Antibiotics have changed the face of medicine, but their impact on the environment is now becoming a real concern. Liquid chromatography-tandem mass spectrometry has the power to shed light on the issue
SCIENTISTS have come a long way since Alexander Fleming’s discovery of penicillin in 1928. Antibiotics are some of the most frequently prescribed drugs used in modern medicine and today’s doctors are armed with a whole suite of them for the purposes of treating bacterial infections in both humans and animals. While the development of antibiotic-resistant bacteria is becoming of increased concern, there is, however, another worry: the fate of antibiotic residues entering the environment (Petrovic et al., 2005).
Pharmaceutical industry wastewater, unused antibiotics that are not disposed of properly and non-metabolised antibiotics excreted by humans can all enter the sewage system at low concentrations. Because sewage treatment plants are rarely equipped to detect or remove these drugs from wastewater, antibiotics may be released into the water system where they can then enter the environment and, ultimately, the drinking water supply. A variety of prescription and over-the-counter drugs have been detected in the water supplies of at least 41 million Americans throughout the United States (Associated Press, 2008). Veterinary antibiotics used in livestock are another major source of contamination: agricultural waste such as manure and water runoff can carry antibiotics into the soil and groundwater.
The effects of antibiotics on the environment are still poorly understood. One major concern is the development of antibiotic-resistant strains of bacteria that can critically disturb natural bacterial ecosystems and lead to a serious threat to human health. In addition, there are concerns that exposure to antibiotic residues in the environment might lead to carcinogenic or allergic reactions in people and present hazards to aquatic and soil organisms (Göbel et al., 2004; Yang et al., 2004).
Sulfonamides (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 animal feeding operations. Of particular interest are sulfamethazine (administered to animals) and sulfamethoxazole (prescribed to humans). They are regarded as emerging contaminants that have been found in modest concentrations in wastewater treatment plants and waterways in the United States, Canada, Korea and Switzerland. Unfortunately, the presence of these drugs in water supplies is not routinely monitored. Furthermore, there is no regulation of their levels in water, sediment and soil, primarily because our knowledge of the input, fate and effect of most pharmaceuticals in the environment is limited. Sensitive and reliable analytical methods are crucial for detecting the characteristically low concentrations of these compounds in wastewater, and for providing insights into what could be an important problem.
Traditionally, the environmental industry relies on liquid chromatography coupled
Figure 1: LC-MS/MS chromatograms of sulfamethoxazole in wastewater sample extract, and of a standard solution of sulfamethoxazole
with tandem mass spectrometry (LC-MS/MS) to identify and quantify pharmaceutical residues such as antibiotics, non-steroidal anti-inflammatory drugs, beta-blockers, psychiatric drugs and lipid-regulating agents in environmental samples. This approach tends to involve extensive offline sample preparation, but is excellent at confirming the presence of a suspected contaminant. Because the compounds of interest are often present in trace amounts, sample preparation usually requires some sort of pre-concentration process (which can be performed online or offline).
Before the advent of modern (tandem) mass spectrometry, quantitation was typically carried out using high-performance liquid chromatography (HPLC) coupled with UV detectors. This approach uses the retention time, peak area and UV spectral character of samples to analyse their make-up, but suffers from a lack of sensitivity and specificity. A more sophisticated method – specifically, tandem mass spectrometry (MS/MS) – is needed to achieve the required sensitivity.
While a single mass analyser can perform quantitation, it often does not provide structural discrimination or the sensitivity needed to analyse trace quantities in complex matrices such as blood or wastewater. Two or more spectrometers working in tandem, however, provide additional selectivity and the ability to identify overlapping mass peaks. In most cases the second spectrometer provides a unique molecular fragment; by combining the specific parent mass and the unique fragment, one can selectively detect the compound under study.
Researchers based at the General Chemical State Laboratory in Athens, Greece, have used LC-MS/MS to successfully detect and analyse sulfonamide compounds in municipal wastewater. The experiments performed by Eleni Botitsi, Charalampia Frosyni and Despina Tsipi, which are described in this article, show that antibiotics can be identified in water samples even when present at very low part-per-trillon (ppt) levels (ng/L).
Botitsi and colleagues have collected secondary effluent samples from sewage treatment plants in Greece and prepared them for analysis using an offline approach. Wastewater samples were vacuum-filtered, diluted with de-ionised water and acidified. A solid phase extraction method was used to isolate and concentrate the sulfonamides, and to reduce the level of organic matter contaminants from the wastewater.
The extracted antibiotics were then analysed using a high-performance liquid
Figure 2: Chemical structures of some sulfonamide compounds
chromatograph ( Thermo Scientific Surveyor HPLC System) and a tandem mass spectrometer operating in a positive-ion, selected-reaction-monitoring (SRM) mode and equipped with an electrospray ionization source (Thermo Scientific, TSQ Quantum Ultra triple stage quadrupole mass spectrometer). The HPLC analysis, which employed a gradient LC method, used water and acetonitrile as the mobile phases flowing at a rate of 0.2mL/minute. Target sulfonamides were identified based on their LC retention times and on the ratio of the two monitored transitions for each compound. The chromatographic response to each antibiotic was calibrated using standard sulfonamide solutions at eight different concentrations ranging from 0.1μg/L to 100μg/L (2pg to 2000pg injected).
Recovery studies were used to determine the accuracy of the solid phase extraction scheme employed to enrich and isolate the antibiotic components. De-ionised water was spiked with appropriate amounts of the sulfonamides at three concentration levels (2ng/L, 20ng/L and 200ng/L) and the rates of recovery were measured. Typically these were greater than 72%, demonstrating the efficiency of the extraction method.
All of the wastewater samples that were investigated revealed traces of sulfonamide antibiotics. In particular, sulfamethoxazole was detected in every extract, with a median concentration of 150ng/L recorded. The SRM chromatograms of sulfamethoxazole in the effluent extract are shown in Figure 2.
Through this study, and the work of others, LC-MS/MS has proven itself to be a powerful analytical tool that enables the very sensitive detection of antibiotics in municipal wastewater, even when they are present in trace amounts (ng/L, or ppt). In LC-MS/MS studies of wastewater, the antibiotic sulfamethoxazole, was detected in all of the effluent samples at low concentrations, a clear indication that sewage treatment plants do not completely eliminate these drugs from the water supply. This type of analysis will offer important insights into the occurrence and distribution of antibiotics (and other pharmaceuticals) in drinking water supplies. Ultimately it could help to point the way towards imposing proper limits on the quantities of these chemicals that are allowed to leech into our environment.