Title: Determination of Pharmaceuticals in Biosolids usingAccelerated Solvent Extraction and LiquidChromatography/Tandem Mass Spectrometry
Authors: Yunjie Ding, Weihao Zhang, Cheng Gu, IreneXagoraraki, Hui Li
Please cite this article as: Y. Ding, W. Zhang, C. Gu, I. Xagoraraki, H. Li, Determinationof Pharmaceuticals in Biosolids using Accelerated Solvent Extraction and LiquidChromatography/Tandem Mass Spectrometry, Journal of Chromatography A (2010),
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain. Determination of Pharmaceuticals in Biosolids using Accelerated Solvent Extraction and Liquid Chromatography/Tandem Mass Spectrometry
Yunjie Ding1, Weihao Zhang2, Cheng Gu1, Irene Xagoraraki3, and Hui Li1,*
Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan
School of Resource and Environmental Science, Wuhan University, Wuhan, Hubei 430079,
Department of Civil and Environmental Engineering, Michigan State University, East Lansing,
An analytical method was developed to quantitatively determine pharmaceuticals in biosolid
(treated sewage sludge) from wastewater treatment plants (WWTPs). The collected biosolid
samples were initially freeze dried, and grounded to obtain relatively homogenized powders.
Pharmaceuticals were extracted using accelerated solvent extraction (ASE) under the optimized
conditions. The optimal operation parameters, including extraction solvent, temperature, pressure,
extraction time and cycles, were identified to be acetonitrile/water mixture (v/v 7:3) as extraction
solvent with 3 extraction cycles (15 minutes for each cycle) at 100 °C and 100 bars. The extracts
were cleaned up using solid-phase extraction followed by determination by liquid
chromatography coupled with tandem mass spectrometry. For the fifteen target pharmaceuticals
commonly found in the environment, the overall method recoveries ranged from 49% to 68% for
tetracyclines, 64% to 95% for sulfonamides, and 77% to 88% for other pharmaceuticals (i.e.
acetaminophen, caffeine, carbamazepine, erythromycin, lincomycin and tylosin). The developed
method was successfully validated and applied to the biosolid samples collected from WWTPs
located in six cities in Michigan. Among the fifteen target pharmaceuticals, fourteen
pharmaceuticals were detected in the collected biosolid samples. The average concentrations
ranged from 2.6 g/kg for lincomycin to 743.6 g/kg for oxytetracycline. These results indicated
that pharmaceuticals could survive wastewater treatment processes, and accumulate in sewage
sludge and biosolids. Subsequent land application of the contaminated biosolids could lead to the
dissemination of pharmaceuticals in soil and water environment, which poses potential threats to
at-risk populations in the receiving ecosystems. 1. Introduction
Administration of pharmaceuticals is the most common practice for human disease
control and treatments. Large fractions of administrated dosage are, in fact, not assimilated or
metabolized in human body, but released into influents to municipal wastewater treatment plants
(WWTPs) as either parent compounds or bioactive metabolites [1-3]. Unfortunately, currently
operating WWTPs are not designed for effective removal of pharmaceuticals; as a result, a
portion of pharmaceuticals could survive wastewater treatment processes, and disseminate to the
environments via effluents from WWTPs in a dissolved form [4,5] or sorbed pharmaceuticals by
sewage sludge . Most sewage sludge is treated to produce biosolid and is subsequently applied
to agricultural lands for its fertilizer value. This practice undoubtedly introduces sludge-born
pharmaceuticals into soil and water environment where their fate and risks remain largely
unknown [7-12]. There is growing concern regarding the potential impacts of these
pharmaceuticals on at-risk populations in the ecosystems.
The information on pharmaceutical compositions and concentrations in land-applied
biosolids is critical to assessing the exposure and potential risks. Analytical methods have been
developed to analyze pharmaceuticals in water [10,11,13,14], animal manure [15-18] and soil
[15,19-21]. In several studies analytical methods have been also reported to identify and quantify
pharmaceuticals in sewage sludge from WWTPs [10,22-26]. These methods are worthy of
further examination for their applicability to multiple classes of pharmaceuticals in sewage
sludge or biosolids . The procedure of the analytical methodology usually consists of
extraction of pharmaceuticals from sludge/biosolid, cleanup of extracts, and analysis using liquid
or gas chromatography. Liquid-solid partitioning is the most common approach utilized in
combination of ultrasonic-assisted extraction , microwave-assisted extraction  and
accelerated solvent extraction (ASE). The extraction efficiency is dependent of sample matrices
and characteristics of target analytes [28-30]. Among these three extraction techniques, ASE
becomes more preferred because it provides apparent advantages such as less solvent assumption,
automatic procedure for simultaneous extraction of multiple samples, short sample preparation
time, and higher extraction recoveries. Solid-phase extraction (SPE) is usually selected as the
cleanup tool for the sludge/biosolid extracts, and the corresponding cartridges commonly used
included Waters Oasis hydrophilic-lipophilic balance (HLB), strong-anion exchange (SAX),
strong cation-exchange (SCX) or their combinations [21,26,31]. The pharmaceuticals are then
analyzed using liquid chromatography with fluorescence or ultraviolet detectors [25,32,33] or
gas chromatography coupled to mass spectrometer . Recently, liquid
chromatography/tandem mass spectrometry (LC-MS/MS) became the most efficient tool for
identifying and quantifying trace levels of pharmaceuticals in environmental samples [11,35-38].
The objective of this study was to develop a robust, sensitive and practical analytical
method to simultaneously identify and quantify multiple classes of pharmaceuticals in biosolids.
The ASE experimental parameters (i.e., extraction solvent, pressure, temperature, extraction time
and cycles) were optimized to achieve the maximum extraction efficiencies for pharmaceuticals.
SPE was used to pre-concentrate and clean up the ASE extracts. The amounts of pharmaceuticals
were quantitated by a LC-MS/MS equipped with electrospray ionization (ESI) source. The
method was validated, and applied to determine multiple classes of pharmaceuticals in biosolid
samples collected from several WWTPs of different locations in Michigan, USA.
Tetracycline, demeclocycline, chlortetracycline, oxytetracycline, meclocycline,
sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole, carbamazepine, acetaminophen,
caffeine, tylosin and lincomycin were purchased from Sigma-Aldrich Chemical Company (St.
Louis, MO, USA). Doxycycline was obtained from Fisher Bioreagents (Pittsburgh, PA, USA).
C6-sulfamethazine was obtained from Cambridge Isotope Laboratories (Andover, MA, USA).
Simeton was purchased from AccuStandard (New Haven, CT, USA). Methanol, sodium
ethylenediaminetetraacetate (EDTA), formic acid, and sulfuric acid were purchased from J.T.
Baker (Phillipsburg, NJ, USA). Acetonitrile was purchased from EMD Chemicals (Gibbstown,
NJ, USA). Waters Oasis hydrophilic-lipophilic balance (HLB) cartridge was purchased from
During the winter season of 2009, six grab biosolid samples were collected from WWTPs
of Lansing, East Lansing, St. Clair, Plainwell, Traverse City, and Imlay City, Michigan, USA.
The biosolids collected from WWTPs of Traverse City, Plainwell, St. Clair, Imlay City, and
Lansing were classified as class B biosolids which meet the criteria for land application. The
biosolids collected from East Lansing WWTP was sent off to landfills. At each sampling site,
one liter of biosolid was collected, and stored in a polypropylene narrow-necked bottle. The
bottles were capped, placed in an iced cooler, and transported immediately to laboratory. The
samples were then freeze-dried to obtain dry solids, ground using a mortar and pestle, passed
through a 0.5 mm sieve, and stored in a refrigerator (-20 ºC) prior to use.
2.3. Sample extraction and cleanup
The dried biosolid sample was weighed (500.0 mg) into cellulose extraction thimble, and
mixed with diatomaceous earth (5.0 g) to prevent aggregation during extraction process. 13C6-
sulfamethazine and meclocycline were used as surrogates for sulfonamide and tetracycline
antibiotics, respectively. The collected biosolid samples had been checked not to contain
meclocycline. 100 µL of 13C6-sulfamethazine (1.0 mg/L) and 200µL of meclocycline (1.0 mg/L)
were spiked into the biosolid samples, which were then placed on a rotator and mixed overnight.
The thimbles were then placed into 22 mL stainless steel extraction cells, and extracted using a
Dionex ASE 200 accelerated solvent extractor (Sunnyvale, CA, USA). The extraction solvent
and operating conditions had been optimized in which acetonitrile and water mixture (v/v = 7:3)
was used. The samples were preheated for 5 min, and then extracted at 100 °C under 100 bars.
The static extraction period was set 15 min with three extraction cycles. The flushing solvent was
100% of cell volume, and extraction cells were purged with N2 for 120 s. The extracts were
collected and transferred to volumetric flasks, and diluted to 100 mL with deionized water. The
pH value of the solution was adjusted to ~3.0 using H2SO4. An aliquot of 10.0 mL of the diluted
solution passed through a preconditioned HLB solid-phase cartridge (Waters Corporation,
Millford, MA, USA) at a rate of 1 mL/min. The analyte-loaded HLB cartridge was rinsed with 3
mL of deionized water, and eluted by 5 mL of methanol-water mixture (v/v = 1:1) containing
150 mg/L EDTA. The elute volume was reduced to 1.0 mL using a gentle N
the samples, which took ~45 min. The prepared samples were transferred to amber LC
autosampler vials, and 10 ng of simeton (internal standard) was added prior to LC-MS/MS
2.4. Liquid chromatography and tandem mass spectrometry
The prepared samples were analyzed using a LC-MS/MS system consisting of a
Shimadzu high-performance liquid chromatography (HPLC, Columbia, MD, USA) fully
integrated with an Applied Biosystems Sciex 3200 triple quardrupole mass spectrometer (Foster
City, CA, USA). A PEAK Scientific gas generator (NM20ZA, Billerica, MA) was used to supply
gases required for the mass spectrometer. Turbo IonSpray source of the mass spectrometer was
operated with electrospray ionization source in positive mode, ionspray voltage at 5500 V and
temperature at 600 °C. Curtain gas pressure was 10 psi, collision gas pressure was 6 psi, and ion
source gas pressure was 10 psi. The compound-dependent mass spectrometer parameters were
optimized for each analyte (Table 1). Pharmaceutical concentrations were quantified using
multiple-reaction monitoring mode. Two pairs of precursor/product ion transitions were
simultaneously monitored during analysis in order to unambiguously identify the target
pharmaceuticals in environmental samples [11,39]. The pair of precursor and product 1 ions
were selected to quantify the analytes because the product ion 1 manifested a relatively greater
The Shimadzu HPLC system consisted of two LC-20AD pumps, a SIL-20A autosampler
and a DGU-20A degasser operated by a CBM-20A controller. The analytes were separated using
a Phenomenex Luna C18 column (150 mm × 4.6 mm, particle size: 3 µm, Torrance, CA, USA). A
gradient mobile phase was applied with a flow rate of 350 µL/min in which phase A
se B (acetonitrile) both contained 0.1 % (v/v) formic acid. The mobile phase
gradient was programmed as: at 0 min 100% phase A; 0 to 2.0 min phase B linearly increased to
12%; 2 to 20 min phase A and phase B held at the ratio of 88:12; 20 to 22 min phase B linearly
increased to 25%; 22 to 35 min phase B increased to 40%; 35 to 40 min phase B increased to
80%, and held at this ratio until 42 min. From 42 to 43 min phase B reduced to 0%, and
equilibrated in the flow system for 5 min before next injection. The injection volume was 10 µL.
After each sample injection, the first three-minute elution was diverted to waste through a built-
in valve, and then switched to the inlet of tandem mass spectrometer.
External standard solutions were prepared in methanol-water mixture (v/v = 1/1)
containing 150 mg/L EDTA. The same background solution was also used to elute analytes from
the HLB cartridge during the cleanup step of sample extracts. Standard curves were established
with the concentrations spanning 2 orders of magnitude. The method sensitivity was determined
by analyzing pharmaceuticals at ~20 g/L for 10 times using the ASE extracts fortified with
target compounds, and standard deviation of these measurements was calculated. Limit of
detection (LOD) was estimated as three times the standard deviation, and limit of quantification
(LOQ) was defined as ten times the standard deviation. To evaluate extraction efficiency and
repeatability of the overall method, the selected target pharmaceuticals and two surrogates (i.e.,
C6-sulfamethazine and meclocycline) were spiked into biosolid samples, and mixed together
overnight. Eight replicates of spiked biosolid samples were prepared followed by accelerating
solvent extraction, SPE cleanup and LC-MS/MS analysis (described above). Biosolid control
samples without spiked pharmaceuticals were also prepared; the amounts of pharmaceuticals
measured in the control were subtracted from the amounts measured in the spiked samples. The
sample matrix effects on the responses of tandem mass spectrometer was evaluated via spiking
known amounts of target pharmaceuticals into the extract of biosolid which was obtained by
ASE followed by the SPE cleanup. The measured pharmaceutical concentrations in the spiked
samples were subtracted by the background pharmaceutical amounts originally present in the
biosolids, and compared with the amounts of the spiked analytes. 3. Results and discussion 3.1. Selection of extraction solvent
The biosolid sample collected from Lansing WWTP was fortified with carbamazepine,
sulfamethazine, sulfamethoxazole, tetracycline, chlortetracycline and oxytetracycline with
concentrations ranging from 780 to 1862 g/kg on dry matter basis, and subjected to ASE using
water and organic solvent mixtures. The extracted concentrations of pharmaceuticals were
subtracted by the measured concentrations of the corresponding compounds in the unfortified
biosolid sample, and reported in Table 2. Extraction solvents used here were aqueous methanol
or acetonitrile mixtures with volume ratios of organic solvent to water 7:3, 5:5 and 3:7. Several
previous studies indicated that polar organic solvents (e.g. acetonitrile, methanol) and water
mixtures manifested the superior capability to extract pharmaceuticals from sewage sludge and
soil [17,26,35]. Among the extraction solvents tested, it appeared that higher extraction
efficiencies were achieved for the mixtures containing greater contents of organic solvent (Table
2). Acetonitrile/water mixture (v/v = 7:3) was more effective to remove pharmaceuticals from
biosolids than methanol/water mixture. Therefore, acetonitrile/water mixture (v/v = 7:3) was
chosen as the extraction solvent in this study. To evaluate the influence of pH on extraction
efficiency, pH of acetonitrile/water mixture (v/v = 7:3) was adjusted to ~2.0 using hydrochloric
acid, and then extracted the selected pharmaceuticals from the biosolid. The use of acidified
extraction solvent, in fact, lowered extraction efficiency by 36% for tetracycline, 42% for
chlortetracycline, and 75% for oxytetracycline compared to unacidified acetonitrile/water
mixture; no apparent discrepancy was observed for extraction of carbamazepine and
sulfonamides. Several previous studies indicated that acidified water-organic solvent mixture
(e.g., pH < 2.5) enhanced extraction recoveries of pharmaceuticals, but the target
pharmaceuticals were not the same as those in this study [25,26]. The acidification of extraction
solvent protonates the acidic functional groups (e.g., carboxylic acids and phenols) in the organic
fractions of sewage sludge/biosolids, and thereby reducing electrostatic interactions between
sewage sludge/biosolid and cationic moiety of tetracyclines. In these previous studies, sand was
used to mix with sewage sludge or soil samples prior to extraction [25,26,40]. Diatomaceous
earth, used as the dispersant of biosolid in our study, could release a certain amount of inorganic
cations (e.g., Na+, Mg2+ etc.) which could effectively suppress the interactions of biosolid with
cationic speciation of tetracyclines hence resulting in a higher extraction efficiency. 3.2. Optimization of ASE operating parameters
High pressure is applied during ASE operation in order to keep extraction solvent in
liquid state while temperature is held at or above the boiling point. The application of high
pressure also facilitates the contact of extraction solvent with analytes via penetrating into the
domains in biosolids where the extraction solvent could not enter under normal atmospheric
pressure. In this study four levels of pressure (55, 80, 100, and 130 bars) were applied to the
extraction, and the corresponding extracted concentrations are reported in Table 3. The
pharmaceutical concentrations at different extraction pressures were compared using a linear-
mixed model (SAS 9.1 software, SAS Institute Inc., Cary, NC, USA). Significant level was set at
p < 0.05 with 95% confidence. The results indicate that the extraction efficiencies at pressure of
100 bars were significantly higher than those conducted at other pressures (p = 0.0049). This
extraction pressure falls within the similar range reported in several previous studies [25,26].
Static extraction with 2 or 3 consecutive cycles was conducted at 100 °C and under 100
bars. Two periods of the static extraction time (15 vs. 25 min) were tested for extraction
efficiency (Table 3). In general, prolonging extraction time and more extraction cycles achieve
greater extraction efficiency [41,42]. For the static time of 15 min vs. 25 min with the same
numbers of extraction cycles, the analysis of the linear-fixed model results indicated no
significant difference (p = 0.897) of extraction efficiencies. This suggests that 15 min of static
period is sufficient for the analytes to approach partition equilibrium between biosolid and
solvent. However, increase of consecutive extraction cycles from 2 to 3 significantly enhanced
the extraction efficiencies for all pharmaceuticals tested (p = 0.0002). In each extraction cycle
the induction of fresh solvent into the sample cell reestablishes new partition equilibrium for the
analytes between the added solvent and the biosolid residues. This partitioning process drives the
release of biosolid-sorbed pharmaceuticals to extraction solvent phase. Taken together, the
observation of enhanced extraction efficiencies of 3 consecutive cycles (versus 2 cycles), and no
apparent difference with prolonged static time (25 vs. 15 min), warrant that the optimal operative
condition consists of 3 extraction cycles with 15 min static time in each cycle.
Increasing temperature in ASE operation resulted in enhanced extraction of the
pharmaceuticals from biosolid (Table 3). The extraction temperature was tested at 50, 75, and
100 °C with the pressure of 100 bars, 3 extraction cycles and static period of 15 min in each
cycle. The extraction efficiencies were observed to significantly increase with increasing
0.0001). Therefore, higher temperature is preferred in the ASE operation.
However, considering thermal stability of analytes, the extraction was not conducted at
temperature >100 °C. In this study, the temperature of 100 °C was selected as optimal operating
condition. The increase of temperature enhances dissolution of pharmaceuticals in extraction
solvent and hence increasing extraction efficiencies. At the same time, the viscosity of extraction
solvent and surface tension on biosolid surfaces decrease as temperature increases, which also
facilitates the solvent to access the domains where the analytes are present. In addition, the
enhanced extraction efficiency could be due to accelerating mass transfer rate from sample
matrices to solvent at increased temperature.
The linear ranges of standard curves and correlation coefficients, along with method
LOD and LOQ values, are reported in Table 4. The standard curves were prepared with two
order of magnitude, and demonstrated an excellent linearity with the correlation coefficients (r2)
>0.999. The described method was tested for simultaneous extraction and determination of 15
pharmaceuticals in biosolid, which manifested varying levels of LOD and LOQ. For the class of
sulfonamides the LOD ranged from 0.6 g/kg (for sulfamethazine) to 15.0 g/kg (for
sulfadiazine) on dry weight basis of biosolid. This method was less sensitive for tetracycline
antibiotics as shown with the LOD ranging from 4.6 g/kg (for doxycycline) to 146 g/kg (for
tetracycline). The overall method recoveries reported were the average of eight replicates of
spiked samples ranging from 49.3% to 94.6%. The standard deviations of the recoveries (n = 8)
were < 10% indicating the reported method achieved satisfactory repeatability. It was noted that
tetracycline antibiotics manifested the relatively lower recoveries ranging from 49% to 68%
(Table 4). This range of recoveries fell within the low end of the recovery range (i.e., 47.2% to
125 %) reported by Jacobsen et al.  in which tetracyclines were analyzed in swine manure
using the similar analytical approach. However, the extraction recovery of tetracycline (53.8%)
measured in this study is significantly greater that (~27%) reported by Lillenberg et al. . The
overall recoveries for sulfonamides (from 63.6 % to 94.6%) are comparable with the results in
several previous studies [17,26,37]. For instance, sulfamethazine manifested the highest recovery
(94.6%) in this study; the similar results were also reported for extraction of sulfamethazine from
swine manure (84.1% to 97.8%) , and from sewage sludge (104%) .
The manifestation of relatively low recoveries (e.g. ~50%) of the overall method for a
certain class of pharmaceuticals such as tetracyclines could be attributed to inefficient extraction
of ASE operation, analyte loss during SPE cleanup, and/or matrix effects on mass spectrometer
responses from extracts. The loss of target analytes during SPE cleanup step was < 11 %
(describe below). To examine sample matrix effects on mass spectrometer responses, standard
addition method was used in which the analytes were spiked into biosolid extracts after ASE
treatment and SPE cleanup. The spiked pharmaceutical concentrations were tested at two levels:
10-23 g/L and 100-232 g/L. The ratios of measured concentrations to the corresponding
spiked concentrations ranged from 0.90 to 1.15, indicating matrix effects caused a minimal
impact to the overall method recovery. Therefore, the low recoveries measured in this study
result primarily from inefficient extraction of pharmaceuticals from ASE procedure, particularly
for the class of tetracycline antibiotics which usually demonstrate a great affinity with sorbents.
In order to evaluate the extent of pharmaceutical loss during SPE cleanup step, the target
compounds were spiked into biosolid extracts after ASE treatment. The solution passed through
the preconditioned HLB cartridge, followed by an elution with methanol-water mixture (v/v =
1:1) containing 150 mg/L EDTA and analyzed by LC-MS/MS. Two concentration levels (10-23
g/L and 98-232 g/L) of pharmaceuticals were spiked into the biosolid extracts (i.e., after ASE
treatment). The measured pharmaceutical concentrations were subtracted by the amount
pharmaceuticals originally present in the samples, and compared to the spiked concentrations
(Table 5). For most pharmaceuticals, the recoveries of spiked amount ranged from 86.8 to 120%,
indicating that the loss of analytes during SPE cleanup step was minimal. Demeclocycline and
lincomycin manifested relatively higher recovery (i.e., 155 % for demeclocycline at 12.7 g/L,
and 176% for lincomycin at 134.6 g/L), which was attributed to the experimental variations
since reasonable recoveries were obtained for these two compounds at another concentration.
Therefore, the relatively low recoveries of the overall method for some analytes (e.g.,
teracyclines) could be attributed to the inefficient extraction from biosolids during the ASE step.
3.5. Application to biosolid samples
Biosolid samples were collected at WWTPs of six cities (Lansing, East Lansing, St Clair,
Plainwell, Traverse City, and Imlay City) in Michigan, USA. These samples were extracted
using the optimized method described above. The samples were conducted in triplicates. The
measured concentrations and the corresponding standard deviations (n = 3) are presented in
Table 6. Among these samples, 14 out of the 15 target pharmaceuticals were found except
tylosin. The average concentrations of the pharmaceuticals manifested a relatively wide range
from 2.6 to 743.6 g/kg (Table 6). The calculated standard deviations for all the measurements
were < 17%, indicating a reasonable repeatability of the analysis. Tetracycline antibiotics were
frequently detected with the concentrations ranging from 36.6 to 743.6 g/kg. Doxycycline was
detected in all 6 biosolid samples with concentrations ranging from 159.9 to 292.4 g/kg.
Relatively higher concentrations of tetracycline (281.9 g/kg) and chlortetracycline (346.6 g/kg)
were found in the sample of Lansing WWTP compared to the samples collected from other five
WWTPs. Oxytetracycline was detected in four biosolid samples from 51.9 to 743.6 g/kg. Trace
amounts of demeclocycline were found in three biosolid samples (i.e., 36.6, 98.2 and 131.2
g/kg) from WWTPs in Plainwell, East Lansing, and St. Clair. Sulfonamides were also
frequently detected, and the corresponding concentrations ranged from 4.8 (sulfamethoxazole) to
668.9 g/kg (sulfamerazine). For other six pharmaceuticals (acetominephen, carbamazepine,
caffeine, erythromycin, lincomycin and tylosin), the measured concentrations spanned from 2.6
to 370.4 g/kg. Caffeine and carbamazepine, both of which are solely used by humans, were
detected in the biosolids from five cities except Imlay City. Interestingly, Imlay City has the least
populations among the six cities; this might be the reason responsible for the least occurrence of
human medicine in the biosolid of Imlay City WWPT . Tylosin is commonly used as
veterinary pharmaceutical to control livestock infection and promote livestock growth. It is
reasonable that no samples were found to contain tylosin in municipal WWTP biosolids.
Acetominephen was detected in three biosolid samples from Lansing, East Lansing and St. Clair
with concentrations of 88.6 to 370.4 µg/kg. In fact, these three cities have more populations than
Plainwell, Traverse City, and Imlay City.
This study describes an efficient and reproducible analytical approach for simultaneous
determination of multiple classes of pharmaceuticals in biosolids. The experimental steps
consisted of accelerated solvent extraction of freeze-dry biosolid sample, and cleanup by solid-
phase extraction followed by simultaneous analysis by liquid chromatography/tandem mass
. The extraction parameters of ASE including extraction solvent, temperature,
pressure, static period and extraction cycle were optimized to achieve satisfactory extraction
efficiency. Overall, this method offers less laborious work, automatic extraction procedure, less
consumption of organic solvent and relatively high extraction efficiency. The method has been
tested successfully for simultaneously determining multiple classes of pharmaceuticals in
WWTP biosolids collected from different locations. The results also suggest that many
pharmaceuticals could survive wastewater treatment processes and accumulate in solid phase
(e.g., biosolids). Land application of the contaminated biosolids could result in the dissemination
of pharmaceuticals in soil and water environment. Chronic exposure to low levels of antibiotics
might exert selection pressure on development of antibiotic resistant bacterial stains in the
This study was supported in part by Agriculture and Food Research Initiative Competitive Grant
2009-65102-05847 from the USDA National Institute of Food and Agriculture, American Water
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Table 1. Precursor, product ions and mass spectrometer parameters used to identify and quantify pharmaceuticals.
declustering potential. b entrance potential. c cell entrance potential. d collision energy. e collision cell exit potential
Table 2. Extracted concentrations of pharmaceuticals vs. spiked concentrations from biosolid by water-organic solvent mixtures using accelerating
acetonitrile/ water (7:3) 947 ± 21b 1008 ± 25 1065 ± 28
Operation conditions: 100 bars, 100°C, 3 × 15 min. b measured concentration: mean ± standard deviation (n = 3)
Table 3. Extraction efficiency of pharmaceuticals under varying accelerating solvent extraction parameters
Table 4. Linear range and correlation coefficient (r2) of standard curves, method limit of detection (LOD), limit of quantification
Table 5. Recoveries of solid-phase extraction for pharmaceuticals spiked into biosolid extracts after accelerating solvent extraction.
Table 6. Measured pharmaceutical concentrations and standard deviations (µg/kg dry matter) in the collected biosolid samples.
Not detected. b Detected but less than limit of quantification.
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