£EPA
United States
Environmental Protection
Agency
Development, and Ground
Truthing, of Analytical Tools for
Measuring Select Emerging
Contaminants (ECs) in a Subset
of Biosolids Collected During
OW's 2006 - 2007 National
Biosolids Survey Project
APM 199
RESEARCH AND DEVELOPMENT
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EPA/600/R-10/108
September 2010
www.epa.gov
Development, and Ground Truthing,
of Analytical Tools for Measuring
Select Emerging Contaminants (ECs)
in a Subset of Biosolids Collected
During OW's 2006 - 2007 National
Biosolids Survey Project
ARM 199
Prepared by
Tammy Jones-Lepp, Research Chemist
U.S. Environmental Protection Agency
National Exposure Research Laboratory
Environmental Sciences Division
Environmental Chemistry Branch
Las Vegas, NV89119
U.S. Environmental Protection Agency
Office of Research and Development
Washington, DC 20460
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The information in this document has been funded by the United States Environmental
Protection Agency. It has been subjected to the Agency 'speer and administrative review
and has been approved for publication as an EPA document.
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CONTENTS
Section pg#
FOREWORD v
EXECUTIVE SUMMARY vi
LIST OF ACRONYMS AND ABBREVIATIONS vii
1.0 INTRODUCTION 1
2.0 SAMPLING 5
3.0 EXPERIMENTAL 5
3.1.1 Sample preparation 5
3.1.2 Optimization of pressurized liquid extraction (PLE)
procedures 6
3.1.2.1 Pre-PLE Extraction Procedures 7
Sample preparation
Extraction Cell preparation
PLE methods 1 and 2
PLE method 3
3.1.2.2 Extraction procedures 8
PLE method 1
PLE methods 2 and 3
Hexane clean-up
3.1.3 Optimization of mass spectrometry detection method 10
3.1.3.1 Liquid chromatography 10
3.1.3.2 ESI-Ion trap mass spectrometry 10
3.1.3.3 Calibration, blanks, and quantitation 11
3.1.4 Safety considerations 11
4.0 RESULTS and DISCUSSION 12
4.1 Extraction methods 12
4.2 Recovery results 13
4.3 Enhancement of chromatography 15
4.4 Analytical concerns 15
4.5 Application of optimized method to nine diverse
biosolids matrices 17
5.0 CONCLUSIONS 18
6.0 FUTURE RECOMMENDATIONS 20
7.0 REFERENCES 21
Appendix
Tables a
Figures f
in
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IV
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FOREWORD
The U.S. Environmental Protection Agency (EPA) is charged by Congress to
protect the nation's natural resources. Under the mandate of national environmental laws,
the EPA strives to formulate and implement actions leading to a compatible balance
between human activities and the ability of natural systems to support and nurture life. To
meet this mandate, the EPA's Office of Research and Development (ORD) provides data
and scientific support that can be used to solve environmental problems, build the
scientific knowledge base needed to manage ecological resources wisely, understand
how pollutants affect public health, and prevent or reduce environmental risks.
The National Exposure Research Laboratory (NERL) is the Agency's center for
investigation of technical and management approaches for identifying and quantifying
exposures to human health and the environment. Goals of the laboratory's research
program are to: (1) develop and evaluate methods and technologies for characterizing and
monitoring air, soil, and water; (2) support regulatory and policy decisions; and (3)
provide the scientific support needed to ensure effective implementation of
environmental regulations and strategies.
This report presents the experimentation, results, findings, and recommendations
of biosolids research conducted from 2006 to 2009. The data from this report will be
transmitted to the Office of Water in support of EPA's statutory requirements under the
Clean Water Act, Section 405(d)(2)(C), to conduct a review of the 40 CFR 503 standards
not less than every two years for purposes of regulating new pollutants where sufficient
data exist.
The author acknowledges the tremendous support of Mr. Thomas Moy, Senior
Environmental Employment Program (SEEP) and Mr. Reza Kazemi (Student Services
Contractor) for all of their laboratory technical support and day-to-day operations,
without whom this research would not have been possible.
The author would also like to acknowledge Dr. Rick Stevens, USEPA, National
Biosolids Program Coordinator, for access to samples and support.
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EXECUTIVE SUMMARY
On February 19, 1993, EPA promulgated the CFR 40 Part 503 Standards for the Use or
Disposal of Sewage Sludge, resulting in numerical standards for ten metals and
operational standards for microbial organisms. The 1993 rule established requirements
for the final use or disposal of sewage sludge when it is: (1) applied to land as a fertilizer
or soil amendment; (2) placed in a surface disposal site, including sewage sludge-only
landfills; or (3) incinerated. These requirements apply to publicly and privately owned
treatment works that generate or treat domestic sewage sludge and to anyone who uses or
disposes of sewage sludge. Under Section 405(d), of the Clean Water Act, EPA
establishes numerical limits and management practices that protect public health and the
environment from the reasonably anticipated adverse effects of chemical and microbial
pollutants in sewage sludge. EPA's statutory requirements under the Clean Water Act,
Section 405(d)(2)(C), is to conduct a review of the 40 CFR 503 standards not less than
every two years for purposes of regulating new pollutants where sufficient data exist.
The purpose of such reviews is to identify additional toxic pollutants and promulgate
regulations for those pollutants consistent with the requirements set forth.
The development and standardization of state-of-the-art science techniques can give
regulators another tool to track, monitor, and measure levels of emerging contaminants in
biosolids that are either land-applied, used as biofuels, or landfilled. The research
presented in this report is a reflection of a lack in the literature regarding a
comprehensive method for the recovery and accurate identification of macrolide
antibiotics and other drugs from biosolids.
A pressurized liquid extraction (PLE), using an in-situ incorporation of cleanup materials
(i.e., fluorosil, alumina) into the PLE cell for biosolids extraction, and high performance
liquid chromatography-electrospray ionization-ion trap mass spectrometer (FtPLC-ESI-
ITMS) analytical method, were optimized and tested on several biosolids matrices. In
this report, not only are the positive results presented, but also negative results are
presented and discussed, offering these as precautionaries to other environmental
analysts.
VI
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LIST OF ACRONYMS AND ABBREVIATIONS
API
AZI
CID
CLA
CLI
CWA
EC
EDC
EPA
BSD
ESI-ITMS
g
HPLC
kg
L
LC
LOD
LOQ
MDL
MDMA
mL
MS
MTBE
n,n'-DMPEA
NERL
NIST
NRC
ORD
OTC
OW
PBDE
PEC
PLE
ppb
PPE
PPCP
ppt
PSEU
RXY
SIM
SEEP
SPE
TNSSS
ML
UPLC
USE
WWTP
active pharmaceutical ingredient
azithromycin
collision induced dissociation
clarithromycin
clindamycin
Clean Water Act
emerging contaminant
endocrine disrupting compound
Environmental Protection Agency
Environmental Sciences Division
electrospray ionization-ion trap mass spectrometer
gram
high performance liquid chromatography
kilogram
liter
liquid chromatograph
limit-of-detection
limit-of-quantitation
minimum detection limit
3,4- methylenedioxymethamphetamine, Ecstasy
milliliter
mass spectrometer
methyl tertbutyl ether
n,n' -dimethylphenethylamine
National Exposure Research Laboratory
National Institute of Standards and Technology
National Research Council
Office of Research and Development
over-the-counter
Office of Water
polybrominated diphenyl ether
predicted environmental occurrence
pressurized liquid extraction
part-per-billion
personal protective equipment
pharmaceutical and personal care product
part-per-trillion
pseudoephedrine
roxithromycin
single ion monitoring
Senior Environmental Employment Program
solid phase extraction
Targeted National Sewage Sludge Survey
microliter
ultra performance liquid chromatography
ultrasonic extraction
wastewater treatment plant
vn
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1.0 INTRODUCTION
Biosolids are defined, for this report, as the solid residue byproducts from wastewater
treatment plants (WWTPs). This biosolids material is usually comprised of human waste
which is a mix of excreta containing bacterial microflora, fats, proteins, pigments, as well
as ingested xenobiotics, such as pharmaceuticals and illicit drugs (excreted unchanged or
as metabolites), personal care products (e.g., shampoos, detergents, cosmetics), along
with other domestic, hospital, and industrial wastes. Some WWTPs mix in municipal
organic solid waste (e.g., yard trimmings, other greenwastes) along with the WWTP-
produced biosolids before composting, landfilling, or marketing. The United States
Environmental Protection Agency (EPA) asked the National Research Council (NRC) to
conduct an independent evaluation of the technical methods and approaches used to
establish the standards for biosolids.(7) Among other things this report identified
pharmaceutical and personal care products (PPCPs) as one category of diverse
compounds that had not yet been studied in biosolids and were especially likely to be
present in domestic biosolids. In response, EPA developed a strategy that while
emphasis was being placed on pathogens to address areas of uncertainty and public
interest, selected new chemicals, of which pharmaceuticals were a set of, would also be
addressed to help determine significant issues and identify information gaps that remain
to be addressed.
After reviewing the literature and various toxicological parameters of some of the
most widely prescribed pharmaceuticals in the US, eight emerging contaminants were
chosen for methodology development. Chosen were four pharmaceuticals: three
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macrolide antibiotics: azithromycin (AZI), clarithromycin (CLA), roxithromycin (RXY);
one lincosamide antibiotic: clindamycin (CLI); two illicit drugs: methamphetamine,
MDMA (Ecstasy); one industrial food additive: n,n'-dimethylphenethylamine (n,n'-
DMPEA) (an isobaric ion to methamphetamine); and one over-the-counter (OTC) drug:
pseudoephedrine (PSEU), which is closely related in chemical structure to MDMA.
While our list of analytes is diverse, the rationale for developing a method is to ultimately
support the EPA's mission to protect the environment and human health.(2-7) For
example, there are reports in the literature of increasing bacterial resistance to multiple
classes of antibiotics.(6, 5) One of the most widely prescribed antibiotics (in the United
States) is the macrolide azithromycin, the annual sales of this drug, in 2009, were
$1,056,715,000 (equating to 49,902,000 prescriptions, see
http://drugtopics.modernmedicine.com/Pharmacy+Facts+&+Figures. McCaig et al.(P)
have reported a correlation between increasing macrolide-resistant pneumococci and the
prescription rate for azithromycin and clarithromycin, both of whose prescription rates
rose by 388%, during the 1990's. While roxithromycin is not prescribed in the United
States, it is widely used in Latin America and Europe; thereby, lending itself as a marker
of the importation of drugs by other than traditional means. The two illicit drugs were
chosen because of limited environmental occurrence data of illicit drugs in environmental
media, and verifiable usage in the United States (last accessed 28-August-2009,
http://www.usdoj .gov/dea/statistics.html).(70, 11)
There are few studies in the literature regarding the analysis of macrolide
antibiotics in WWTP-produced sewage sludges/biosolids. Nieto et al.(72) published a
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method for the quantitative determination of three macrolides (erythromycin,
roxithromycin, and tylosin), five sulfonamides, ranitidine, omeprazole and trimethoprim
in sewage sludge samples, using pressurized liquid extraction (PLE) and high-
performance liquid chromatography-electrospray ionization-mass selective detector
(HPLC-ESI-MSD). One potential issue with this method is that the authors used single
ion monitoring (SUV!) with source voltage induced fragmentation. Their technique is not
as robust, nor definable as other HPLC-ESI methods that use collision induced
dissociation mode (CID) in conjunction with ion traps, or triple quadrupoles, or other
mass spectrometers that are capable of inducing multiple fragmentation product ions
from a precursor ion (referred to as "MS/MS" mode). For example, there are many high
molecular weight compounds (e.g., nonylphenol ethoxylates, ionic surfactants) that can
give ions at the correct molecular weight range as the macrolides, or other higher
molecular weight Pharmaceuticals, but without confirmation by MS/MS there can be
some doubt as to the correct identification and subsequent quantification of the unknown
contaminant.(73) Gobel et al.(7¥) developed a PLE for several classes of antibiotics,
including macrolides from biosolids. In their method, they first performed a PLE
extraction on the biosolids, then a subsequent cleanup of the PLE extract, by passing the
extract through a solid phase extraction (SPE) cartridge, concentrating the SPE extract
and analysis by LC-ESI-MS/MS (triple quadrupole mass spectrometer). Their average
absolute % recoveries (absolute recovery as defined as the amount of material spiked vs
the amount of material measured in final extract, without correction of labeled
surrogates) of AZI, RXI, CLA were 29, 45, and 33% (n = 4), respectively. Radjenovic et
al.(75) developed a PLE to extract pharmaceuticals, including AZI, from freeze-dried
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sewage sludges, followed by SPE cleanup, and analysis by HPLC-MS/MS. In this
method, they use both labeled surrogates and labeled internal standards to compensate for
matrix effects and ion signal suppression because of co-extracted interfering materials in
the biosolids. Their relative % recovery (relative % recovery is defined as labeled
surrogate corrected recoveries of the spiked materials, as opposed to absolute %
recoveries) of AZI was 81% (n= 3). In another recent publication, Jelic et al.(7<5), using
the same method as in Radjenovic et al.(75), obtained relative % recoveries for RXI and
CLA at 146 and 38% (n = 3), respectively.
In this report, an optimized analytical method for the detection of several
Pharmaceuticals and drugs in biosolids and its application to real-world biosolids samples
is presented. The method developed uses a PLE technique, with an in-situ extraction cell
clean-up (fluorosil and alumina packed into the PLE extraction cell), spike correction for
matrix effects, and a definitive detection method using HPLC-ESI-ITMS, in the MS/MS
mode.
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2.0 SAMPLING
This has been described in greater detail elsewhere.(7 7) Briefly, grab samples of
biosolids were collected and composited from various WWTPs around the US (Table 1,
Figure 1). Subsets of the samples were split between OW and ORD/NERL-ESD/Las
Vegas, with six samples from various capacity and types of WWTPs were sent to
ORD/NERL-ESD/Las Vegas. The samples were stored at the Las Vegas laboratory,
along with three other biosolids samples collected, by Las Vegas, at an earlier date (18).
The samples were stored at < -4° C until sample preparation. For most of the samples,
there was a lag time of over two years between sample collection and preparation for
analysis.
3.0 EXPERIMENTAL
The steps in environmental method development involve assessing: (1) the ability
to extract the analytes of interest with some degree of precision and accuracy from an
environmental matrix; and (2) the ability to accurately identify and measure at low
(environmentally-relevant) concentrations the analytes of interest. The ability to
correctly identify and measure the analytes identified in this research has previously been
published (JO, 79); therefore, we will focus the experimental section on the development
of the extraction and chromatographic procedures.
3.1.1 Sample preparation. All forms of biosolids have physical properties that
pose challenges for analytical chemistry methods development. Biosolids are made up of
9 1
particles with large surface areas (0.8 - 1.7 m g" ), have negative surface charges, and
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have extensive interstitial spaces; of which these physical properties can promote
sorption, occlusion into the biomass, and strong bonding between charged species and the
particulate surfaces. Further challenges are created by the WWTP-addition of chemical
additives, such as, ferric chloride, lime, and cationic polyacrylamide polymers (the most
widely used polymers for conditioning) during the biosolids conditioning steps.(20)
Therefore, due to the complexity and variable sizes of biosolids particulates, they need to
be homogenized before extraction.
Batches of biosolids were pre-dried by spreading them around the bottom of a
large beaker and leaving it open to air and light in the laboratory before homogenization
could proceed. The dried biosolids were placed in 25 ml zirconium oxide/steel jacketed
grinding jars, along with one zirconium oxide grinding ball, and ground to a fine powder
using a high impact ball mill (mixer mill 301, Retsch Inc, Newtown, PA) for 3 minutes at
a frequency of 20 s"1.
3.1.2 Optimization of pressurized liquid extraction (PLE) procedures. Initially,
both ultra-sonic extraction (USE) and PLE were investigated as extraction techniques.
Lower recoveries were obtained from USE than from PLE; therefore, only the three PLE
methodologies (PLE 1, PLE 2, and PLE 3) will be explored in detail. Several PLE
variables: temperature, solvents, pressures, and cell matrix materials were examined, and
are listed in Table 2.
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3.1.2.1 Pre-PLE Extraction Procedures.
Sample preparation. For PLE methods 1 and 2, a 1.0 g aliquot of the pre-
dried homogenized biosolids was weighed out, placed in a mortar, along with 5 g
of Hydromatrix™. The subsequent mixture was homogenized, using a silanized
pestle, until a free-flowing powder was achieved. For PLE 3, a 1.0-g aliquot of
the pre-dried homogenized biosolids was weighed out, placed in a mortar along
with 1 g of Hydromatrix™, and thoroughly mixed with a glass-stirring rod, finally
a 5-g aliquot of alumina was added and completely mixed in.
Extraction Cell preparation. A 22-mL stainless steel extraction cell was
prepared by inserting a cellulose filter at the bottom of the cell (capped on one
end).
For PLE methods 1 and 2: approximately 10 g of Hydromatrix™ was
added to the bottom of the extraction cell, the biosolids homogenate was
transferred on top of Hydromatrix™ layer, using a teflon lined funnel,
tapping the cell to ensure no air pockets were present. The remaining
volume was filled with Hydromatrix™. Another cellulose filter was
placed on top of the material inside the extraction cell, the cell was capped
and the cap was screwed on tightly.
For PLE method 3: a 5-g aliquot of fluorosil was funneled into the cell,
tapping the cell to ensure no air pockets were present, followed by 5-g of
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alumina, again gently tapping the cell to reduce the air pockets. The
biosolids homogenate was transferred on top of the alumina/fluorosil
layer, using a teflon lined funnel, tapping the cell to ensure no air pockets
were present, and the remaining volume was filled with Hydromatrix™.
Another cellulose filter was placed on top of the material inside the
extraction cell, the cell was capped and the cap was screwed on tightly.
3.1.2.2 Extraction procedures
For PLE method 1. The extraction cells were loaded into the PLE system and
extraction was performed using the following PLE extraction conditions: 99%
methanol/1% acetic acid as the extracting solvent; 2-cycles; 2800 psi; extraction
temperature: 50°C. After a static period of 15 minutes the eluant (approximately
40 mLs) was purged into a clean collection vial. Leaving the extracts in-situ in
the Turbovap® tubes, the tubes were removed from the Turbovap® for hexane
cleanup.
For PLE method 2 and method 3. The extraction cells were loaded into the
PLE system and two extractions were performed using the following PLE
extraction conditions. First, a mixture of methyl tertbutyl ether (MTBE):methanol
(90:10 v/v) was flushed at 80% of cell volume, at 50° C and 1500 psi; after a static
period of 15 minutes the eluant (approximately 40 mLs) was purged into a clean
collection vial. A subsequent extraction was performed on the same biosolids
sample, with a solvent mixture of methanol:l% acetic acid, flushed at 80% of cell
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volume, at 80° C and 2800 psi; after a static period of 15 minutes the eluant
(approximately 40 mLs) was purged into a separate, second collection vial. The
MTBE:methanol extracts were placed into a Turbovap® (Caliper Life Sciences,
MA) evaporation tube and concentrated to 5 mL (Turbovap® settings: 5 psi N2,
23° C). The methanol: l%acetic acid extract was combined with the MTBE
extract and concentrated until a total combined extract volume of 5 mL was
reached. Leaving the extracts in-situ in the Turbovap® tubes, the tubes were
removed from the Turbovap® for hexane cleanup.
Hexane cleanup. Whether using PLE 1, 2, or 3, the resultant PLE extracts were
rinsed several times with hexane: 2-mLs of hexane was pipetted into the extract,
ensuring mixing occurs, the hexane was allowed to settle out on top of the extract,
this hexane layer was removed and discarded (via pipetting), this procedure was
repeated until a clear yellowish color was obtained. The number of hexane rinses
varies from one biosolids matrix to the next, but this procedure was performed as
many times as necessary (up to 6 or 7 times) in order to clean the sample of much
of the undesirable compounds, such as fats and waxy materials. The cleaned
extract was placed back into the TurboVap® and further concentrated. Solvent
exchanging with methanol: 1% acetic acid until a 0.5 mL endpoint was achieved;
the extract was transferred to a 1.8 mL autosampler vial and ready for analysis by
LC-MS/MS. Early in the stages of method development, the loss of the analytes
of interest to the hexane was tested and was found to be minimal (< 1%).
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3.1.3 Optimization of mass spectrometry detection method
3.1.3.1 Liquid Chromatography. HPLC separations were performed using
an Ascentis Express Cig (Supelco-Aldrich, Bellefonte, PA), 2.7 |im particle
size, 3 cm x 2.1 mm, coupled to a MetaGuard Pursuit XRs 2.0 mm 3|im Cig
guard column (Varian Inc., Palo Alto, CA). Gradient elution conditions were
as follows: Mobile phase A 100%, hold for 2 min, 3 min gradient to 30%
A:70% B, hold for 5 min, then a 3 min gradient to 100% A, hold for 2 min,
followed by a 5 min equilibration time before the next injection. The mobile
phases were composed of the following: mobile phase A: de-ionized
water/0.5% formic acid; mobile phase B: 82% methanol/18%
acetonitrile/0.5% formic acid.
3.1.3.2 ESI-Ion Trap Mass Spectrometry. Data were acquired with a
Varian 500MS ion trap mass spectrometer (Varian Inc., Palo Alto, CA). The
500MS was operated in the positive ionization mode, with the following
conditions:
• ESI needle voltage: 5 kV
• Drying gas: 20 psi and 200° C
• Housing chamber: 50° C
• Nebulizer gas at 50 psi
• Spray shield at 600V
• Capillary voltages were individually set per analyte, dependent upon
the optimized response of the product ions of interest.
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Due to the extremely large amounts of interfering materials that were co-
extracted with the Pharmaceuticals, the analyses were performed in the
MS/MS mode, using CID in the ion trap, for both identification and
quantitation of the macrolides and illicit drugs. Two to three product ions
were used for identification and the most abundant product ion for
quantification. The precursor ions, product ions and limits-of-detection
(LODs) used to identify and quantify the analytes have been previously
reported.(70, 19}
3.1.3.3 Calibration, blanks, and LC-ES-ITMS quantitation. The methods
for determining the LODs and limits-of-quantitation (LOQs) have
been previously reported.(JO, 19)
3.1.4 Safety Considerations. The pressurized liquid extractor can rise to very
high pressures. The septa on the rinse vials should be changed daily; if
not this could possibly lead to an accidental explosion of the rinse vial.
All chemicals should be handled with caution and personal protective
equipment (PPE) should be used.
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4.0 Results and Discussion
The steps in environmental method development involve assessing: (1) the ability
to extract the analytes of interest with some degree of precision and accuracy from an
environmental matrix; and (2) the ability to accurately identify and measure at low
(environmentally-relevant) concentrations the analytes of interest. The ability to
correctly identify and measure the analytes identified in this research has previously been
published.(10, 19) Therefore, we will focus the results and discussion section on the
development of the extraction and chromatographic procedures and then on the results of
the application of the finalized method to nine different biosolids matrices.
4.1 Extraction methods. Initially, both ultra-sonic extraction and PLE were
investigated as extraction techniques. Three materials; sand, Milorganite® (commercially
available Class A biosolids from Milwaukee, WI), and a Class A biosolids from the
Southern California Los Angeles Hyperion WWTP were tested with the USE and
simplified PLE 1. Since lower recoveries were obtained from USE than from PLE, we
only pursued further development of the PLE technique. (18)
Subsequently, we added several other biosolids materials collected during EPA's
2006-2007 Targeted National Sewage Sludge Survey (TNSSS),(77) for a total of nine
different biosolids matrices to be studied by PLE 2 and 3 (Table 1). The recoveries were
very different dependent upon the materials tested.
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4.2 Extraction recovery results. Initially, only the recoveries of AZI, RXY, and CLA
from the Milorganite® and Hyperion biosolids were investigated using a simplified PLE.
At the time of the initial methods development neither the other emerging contaminants
nor the other seven biosolids matrices were available.(75) The recoveries from the earlier
study, using PLE method 1, were as follows: AZI: 24% (n=6); CLA: 40% (n=6); and
RXI: 13% (n=6). While the overall recoveries were low, they were not discouraging. It
was interesting to note that when comparing the recoveries between the two different
biosolids, they were distinctly different. Milorganite® gave the resulting recoveries: AZI:
28% (n=4); CLA: 54% (n=4); and RXI: 19% (n=4); while the Hyperion biosolids
recoveries were: AZI: 16% (n=2); CLA: 13% (n=2); and RXI: 1% (n=2). This difference
in recoveries between biosolids matrices is the result of the variations in the unique
physical and chemical compositions of each of the biosolids matrices, e.g., varying levels
of lipids, de-watering processes, chemical stabilizers and chemical additives. For
example, Brumley et al.(27) report high levels, part-per-billion (ppb), of surfactants
(nonylphenol ethoxylates) and polybrominated diphenyl ethers (PBDEs) in these same set
of biosolids, which gave rise to analytical interferences, as the pharmaceutical
concentrations were in the part-per-trillion (ppt) range.
In searching for improvements in recoveries to the original PLE (method 1), PLE 2
and subsequently PLE 3, were developed. There were two major differences between
method PLE 2 and PLE 3. In PLE 3, a layer of fluorosil and alumina were added to the
bottom of the extraction cell and thel-g of biosolids material was mixed with alumina
and hydromatrix, not hydromatrix alone as in PLE 2. All other PLE parameters (e.g.,
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solvents, pressures, temperatures) were kept the same for PLE 2 and PLE 3 (Table 2).
The spiked recoveries of all seven analytes, from all nine matrices, were compared
and the results are presented in Table 3. Overall, the spiked recoveries from PLE 3 were
substantially better than those from PLE 2. The average recovery of the three macrolides,
AZI, RXI, and CLA were: 17, 8, and 11%; and 57, 16, and 61% recovery, PLE 2 vs PLE
3, respectively.
Better recoveries were obtained for the lower molecular weight emerging
contaminants from PLE 3. The recoveries for methamphetamine, MDMA, and
pseudoephedrine, and n,n'-DMPEA were: 15, 15, 25 and 26% vs 53, 55, 67 and 57%,
PLE 2 vs. PLE 3, respectively. For the lincosamide clindamycin, there was a slight
increase in recovery when using PLE 3; 38% (PLE 2) vs 46% (PLE 3).
It is not surprising that PLE 3 provided better recoveries than PLE 2 as fluorosil
and alumina were used in situ during the extraction procedure; thereby, providing
removal of some of the interfering substances. This was evidenced by only one instance
of matrix interference from an overlapping ion during the mass spectrometric analyses in
comparison to multiple instances of interfering overlapping ions when analyzing extracts
produced from PLE 2 (Table 3). Also, there was less drop off in mass spectral sensitivity
due to fewer instances of the electrospray shield getting dirty between analyses when
analyzing extracts produced from PLE 3. However, the macrolides still had drifting
chromatographic retention times no matter which PLE method was used.
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4.3 Enhancement of chromatography. The HPLC Cig column used for this method
incorporated what was termed "fused-core" silica particles. This type of silica particle is
a solid particle with the Cig moiety bound to the surface; there are no interstitial spaces
inside the particle. This type of particle enhanced our chromatographic performance by
increasing the number of theoretical plates, on the order of ultrahigh performance liquid
chromatography (UPLC) technology, without giving the high back pressures that UPLC
gives. Previously, standard particle HPLC Cig columns were used in our studies with the
total chromatographic analysis times ran upwards of 30 minutes.(19) With this newer
particle type column, the total chromatographic analyses were shorter at 15 minutes.
Another difference between this column and the normal Cig was the ability to recover this
column's chromatography back to its initial chromatographic conditions even after
injecting several biosolids extracts onto the column. The chromatographic recovery was
possible by back-flushing the column with 100% organic phase (82% methanol/18%
acetonitrile/0.5% formic acid) for 10 min. After several weeks of biosolids analysis, this
solid particle column was still operational, whereas the traditional Cig column previously
used in our studies was not and had to be replaced more frequently.
4.4 Analytical concerns. The biosolids extracts produced with PLE 3 were cleaner
than those produced from the other methods. However, there were still residual
surfactants and other unidentifiable materials present in some of the extracts. These co-
extracted interferents can interact with the Cig and silica moieties on the chromatographic
column, sometimes irreversibly binding to active sites. These interfering materials can
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cause changes in the retention times of the analytes between analyses at the beginning
versus those extracts and standards analyzed at the end of an analytical day (typically 8
hrs). As an example, it was observed, in several biosolids extracts, that the azithromycin
present appeared as a very small chromatographic peak in the first sample extract
injection. In the second injection of the same extract a large peak attributable to
azithromycin appeared. A third injection of an instrument blank (methanol) was made
immediately after the 2n injection, and again a large peak attributable to azithromycin
appeared (Figure 2). An assumption can be made that the azithromycin in the 1st
injection of the extract was actually eluting out in the 2n sample injection, and that the
azithromycin present in the 2nd injection of the extract carries over into the methanol
blank injection. This hypothesis was tested and proven by showing that the
chromatographic peak carried over into the blank was attributable to azithromycin not
only by the large area counts under the peak, but also through spectral confirmation. The
azithromycin peak that was present in the 2nd injection and blank chromatogram had the
presence of the three product ion masses for azithromycin: 591.4 m/z, the most
predominant ion (and quantitation ion), as well as masses 573.4 m/z and 434.4 m/z, two
minor product ions. The product ions are in the correct mass isotope ratios to each other,
in both the samples and blanks; therefore, this finding confirmed that the
chromatographic peak was azithromycin.
To prove that the methanol blank was not previously contaminated, several
experiments were performed. A methanol blank, injected after every calibration
standard, was always blank after the standard injection. However, the methanol blank
16
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injected after the biosolids sample injection would show azithromycin eleuting in the
blank injection. These two experiments showed that the methanol blank was not
contaminated, but instead that the azithromycin was being bound onto something else in
the column and not eluted from the column until after a second injection was made. It is
believed that this phenomenon is a by-product of the residuals found in biosolids. For
example, it has previously been observed that the chromatographic peaks of azithromycin
do not drift when environmental water (e.g., wastewater, source water) extracts are
injected, nor is this phenomenon observed during multiple injections of calibration
standards. This drifting peak phenomenon does not occur with all biosolids, but it did
occur with several of the biosolids matrices received from the TNSSS study. The drifting
peak phenomenon seemed to be limited to the macrolide antibiotics (i.e., AZI, RXI,
CLA), as this phenomenon was not observed to occur with the smaller molecules (i.e.,
methamphetamine, n,n'-dmpea, MDMA, and clindamycin). The binding of the
macrolides to the column is almost certainly due to certain functional groups located on
the macrolides (e.g., ethyl aldehyde at Ce azithromycin, saccharide branch at Cs
azithromycin )(22), as well as their cage-like chemical structures that encourages binding
(Figure 3).
4.5 Application of optimized method to nine diverse biosolids matrices. Table 4
shows the results of the application of the optimized method to the nine different
biosolids matrices, collected from nine different WWTPs across the US. Although
sample #7, LVBIO007, was part of the spiking study, we were unfortunately unable to
process the unspiked matrix of LVBIO007 at the time of this report. This event was due
17
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to our building being shut down for nine months and LVBIO007 was one of the last
unspiked samples to be processed with method PLE 3. However, the other 8 samples are
reported in Table 4 and cross-correlated with the data collected from the TNSSS.(2 ¥)
Although the methods used to study the TNSSS biosolids were different between
the EPA contractor (Method 1694) (24) and our method (PLE method 3) we tried to
make some comparisons between the two methods using samples LVBIO- 004, 005, 006,
008 and 009 (Table 5). Only one of the clarithromycin results corresponded, both being
non-detects, perhaps due to the delay between collection and extraction on our part. For
roxithromycin, 5 of the 6 results matched up, as non-detects. In only one instance did the
TNSSS contractor detect roxithromycin, in sample LVBIO006. However, in this
particular biosolids matrix, we were only able to obtain a recovery of 16% for
roxithromycin from the spiked matrix. This made it unlikely that our method would
detect the low levels, 14 ng/g, that they detected. It should be noted that the amount of
roxithromycin detected in the TNSSS study, 14 ng/g, is at, or near, the LOD for Method
1694, as well as our method's LOD for this compound, and should be treated as suspect.
5.0 CONCLUSIONS
The finalized method presented here, PLE 3, provides an efficient in situ cleanup of dirty
environmental matrices, like biosolids. Overall, the recoveries of the analytes are better
and there are less analytical difficulties from interfering matrix ions. In comparing our
method to Gobel's et al. (14), which uses absolute recoveries like ours, we obtain better
recoveries for AZI and CLA: 57% and 61% (n=9) vs. Gobel's, 29% and 33% (n=4).
18
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However, Gobel's method obtained better results for RXI: 45% vs. our 16%. In the US
the two most widely prescribed macrolide antibiotics are AZI and CLA, while RXI is not
prescribed in the US, it is in Latin America and the European Union. We cannot compare
our results to those of Jelic, et al. (16), nor Radjenovic, et al. (75), nor US EPA Method
1694(23), as they use a relative % recovery method, a method that uses carbon-labeled
standards. While using labeled standards may give a more accurate sense of recoveries
and negate the issue of matrix interferences, the disadvantages are that labeled standards
are difficult to find, expensive, and/or difficult to have synthesized.
It was not surprising to detect azithromycin in all of the biosolids matrices, as the
production levels of the active pharmaceutical ingredient (API) for azithromycin is
roughly 219,000 kg/yr. Loganathan et al.(7P), using this API, calculated a predicted
environmental occurrence (PEC) at approximately 3000 ng/L. However, there is a 10-
fold difference between what was found in WWTP effluent (water column) and what was
predicted (19), pointing to other environmental sinks of azithromycin, of which biosolids
are one.
The extraction and detection approach presented in this report may not be as
precise as an absolute recovery method that uses labeled standards; however, it provides a
much simpler and more cost effective approach. The drawback to the method PLE 3,
presented in this report, is that in order for it to be effective, every unique batch of
biosolids matrices from each WWTP, must include a spiked matrix in order to correct for
the extraction efficiency from each distinctive biosolids matrix.
19
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6.0 FUTURE RECOMMENDATIONS
The current standards for the use of biosolids in the U.S. are science-based risk
assessments. With respect to emerging contaminants in biosolids, more accurate data on
biosolids uses, advances in chemical analytical methodology, survival efficiencies in
wastewater treatment facilities, environmental fate and transport, and the potential for
effects in humans and the environment are required to conduct reliable exposure and
hazard assessments. Sufficient data to conduct an exposure and hazard assessment
include unbiased national estimates of concentrations, environmental fate and transport,
plausible effects end-points for humans and ecological receptors, and other relevant
information for pollutants in biosolids. An even more important question that should be
asked is whether these emerging contaminant residues are bioavailable, and if so, then
what will be the environmental impact. Therefore, possible future research efforts could
be directed towards crop uptake studies from biosolids treated fields, weathering of and
in situ breakdown of emerging contaminants in biosolids residues, and other studies that
would look at the emerging contaminants leaching from, or still available in, the biosolids
matrix, dependent upon their final treatment (e.g., landfilling, biofuels, field amendments,
composting).
20
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REFERENCES
1. National; Research; Council Biosolids applied to Land: Advancing Standards and
Practices. ; National Research Council: 2002.
2. Chenxi, W.; Spongberg, A. L.; Witter, J. D., Determination of the persistence of
Pharmaceuticals in biosolids using liquid-chromatography tandem mass
spectrometry. Chemosphere 2008, 73, (4), 511-518.
3. Daughton, C. G., Pharmaceuticals and personal care products in the environment:
Overarching issues and overview. In Pharmaceuticals and Personal Care
Products in the Environment: Scientific and Regulatory Issues, Daughton, C. G.;
Jones-Lepp, T. L., Eds. American Chemical Society: Washington, DC, 2001; pp
2-38.
4. Isidori, M.; Lavorgna, M.; Nardelli, A.; Pascarella, L.; Parrella, A., Toxic and
genotoxic evaluation of six antibiotics on non-target organisms. Science of the
Total Environment 2005, 346, (1-3), 87-98.
5. Sanderson, H.; Brain, R. A.; Johnson, D. J.; Wilson, C. J.; Solomon, K. R.,
Toxicity classification and evaluation of four pharmaceuticals classes: antibiotics,
antineoplastics, cardiovascular, and sex hormones. Toxicology 2004, 203, (1-3),
27-40.
6. Schwartz, T.; Volkmann, H.; Kirchen, S.; Kohnen, W.; Schon-Holz, K.; Jansen,
B.; Obst, U., Real-time PCR detection of Pseudomonas aeruginosa in clinical and
municipal wastewater and genotyping of the ciprofloxacin-resistant isolates.
FEMSMicrobiology Ecology 2006, 57, (1), 158-167.
7. Xia, K.; Bhandari, A.; Das, K.; Pillar, G., Occurrence and fate of pharmaceuticals
and personal care products (PPCPs) in biosolids. Journal of Environmental
Quality 2005, 34, (1), 91-104.
8. Schwartz, T.; Kohnen, W.; Jansen, B.; Obst, U., Detection of antibiotic-resistant
bacteria and their resistance genes in wastewater, surface water, and drinking
water biofilms. FEMS Microbiology Ecology 2003, 43, (3), 325-335.
9. McCaig, L. F.; Besser, R. E.; Hughes, J. M., Antimicrobial Drug Prescriptions in
Ambulatory Care Settings, United States, 1992-2000. Emerging Infectious
Diseases 2003, 9, (4).
10. Jones-Lepp, T. L.; Alvarez, D. A.; Petty, J. D.; Huckins, J. N., Polar Organic
Chemical Integrative Sampling and Liquid Chromatography-Electrospray/Ion-
Trap Mass Spectrometry for Assessing Selected Prescription and Illicit Drugs in
Treated Sewage Effluents. Archives of Environmental Contamination and
Toxicology 2004, 47, (4), 427-439.
11. Zuccato, E.; Chiabrando, C.; Castiglioni, S.; Calamari, D.; Bagnati, R.; Schiarea,
S.; Fanelli, R., Cocaina nelle acque di superficie: Un nuovo strumento evidence-
based per monitorare 1'abuso comunitario di sostanze [Cocaine in surface waters:
New evidence-based tool to monitor community drug abuse] Environmental
Health Journal: A Global Access Science Source 2005, 4, (14), 103-112.
12. Nieto, A.; Borrull, F.; Marce, R. M.; Pocurull, E., Selective extraction of
sulfonamides, macrolides and other pharmaceuticals from sewage sludge by
pressurized liquid extraction. Journal of Chromatography A 2007, 1174, (1-2),
125-131.
21
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13. Jones-Lepp, T. L.; Alvarez, D. A.; Englert, B.; Batt, A. L., Pharmaceuticals and
Hormones in the Environment. In Encyclopedia of Analytical Chemistry., Meyers,
R. A., Ed. John Wiley and Sons: City Unknown, 2009; p 59.
14. Gobel, A.; Thomsen, A.; McArdell, C. S.; Alder, A. C.; Giger, W.; Thei, N.;
Loffler, D.; Ternes, T. A., Extraction and determination of sulfonamides,
macrolides, and trimethoprim in sewage sludge. Journal of Chromatography A
2005, 1085, (2), 179-189.
15. Radjenovic, J.; Jelic, A.; Petrovic, M.; Barcelo, D., Determination of
Pharmaceuticals in sewage sludge by pressurized liquid extraction (PLE) coupled
to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analytical
andBioanalytical Chemistry 2009, 393, (6), 1685-1695.
16. Jelic, A.; Petrovic, M.; Barcelo, D., Multi-residue method for trace level
determination of pharmaceuticals in solid samples using pressurized liquid
extraction followed by Liquid Chromatography/Quadrupole-Linear Ion Trap
Mass Spectrometry. Talanta2009, 80, (1), 363-371.
17. USEPA, Targeted National Sewage Sludge Survey: Overview Report. In US
Environmental Protection Agency: Washington, DC, 2009; p 9.
18. Jones-Lepp, T. L.; Stevens, R., Pharmaceuticals and personal care products in
biosolids/sewage sludge: the interface between analytical chemistry and
regulation. Analytical and Bioanalytical Chemistry 2007, 387, (4), 1173-1183.
19. Loganathan, B.; Phillips, M.; Mowery, H.; Jones-Lepp, T. L., Contamination
profiles and mass loadings of select macrolide antibiotics and illicit drugs from a
small urban wastewater treatment plant. Chemosphere 2009, 75, (1), 70-77.
20. McFarland, M., Biosolids Engineering. McGraw-Hill: New York, 2001.
21. Brumley, W. C. V., Katrina E.; Riddick, L. A., Partial Characterization Of
Biosolids: Lipophilic Organic Components. Environmental Science: An Indian
Journal 2008,3,3.
22. Hansen, J. L.; Ippolito, J. A.; Ban, N; Nissen, P.; Moore, P. B.; Steitz, T. A., The
Structures of Four Macrolide Antibiotics Bound to the Large Ribosomal Subunit.
Molecular Cell 2002, 10, 117-128.
23. USEPA, Method 1694: Pharmaceuticals and Personal Care Products in Water,
Soil, Sediment, and Biosolids by HPLC/MS/MS. In Office of Water; Office of
Science and Technology; Engineering and Analysis Division (4303T), Eds. US
Environmental Protection Agency: Washington, DC, 2007; p 77.
24. USEPA, Targeted National Sewage Sludge Survey - Statistical Analysis Report.
EPA External Report September 30, 2008.
22
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Tables.
1) Biosolids matrices: identification numbers, WWTP types, mgd, biosolids type,
final deposition.
2) Experimental parameters for methods PLE 1, PLE 2, and PLE 3.
3) Spiked biosolids recovery: PLE 2 versus PLE 3.
4) Concentrations of targeted Pharmaceuticals and drugs from nine US WWTPs
using PLE 3.
5) Comparison of concentrations of targeted pharmaceuticals PLE 3 vs. TNSSS.
-------
Table 1. Biosolids matrices: identification numbers, WWTP types, mgd, biosolids type, final deposition.
Sample ID
LVBIO001
LVBIO002
LVBIO003
LVBIO004
LVBIO005
LVBIO006
LVBIO007
LVBIO008
LVBIO009
WWTP type
Tertiary
Tertiary
Tertiary
Secondary
Tertiary
Secondary (?)
Tertiary
Secondary
Secondary
mgd
91
250
450
3.3
>100
5
47
20
1
Biosolids type
Class B
Class A
Class A
Class B anaerobic digestion
Class B anaerobic mesophilic
digestion
Class B anaerobic digestion
Class B anaerobic digestion
Class B activated sludge
Class B anaerobic digestion
Final deposition
Landfill
Commercial product, land application
Composting, land application, biofuels
on-site use
Incineration, landfill
Land application
Mixed with green waste, sold as compost
to public
Land application
Landfill
Land application
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Table 2. Experimental parameters for methods PLE 1, PLE 2, and PLE 3.
Hydromatrix
Fluorosil
Alumina
Sample/hydromatrix mix
Sample/ Alumina mix
Extracting Solvent(s)
Pressure (psi)
Extraction Temp. °C
Static time (min)
PLE1
Yes
No
No
Yes
No
99% methanol/1% acetic
acid
2800
50
15
PLE 2
Yes
No
No
Yes
No
Two extractions:
(1) MTBE:methanol
(90: lOv/v) is flushed at
80% of cell volume; (2)
methanol/1% acetic acid
1) 1500
2) 2800
1)50
2)80
(1) and (2) 15
PLE 3
Yes
Yes (5 g, bottom layer)
Yes (5 g, 2nd layer)
No
Yes
Two extractions:
(1) MTBE:methanol
(90: lOv/v) is flushed at
80% of cell volume; (2)
methanol/1% acetic acid
1) 1500
2) 2800
1)50
2)80
(1) and (2) 15
-------
Table 3. Spiked biosolids recovery: PLE 2 versus PLE 3.
Sample ID
LVBIO001
LVBIO002
LVBIO003
LVBIO004
LVBIO005
LVBIO006
LVBIO007
LVBIO008
LVBIO009
Average
% recovery
Spiked biosolids, % recovery: PLE 2 vs PLE 3
AZI
PLE
2
4
17
26
7
11
21
25
14
24
17
PLE
3
41
94
55
46
37
77
45
86
28
57
RXI
PLE
2
33
1
**
5
16
0
0
1
}
8
PLE
3
1
*
39
6
2
16
5
5
57
16
CLA
PLE
2
6
34
27
1
11
7
0
2
«
11
PLE
3
35
93
48f
58
41
76
87
51
56
61
CLI
PLE
2
9
43
m
29
64
29
57
37
37
38
PLE
3
ttt
53
37f
29
51
58
17
79
40
46
METH
PLE
2
18
18
16
27
12
1
5
5
31
15
PLE
3
92
44
31
39
46
110
25
36
53
53
MDMA
PLE
2
18
16
15
25
9
10
6
8
26
15
PLE
3
87
75
39
54
68
57
13
45
59
55
d5-MDMA
PLE
2
40
na
37
57
31
26
15
13
43
33
PLE
3
72
97
37
50
65
71
27
62
51
59
n,n'-DMPEA
PLE
2
15
38
43r
64f
9
14
5
7
38
26
PLE
3
101
41
60
25f
74
81
33
50
49
57
PSEU
PLE
2
25
31
13
30
23
29
19
25
27
25
PLE
3
94
82
58
67
64
76
34
64
66
67
Data unusable due to poor chromatography from interfering unknown compounds. ^Interference from overlapping ion isotopes from unknown
analyte mass 749.8 da; ^Overlapping ion isotopes from unknown mass 589.5 da; ^Overlapping ion isotopes from unknown mass 376 da ; ^Due to
the large amounts (> 100 ng/g) of native analyte found in the original sample the spiked recoveries values were corrected to reflect that, na =
labeled standard was not available for spiking for this sample.
-------
Table 4. Concentrations of targeted Pharmaceuticals and drugs from nine U.S. WWTPs using PLE 3.
Sample ID
LVBIO001
LVBIO002
LVBIO003
LVBIO004
LVBIO005
LVBIO006
LVBIO008
LVBIO009
Average amount detected*, ng/g (dry wt)
AZI
150
52
130
170
200
53
180
105
CLA
nd
nd
310
nd
nd
nd
nd
190
CLI
nd
nd
2100
nd
nd
nd
nd
nd
METH
31
nd
nd
nd
nd
nd
nd
nd
n,n'-DMPEA
nd
nd
nd
630
nd
nd
35
nd
PSEU
nd
nd
nd
nd
nd
nd
nd
140
(n=2), corrected values from matrix spikes recoveries, nd = not detected. Note: LVBIO007 was not analyzed.
Table 5. Comparison of concentrations of targeted pharmaceuticals PLE 3 vs TNSSS.'
Sample ID
LVBIO004
LVBIO005
LVBIO006
LVBIO008
LVBIO009
Average amount detected
ng/g (dry wt)
AZI
PLE vs TNSSS
170-1180
200 - 392
53-63
180-548
105- 157
RXI
PLE vs TNSSS
nd - nd
nd - nd
nd- 14
nd - nd
nd - nd
CLA
PLE vs TNSSS
nd- 141
nd- 19
nd - nd
nd-53
190 -nd
EPA TNSSS results were taken from reference (24). nd = not detected.
-------
Figures
1. TNSSS National Survey Sites.
2. Chromatograms of drifting retention times of azithromycin.
3. Chemical structures of three antibiotics and two illicit drugs.
-------
Figure 1. TNSSS Survey sites.
•agansett
Facility sampled during the 2006 TNSSS
Facility sampled during the 2006 TNSSS, and analyzed by NERL-LV
Facility not part of the 2006 TNSSS, but collected and analyzed by NERL-LV
0 50100 200 300 400
Miles
Map provided by USEPA/OW
-------
Figure 2. Chromatograms of drifting retention times of azithromycin.
(a) Calibration standard: injection time: 8:03 am; apex of peak: 7.2 min
(b) Biosolids sample 1st inj: injection time: 9:26 am; apex of peak: 7.1 min
(c) Biosolids sample 2nd inj: injection time: 9:46 am; apex of peak: 10.1 min
(d) Methanol blank inj: injection time: 10:07 am; apex of peak: 9.1 min
(e) Calibration standard, end of day: injection time: 1:29 pm; apex of peak: 7.2 min
Scan Range: 1 - 1784 Time Range: 0 .00 - 14.99 rnin.
Sample Holes: caiib std 020209
Date: 2/4/2009 8:03 AM
-.Pt:<:7.i97 niln.
-rf a: 37029
Scan Range: 1 - 1810 Time Range: 0.00 - 14.Q9 min.
Sample Notes: ase #6 Iv0sed004 011309
Date: 2/4/2009 9:26 AM
t
(b)
-------
in Range : 1 - 1812 Time Range : D .DD - 1-4.!
nple Nctes: a se #6 lvQsedQQ4 01 1 309
Date: 2/-4/2DD9 9 :-*6 AM
10 JOE 3 in II
(c)
Scan Range : 1 - 1793 Time Range: 0 .00 - 1 4.99 min .
Sample Notes: meoh
Date: 2/4/2009 10:07 AMI
p.|ianii.lO—.T..1 C; tan i : 5* I .3
(d)
Scan Range: 1 - 1781 Time Range: D .DO - 14.99 min.
Sample Notes: calib std 020209
Date: 2/4fflDD9 1 :29 PM
(e)
-------
Figure 3. Chemical structures of three antibiotics and two illicit drugs
H,C
cm
H3C
H,C
\
CH3 N —CH3
HO-
H3C.
H,C
CH3
Azithromycin mw = 748 Da
CH,
OH
Roxithromycin mw = 837 Da
H3C,
H3C
CH3
QCH3 H3C CH3
OH N
UM3
O- -- -O
Methamphetamine mw = 149 Da
O
Clarithromycin mw = 747 Da
MDMA mw = 193 Da
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