DSpace at VNU: Screening determination of pharmaceutical pollutants in different water matrices using dual-channel capillary electrophoresis coupled with contactless conductivity detection

Author’s Accepted Manuscript Screening determination of pharmaceutical pollutants in different water matrices using dualchannel capillary electrophoresis coupled with contactless conductivity detection Minh Duc Le, Hong Anh Duong, Manh Huy Nguyen, Jorge Sáiz, Hung Viet Pham, Thanh Duc Mai PII: DOI: Reference: www.elsevier.com/locate/talanta S0039-9140(16)30524-0 http://dx.doi.org/10.1016/j.talanta.2016.07.032 TAL16729 To appear in: Talanta Received date: May 2016 Revised date: 11 July 2016 Accepted date: 12 July 2016 Cite this article as: Minh Duc Le, Hong Anh Duong, Manh Huy Nguyen, Jorge Sáiz, Hung Viet Pham and Thanh Duc Mai, Screening determination of pharmaceutical pollutants in different water matrices using dual-channel capillary electrophoresis coupled with contactless conductivity detection, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.07.032 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 galley proof before it is published in its final citable form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain -1Screening determination of pharmaceutical pollutants in different water matrices using dualchannel capillary electrophoresis coupled with contactless conductivity detection Minh Duc Le1, Hong Anh Duong1, Manh Huy Nguyen 1, Jorge Sáiz2, Hung Viet Pham1*, Thanh Duc Mai1 * Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Nguyen Trai Street 334, Hanoi, Viet Nam Institute of General Organic Chemistry (IQOG), Spanish National Research Council (CSIC) Calle Juan de la Cierva, 3, 28006 Madrid, Spain e-mail: maithanhduc83@gmail.com; Tel : ++33 651 37 79 49; phamhungviet@hus.edu.vn ; Fax: +84 3858 8152 website: www.CE-Vietnam.com Keywords: capacitively coupled contactless conductivity detection (C4D); capillary electrophoresis (CE); dual-channel CE; water analysis,;pharmaceutically active compounds (PhACs); water samples -2Abstract In this study, the employment of purpose-made dual-channel compact capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C4D) as a simple and inexpensive solution for screening determination of various pharmaceutical pollutants frequently occurring in surface water and hospital wastewater in Hanoi, Vietnam is reported Five negatively charged pharmaceutically active compounds, namely ibuprofen, diclofenac, bezafibrate, ketoprofen and mefenamic acid were determined using the first channel whereas three positively charged ones, namely diphenhydramine, metoprolol and atenolol were determined with the second channel of the CE-C4D instrument Two different background electrolytes (BGEs) were used in these two CE channels independently The best detection limits achieved were in the range of 0.2 - 0.8 mg/L without sample pre-concentration Enrichment factors up to 200 were obtainable with the inclusion of a solid phase extraction step Good agreement between results obtained from CE-C4D and those with the standard confirmation method (HPLC-DAD) was achieved, with correlation coefficients higher than 0.98 -31 Introduction Many pharmaceutically active compounds are classified as environmental contaminants due to their low biodegradability and their potential to cause undesirable ecological and human health effects [1-5] While having positive effects on the treatment of various pathologies and diseases, the use of pharmaceutical products results in the contamination of the aquatic environment, mainly through municipal and hospital effluents [6-8] The occurrence of a large number of pharmaceutical contaminants in the environment has been reported for different developed countries (see some recent examples in [7, 9-12]) In emerging countries, although the situation is often worse, only limited information is available as the regulations for environmental protection are not well established In Vietnam, the first study on pharmaceutically active compounds contamination was recently reported by Tran et al., focusing on hospital wastewater and surface water in Hanoi [13] Water contamination in Hanoi caused by direct discharge of domestic and industrial wastewater to rivers and lakes poses a big threat to public health This leads to an urgent need for regular control of these contaminants in different water sources So far, various analytical methods for determination of pharmaceutical contaminants in different environmental matrices have been proposed [14, 15] Among all techniques, high performance liquid chromatography (HPLC) is the most common due to its high degree of confidence, reliability and reproducibility The utilization of HPLC nevertheless requires long analysis time, a large amount of costly HPLC-grade solvents as mobile phases as well as high setup and maintenance costs (especially for the high pressure pumps and accessories) In Vietnam, only a few central environmental monitoring agencies or companies with abundant funding and sufficient expertise can afford the installation, long-term operation and maintenance of such instruments On the other hand, capillary electrophoresis (CE), relying -4on a high voltage rather than high pressure for driving the analytes in a micro separation channel, offers a more economic and higher-throughput alternative The determination of pharmaceutical residues in water with CE using UV or mass-spectrometric detection has been reported repeatedly (see refs [16-22] for example) Three additional positive features of CE that make it even more suitable for screening analysis purposes are portability for mobile deployment (see [23-26] and references listed therein), customer-oriented CE configuration for adaptation to different financial and expertise situations [27, 28] and the possibility of using dual-channel setup for the concurrent determination of positively and negatively charged analytes [29-31] The portability and the use of more than one channel were enabled when CE was used together with capacitively coupled contactless conductivity detectors (C4D) In these detectors, the difference between the conductivities of the analytes and that of the background electrolyte (BGE) can be measured by employing a pair of tubular electrodes fitted around the capillary wall Some notable advantageous features of C4D include high versatility, ease in construction and operation, low power consumption and the potential of miniaturization (for more details see [32, 33] and the references listed therein) CE - C4D has been repeatedly used by Richter et al for determinations of active ingredients together with their counter ions or degradation products in pharmaceutical formulations [34-39] Simultaneous determination of cations and anions in these pharmaceutical applications were implemented through the use of BGEs with pH above to produce high magnitudes of electro-osmotic flow (EOF) in fused silica capillaries for sweeping the anions with low and opposite electrophoretic mobilities towards the detector.In environmental applications, the employment of CE-C4D has been communicated for separation of negatively and positively charged pharmaceutical contaminants in standard solutions [40] and for tracing of some negatively charged ones in a -5hospital wastewater sample [41] Only one single CE channel was used and no cross check with another well established analytical method was realized in both cases Herein we report a straightforward and cost-effective method based on purpose-made dualchannel compact CE-C4D for concurrent screening determination of different positively charged pharmaceutical residues (diphenhydramine, metoprolol and atenolol) and negatively charged ones (ibuprofen, diclofenac, bezafibrate, ketoprofen and mefenamic acid) in various water matrices in Hanoi, Vietnam Compared to the recently reported dual-channel CE instruments that share a common BGE for both channels [31, 42-44], the system reported herein allows the employment of two different BGEs, allowing the separations of analytes belonging to different categories Experimental 2.1 Chemicals and Materials All chemicals were of analytical or reagent grade and purchased from Fluka (Buchs, Switzerland) or Merck (Darmstadt, Germany) Stock solutions (1 mM) of diphenhydramine, metoprolol, atenolol, ibuprofen, diclofenac, ketoprofen, bezafibrate and mefenamic were used for the daily preparation of the standard solutions Chemicals used for the preparation of BGEs included: Acetic acid (Ace), histidine (His), 2-(N-morpholino)ethanesulfonic acid (MES), lactic acid (Lac), tris(hydroxymethyl)aminomethane (Tris), 3-(Nmorpholino)propanesulfonic acid (MOPS) and hydroxypropyl-beta-cyclodextrin (HP-β-CD) Fused silica capillaries of 50 µm ID and 365 µm OD were obtained from Polymicro Technologies (Phoenix, AZ, USA) Prior to their use, the capillaries were pre-conditioned with M NaOH for 10 and de-ionized water for 10 min, followed by flushing with the BGE The capillaries were then used continuously for successive separations De-ionized -6water, purified using a water purification system from Millipore - model Simplicity UV (Bedford, MA, USA), was used for the preparation of all standard solutions and for sample dilution if required Commercial solid phase extraction (SPE) cartridges, including i) LiChrolut RP-18 with the cartridge volume of mL containing 500 mg sorbents (Merck) and ii) hydrophilic modified, styrene-based polymer (hydrophilic lipophilic balance, HLB) SPE cartridges (200 mg sorbents per cartridge, 30 µm particle size, Waters Corporation), as well as the SPE vacuum manifold (Visiprep 5-7030, Sulpelco) were used for sample treatment and pre-concentration Cross-checking was carried out using an HPLC instrument (LC-20AB) equipped with a UV-VIS-based diode array detector (DAD) from Shimadzu Corp (Japan) 2.2 Instrumentation All experiments were performed on a purpose-made dual-channel CE instrument The high voltage (HV) modules (DX250 series) capable of providing up to 25 kV were obtained from EMCO (Sutter Creek, CA) The HV end of the capillary was isolated with a safety cage made from Perspex, which was equipped with a microswitch to interrupt the HV on opening The miniature membrane pumps (NF-5-DCB) for sample aspiration were purchased from KNF (Balterswil, Switzerland) Micro-graduated needle valves were obtained from IDEX (P-470, Oak Harbor, WA) and solenoid valves from NResearch (product nos 116T021 and 116T031, West Caldwell, NJ) All fluidic connections were made with 0.02" I.D and 1/16" O.D Teflon tubing and with polyether ether ketone (PEEK) flangeless nuts and ferrules 10-32 or ¼-28 UNF (IDEX) The injection interface that accommodates the grounded end of the capillary and the ground electrode was machined from a Perspex block (2 cm × cm × cm) Detection was carried out with in-house built miniature HV - C4D according to a design reported previously [43, 45].The resulting signals were recorded with an ADC-20 data acquisition system (Pico Technology, St Neots, UK) connected to the USB-port of a personal -7computer A lithium battery pack of 14.8 V (CGR 18650CG 4S3P, Contrel, Hünenberg, Switzerland) and a separate pair of smaller Li-ion batteries (CGR 18659CG 4S1P, Contrel) fitted with 12 V regulators of appropriate polarities were used for powering the CE-C4D system Mains power can be utilized whenever available 2.3 Field sampling The sampling sites are located in Hanoi - the capital of Vietnam (see details in Fig S1) Surface water samples were collected from the Nhuệ river (SN1, SN2 and SN3), Tô Lịch river (TL1 - TL5), Lừ river (SL1), Sét river (SS1), Kim Ngưu river (KN1), the lake near Hanoi Medical University (YHN) and West Lake (HT2) The distances between the sampling sites from the same river or lake were at least 500 m The samples from these rivers and lakes were collected near the municipal discharges at the distance of m from the borders and at the depth of 20 - 30 cm below the surface Untreated and treated wastewater samples were collected from the influents and effluents of wastewater treatment plants of the central pharmaceutical manufactory (TW1 and TW2, respectively) and the VCP pharmaceutical manufactory (VCP1 and VCP2, respectively) Water samples from discharges to the receiving points in residential zones were also collected These samples came from a wastewater discharge (NCT2), Vietnam sports hospital (TT1) and Vietnam national hospital of pediatrics (NH) Totally 20 samples representative for different surface water bodies in Hanoi that may be contaminated by pharmaceutical pollutants were collected for analyses Water samples were firstly filtered with 0.45 µm membrane filters (Sartorius, Gưttingen, Germany), then collected in amber glass bottles and stored at °C (up to one week) The collected water samples, especially wastewater samples may contain microorganisms whose activity can lead to modification of the concentrations of pharmaceutical pollutants via microbial degradation of pharmaceutical compounds [46] The samples therefore were stored at 4°C rather than at -8room temperature in order to inhibit / minimize the activity of microorganisms Operations with CE-C4D and HPLC were carried out immediately upon conclusion of the sampling campaign 2.4 Analytical procedures For pre-concentration of negatively charged pharmaceutical pollutants (i.e ibuprofen, diclofenac, bezafibrate, ketoprofen and mefenamic acid), the surface water and wastewater samples were passed through RP-C18 extraction cartridges at a flow rate of - mL / Before sample loading, SPE cartridges were conditioned with mL acetonitrile, mL deionized water, mL buffer composed of mM Tris adjusted to pH with lactic acid and finally ml water Samples or standard solutions were first acidified to pH - in order to preserve the target pharmaceutical compounds in the protonated forms and then loaded through the SPE cartridges The loading volumes were adjusted from 200 to 900 mL depending on the sample matrices Upon completion of the extraction, the SPE cartridges were flushed with mL buffer of 9mM Tris / Lactic acid (pH 8) and vacuum dried for 30 Elution was then implemented with mL eluent composed of mM Tris / lactic acid, pH (40 % by volume) and acetonitrile (60 % by volume) The pre-concentrated samples were subsequently analyzed with CE-C4D and with HPLC- DAD for cross-checking For pre-concentration of positively charged pharmaceutical pollutants (i.e diphenhydramine, metoprolol and atenolol), the samples or standard solutions were acidified to pH with hydrochloric acid (8 M) The acidified sample solution (200 mL) was then passed at a flow rate of 1drop / second through a HLB SPE column that had been conditioned with mL of methanol and mL of deionized water The HLB SPE column was then flushed with deionized water and air-dried for before elution was carried out with 10 mL of formic -9acid 0.1 % (v/v) in methanol The eluent was subsequently dried with nitrogen and filled to mL with a 2.5 mM His / 2.5 mM MOPS (pH 6.5) solution Shaking was needed for complete dissolution of the eluted analytes in this His / MOPS medium For CE-C4D separation of negatively charged pharmaceuticals, a high voltage of 15 kV was applied over a capillary with 75 cm total length (Lt) and 65 cm effective length (Leff), The optimal BGE composition found was composed of 36 mM Tris and 0.5 mM HP-β-CD adjusted to pH with lactic acid As for the positively charged species, the CE-C4D analyses were carried out using the optimized BGE composed of 25mM His, 25 mM MOPS and 1mM HP-β-CD (pH 6.5) A high voltage of 15 kV was applied over a capillary with 80 cm Lt and 70 cm Leff The HPLC - DAD procedure (detection at 260 nm) for cross-checking was developed according to a protocol reported elsewhere [47] Results and Discussion 3.1 Dual-channel CE setup using individual BGEs for analytical throughput improvement As numerous medicinal compounds can be present in the aqueous environment [10, 12, 13], it is desirable to develop a screening method that allows simultaneous determination of a wide range of analytes in one single run In the first work on determination of pharmaceutical contaminants with CE-C4D, 13 pharmaceutical compounds in both anionic and cationic forms in standard solutions (artificial samples) were successfully separated [40] The satisfactory separation resolution for these standards, with the cationic species being incompletely separated, however could not be achieved with real samples having complex matrices, at least in our hands To our experience, separating many species having close electrophoretic mobilities in a single CE profile is not always the best approach for increasing analysis 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-CD (pH 6.5); voltage: 15kV; capillary: uncoated fused-silica, 50 µm id, Lt = 80 cm (Leff = 70 cm) Correlation of Range Analyte LODb (mg RSDn=4 % RSDn=4 % / L) (peak area) (migration time) determination (mg / L)a r Ibuprofen 1-20 0.995 0.20 1.96 0.43 Bezafibrate 1-20 0.997 0.29 2.74 0.39 Mefenamic 1.5-10 0.998 0.44 5.29 0.94 Ketoprofen 1.5-10 0.997 0.41 4.83 0.73 Diclofenac 1.5-20 0.998 0.39 4.53 0.59 Diphenhydramine 2.5-50 0.997 0.81 5.51 0.98 Metoprolol 2.0-50 0.56 2.39 0.75 Atenolol 1-50 0.999 0.29 2.16 0.44 a concentrations b Based on peak heights corresponding to times the baseline noise -28Table Concentrations of pharmaceutical pollutants in different water matrices determined with CE-C4D and cross-checked with HPLC-DAD Ibuprofen Sample CE HPLC (µg/L) (µg/L) SN1 < 2.0