Optimizing a Generic Approach to Analyzing PPCPs in River Water
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1 ptimizing a Generic Approach to Analyzing PPCPs in River Kelly Munro, 1 Anthony Edge, 2 Claudia Martins, 3 David Cowan 4 and Leon Barron 1 1 Analytical & Environmental Sciences Division, King s College London, London, UK, 2 Thermo Fisher Scientific, Tudor Road, Runcorn, UK, 3 Thermo Fisher Scientific, Villebon, France, 4 Drug Control Centre, King s College London, London, UK
2 Results & D The occurrence of pharmaceutically-related contaminants within the environment continues to be a research area which generates great interest. The full environmental effects of chronic exposure of such pollutants have yet to be fully understood. As such more knowledge is sought on the presence of these contaminants within the environment. Traditionally, a targeted multi-residue analytical approach is applied to the analysis of environmental waters. A consequence of this can be a somewhat limited estimation of the true breadth of occurrence of pharmaceutically-related drug residues within such waters. Recent advances have seen non-targeted methods proposed as valid alternatives to the traditional approach. 1. SPE Method The recoveries of c sample volumes. F compounds were o sorbent with a 1 FIGURE 2. Absolu mode cation exch A semi-targeted analytical approach is presented herein for the detection of a range of over-the-counter, prescribed and illicit drugs in environmental waters using mixed mode solid phase extraction (SPE) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The potential to perform retrospective non-target analysis is also presented. FIGURE 1. Schematic showing developed semi-targeted analytical approach. Sample Collection 1 ml ph 2 adjustment Filtration, GF/F.7 µm Condition: 4 ml MeH, 4 ml Wash: 4 ml 5%MeH in Elution: 4 ml MeH SPE HyperSep Retain PEP 2 mg Evaporated to dryness at C under nitrogen Reconstituted in 1 μl of mobile phase A 1 5 Salicylic Acid A broad analytical screening method, shown in Figure 1, was developed using a selection of structurally diverse species which represented a variety of compounds classes, functional groups, pka and log P values as well as reported environmental occurrences. % A bsolut e Recovery Experimental 15 Introduction 2. ʻSemi-Target The developed ana influent wastewate reference standard FIGURE 3. Cocain BP: Base Peak ac LC -HRMS 1 LC-HRMS was performed using a Thermo Scientific Q Exactive system (Thermo Fisher Scientific, Bremen, Germany), and the chromatographic and MS conditions are detailed in Table Table 1. Chromatographic and MS conditions used. 2 Thermo Scientific Accucore Column C µm (15 x 2.1 mm) ptimizing a Generic Approach to Analyzing PPCPs in River Mobile Phase A: 9:1 + 1 mm Ammonium Acetate : % Relative Abunda 1 5
3 detailed in Table Table 1. Chromatographic and MS conditions used. LC % Relative Abunda Thermo Scientific Accucore Column C µm (15 x 2.1 mm) Mobile Phase A: 9:1 + 1 mm Ammonium Acetate : Acetonitrile B: 2:8 + 1 mm Ammonium Acetate : Acetonitrile Injection volume 2 μl Fragmentation Mode 4 μl/min 3 +ve. 4.5 kv +ve V -ve. 3 kv -ve V +ve. 1 V -ve. 1 V 5, FWHM m/z: 1-1 1,, 1 ms HCD (2eV) Week-long qualitat targeted compound quantitative analys FIGURE 4. Weekly 1E+7 1E+6 HRMS Flow Rate Capillary Temp ( C) Heater Temp ( C) Spray Voltage Capillary Voltage Tube Lens Voltage Resolution Scan Range AGC Target Max. Inject Time 1E+5 1E+4 1E+3 Thermo Scientific Poster ote P296_HPLC_214_E_5/14S 3 W ed
4 Results & Discussion the analysis of ed estimation of the within such waters. alternatives to the 1. SPE Method Development FIGURE 5. We The recoveries of compounds were evaluated for two different SPE sorbents using different sample volumes. Figure 2 shows that optimized absolute recoveries for the majority of compounds were obtained using the Retain PEP-functionalized polystyrene-divinylbenzene sorbent with a 1 ml sample when adjusted to ph 2. 1E+8 1E+7 FIGURE 2. Absolute recoveries obtained using a PS-DVB sorbent (PEP) and a mixed mode cation exchange sorbent (CX) with a sample adjusted to ph 2. ion of a range of sing mixed mode n mass arget analysis is 1mL PEP 15 1L PEP 1mL CX 1L CX nvironment ll environmental rstood. As such in the Levels of the m fluctuations bei been seen that 1E+6 1E Mephedro T emazepam Ketamine Propranolol Carbamazepine C ompound I buprofen Diclofenac Cocaine W arfarin Bezafibrate Ketoprofen T rimethoprim m Clofibric Acid Salicylic Acid SM Z Using the abov methylmethcat SM X al approach. 1 d using a selection mpounds classes, ntal occurrences. % A bsolut e Recovery 1E+5 2. ʻSemi-Targetedʼ Screening of Real Samples s at C under μl of mobile The developed analytical method was applied to the analysis of both Thames river water and influent wastewater. The presence of an analyte was confirmed by comparison with a reference standard. As and example the presence of cocaine is shown in Figure 3. FIGURE 3. Cocaine confirmation. tr: retention time; AA: Peak Area; AH: Peak Height; BP: Base Peak accurate mass. 1 ystem (Thermo S conditions are t R : 8.96 AA: AH: BP: olumn C18 % Relative Abundance 1 t R : 8.95 AA: AH: BP: % Relative Ab tr : AA: 24 AH: 1 5 BP: Fragment in tr : 6.9 AA: 11 AH: 1 BP: 17 1 t R : 9.9 AA: AH: BP: nium Acetate : µg/l 5 5 FIGURE 6. Ch within river wa 5 m/z River Sample t R : ptimizing a Generic Approach to Analyzing PPCPs in River AA: AH: Figure 6 shows
5 s at C under μl of mobile influent wastewater. The presence of an analyte was confirmed by comparison with a reference standard. As and example the presence of cocaine is shown in Figure 3. FIGURE 3. Cocaine confirmation. tr: retention time; AA: Peak Area; AH: Peak Height; BP: Base Peak accurate mass. t R : 8.96 AA: AH: BP: stem (Thermo conditions are t R : 8.95 AA: AH: BP: Fragment in tr : 6.9 AA: 116 AH: 1 BP: t R : 9.9 AA: AH: BP: % Relative Abundance 1 5 t R : 9.8 AA: AH: BP: m/z River Sample 5 nium Acetate : 5 % Relative Abu tr : AA: 24 AH: 1 5 BP: nium Acetate : 1 olumn C µg/l m/z Fragment Confirmation in River Time (min) Week-long qualitative studies of both river water and influent showed that the majority of the targeted compounds were present. This is shown in Figures 4 and 5 respectively. A quantitative analysis is now in preparation. FIGURE 4. Weekly variation of identified compounds in Thames river water. 1E+7 1E+6 Figure 6 shows 8.6 min which h a blank sample whereas the un the river water. ions (m/z 16.1 retention time to optimization of s method in a qua determined bas Conclusion A developed se medicinal and il target retrospec mephedrone wi Bezafibrate Carbamazepine Cocaine 1E+5 Propranolol T emazepam 1E+4 T rimethoprim W arfarin 1E+3 W ed T hurs Fri Sat Sun M on 214 Thermo Fish Fisher Scientific and This information is n the intellectual prop T ues Thermo Scientific Poster ote P296_HPLC_214_E_5/14S 5
6 Levels of the majority of compounds remain consistent across the week, with the biggest fluctuations being observed in river water, in particular for cocaine and diazepam. It can also been seen that levels were approximately ten fold higher in influent for several compounds. FIGURE 5. Weekly variation of identified compounds in influent wastewater. ents using different he majority of ene-divinylbenzene 1E+8 1E+7 X 1L CX PEP) and a mixed. Bezafibrate Carbamazepine Cocaine 1E+6 Ketamine Propranolol 1E+5 T emazepam T rimethoprim 1E+4 W ed T hurs Fri Sat Sun M on T ues 3. Mephedrone in the Environment Using the above approach, it was also possible to identify the illicit drug, mephedrone (4methylmethcathinone) in both river and wastewater. 1 ames river water and arison with a n Figure 3. AH: Peak Height; liq 3 FIGURE 6. Chromatograms indicating the presence of the illicit drug mephedrone within river water and influent wastewater H 1 µg/l tr : 6.8 AA: AH: tr : 8.6 AA: AH: tr : 6.75 AA: AH: m/z River Spike tr : 8.62 AA: % Relative AbundanceAH: 5852 tr : AA: AH: BP: tr : 8.1 AA: AH: tr : 6.99 AA: AH: ptimizing a Generic Approach to Analyzing PPCPs in River 5 1 River Blank Influent Blank Confirmed with m/z 16 and 145 fragments 15 2 Time (min) 25 3
7 Using the above approach, it was also possible to identify the illicit drug, mephedrone (4methylmethcathinone) in both river and wastewater. FIGURE 6. Chromatograms indicating the presence of the illicit drug mephedrone within river water and influent wastewater. tr : 6.75 AA: AH: tr : 6.8 AA: AH: tr : 8.6 AA: AH: AH: Peak Height; 1 µg/l 5 mes river water and arison with a Figure H m/z River Spike tr : 8.62 AA: % Relative AbundanceAH: 5852 tr : AA: AH: BP: iq tr : 8.1 AA: AH: Influent Blank tr : 6.99 AA: AH: River Blank 5 Confirmed with m/z 16 and 145 fragments Time (min) 25 3 t the majority of the ectively. A Figure 6 shows the presence of mephedrone in river water, along with an unknown peak at 8.6 min which has the same accurate mass as mephedrone. Comparing a spiked sample with a blank sample it is clear that the intensity of the mephedrone peak increases accordingly whereas the unknown peak stays constant, confirming the presence of mephedrone within the river water. Mephedrone was also detected in influent water, with confirmatory fragment ions (m/z and ). Again, an unknown peak was present at a similar retention time to that observed in river water. Therefore, this shows that even with HRMS, the optimization of separation conditions is still very important. ngoing efforts aim to apply this method in a quantitative analysis of both sample types once a complete analyte list is determined based on actual occurrence data. r water. Conclusion yline ate azepine m yline olol pam oprim A developed semi-targeted analytical method was used to confirm the presence of several medicinal and illicit species in both river water and influent wastewater. The potential of nontarget retrospective analysis was also highlighted with the detection of the illegal drug mephedrone within environmental waters Thermo Fisher Scientific Inc. All rights reserved. IS is a trademark of the International s rganization. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. ot all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientific, Sunnyvale, CA USA is IS 91:28 Certified. Africa Scientific 62 6 Japan All trademarks Russia/CISare +43the of Thermo 214Denmark Thermo+45 Fisher Inc. All rights reserved. property Australia Fisher Europe-ther +43its Korea Singapore Scientific and subsidiaries. Austria Finland Latin America Sweden Belgium This information France +33 is Middle East products Switzerland might infringe not92intended to encourage use1 333 of these in any+41 manners that Brazil Germany of others. etherlands Taiwan the intellectual property rights Canada India ew Zealand UK/Ireland China (free call domestic) Italy orway USA P296_E 5/14S
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