Quantitative Raman assays for on-site analysis of stockpiled drugs.

We present a rapid Raman assay for on-site analysis of stockpiled drugs in aqueous solution. This approach was tested on Tamiflu (oseltamivir phosphate). Tamiflu is a drug approved by the FDA for treatment of influenza and is the most common antiviral included in stockpiles for use in the event of a national emergency. Rapid assays were performed on three concentrations (30, 45, and 75 mg) of oseltamivir using three different portable & handheld Raman instruments. PLS regression models were developed to establish a calibration curve and applied to the Tamiflu samples. Raman assay values were compared against the standard HPLC assay to demonstrate the viability of this approach, yielding an average assay value within 0.3% of that obtained from the HPLC analysis for the 35 different capsules analyzed. The Raman method demonstrates the potential for rapid screening of stockpiled pharmaceuticals on-site using portable Raman instrumentation and readily available consumables for sample preparation. In addition to routine screening to ensure product quality past the expiration date, this approach could also be used to assist in rapid deployment of such medications in the case of a national emergency.

A Lateral Flow Immunochromato-graphic Strip Test for Rapid Detection of Oseltamivir Phosphate in Egg and Chicken Meat.

A lateral flow immunochromatographic strip test (LFIST) based on a competitive format was developed for rapid and sensitive on-site detection of oseltamivir phosphate (OP) residues in poultry product. The sensitivity (half inhibitory concentration, IC50) of the LFIST in the detection of egg and chicken meat samples was confirmed to be 2.56 and 2.63 µg/kg, and the limit detection (LOD) value were 0.43 and 0.42 µg/kg, respectively. For intra-assay and inter-assay reproducibility, recoveries of OP spiked samples ranged between 82.8% and 91.2% with coefficients of variations (CV) less than 5.67% (intra-assay) and 6.52% (inter-assay). The performance of LFIST was comparable to high-performance liquid chromatography (HPLC) in a parallel testing of egg samples and chicken samples. LFIST takes less than 5 minutes, eliminates the dependency on professional personnel, and thus can be used as a surveillance tool for on-site detection of OP residues.

Reductive amination assistance for quantification of oseltamivir phosphate and oseltamivir carboxylate by HPLC-MS/MS.

Oseltamivir phosphate (OP) is the first line therapy for influenza, and its primary metabolite oseltamivir carboxylate (OC) is the active agent via inhibition of neuraminidase of influenza virus. Dosages of OP and OC might affect human causing nausea and vomiting and it is therefore necessary to evaluate their toxicity and safety. The separation system: liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique to monitor OP and OC. However, quantification of OP and OC needs isotopic analogs as internal standards to monitor the stability of the sample pretreatment procedures and instruments. In this study, we demonstrated a modified method (i.e., reductive amination) to synthesize OP and OC deuterated and hydrogenated analogs as internal standards (ISs) and for illustration of calibration curves, respectively. This modification allowed to overcome ISs selection and to enhance the signal intensities via high yield reductive amination in MS detection. We utilized the multiple reaction monitoring (MRM) mode to target m/z values of precursor and product ions. N-dimethylated OP and N-dimethylated OC showed linearity ranging from 1 to 1000 ng/mL with coefficient of determination (R2) values of 0.9995 and 0.9999, respectively. Additionally, the relative standard deviations (RSD) of intra-day ranged from 0.3% to 5.2%, and the RSD of inter-day ranged from 2.0% to 18.8%, respectively. This quantitative method utilized spiked OP and OC at low (20 ng/mL), intermediate (100 ng/mL), and high (500 ng/mL) concentrations in human serum samples. The average recoveries for OP and OC were 84.6%-107.7% and 94.9%-98.5%, respectively.

Development and application of an in-cell cleanup pressurized liquid extraction with ultra-high-performance liquid chromatography-tandem mass spectrometry to detect prohibited antiviral agents sensitively in livestock and poultry feces.

An in-cell cleanup pressurized liquid extraction was developed to analyze prohibited antiviral agents in livestock and poultry feces. Extraction and cleanup were integrated into one step. The extraction was performed using methanol-acetonitrile (1:1, v/v) with 0.5% glacial acetic acid at 90°C, and 0.75g of PSA was used as the adsorbent during the extraction procedure. Under optimal conditions, the average recoveries for 11 antiviral drugs were 71.5-112.5% at three spiked levels (20, 40, and 100µgkg-1). The detection limits and detection quantitations of the analysis method for the eleven antiviral drugs were 0.6-1.4 and 1.4-4.7µgkg-1, respectively. Finally, the method was applied to analyze amantadine, oseltamivir and its metabolites oseltamivir acid in duck feces based on an experiment of an oral dose of two antiviral drugs in duck. The amantadine, oseltamivir and oseltamivir acid can be detected in feces within approximately four weeks after amantadine and oseltamivir were orally administered. The results indicate that the residue analysis in feces is a noninvasive method to monitor inhibited antiviral agents efficiently in livestock and poultry breeding.

Transplacental transfer of oseltamivir phosphate and its metabolite oseltamivir carboxylate using the ex vivo human placenta perfusion model in Chinese Hans population.

OBJECTIVE: The objective of the study was to determine transplacental of oseltamivir phosphate (OP) and its active metabolite oseltamivir carboxylate (OC) using the ex vivo human placental perfusion model in Chinese Hans population. STUDY DESIGN: Perfusion studies were performed on 20 placentas from healthy term pregnancies. Concentrations typical for 75 mg, twice-daily oral dose were tested (OP 65.2 ng/ml and OC 348 ng/ml), along with the positive control antipyrine 0.1 mg/ml. Each perfusion experiment was conducted for 180 min while samples were taken from both maternal and fetal compartments. Concentrations of OP and its metabolite OC were determined by ultrafast-performance liquid chromatography/tandem mass spectrometry. Integrity and viability of the placenta were determined by measuring fetal volume loss, pH, pO2, ΔhCG, glucose consumption and lactate production during the perfusion experiments. RESULTS: Following 3 h of perfusion, the fetal transfer rates of OP and its metabolite OC were 12.39%±3.26%, 10.17%±2.03%, respectively. The clearance indexes of OP and OC were 0.36 ± 0.11 and 0.29 ± 0.06, respectively. CONCLUSION: These data suggest that OP and its metabolite OC pass through the healthy term placenta at a small amount according to the ex vivo human placental perfusion model, fetal exposure must be considered when treating pregnant women with OP.

Detection of peramivir and laninamivir, new anti-influenza drugs, in sewage effluent and river waters in Japan.

This is the first report of the detection of two new anti-influenza drugs, peramivir (PER) and laninamivir (LAN), in Japanese sewage effluent and river waters. Over about 1 year from October 2013 to July 2014, including the influenza prevalence season in January and February 2014, we monitored for five anti-influenza drugs-oseltamivir (OS), oseltamivir carboxylate (OC), zanamivir (ZAN), PER, and LAN-in river waters and in sewage effluent flowing into urban rivers of the Yodo River system in Japan. The dynamic profiles of these anti-influenza drugs were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage effluents and river waters were, respectively, 82 and 41 ng/L (OS), 347 and 125 ng/L (OC), 110 and 35 ng/L (ZAN), 64 and 11 ng/L (PER), and 21 and 9 ng/L (LAN). However, application of ozone treatment before discharge from sewage treatment plants was effective in reducing the levels of these anti-influenza drugs in effluent. The effectiveness of the ozone treatment and the drug dependent difference in susceptibility against ozone were further evidenced by ozonation of a STP effluent in a batch reactor. These findings should help to promote further environmental risk assessment of the generation of drug-resistant influenza viruses in aquatic environments.

Photocatalytic degradation of the antiviral drug Tamiflu by UV-A/TiO2: Kinetics and mechanisms.

The photocatalytic degradation of the antiviral drug Tamiflu (oseltamivir phosphate, OP) by TiO2 - P25, ST-01 and ATO was investigated in aqueous solution under ultraviolet (UV-A) irradiation. The photocatalysis of OP is well described by pseudo-first-order kinetics with r2>98.0% for all cases. The kinetic constant of P25 with 80% anatase and 20% rutile (0.040 min(-1)) is 4 and 10 times higher than that of ATO and ST-01 with 100% purity of anatase, respectively. We examined the effects of the catalyst loading and initial OP concentration on the photodegradation of OP, and used potassium iodine, isopropanol, and calcium fluorine as radical quenchers to evaluate the contributions of the hydroxyl radical (OH) and photo hole (h+) in the photodegradation. Results confirmed that 80% of the contribution came from the OH species. Although more than 95% of the OP (21 µM) was removed after 80 min of UV-A irradiation with 20 and 100 mg L(-1) P25, the removal efficiencies of total organic carbon (TOC) were only 45.6% and 67.0%, respectively, after 360 min UV-A irradiation. Based on an intermediate analysis by HPLC coupled with a triple quadrupole spectrometer and an ion trap mass spectrometer, typical intermediate species such as hydration derivatives, hydroxyl substitutes and keto-derivatives were identified and possible degradation pathways of OP by P25 were proposed.

A novel coated platinum electrode for oseltamivir determination in pharmaceuticals.

New coated platinum selective electrodes have been prepared and used for the determination of oseltamivir phosphate (OSL) in bulk drug solutions and in pharmaceutical preparations. Electrodes were using plasticized PVC membranes doped with ion-pair complexes based on drug-phosphomolybdate and drug-tetraphenylborate as electroactive materials. The influence of membrane composition (plasticizers and ion-pair complexes) has been investigated. Optimum performance was obtained for two polymeric membranes: PVC:o-NPPE:OSL-TPB in the ratio of 30:68:2 (%, w:w:w) and PVC:DPP:OSL-PMA in the ratio of 30:68:2 (%, w:w:w). The electrodes exhibited linear responses over large concentration ranges (1.0×10(-5)-1.0×10(-2) and 5.0×10(-5)-5.0×10(-2)M, respectively) with near-Nernstian responses (58.9 and 57.3mV/decade, respectively). The selectivity coefficients indicated good selectivity for OSL drug over a large number of organic compounds and some inorganic cations. The proposed electrodes were successfully applied to the determination of OSL in raw material and in pharmaceutical formulations. The results were validated by comparison with a capillary electrophoresis method.

Mass balance of anti-influenza drugs discharged into the Yodo River system, Japan, under an influenza outbreak.

In February 2011, at the peak of an influenza outbreak, we performed a comprehensive analysis of the mass balances of four anti-influenza drugs-oseltamivir (OS), oseltamivir carboxylate (OC), amantadine (AMN), and zanamivir (ZAN)-in the urban area of the Yodo River system. This area includes three main river catchments (the Katsura, Uji, and Kidzu Rivers) and is home to 12 million people, about 10% of Japan's population. Water was sampled at six main rivers and 13 tributary sites and eight sewage treatment plants (STPs). We concluded that the STP effluents were the major sources of the anti-influenza drug load in the Yodo River system (68-94% of total mass fluxes). Extended measurement throughout the Yodo River system further showed only small fluctuations of the ratio of OS to OC from 0.2 to 0.3, suggesting that OS and its metabolite are environmentally stable. The results also clearly showed the importance of reducing the levels of anti-influenza drugs in the water environment by reducing their emission at STPs.

Compliance to oseltamivir among two populations in Oxfordshire, United Kingdom affected by influenza A(H1N1)pdm09, November 2009--a waste water epidemiology study.

Antiviral provision remains the focus of many pandemic preparedness plans, however, there is considerable uncertainty regarding antiviral compliance rates. Here we employ a waste water epidemiology approach to estimate oseltamivir (Tamiflu®) compliance. Oseltamivir carboxylate (oseltamivir's active metabolite) was recovered from two waste water treatment plant (WWTP) catchments within the United Kingdom at the peak of the autumnal wave of the 2009 Influenza A (H1N1)pdm09 pandemic. Predictions of oseltamivir consumption from detected levels were compared with two sources of national government statistics to derive compliance rates. Scenario and sensitivity analysis indicated between 3-4 and 120-154 people were using oseltamivir during the study period in the two WWTP catchments and a compliance rate between 45-60%. With approximately half the collected antivirals going unused, there is a clear need to alter public health messages to improve compliance. We argue that a near real-time understanding of drug compliance at the scale of the waste water treatment plant (hundreds to millions of people) can potentially help public health messages become more timely, targeted, and demographically sensitive, while potentially leading to less mis- and un-used antiviral, less wastage and ultimately a more robust and efficacious pandemic preparedness plan.

Synchronous dynamics of observed and predicted values of anti-influenza drugs in environmental waters during a seasonal influenza outbreak.

Time-dependent dynamics in the concentrations of four anti-influenza drugs (oseltamivir, oseltamivir carboxylate, zanamivir, and amantadine) in environmental waters collected from the Yodo River basin, Japan, were monitored for the first time over a 1 year period (July 2010 to June 2011). The clear, convex dynamic profiles of oseltamivir, oseltamivir carboxylate, and zanamivir during a 3 month seasonal influenza outbreak (January to March 2011) were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage treatment plants (STPs) and river waters were, respectively, 177 and 60 ng/L (oseltamivir), 827 and 288 ng/L (oseltamivir carboxylate), and 30 and 15 ng/L (zanamivir). Fixed levels of amantadine were detectable year-round (100-200 ng/L in the STPs and 10-30 ng/L in river waters). The predicted convex profiles of oseltamivir, oseltamivir carboxylate, and zanamivir in both STPs and river waters were significantly correlated (0.714 < R < 0.932) with the observed values. The profiles were predicted successfully by simple mathematical principles, taking the number of influenza patients, quantities of Tamiflu and Relenza used, dilution by drainwaters passing through STPs, removal rates at STPs, dilution rates in river effluents, and attenuation rates in rivers into consideration.

Spectrofluorimetric determination of oseltamivir phosphate through derivatization with o-phthalaldehyde. Application to pharmaceutical preparations with a preliminary study on spiked plasma samples.

A simple and sensitive spectrofluorimetric method has been developed and validated for the determination of oseltamivir phosphate (OST) in pharmaceutical preparations. The method is based on the reaction between oseltamivir phosphate and o-phthalaldehyde in presence of 2-mercapto-ethanol in borate buffer, pH 10.8, to give a highly fluorescent product measured at 450 nm after excitation at 336 nm. The different experimental parameters affecting the development and stability of the reaction product were studied and optimized. The fluorescence intensity-concentration plot is rectilinear over the range 0.05-1.0 µg/mL, with a lower detection limit of 5 ng/mL and limit of quantitation of 16 ng/mL. The developed method was successfully applied to the analysis of the drug in its commercial capsules and suspension, mean recoveries of OST were 99.97 ± 1.67% and 100.17 ± 1.18%, respectively (n = 3). Statistical comparison of the results obtained by the proposed and comparison method revealed no significant difference in the performance of the two methods regarding accuracy and precision. The proposed method was further extended to in vitro determination of the studied drug in spiked human plasma as a preliminary investigation; the mean recovery (n = 3) was 98.68 ± 5.8%. A reaction pathway was postulated.

Removal of the antiviral agent oseltamivir and its biological activity by oxidative processes.

The antiviral agent oseltamivir acid (OA, the active metabolite of Tamiflu(®)) may occur at high concentrations in wastewater during pandemic influenza events. To eliminate OA and its antiviral activity from wastewater, ozonation and advanced oxidation processes were investigated. For circumneutral pH, kinetic measurements yielded second-order rate constants of 1.7 ± 0.1 × 10(5) and 4.7 ± 0.2 × 10(9) M(-1) s(-1) for the reaction of OA with ozone and hydroxyl radical, respectively. During the degradation of OA by both oxidants, the antiviral activity of the treated aqueous solutions was measured by inhibition of neuraminidase activity of two different viral strains. A transient, moderate (two-fold) increase in antiviral activity was observed in solutions treated up to a level of 50% OA transformation, while for higher degrees of transformation the activity corresponded to that caused exclusively by OA. OA was efficiently removed by ozonation in a wastewater treatment plant effluent, suggesting that ozonation can be applied to remove OA from wastewater.

Environmental release of oseltamivir from a Norwegian sewage treatment plant during the 2009 influenza A (H1N1) pandemic.

During the 2009 influenza type A(H1N1) pandemic, the antiviral drug oseltamivir (OP, Tamiflu®) was extensively used for treatment and prophylaxis after recommendation from World Health Organisation (WHO). Previous studies have indicated that the pharmaceutically active metabolite of OP, oseltamivir carboxylate (OC), is not readily degraded in sewage treatment plants (STPs) and therefore will be released into receiving waters in elevated concentrations during a pandemic outbreak of influenza. A method for analyzing OP and OC in wastewater by UPLC-TOF has been developed and validated. This analytical method has been used to study the release of OP and OC from a sewage treatment plant outside Oslo, Norway during the 2009 pandemic. Daily flow-proportional influent and effluent samples from 11 weeks covering the main wave of the influenza pandemic were analyzed, and the observed trend in OP and OC concentrations closely followed the trend in percentage of medical consultations caused by influenza-like illness. Concentrations in wastewater influent were in the range of 5-529 ng/L and 28-1213 ng/L for OP and OC, respectively. Concentration data from the 54 influent/effluent sample sets suggest STP removal in the range of -0.8% to 8% for OP and -14% to 0.6% for OC. Statistical analysis of the data sets was inconclusive in determining a removal rate different from 0.

Optimization of polymerization parameters for the sorption of oseltamivir onto molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) are tailor-made polymers with high selectivity for a given analyte, or group of structurally related compounds. The influence of the process parameters (the moles of functional monomer and cross-linker, the selection of functional monomer and solvent) on the preparation of oseltamivir (OS)-imprinted polymers was investigated. A mathematical method for uniform design to optimize these selected parameters and to increase the MIP selectivity for template molecules was applied. The optimal conditions to synthesize MIP were 0.69 mmol 30% acrylamide (AA) + 70% 4-Vinylpyridine (4-VP) and 5.0 mmol ethylene glycol dimethacrylate (EGDMA) copolymerized in 5 ml toluene in the presence of 0.1 mmol OS. MIP showed high affinity and selectivity for separation of the template molecule from other compounds. In the present study, we have established an effective LC-MS/MS method to identify and quantify OS with good sensitivity, accuracy and precision.

Photofate of oseltamivir (Tamiflu) and oseltamivir carboxylate under natural and simulated solar irradiation: kinetics, identification of the transformation products, and environmental occurrence.

In this work the photodegradation pathways and rates of oseltamivir ester (OE) and oseltamivir carboxylate (OC) were studied under artificial and natural solar irradiation with the goal of assessing the potential of photolysis as a removal mechanism in aquatic environments. The structures of the photoproducts of OE, elucidated by ultra performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-QToF-MS), were proposed to originate from hydration of the cyclohexene ring (TP330), ester hydrolysis (TP284), a combination thereof (TP302), intramolecular cyclization involving the ester (TP312), and cleavage of the ethylpropoxy side chain (TP226). The only photoproduct detected in case of OC was postulated to correspond to the hydration of the α,ß-unsaturated acid (TP302). Under simulated solar irradiation the degradation rate of OC was approximately 10 times slower than that of OE, with half-lives ranging from 48 h in ultrapure water to 12 h in surface water from Sant Joan Despi, Llobregat river. The photodegradation under natural solar irradiation during the season of pandemic Influenza peak incidence was about 150 days for OC and 15 days for OE. In general, the photoproducts proved to be more resistant toward further photodegradation than the parent antivirals. In a monitoring survey of surface waters from the Ebro river (NE Spain), OC and OE were detected along with the photoproducts TP330 and 312.

Pandemic pharmaceutical dosing effects on wastewater treatment: no adaptation of activated sludge bacteria to degrade the antiviral drug oseltamivir (Tamiflu®) and loss of nutrient removal performance.

The 2009-2010 influenza pandemic saw many people treated with antivirals and antibiotics. High proportions of both classes of drugs are excreted and enter wastewater treatment plants (WWTPs) in biologically active forms. To date, there has been no study into the potential for influenza pandemic-scale pharmaceutical use to disrupt WWTP function. Furthermore, there is currently little indication as to whether WWTP microbial consortia can degrade antiviral neuraminidase inhibitors when exposed to pandemic-scale doses. In this study, we exposed an aerobic granular sludge sequencing batch reactor, operated for enhanced biological phosphorus removal (EBPR), to a simulated influenza-pandemic dosing of antibiotics and antivirals for 8 weeks. We monitored the removal of the active form of Tamiflu(®), oseltamivir carboxylate (OC), bacterial community structure, granule structure and changes in EBPR and nitrification performance. There was little removal of OC by sludge and no evidence that the activated sludge community adapted to degrade OC. There was evidence of changes to the bacterial community structure and disruption to EBPR and nitrification during and after high-OC dosing. This work highlights the potential for the antiviral contamination of receiving waters and indicates the risk of destabilizing WWTP microbial consortia as a result of high concentrations of bioactive pharmaceuticals during an influenza pandemic.

Occurrence and fate of oseltamivir carboxylate (Tamiflu) and amantadine in sewage treatment plants.

We investigated the occurrence and fate of the two antiviral drugs oseltamivir carboxylate (OC)-the active metabolite of Tamiflu-and amantadine (AMT) at three sewage treatment plants (STPs) during the 2008-2009 and 2009-2010 influenza seasons in Japan. Both compounds were detected in all samples analyzed. The concentrations in raw influents at the STPs ranged from 140 to 460 ng L(-1) OC and from 184 to 538 ng L(-1) AMT. Primary treatment gave no substantial removal of the drugs (OC, 2-9%; AMT, 7-17%). Biological nutrient-removal-based secondary treatment (anoxic-oxic-anoxic-oxic and anaerobic-anoxic-oxic) removed 20-37% of OC, whereas extended-aeration-based conventional activated sludge treatment removed <20%. STPs using primary plus biological secondary treatment removed <50% of the drugs. The incorporation of tertiary treatment by ozonation removed >90%. Ozonation after secondary treatment in STPs will be necessary during an influenza pandemic to reduce the risks associated with the widespread use of antiviral drugs.

Removal of oseltamivir (Tamiflu) and other selected pharmaceuticals from wastewater using a granular bioplastic formulation entrapping propagules of Phanerochaete chrysosporium.

The capacity of the ligninolytic fungus Phanerochaete chrysosporium to degrade a wide variety of environmentally persistent xenobiotics has been largely reported in the literature. Beside other factors, one barrier to a wider use of this bioremediation fungus is the availability of effective formulations that ensure easy preparation, handling and application. In this series of laboratory experiments, we evaluated the efficiency of a granular bioplastic formulation entrapping propagules of P. chrysosporium for removal of four selected pharmaceuticals from wastewater samples. Addition of inoculated granules to samples of the wastewater treatment plant of Bologna significantly increased the removal of the antiviral drug oseltamivir (Tamiflu), and the antibiotics, erythromycin, sulfamethoxazol, and ciprofloxacin. Similar effects were also observed in effluent water. Oseltamivir was the most persistent of the four active substances. After 30d of incubation, approximately two times more oseltamivir was removed in bioremediated wastewater than controls. The highest removal efficiency of the bioplastic formulation was observed with the antibiotic ciprofloxacin. Microbiological DNA-based analysis showed that the bioplastic matrix supported the growth of P. chrysosporium, thus facilitating its adaptation to unusual environment such as wastewater.

Dissipation and removal of oseltamivir (Tamiflu) in different aquatic environments.

The antiviral drug oseltamivir (Tamiflu) has received recent attention due to the potential use as a first-line defense against H5N1 and H1N1 influenza viruses. Research has shown that oseltamivir is not removed during conventional wastewater treatments, thus having the potential to enter surface water bodies. A series of laboratory experiments investigated the fate and the removal of oseltamivir in two surface water ecosystems of Japan and in a municipal wastewater treatment plant located in Northern Italy. Persistence of oseltamivir in surface water ranged from non-detectable degradation to a half-life of 53d. After 40d, <3% of radiolabeled oseltamivir evolved as (14)CO(2). The presence of sediments (5%) led to a significant increase of oseltamivir degradation and mineralization rates. A more intense mineralization was observed in samples of the wastewater treatment plant when applying a long incubation period (40d). More precisely, 76% and 37% of the initial radioactivity applied as (14)C-oseltamivir was recovered as (14)CO(2) from samples of the biological tank and effluent water, respectively. Two bacterial strains growing on oseltamivir as sole carbon source were isolated and used for its removal from synthetic medium and environmental samples, including surface water and wastewater. Inoculation of water and wastewater samples with the two oseltamivir-degrading strains showed that mineralization of oseltamivir was significantly higher in both inoculated water and wastewater, than in uninoculated controls. Denaturing gradient gel electrophoresis and quantitative PCR analysis showed that Tamiflu would not affect the microbial population of surface water and wastewater.