Introduction
Antibiotics are widely used in human and veterinary
medicine for the prevention and treatment of bacterial
infectious diseases. An important but often
disregarded aspect of antibiotic use is the fate of
antibiotic residues entering the environment.1
Pharmaceutical industry wastewater, improperly
disposed of unused antibiotics and non-metabolized
antibiotics excreted by humans can all enter the sewer
system in low concentrations. Because sewage
treatment plants are rarely equipped to remove these
drugs from wastewater, antibiotics are released into
the water system where they can enter the
environment and eventually the drinking water supply.
Veterinary antibiotics used in livestock operations are
another major source of contamination in the
environment. Agricultural waste such as manure and
water run-off can carry these antibiotics into the soil
and groundwater.
The effects of antibiotics in the environment are still
poorly understood. One major concern is the
development of antibiotic-resistant strains of bacteria
that could critically disturb the natural bacteria
ecosystems and lead to a serious threat to human health. There are also concerns that exposure to environmental
antibiotic residues might lead to carcinogenic or allergic
reactions in humans and create hazards to aquatic and soil organisms.2,3
The presence of antibiotics and other drugs in the
environment has garnered international attention recently. The
Associated Press (AP) reported in March 2008 that a variety of
prescription and over-the-counter drugs have been found in
the drinking water supplies of at least 41 million Americans
living throughout the US.4 Achieving low limits of detection
(LODs) of pesticides, antibiotics and veterinary residues in
drinking water is of paramount importance to monitor the
regulatory levels as stated by US, Canadian, Japanese and
European environmental and water directives. Because many of
these substances may pose a significant health threat, they
need to be accurately detected.
Traditionally, liquid chromatography coupled with tandem
mass spectrometry (LC–MS–MS) has been used by the
environmental industry for the identification and quantification
of these residues. This methodology typically requires extensive
off-line sample preparation. Additionally, the compounds of
interest are generally present at trace levels, so the sample
preparation method requires preconcentration. Researchers
have recently developed an on-line preconcentration method
for sample preparation of water samples that overcomes
challenges related to sample preparation of water samples.5
Figure 1
|
Sulphonamides (Figure 1) are a common class of synthetic
antimicrobials that are widely used in human and veterinary
medicine and as feed additives to promote growth in
concentrated animal feeding operations. They are regarded as
emerging contaminants that are introduced into the
environment predominantly in the US and Europe. There is no
regulation of the levels of these compounds in environmental
matrices (water, sediment, soil). This is probably because of the
limited knowledge of the input, fate and effects of most
pharmaceuticals in the environment. Therefore, sensitive and
reliable analytical methods for detection of low concentrations
(ng/L) of these compounds are needed.
Experimental Conditions
Sample preparation: Samples of secondary effluent were
collected from sewage treatment plants in Greece and then
vacuum filtered. Each 50 mL sample was diluted with 200 mL
deionized water. After acidification to pH 4, 5 ng of the
surrogate standard d4-sulphamethoxazole (d4-SMX) was
added before enrichment to assess possible losses during the analytical procedure. The effluent samples were enriched by
solid-phase extraction (SPE). The diluted wastewater samples
were percolated through the cartridges at a flow-rate of
5 mL/min. The cartridges were then washed with 5 mL
deionized water. Wastewater organics were eluted with 2 X
4 mL methanol. The solvents were evaporated under a stream
of nitrogen gas and then the extracts were redissolved in
0.5 mL mobile phase A (0.1% formic acid in water).6 While this
method employed the more traditional off-line method of
sample preparation, other researchers have successfully
employed an on-line preconcentration method for sample
preparation.5
HPLC: HPLC analysis was performed using the Surveyor HPLC
system (Thermo Fisher Scientific, San Jose, California, USA).
Each 20 μL sample was injected directly onto a 150 3 2.1 mm,
3.5 μm, C18 HPLC column. A gradient LC method used mobile
phases A (0.1% formic acid in water) and B (0.1% formic acid
in acetonitrile) at a flow-rate of 0.2 mL/min.
MS: MS analysis was performed on a TSQ Quantum Ultra triple
stage quadrupole mass spectrometer with an electrospray
ionization source (Thermo Fisher Scientific, San Jose, California,
USA). The MS conditions were as follows:
Ion source polarity: Positive ion mode
Sheath gas pressure (N2): 40 units
Ion transfer tube temperature: 350 °C
Collision gas pressure (Ar): 1.0 mTorr
Q1 resolution: 0.2 FWHM
Q3 resolution: 0.7 FWHM
Dwell time: 0.2 s
Scan type: SRM
Table 1 summarizes the SRM transitions that were
monitored. MS detection of the target compounds was divided
into three time segments on the basis of their retention times
during chromatography. The protonated molecular ion of the
compound [M 1 H]1 was selected as the precursor ion.
Detection was performed in the multiple reaction monitoring
mode using, usually, the two most intense and characteristic
precursor/product-ion transitions obtained from the MS–MS
optimization procedure. Identification criteria for the target
compounds were based on the LC retention time (tR) and on
the ratio of the two monitored transitions for each compound.
Method accuracy and precision were evaluated by recovery
studies, using deionized water spiked with appropriate
amounts of the sulphonamides at three concentrations (2 ng/L,
20 ng/L and 200 ng/L). Calibration plots were obtained by
analysis of standard solutions at eight concentrations in the
range 0.1–100 μg/L (2–2000 pg injected).
