract is 100-fold concentrated during extraction and purifica-
tion. Therefore, the concentration of drug in the influent sample
is the instrumental readout divided by 100. In the case of SPM
190 Katelyn S. Foppe and Bikram Subedi
and sludge, the concentration readout is the concentration of
drug in 1 mL of the final purified extract from 0.1 g of dried
SPM or sludge. Calculate the concentration of each analyte per
g of dried SPM and dried sludge and convert it to the concentra-
tion of each analyte per g of wet SPM and per g of wet sludge
utilizing % moisture as C wet = % moisture × C dry .
100
4 Notes
1. The matrix-match calibration curve may be required to mini-
mize the effect of matrix components and abide by QAQC
criteria. Matrix-match calibration curves can be prepared by
analyzing seven to nine aliquots of wastewater extracts spiked
with increasing known levels of analytes but a constant level of
internal standard.
2. Neglect the calibration level(s) in the calibration curve that pro-
vides S/N < 3. Typically, the coefficient of determination (R2)
of >0.99 in quadratic or linear regression fits is satisfactorily.
3. Samples are recommended to be extracted within 24 h to
ensure sample integrity. Samples should be stored ≤4 °C until
extraction.
4. Any water droplets left over can compete with eluent; there-
fore, make sure that the cartridge is dried well before elution.
5. Concentration of eluate to complete dryness or concentration
at >35 °C potentially results in a loss in target analytes.
6. Typically, recovery of ±25% can be considered satisfactory for
illicit drugs in wastewater.
7. Duration of sampling events and the sample collection point at
WWTP depend on a research question. For example, wastewa-
ter influent is required to determine the mass load of drug and
the drug usage in a community while the effluent and sludge
are required to determine the amount of drug discharged into
the environment.
8. There are reports of pH adjustments in collected wastewater
samples. It is recommended that the pH for optimum stability
of drugs in wastewater from a WWTP be determined.
9. Depending on a research question, additional information may
need to be collected while sampling, such as flow rate of influ-
ent (an average for sampling periods), population served by
the WWTP, the total amount of the sludge produced per day,
and sludge retention time.
Analysis of Illicit Drugs in Wastewater Using High-Performance Liquid… 191
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