- Email: [email protected]
Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry
G Model CHROMA-358884; No. of Pages 8
ARTICLE IN PRESS Journal of Chromatography A, xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Full length article
Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry S. Poirier Larabie, M. Houde, C. Gagnon ∗ Aquatic Contaminants Research Division, Science and Water Technology Directorate, Environment and Climate Change Canada, 105 McGill Street, Montréal, Québec, H2Y 2E7, Canada
a r t i c l e
i n f o
Article history: Received 17 March 2017 Received in revised form 21 September 2017 Accepted 23 September 2017 Available online xxx Keywords: Fish Plasma Pharmaceuticals LC–MS/MS Surface waters Environment
a b s t r a c t Aquatic systems near major urban centers are constantly contaminated with effluent from wastewater treatment plants. Pharmaceuticals are part of the contamination and several classes of drugs have been detected in surface waters in the last decade. To better understand the impact of those pharmaceuticals in ecosystems, the exposure to aquatic species needs to be investigated. This study presents a new simple and rugged quantitative method for the determination of several classes of drugs using 100 L of plasma from fish environmentally exposed to a major but highly diluted urban effluent. Six common drugs (i.e., diclofenac, ibuprofen, naproxen, salbutamol, sulfamethoxazole and trimethoprim) and one major metabolite (2-hydroxy-ibuprofen), present in significant amount in impacted waterways have been selected for the development and validation of the method. First, all drugs were extracted using cation exchange solid phase extraction (SPE) and eluted with two solvent mixtures. Then, the extracts ® were analyzed using a reverse-phase analytical column Waters CORTECS C18 + (150 × 2.1 mm, 2.7 m) within 14 min. MS/MS was performed with an electrospray (ESI) interface in positive ion mode, with multiple reaction monitoring (MRM) experiment acquiring two product ions per drugs. Quantification has been made with standard curves for each analyte using isotopically labeled internal standards. This method has high sensitivity with limits of quantification of 1 ng mL−1 for each drug, except for ibuprofen and its metabolite 2-hydroxy-ibuprofen at 2 ng mL−1 . The precision of the method was below 11%, the accuracy between 94 and 105% and overall recovery between 94 and 111% for all drugs, with high selectivity. Application of the method to plasma samples from wild northern pike inhabiting the St. Lawrence River collected over a three-year period showed the presence of naproxen, diclofenac, trimethoprim and salbutamol at very low concentrations (around 1 ng mL−1 ). Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved.
1. Introduction Pharmaceuticals consumed by humans are continuously discharged in aquatic environments through urban effluents. Several classes of pharmaceuticals such as antibiotics, non-steroidal anti-inflammatory drugs, -blockers, anti-psychotics, cholesterol lowering drugs, anticonvulsants, and hormones have indeed been widely detected in surface waters near major cities around the world (e.g., Montreal, QC, Canada; Buffalo, TX, U.S.; Danube river basin, Romanian territory; KwaZulu-Natal, South Africa) [1–6]. The presence of pharmaceuticals in municipal wastewater effluents and their potential impacts on aquatic ecosystems are of growing con-
∗ Corresponding author. E-mail address: [email protected] (C. Gagnon).
cerns [7]. Aquatic organisms are indeed chronically exposed to these drugs in their natural habitats [8] and absorption pathways are multiple [9]. The absorption through gills in fish allow the direct passage of pharmaceutical in blood, bypassing the digestive system. This suggests that pharmaceuticals could be found in their original form in fish blood. This absorption can vary depending on physico-chemical factors of the surrounding water, mainly pH, thus affecting the bioavailability of pharmaceuticals [2,10]. Fish plasma concentrations may also vary depending on the pharmaceutical type, its ionization and its clearance [11]. Ionized molecules usually have a lower bioconcentration potential than neutral molecules [12]. In addition, pharmaceuticals are relatively polar and mostly ionized molecules in environmental media, in contrast to non-polar contaminants (e.g., polychlorinated biphenyls, dioxins and furans), which could lead to their circulation in aqueous body fluids rather than accumulation in more lipid-rich organs. Plasma is therefore an
https://doi.org/10.1016/j.chroma.2017.09.055 0021-9673/Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model CHROMA-358884; No. of Pages 8 2
ARTICLE IN PRESS S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
indicated medium to investigate the bioavailability of pharmaceuticals as it is the carrier of the majority of drugs to their target tissues and organs (muscles, brain, liver, kidney) [9]. Moreover, human pharmaceuticals are synthetized to reach specific target tissues that can be found in fish as well [11,13]. Juvenile rainbow trout have been experimentally exposed in Sweden for different periods of time (12 days, 13 days and 6 weeks) to aerated undiluted treated effluents from three wastewater treatment plants (WWTP). Plasma concentration of pharmaceuticals in these rainbow trout ranged from 2.2 to 20 ng mL−1 for diclofenac, 5.5 to 102 ng mL−1 for ibuprofen, and 33 to 46 ng mL−1 for naproxen [14]. In Texas, levels of several pharmaceuticals in four rivers highly impacted by urban effluents were determined as well as in different fish species [2]. The authors found the accumulation of the lipid regulator gemfibrozil, the antihistamine diphenhydramine, and the calcium channel blocker diltiazem in plasma of fish at concentrations around undetected level to ng mL−1 . In addition, plasma concentrations in fish were 3 orders of magnitude higher than those found in water. Since the flow of these receiving rivers was strongly influenced by the effluent flow of urban waste from neighboring towns, one can expect that pharmaceutical concentrations were higher than in a river where the water flow would have been higher and allowed much greater dilution of the effluents. While there are many studies on the bioaccumulation of pharmaceuticals in fish highly exposed, few studies have however measured concentrations of drugs in plasma of wild fish chronically exposed to low concentrations of drugs under environmental conditions. Hence, lower plasma concentrations are expected in fish naturally exposed compared to experiments in controlled environments where fish are exposed to steady concentrations (i.e., cage experiments) or without effluent dilution. Published methods currently available for the extraction of pharmaceuticals in fish plasma use polymeric adsorbents HLB type with a hydrophilic-lipophilic selectivity ideal for purification by neutral interactions. Also, the cation selectivity using a mixedmode type adsorbent, reversed-phase/strong cation-exchange, water-wettable polymer which is suitable for the purification of basic and neutral molecules. None of these methods however allow a simultaneous investigation of selected drugs such as ibuprofen (IBU), 2-hydroxy-ibuprofen (2-OH-IBU), diclofenac (DCF), naproxen (NAP), sulfamethoxazole (SMX), trimethoprim (TRIM) and salbutamol (SAL) in fish plasma. The aim of this study was to develop a robust method with maximum sensitivity by ESI-LC-QqQMS to simultaneously determine concentrations of DCF, IBU, 2-OH-IBU, NAP, SAL, SMX and TRIM in plasma of wild fish exposed to low environmental levels of pharmaceuticals. In order to apply the present sensitive method developed, plasma of northern pikes (Esox lucius) environmentally exposed in the St. Lawrence River to a major (flow of 2.5 million m3 d−1 ) but highly diluted (160×) urban effluent (advanced physico-chemical treatments) was investigated. Therefore the analytical emphasis was on the extraction and purification of samples to get minimal matrix effect and maximum and reproducible recovery so that quantification can be carried out on routine LC–MS/MS instruments. For the development of a less-invasive method to preserve life of fish, the plasma volume was set at a maximum of 100 L, which increases the challenge of sensitivity with moderately contaminated environmental samples. The choice of studied drugs was based on their widespread use and their presence in high concentrations in urban effluent and surface waters [1,6,15]. The selected pharmaceuticals have contrasting physico-chemical properties; acids and bases with pKa values from 1 to 17 (Table 1) as the functional groups and the combined plasma extraction thereof becomes a challenge. To combine in a single method all selected drugs, a specific extraction was developed to achieve optimum recovery, and a chromatography showing all peaks resolved min-
imizing the matrix effect by electrospray (ESI) LC-QqQMS, while having a maximum sensitivity without heated electrospray (HESI) source.
2. Material and methods 2.1. Material Standards of diclofenac sodium salt (DCF), 2-hydroxy-ibuprofen (2-OH-IBU), naproxen (NAP), salbutamol (SAL), sulfamethoxazole (SMX) and trimethoprim (TRIM) were purchased from Sigma-Aldrich Canada (Oakville, ON). Standard of ibuprofen (IBU) was purchased from Toronto Research Chemical (Toronto, ON). Isotopically-labeled standard of DCF (acetophenyl ring 13 C6 ), IBU (d3), SMX (phenyl 13 C6 ) and TRIM (13 C6 ) were purchased from Sigma-Aldrich Canada (Oakville, ON) and used as internal standards. Isotopically-labeled standard of NAP (methoxy-d3) and SAL (3-hydroxymethyl-d2; ␣-d1) were purchased from C/D/N ISOTOPES (Montréal, QC) and used as internal standards. Isotopically-labeled standard of 2-OH-IBU (d6) was purchased from Toronto Research Chemical (Toronto, ON) and used as internal standard. LC–MS grade water (H2 O), methanol (MeOH), isopropanol (IPA) and acetonitrile (ACN) were purchased from Fisher Scientific Canada (Ottawa, ON). Dichloromethane (DCM) was purchased from Caledon (Georgetown, ON). Methyl-tert-butyl-ether (MTBE), citric acid, formic acid, ammonium acetate and ammonium hydroxide (NH4 OH) were purchased from Sigma-Aldrich Canada (Oakville, ON). Deionized water (DI-H2 O) passed through a Milli-Q Advantage A10 system (Millipore, Billerica, MA) was used for method blanks. This system is equipped with activated carbon, an ion exchange resin and a UV lamp to reduce total organic carbon (TOC) to ≤5 ppb and increase resistivity (≥18.0 M cm). Wild salmon plasma used for method development was purchased from BioreclamationIVT (NY).
2.2. Preparation of standard solutions Individual stock solutions (1 mg mL−1 ) of each standard and internal standard (IS) were all prepared by dissolving appropriate amount of the standard in ACN/H2 O (65:35, v/v), and stored at 4 ◦ C. Analytical standard mixture solutions (10 g mL−1 ) and analytical standard mixture solutions of IS (5 g mL−1 ) were all prepared by diluting appropriate volume of stock solutions in ACN/H2 O (65:35, v/v), and stored at 4 ◦ C. Working solutions for the standard curves at different concentrations, spiking solutions for recovery test, and working solution for the internal standards (WIS) (100 ng mL−1 ) were prepared by diluting appropriate volume of analytical standard mixture solution in ACN/H2 O (20:80, v/v), and stored at 4 ◦ C.
2.3. Sample collection and preservation Wild northern pikes (n = 49; length = 342–930 mm) were collected, using a beach seine, at two distinct sites located upstream and downstream of a municipal wastewater discharge in the St. Lawrence River, Montréal, QC, Canada in May and June of 2014, 2015 and 2016. Pike were euthanized in a 250 mg L−1 solution of clove oil. Protocols were approved by Environment and Climate Change Canada’s animal care committee working under the Canadian council on animal care. Blood was drawn from the dorsal artery in EDTA tubes, and kept on ice until centrifugation (14,000 rpm, 10 min) in the laboratory to obtain plasma. Plasma was stored at −80 ◦ C until chemical analysis.
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model CHROMA-358884; No. of Pages 8
ARTICLE IN PRESS S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
3
Table 1 Physicochemical properties of selected pharmaceuticals. Drugs
Chemical Formula
Molecular Mass g/mol
Exact Mass g/mol
pKa 1
pKa 2
Log Kow (logP)
Diclofenac Sulfamethoxazole Trimethoprim Ibuprofen 2-OH-Ibuprofen Naproxen Salbutamol
C14 H11 Cl2 NO2 C10 H11 N3 O3 S C14 H18 N4 O3 C13 H18 O2 C13 H18 O3 C14 H14 O3 C13 H21 NO3
296.15 253.28 290.32 206.28 222.28 230.26 239.31
295.0167 253.0521 290.1379 206.1307 222.1256 230.0943 239.1521
COOH = 4.15 Ar-NH2 = 5.7 N = 7.12 COOH = 4.91 COOH = 4.63 COOH = 4.15 NH = 9.4
NH = 1 SO2-NH = 1.6 NH2 = 17.33
4.26 0.79 1.28 3.84
2.4. Sample extraction optimization In the present study, two SPE cartridges were tested on a Vac Master Sample Processing Station from International Sor® ® bent Technology purchased from Biotage (Charlotte, NC) for the extraction of the seven selected analytes using 100 uL of ® fish plasma. First, the Waters Oasis PRiME HLB SPE cartridge, which requires no conditioning step, was tested. These cartridges contain 60 mg of Oasis PRiME HLB water-wettable polymeric sorbent. Sample pH was adjusted by adding 300 L of 1% formic acid to 100 L of plasma sample and 15 L of internal standard solution at 100 ng mL−1 . Sample load flow rate was about 2–4 mL min−1 . After sample loading, cartridges were washed with 1 mL of MeOH/H2 O (5:95) solution. Residual H2 O was removed from the cartridges using a manifold at maximum vacuum −0.5 bar. Analytes were eluted with 2 × 0.5 mL of ACN/IPA/DCM (80:10:10, ® v/v/v). Second, the Waters Oasis MCX mixed-mode cartridge (reverse phase/strong cation-exchange) which contains 30 mg of water-wettable polymeric sorbent with a particle size of 30 m was tested. SPE was first conditioned with 1 mL of MeOH followed by 1 mL of DI-H2 O. Sample pH was adjusted by adding 100 uL of a solution of 10 mM citric acid (pH 2) to 100 uL of plasma sample and 15 L of internal standard solution at 100 ng mL−1 . Sample load flow rate was about 2–4 mL min−1 . After sample loading, cartridges were washed with 1 mL of MeOH/H2 O (5:95) solution. Residual H2 O was removed from the cartridges using the manifold at maximum vacuum −0.5 bar. Analytes were eluted with 1 mL of ACN/IPA/DCM/MTBE (70:10:10:10, v/v/v/v) followed by 1 mL of ACN/IPA/DCM/MTBE/NH4OH (68:9:9:9:5, v/v/v/v/v). SPE extracts were collected in 10 mL centrifuge tubes and evaporated to dryness under a gentle stream of nitrogen, in a dry bath set to 50 ◦ C. Evaporated extracts were then reconstituted to 100 L with MeOH/H2 O/NH4 OH (5:90:5, v/v/v), vortexed for 10 s and transferred to 2-mL glass vials for LC-QqQMS analysis. 2.5. Identification and quantification of target analytes using LC-QqQMS Three chromatographic columns were tested for the separation of compounds, two HILIC columns and a C18 column. Mass spectrometry sensitivity was evaluated for each drug with various mobile phase pH and solvent. Optimized MS parameters are presented in Table 2. To gain sensitivity in ESI, HILIC chromatography was tested, with a gradient of ACN/MeOH mixture, at different pHs. The follow® ing columns were evaluated: YMC Triart Diol-HILIC 150 × 3 mm, ® 3 m, and Waters XBridge Amide HILIC 580 × 2.1 mm, 3.5 m. ® Waters CORTECS C18 + Column, 150 × 2.1 mm, 2.7 m was also tested with a gradient of ACN/H2 O and MeOH/H2 O at different pH values. This column contains a positively charged surface that allows better peak shape for basic compounds. Since the study investigates both acid and basic compound, this column has a better potential than conventional C18 . Optimum separation was achieved ® with Waters CORTECS C18 + Column using a gradient with sol-
OH = −2.7 OH = 10.12
2.99 0.34
vent (A) 5 mM ammonium acetate buffer at pH 5.5 in MeOH/H2 O (5:95, v/v) and solvent (B) 5 mM ammonium acetate buffer at pH 5.5 in MeOH/H2 O (95:5, v/v). Analysis of samples was done using a 1200 Series liquid chromatographer coupled to an electrospray triple-quadrupole mass spectrometer 6410 both manufactured by Agilent (Santa Clara, CA). The injection volume was 10 L, the flow rate was 400 L min−1 and the column was set at 40 ◦ C. The binary LC solvent gradient was: (%B): 0 min (5%), 2 min (5%), 6 min (60%), 9 min (60%), 10 min (80%), 11 min (80%), 12 min (5%), 14 min (5%). Ionization was performed with an electrospray ionization (ESI) source in positive mode. Source parameters were the following: gas temperature 350 ◦ C; drying gas flow 7 L min−1 , fragmentor 160 V; skimmer 60 V and capillary voltage 3000 V. LC–MS raw data obtained from LC-QqQMS analysis of plasma samples were analysed with MassHunter quantitative software manufactured by Agilent (Santa Clara, CA). Two MRM transitions were selected for each analyte, precursor ion and two fragments ions, the first with highest signal for quantification and a second one for qualitative identification confirmation. 2.6. Validation The method was first developed with commercially available wild salmon plasma, fished far from urban effluent, in order to make a complete validation in the matrix. Wild salmon is also less fat than culture salmon, which is more similar to pike. Validation was also performed in a wild salmon plasma matrix, spiked with a maximum of 5% of spiked solution at various concentrations which solvent was ACN/H2 O (20:80, v/v), in order to minimize protein plasma precipitation and solvent effects on the solubility of drugs in plasma. Quantification was done with standard curves for each analyte based on relative responses of the peak area ratio to their respective internal standard versus the corresponding concentration ratio. Validation parameters were determined according to FDA guidance for bioanalytical method validation [16]. 2.6.1. Linearity and limit of quantification Linearity was evaluated with a 5 point standard curve from 1 to 100 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and from 2 to 200 ng mL−1 for IBU and 2-OH-IBU with fixed concentration of their respective labeled internal standard. Linear regression was chosen in such a way that the correlation coefficients were ≥0.995 for all analytes. Lower limit of quantification (LLOQ) and higher limit of quantification (HLOQ) were evaluated on triplicate respectively at the lowest and highest point of the standard curve. The relative standard deviation (RSD) should not exceed 20% for LLOQ and 15% for HLOQ for the precision. The mean value should not exceed 20% of the theoretical value for the LLOQ and 15% for HLOQ for the accuracy. 2.6.2. Precision and accuracy Precision and accuracy were evaluated with triplicates of quality controls using plasma spiked at 3 different concentrations (lowmid-high) within the standard curves, namely 3, 30 and 60 ng mL−1
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model
ARTICLE IN PRESS
CHROMA-358884; No. of Pages 8
S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
4 Table 2 MS parameters for all pharmaceutical residues. Drugs
RT (min)
Quant/Qual
Prec Ion
Prod Ion
Frag (V)
CE (V)
Polarity
Sulfamethoxazole
2.77
SMX-13 C6 Salbutamol
2.77 4.64
Albuterol-d3 2-OH-ibuprofen + NH4+
4.64 5.25
2-OH-Ibuprofen-d6 Trimethoprim
5.25 6.97
Trimethoprim-d3 Naproxen
6.97 7.44
Naproxen-d3 Diclofenac
7.44 8.55
Diclofenac-13 C6 Ibuprofen + NH4+
8.55 8.88
Ibuprofen-d3 + NH4+
8.88
Quan Qual Quan Quan Qual Quan Quan Qual Quan Quan Qual Quan Quan Qual Quan Quan Qual Quan Quan Qual Quan
254.2 254.2 260.1 240.2 240.2 243.2 240.2 240.2 246.1 291.1 291.1 294.2 231.1 231.1 234.1 296.1 296.1 302.1 224.2 224.2 227.2
108.1 156 114 148 165.9 151.1 163 205.1 211.1 230.2 261.2 230.1 170.1 185.1 188.1 215.1 250.1 221.0 161.1 207.1 164.0
106 106 106 96 96 96 70 70 70 149 149 149 70 70 70 90 90 90 65 65 65
13 13 13 9 9 9 9 9 7 15 15 15 5 5 5 5 5 5 0 0 0
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
Abbreviations: Quan: Quantitative ion; Qual: Qualitative ion; Prec Ion: Precursor ion; Prod Ion: Product Ion; Frag : Fragmentation voltage; CE : collision energy.
for DCF, NAP, SMX, TRIM and SAL and at 6, 60 and 120 ng mL−1 for IBU and 2-OH-IBU. The RSD of the mean for each concentration should not exceed 15% for the precision. The mean value should not exceed 15% of the theoretical value for the accuracy. 2.6.3. Recovery/extraction efficiency/matrix effect The recovery, matrix effect and extraction efficiency were all evaluated in a same extraction experiment. Parameters were evaluated with triplicates of quality controls spiked at 3 different concentrations (low-mid-high) within the standard curve, namely 3, 30 and 60 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and 6, 60 and 120 ng mL−1 for IBU and 2-OH-IBU, before and after extraction on SPE. Reference solutions at the same concentrations are also used to calculate overall recovery. The recovery tests were calculated as follow: Extraction efficiency = QC spiked before extraction/QC spiked after extraction
Matrix effect = QC spiked after extraction/standard solution
Recovery = QC spiked before extraction/standard solution
3. Results and discussion 3.1. Extraction of analytes from fish plasma The methods currently published for the extraction of pharmaceuticals in fish plasma use polymeric adsorbent type Oasis HLB ® ® cartridges from Waters or Strata-X cartridges from Phenomenex because of their hydrophilic-lipophilic selectivity. This type of adsorbent was used for the analysis of DCF by ESI-LC–MS/MS [17], DCF, SMX and TRIM by ESI-LC–MS/MS [2], NAP, IBU and DCF by GC–MS [14], NAP and DCF by LC–MS/MS [18] and NAP, IBU ® and DCF by heated electrospray (HESI)-LC–MS/MS [19]. Waters Oasis MCX SPE also uses a mixed-mode type adsorbent, reversedphase/strong cation-exchange water-wettable polymer which is suitable for the purification of basic and neutral molecules. As an example, this adsorbent was used for DCF, NAP, IBU, 2-OH-IBU and SAL by LC–MS/MS [20]. However, none of these studies have covered the analyses of the present selected drugs representing a range of physico-chemical properties. In the present study, two SPE car-
tridges were tested for the extraction of selected the seven analytes using 100 uL of fish plasma. ® The Waters Oasis PRiME HLB SPE cartridge removes phospholipids and in turn, most of matrix effects in LC–MS/MS. In addition, the Oasis PRiME HLB SPE requires no conditioning. Different pH values at the loading step, and several washing and elution solvents were tested. This SPE cartridge is neutral but the pH at the load step was influencing the retention of drugs. At acidic pH, all ® drugs were retained except SAL. The Waters Oasis MCX mixedmode cartridge (reverse phase/strong cation-exchange) was also tested at different pH values during the loading step, and several different washing and elution solvents. Loading at pH 2 with 10 mM citric acid was successful for all drugs with an optimal recovery. The elution solvent was optimized for the recovery and to reduce matrix effect of each drug. IBU, 2-OH-IBU and SAL were retained by hydrophilic and lipophilic interactions and eluted with a solvent mixture, while DCF, SMX, NAP and TRIM were retained by ionic exchange interactions with SO3 − contained in the MCX and required the addition of a strong base, ammonium hydroxide, to be eluted. A solvent mixture was developed to achieve optimal recovery for all drugs, since with ACN or MeOH alone, or in combination, the recovery for SAL, DCF and SMX was not optimal. The addition of 10% isopropanol (IPA) increased the recovery of SAL, the addition of 10% methylene chloride (DCM) increased the recovery of DCF and the addition of 10% methyl tert-butyl ether (MTBE) increased the recovery of SMX. IBU, 2-OH-IBU and SAL were eluted with a mixture of ACN/IPA/DCM/MTBE (70:10:10:10, v/v/v/v), while DCF, SMX, NAP and TRIM were eluted with a mixture of ACN/IPA/DCM/MTBE/NH4 OH (68:9:9:9:5, v/v/v/v/v).
3.2. Chromatography and mass spectrometry Separation and retention of analytes were not possible with an HILIC chromatography because of the limited retention of compounds such as DCF, SMX, and IBU. The separation of analytes with ® good retention was efficiently performed using a Waters CORTECS C18 + column (150 × 2.1 mm, 2.7 m, 90 Å) (Fig. 1). This column containing a positively charged surface allowed better peak shape for basic compounds such as SAL and TRIM. A gradient composed of methanol and ammonium acetate buffer at pH 5.5 allowed the separation of all compounds in a run time of 10 min plus an equilibration time of 4 min. Maximum sensitivity in MS was achieved by select-
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model CHROMA-358884; No. of Pages 8
ARTICLE IN PRESS S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
5
Fig. 1. MRM chromatogram corresponding to the analysis of salmon plasma spiked with all analytes at 100 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and at 200 ng mL−1 for IBU and 2-OH-IBU.
ing collision energy providing best signal of product ions used for quantification. A second ion product was chosen to confirm the identity of the analytes. 3.3. Method validation parameters Quantification was done with relative responses based on the peak area ratio of analytes to their respective internal standard versus the corresponding concentration ratio. All validation results follow FDA guidelines for bioanalysis [16], except for matrix effect and QC’s below LLOQ. Matrix effects were higher than values permitted in FDA guidelines, but since internal standard are isotopically labeled drugs, they are corrected efficiently. All validation results are given in Table 3. 3.3.1. Linearity/LLOQ/HLOQ Linear range was evaluated with 5 calibration points from 1 to 100 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and from 2 to 200 ng mL−1 for IBU and 2-OH-IBU. Standard curves were linear (R2 > 0.995) for all drugs. The LLOQ was evaluated on triplicates and valued at 1 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and 2 ng mL−1 for IBU and 2-OH-IBU and was reproducible for all drugs with precision from 0.3% to 9% and an accuracy from 99% to 110%. Other studies have also evaluated LOQ (or MQL as Method Quantification Limit) as 10 times the signal to noise ratio (s/n) of target analytes. MQL of naproxen (2.3 pg/L), ibuprofen (2.1 pg/L) and diclofenac (1.6 pg/L) have been evaluated by [19] in 20 L of zebrafish plasma. LOQ of multiple drugs [14] have been evaluated in rainbow trout plasma exposed to undiluted effluent from three different stations. The reported LOQs varied from 0.05 to 5 ng/mL for 0.5 mL of extracted plasma. LOQ of carbamazepine, bisoprolol, diclofenac, ibuprofen and naproxen have also been evaluated by [21] and ranged from 0.86 to 20 ng/mL without clarifying single values, in 300 L of rainbow trout plasma. LLOQ results from the present work are thus lower than previously published studies considering the volume of plasma used, and results have been
proven to be precise, accurate and reproducible. The higher limit of quantification (HLOQ) was evaluated on triplicates and valued at 100 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and 200 ng mL−1 for IBU and 2-OH-IBU with a precision from 0.2% to 2.3% and an accuracy from 99% to 106%.
3.3.2. Precision/accuracy Precision results ranged between 3% to 11% while accuracy values ranged between 94% to 105% for all drugs. Since results from pike samples were all below LLOQ, additional QC’s were evaluated at 0.1, 0.3, and 0.5 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and 1 ng mL−1 for IBU and 2-OH-IBU. Precision results ranged between 4% to 27% while accuracy values ranged between 84% to 147% for all drugs at determined concentrations, except IBU which was not detectable below LLOQ.
3.3.3. Recovery/matrix effect/extraction efficiency The recovery values were between 94 to 111% for all drugs. Matrix effect values ranged from 15% to 73% of signal suppression and extraction efficiency values ranged from 57 to 89%. Results demonstrated that the extraction efficiency did not take into account the matrix effect and signal suppression generated by matrix effect was reflected in the extraction efficiency. The recovery took into account both effects and confirmed that quantification by ratio with internal standard was effective to counterbalance the matrix effect.
3.3.4. Selectivity The selectivity was evaluated on triplicate blank salmon plasma. The matrix blanks contained no peak of all drugs except 4% of LLOQ for DCF and 8% of LLOQ for TRIM, which is below the value of 20% accepted by FDA guidelines.
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model
ARTICLE IN PRESS
CHROMA-358884; No. of Pages 8
S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
6 Table 3 Validation results of all pharmaceuticals residues. Validation Results
IBU
2-OH-IBU
DCF
NAP
SMX
SAL
TRIM
Linearity (Concentration) Linearity R2 LLOQ concentration (ng/mL) LLOQ (Precision; n = 3) LLOQ (Accuracy; n = 3) HLOQ concentration (ng/mL) HLOQ (Precision; n = 3) HLOQ (Accuracy; n = 3) Selectivity (% of LLOQ; n = 3) Precision (3 QC low-mid-high; n = 3) Accuracy (3 QC low-mid-high; n = 3) Extraction efficacity (3 QC low-mid-high) Matrix effect (3 QC low-mid-high) Recovery (3 QC low-mid-high; n = 3) QC below LLOQ (ng/mL) Precision (n = 3) Accuracy (n = 3)
2–200 ng/mL >0.996 2 7.5% 103% 200 0.6% 106% 0% <11% 102–105% 82–87% 122% ± 6% 98–107% 1 NA NA
2–200 ng/mL >0.997 2 3.9% 100% 200 2.3% 102% 0% <6% 94–103% 79–89% 115% ± 4% 94–100% 1 20% 104%
1–100 ng/mL >0.998 1 2.8% 109% 100 0.9% 100% 4% <5% 97–100% 79–84% 128% ± 3.9% 101–104% 0.3 11% 84%
1–100 ng/mL >0.999 1 4.8% 107% 100 1.3% 100% 0% <4% 98–101% 82–84% 126% ± 0.6% 104–105% 0.3 11% 137%
1–100 ng/mL >0.999 1 2.0% 99% 100 1.2% 100% 0% <4% 96–101% 72–74% 146% ± 0.5% 104–111% 0.5 6% 147%
1–100 ng/mL >0.999 1 8.8% 110% 100 0.2% 100% 0% <3% 99–102% 57–62% 173% ± 6.7% 100–104% 0.3 4% 137%
1–100 ng/mL >0.999 1 0.3% 105% 100 0.2% 99% 8% <3% 99–102% 76–78% 130% ± 1.9% 100% 0.1 27% 133%
Abbreviation: LLOQ: Lower Limit of Quantification; HLOQ: Higher Limit of Quantification; QC: Quality Control at low. mid and high concentration.
3.4. Application to environmental samples Samples of wild pike plasma (n = 49), collected over a three-year period downstream and upstream of a wastewater discharge in the St. Lawrence River, were analyzed in duplicate for IBU, 2-OH-IBU, DCF, SMX, SAL, NAP, and TRIM, and results are shown in Table 4. The detected levels for all samples were below the limit of quantification (LLOQ) validated in the method. However, chromatographic peaks were present for DCF, NAP, TRIM and SAL in several samples. Quantification of these samples would be an extrapolation of the linearity of the quantification curve. Three additional QCs were therefore extracted at concentrations below LLOQ (i.e., 0.5, 0.3 and 0.1 ng mL−1 ) to determine the precision and accuracy for each drug. The results (Table 3) showed that the precision was variable and the accuracy was lower below the LLOQ but allow the interpretation of results obtained in fish plasma. Several results presented in Table 4 have values below the concentrations used for QC evaluated below LLOQ, but duplicate relative standard deviation (RSD) values were presented in order to give an overall picture for a maximum of specimens. The selected pharmaceuticals were detected in St. Lawrence pike, however no pattern of high contamination could be observed through the years or among individuals and sites. When extrapolating concentrations, NAP was found in fish collected at the downstream site in 2014 with a RSD of 43% and in 2016 fish from the upstream and downstream sites with RSD between 5 and 31%. SAL was found in a single sample collected downstream of the effluent outfall in 2014 with a RSD of 20%. DIC and TRIM were detected in fish plasma samples in 2014 and only DIC was detected in collected samples downstream in 2016. None of the investigated drugs were detected in samples from 2015. Overall, there was no significant difference between the samples collected downstream and upstream of the effluent discharge point. Pike are known to be sedentary to these specific sites. Also, when comparing plasma concentrations to those measured in the surface water samples collected by [22] in the St. Lawrence River downstream of the same effluent, levels for NAP (approx. 4 ng L−1 ), 2-OH-IBU (approx. 100 ng L−1 ) and IBU (approx. 25 ng L−1 ) as well as SMX (approx. 12 ng L−1 ), and TRIM (approx. <10 ng L−1 ) [6] were less concentrated in fish than in water. These results may indicate that the high flow of the river which causes a major dilution of the effluent could limit the exposure of fish to the pharmaceuticals and explain the lack of differences in pharmaceutical concentrations found in fish samples upstream and downstream of the effluent discharge point.
The ionization of the drugs could not solely explain the differences between the results for DIC, NAP, SMX, IBU, and 2-OH-IBU, which are negatively ionized and SAL which is positively ionized in the effluent plume at a stable pH value of 8.1. The chosen drugs were thus all ionized under environmental conditions except TRIM, which was predominantly neutral. Several studies investigated the absorption and the metabolism of pharmaceuticals in fish in comparison to humans [11,13,23] and differences in cytochrome (CYP) activities were observed. The habitat and feeding ecology can also influence the absorption and metabolism of pharmaceuticals in fish, and consequently, drug clearance. The plasma samples from wild pike were little contaminated with the investigated drugs, but the method still allowed record of signal for lower concentrations. Values previously published by [2,9] were indeed much higher in other rivers influenced by the relatively high flow of urban effluents which were less diluted by the river flow. The present developed method could be easily utilized in more contaminated environments, such as rivers in Texas [2] and Japan [9], to characterize the plasma concentrations of validated drugs and possibly several other pharmaceuticals with further validation. This developed method can indeed allow the detection of very low concentrations of pharmaceuticals of environmental interest in a relevant biological matrix. 4. Conclusion An analytical method based on SPE and LC-QqQMS was developed to identify and analyze DCF, IBU, 2-OH-IBU, NAP, SMX, SAL, and TRIM in plasma of wild pikes exposed in their natural habitat to municipal wastewater. The advantages of this method were the simultaneous analysis of six polar drugs and a common metabolite (2-OH-IBU) (log Kow = 0.79–4.26) in a small volume of 100 L of fish plasma with low LLOQ. Simple SPE extraction and chromatography with an ESI source in LC–MS/MS were successfully developed. Method sensitivity has been determined with a LLOQ of 1 ng mL−1 for DCF, NAP, SMX, SAL and TRIM and a LLOQ of 2 ng mL−1 for IBU and 2-OH-IBU in reproducibly manner with high precision and accuracy. Moreover, the analysis of plasma represents a less-invasive approach to collect small biological samples and monitor pharmaceuticals in fish. Thus, the developed method allowed the quantification of low concentrations of pharmaceuticals in fish plasma with better LLOQ than those reported in previous published studies, with high precision, accuracy and recovery. Therefore this method allowed for quantification of the selected drugs in plasma of wild pike chronically exposed to low levels of environmental contamination. This method could easily be used in more contam-
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
TRIM
NAP
Site
ID
Concentration
RSD
Concentration
RSD
Concentration
2014
IB
1 3 5 9 10 1 2 3 4 5 6 7 8 9 10
0.29 0.30 0.48 ND 0.14 0.16 0.15 0.42 0.15 ND ND ND ND ND ND
7% 32% 16%
0.13 0.13 0.14 0.14 0.12 0.17 0.13 0.13 ND ND ND ND ND ND 0.03
11% 17% 4% 14% 3% 18% 21% 19%
ND ND ND ND ND ND ND ND ND ND ND ND ND 0.10 ND
4 6 8 9 10 12 13 14 3 7 9 10 11 12 13 14 15
ND ND ND ND ND ND ND ND ND 0.20 ND ND ND ND 0.30 ND ND
0.12 0.10 0.12 0.11 0.09 0.13 0.10 ND 0.03 ND ND ND 0.04 ND 0.05 ND ND
3% 2% 40% 10% 41% 24% 3% – 6%
IV
2016
IB
IV
33% 29% 5% 11% 4%
36%
– – 23%
39%
26% 39%
0.20 ND 0.21 0.23 ND 0.18 0.19 0.21 ND 0.19 ND 0.18 0.18 0.20 0.16 0.15 0.22
RSD
43% 17% – 20% 11% 6% 8% 9% 5% 15% 22% 13% 7% 15% 31%
Concentration ND ND ND ND ND ND 0.09 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
SMX RSD
20%
Concentration
2-OH-IBU RSD
Concentration
IBU RSD
Concentration
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
RSD
Abbreviations: RSD: relative standard deviation; ND: Not detected. Concentrations in italics were below the LLOQ but the RSD was still acceptable. Site: IB: Îles de Boucherville, Upstream; IV: Îlet Vert, Downstream. a Data in 2015 were all ND.
ARTICLE IN PRESS
Year
SAL
G Model
CHROMA-358884; No. of Pages 8
DCF
S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
Table 4 Mean concentrations (ng/mL) and RSD (%) of selected pharmaceuticals in plasma of northern pike.a
7
G Model CHROMA-358884; No. of Pages 8
ARTICLE IN PRESS S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
8
inated environments for the characterization of validated drugs in fish plasma and potentially other drugs with further validation. Furthermore, the chromatographic and mass spectrometry conditions developed could be further used to analyze these drugs in other fish tissues and allow a better understanding of the overall body distribution of pharmaceuticals in these vertebrates.
[10]
[11]
Acknowledgments
[12]
We are grateful to Mélanie Douville, Martin Pilote, Julie Reinling, Guillaume Cottin, and Alexandre Bernier-Graveline for their help in the field. This work was supported by the St. Lawrence Action Plan.
[13]
[14]
References [1] L. Viglino, K. Aboulfadl, A.D. Mahvelat, M. Prévost, S. Sauvé, On-line solid phase extraction and liquid chromatography/tandem mass spectrometry to quantify pharmaceuticals, pesticides and some metabolites in wastewaters, drinking, and surface waters, J. Environ. Monit. JEM 10 (2008) 482–489. [2] W.C. Scott, B. Du, S.P. Haddad, C.S. Breed, G.N. Saari, M. Kelly, L. Broach, C.K. Chambliss, B.W. Brooks, Predicted and observed therapeutic dose exceedances of ionizable pharmaceuticals in fish plasma from urban coastal systems, Environ. Toxicol. Chem. 35 (2016) 983. [3] D. Fatta-Kassinos, S. Meric, A. Nikolaou, Pharmaceutical residues in environmental waters and wastewater: current state of knowledge and future research, Anal. Bioanal. Chem. 399 (2011) 251–275. [4] F.O. Agunbiade, B. Moodley, Occurrence and distribution pattern of acidic pharmaceuticals in surface water, wastewater, and sediment of the Msunduzi River, Kwazulu-Natal, South Africa, Environ. Toxicol. Chem. 35 (2016) 36–46. [5] C.L. Chitescu, G. Kaklamanos, A.I. Nicolau, A.A.M. Stolker, High sensitive multiresidue analysis of pharmaceuticals and antifungals in surface water using U-HPLC-Q-Exactive Orbitrap HRMS. Application to the Danube river basin on the Romanian territory, Sci. Total Environ. 532 (2015) 501–511. [6] C. Robert, Bolduc, Anouk, DeBlois, Christian, Résultats du suivi des produits pharmaceutiques et de soins personnels ainsi que des hormones dans des eaux usées, de l’eau de surface et de l’eau potable au Québec, MDDEFPQ, Québec, 2009, pp. 115. [7] V. Christen, S. Hickmann, B. Rechenberg, K. Fent, Highly active human pharmaceuticals in aquatic systems: a concept for their identification based on their mode of action, Aquat. Toxicol. 96 (2010) 167–181. [8] A.J. Ramirez, R.A. Brain, S. Usenko, M.A. Mottaleb, J.G. O’Donnell, L.L. Stahl, J.B. Wathen, B.D. Snyder, J.L. Pitt, P. Perez-Hurtado, L.L. Dobbins, B.W. Brooks, C.K. Chambliss, Occurrence of pharmaceuticals and personal care products in fish: results of a national pilot study in the united states, Environ. Toxicol. Chem. 28 (2009) 2587–2597. [9] R. Tanoue, K. Nomiyama, H. Nakamura, J.-W. Kim, T. Isobe, R. Shinohara, T. Kunisue, S. Tanabe, Uptake and tissue distribution of pharmaceuticals and
[15]
[16] [17]
[18]
[19]
[20]
[21]
[22]
[23]
personal care products in wild fish from treated-wastewater-impacted streams, Environ. Sci. Technol. 49 (2015) 11649–11658. T.W. Valenti Jr., P. Perez-Hurtado, C.K. Chambliss, B.W. Brooks, Aquatic toxicity of sertraline to Pimephales promelas at environmentally relevant surface water pH, Environ. Toxicol. Chem. 12 (2009) 2685–2694. K.A. Connors, B. Du, P.N. Fitzsimmons, A.D. Hoffman, C.K. Chambliss, J.W. Nichols, B.W. Brooks, Comparative pharmaceutical metabolism by rainbow trout (Oncorhynchus mykiss) liver S9 fractions, Environ. Toxicol. Chem. 32 (2013) 1810–1818. J.N. Brown, N. Paxéus, L. Förlin, D.G.J. Larsson, Variations in bioconcentration of human pharmaceuticals from sewage effluents into fish blood plasma, Environ. Toxicol. Pharmacol. 24 (2007) 267–274. T.H. Hutchinson, J.C. Madden, V. Naidoo, C.H. Walker, Comparative metabolism as a key driver of wildlife species sensitivity to human and veterinary pharmaceuticals, Philos. Trans. R. Soc. B Biol. Sci. 369 (2014). J. Fick, R.H. Lindberg, J. Parkkonen, B. Arvidsson, M. Tysklind, D.G.J. Larsson, Therapeutic levels of levonorgestrel detected in blood plasma of fish: results from screening rainbow trout exposed to treated sewage effluents, Environ. Sci. Technol. 44 (2010) 2661–2666. C. Gagnon, A. Lajeunesse, Low removal of acidic and hydrophilic pharmaceutical products by various types of municipal wastewater treatment plants, J. Xenobiotics 2 (2012) 13–17. Guidance for Industry Bioanalytical Method Validation, U.S. Department of Health and Human Services Food and Drug Administration, 2001. B. Du, S.P. Haddad, A. Luek, W.C. Scott, G.N. Saari, L.A. Kristofco, K.A. Connors, C. Rash, J.B. Rasmussen, C.K. Chambliss, B.W. Brooks, Bioaccumulation and trophic dilution of human pharmaceuticals across trophic positions of an effluent-dependent wadeable stream, Philos. Trans. R. Soc. Lond. B Biol. Sci. 369 (2014). F. Cuklev, E. Kristiansson, J. Fick, N. Asker, L. Förlin, D.G.J. Larsson, Diclofenac in fish: blood plasma levels similar to human therapeutic levels affect global hepatic gene expression, Environ. Toxicol. Chem. 30 (2011) 2126–2134. F. Chen, Z. Gong, B.C. Kelly, Rapid analysis of pharmaceuticals and personal care products in fish plasma micro-aliquots using liquid chromatography tandem mass spectrometry, J. Chromatogr. A 1383 (2015) 104–111. Y. Aminot, Etude de l’impact des effluents urbains sur la qualité des eaux de la Garonne estuarienne: application aux composés pharmaceutiques et aux filtres UV, UMR EPOC, Universite Bordeaux 1, Bordeaux, 2013. M. Lahti, J.-M. Brozinski, A. Jylhä, L. Kronberg, A. Oikari, Uptake from water, biotransformation, and biliary excretion of pharmaceuticals by rainbow trout, Environ. Toxicol. Chem. 30 (2011) 1403–1411. A. Lajeunesse, C. Gagnon, Determination of acidic pharmaceutical products and carbamazepine in roughly primary-treated wastewater by solid-phase extraction and gas chromatography–tandem mass spectrometry, Int. J. Environ. Anal. Chem. 87 (2007) 565–578. E.M. Smith, F.I. Iftikar, S. Higgins, A. Irshad, R. Jandoc, M. Lee, J.Y. Wilson, In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes, Aquat. Toxicol. 109 (2012) 259–266.
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
ARTICLE IN PRESS Journal of Chromatography A, xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Full length article
Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry S. Poirier Larabie, M. Houde, C. Gagnon ∗ Aquatic Contaminants Research Division, Science and Water Technology Directorate, Environment and Climate Change Canada, 105 McGill Street, Montréal, Québec, H2Y 2E7, Canada
a r t i c l e
i n f o
Article history: Received 17 March 2017 Received in revised form 21 September 2017 Accepted 23 September 2017 Available online xxx Keywords: Fish Plasma Pharmaceuticals LC–MS/MS Surface waters Environment
a b s t r a c t Aquatic systems near major urban centers are constantly contaminated with effluent from wastewater treatment plants. Pharmaceuticals are part of the contamination and several classes of drugs have been detected in surface waters in the last decade. To better understand the impact of those pharmaceuticals in ecosystems, the exposure to aquatic species needs to be investigated. This study presents a new simple and rugged quantitative method for the determination of several classes of drugs using 100 L of plasma from fish environmentally exposed to a major but highly diluted urban effluent. Six common drugs (i.e., diclofenac, ibuprofen, naproxen, salbutamol, sulfamethoxazole and trimethoprim) and one major metabolite (2-hydroxy-ibuprofen), present in significant amount in impacted waterways have been selected for the development and validation of the method. First, all drugs were extracted using cation exchange solid phase extraction (SPE) and eluted with two solvent mixtures. Then, the extracts ® were analyzed using a reverse-phase analytical column Waters CORTECS C18 + (150 × 2.1 mm, 2.7 m) within 14 min. MS/MS was performed with an electrospray (ESI) interface in positive ion mode, with multiple reaction monitoring (MRM) experiment acquiring two product ions per drugs. Quantification has been made with standard curves for each analyte using isotopically labeled internal standards. This method has high sensitivity with limits of quantification of 1 ng mL−1 for each drug, except for ibuprofen and its metabolite 2-hydroxy-ibuprofen at 2 ng mL−1 . The precision of the method was below 11%, the accuracy between 94 and 105% and overall recovery between 94 and 111% for all drugs, with high selectivity. Application of the method to plasma samples from wild northern pike inhabiting the St. Lawrence River collected over a three-year period showed the presence of naproxen, diclofenac, trimethoprim and salbutamol at very low concentrations (around 1 ng mL−1 ). Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved.
1. Introduction Pharmaceuticals consumed by humans are continuously discharged in aquatic environments through urban effluents. Several classes of pharmaceuticals such as antibiotics, non-steroidal anti-inflammatory drugs, -blockers, anti-psychotics, cholesterol lowering drugs, anticonvulsants, and hormones have indeed been widely detected in surface waters near major cities around the world (e.g., Montreal, QC, Canada; Buffalo, TX, U.S.; Danube river basin, Romanian territory; KwaZulu-Natal, South Africa) [1–6]. The presence of pharmaceuticals in municipal wastewater effluents and their potential impacts on aquatic ecosystems are of growing con-
∗ Corresponding author. E-mail address: [email protected] (C. Gagnon).
cerns [7]. Aquatic organisms are indeed chronically exposed to these drugs in their natural habitats [8] and absorption pathways are multiple [9]. The absorption through gills in fish allow the direct passage of pharmaceutical in blood, bypassing the digestive system. This suggests that pharmaceuticals could be found in their original form in fish blood. This absorption can vary depending on physico-chemical factors of the surrounding water, mainly pH, thus affecting the bioavailability of pharmaceuticals [2,10]. Fish plasma concentrations may also vary depending on the pharmaceutical type, its ionization and its clearance [11]. Ionized molecules usually have a lower bioconcentration potential than neutral molecules [12]. In addition, pharmaceuticals are relatively polar and mostly ionized molecules in environmental media, in contrast to non-polar contaminants (e.g., polychlorinated biphenyls, dioxins and furans), which could lead to their circulation in aqueous body fluids rather than accumulation in more lipid-rich organs. Plasma is therefore an
https://doi.org/10.1016/j.chroma.2017.09.055 0021-9673/Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model CHROMA-358884; No. of Pages 8 2
ARTICLE IN PRESS S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
indicated medium to investigate the bioavailability of pharmaceuticals as it is the carrier of the majority of drugs to their target tissues and organs (muscles, brain, liver, kidney) [9]. Moreover, human pharmaceuticals are synthetized to reach specific target tissues that can be found in fish as well [11,13]. Juvenile rainbow trout have been experimentally exposed in Sweden for different periods of time (12 days, 13 days and 6 weeks) to aerated undiluted treated effluents from three wastewater treatment plants (WWTP). Plasma concentration of pharmaceuticals in these rainbow trout ranged from 2.2 to 20 ng mL−1 for diclofenac, 5.5 to 102 ng mL−1 for ibuprofen, and 33 to 46 ng mL−1 for naproxen [14]. In Texas, levels of several pharmaceuticals in four rivers highly impacted by urban effluents were determined as well as in different fish species [2]. The authors found the accumulation of the lipid regulator gemfibrozil, the antihistamine diphenhydramine, and the calcium channel blocker diltiazem in plasma of fish at concentrations around undetected level to ng mL−1 . In addition, plasma concentrations in fish were 3 orders of magnitude higher than those found in water. Since the flow of these receiving rivers was strongly influenced by the effluent flow of urban waste from neighboring towns, one can expect that pharmaceutical concentrations were higher than in a river where the water flow would have been higher and allowed much greater dilution of the effluents. While there are many studies on the bioaccumulation of pharmaceuticals in fish highly exposed, few studies have however measured concentrations of drugs in plasma of wild fish chronically exposed to low concentrations of drugs under environmental conditions. Hence, lower plasma concentrations are expected in fish naturally exposed compared to experiments in controlled environments where fish are exposed to steady concentrations (i.e., cage experiments) or without effluent dilution. Published methods currently available for the extraction of pharmaceuticals in fish plasma use polymeric adsorbents HLB type with a hydrophilic-lipophilic selectivity ideal for purification by neutral interactions. Also, the cation selectivity using a mixedmode type adsorbent, reversed-phase/strong cation-exchange, water-wettable polymer which is suitable for the purification of basic and neutral molecules. None of these methods however allow a simultaneous investigation of selected drugs such as ibuprofen (IBU), 2-hydroxy-ibuprofen (2-OH-IBU), diclofenac (DCF), naproxen (NAP), sulfamethoxazole (SMX), trimethoprim (TRIM) and salbutamol (SAL) in fish plasma. The aim of this study was to develop a robust method with maximum sensitivity by ESI-LC-QqQMS to simultaneously determine concentrations of DCF, IBU, 2-OH-IBU, NAP, SAL, SMX and TRIM in plasma of wild fish exposed to low environmental levels of pharmaceuticals. In order to apply the present sensitive method developed, plasma of northern pikes (Esox lucius) environmentally exposed in the St. Lawrence River to a major (flow of 2.5 million m3 d−1 ) but highly diluted (160×) urban effluent (advanced physico-chemical treatments) was investigated. Therefore the analytical emphasis was on the extraction and purification of samples to get minimal matrix effect and maximum and reproducible recovery so that quantification can be carried out on routine LC–MS/MS instruments. For the development of a less-invasive method to preserve life of fish, the plasma volume was set at a maximum of 100 L, which increases the challenge of sensitivity with moderately contaminated environmental samples. The choice of studied drugs was based on their widespread use and their presence in high concentrations in urban effluent and surface waters [1,6,15]. The selected pharmaceuticals have contrasting physico-chemical properties; acids and bases with pKa values from 1 to 17 (Table 1) as the functional groups and the combined plasma extraction thereof becomes a challenge. To combine in a single method all selected drugs, a specific extraction was developed to achieve optimum recovery, and a chromatography showing all peaks resolved min-
imizing the matrix effect by electrospray (ESI) LC-QqQMS, while having a maximum sensitivity without heated electrospray (HESI) source.
2. Material and methods 2.1. Material Standards of diclofenac sodium salt (DCF), 2-hydroxy-ibuprofen (2-OH-IBU), naproxen (NAP), salbutamol (SAL), sulfamethoxazole (SMX) and trimethoprim (TRIM) were purchased from Sigma-Aldrich Canada (Oakville, ON). Standard of ibuprofen (IBU) was purchased from Toronto Research Chemical (Toronto, ON). Isotopically-labeled standard of DCF (acetophenyl ring 13 C6 ), IBU (d3), SMX (phenyl 13 C6 ) and TRIM (13 C6 ) were purchased from Sigma-Aldrich Canada (Oakville, ON) and used as internal standards. Isotopically-labeled standard of NAP (methoxy-d3) and SAL (3-hydroxymethyl-d2; ␣-d1) were purchased from C/D/N ISOTOPES (Montréal, QC) and used as internal standards. Isotopically-labeled standard of 2-OH-IBU (d6) was purchased from Toronto Research Chemical (Toronto, ON) and used as internal standard. LC–MS grade water (H2 O), methanol (MeOH), isopropanol (IPA) and acetonitrile (ACN) were purchased from Fisher Scientific Canada (Ottawa, ON). Dichloromethane (DCM) was purchased from Caledon (Georgetown, ON). Methyl-tert-butyl-ether (MTBE), citric acid, formic acid, ammonium acetate and ammonium hydroxide (NH4 OH) were purchased from Sigma-Aldrich Canada (Oakville, ON). Deionized water (DI-H2 O) passed through a Milli-Q Advantage A10 system (Millipore, Billerica, MA) was used for method blanks. This system is equipped with activated carbon, an ion exchange resin and a UV lamp to reduce total organic carbon (TOC) to ≤5 ppb and increase resistivity (≥18.0 M cm). Wild salmon plasma used for method development was purchased from BioreclamationIVT (NY).
2.2. Preparation of standard solutions Individual stock solutions (1 mg mL−1 ) of each standard and internal standard (IS) were all prepared by dissolving appropriate amount of the standard in ACN/H2 O (65:35, v/v), and stored at 4 ◦ C. Analytical standard mixture solutions (10 g mL−1 ) and analytical standard mixture solutions of IS (5 g mL−1 ) were all prepared by diluting appropriate volume of stock solutions in ACN/H2 O (65:35, v/v), and stored at 4 ◦ C. Working solutions for the standard curves at different concentrations, spiking solutions for recovery test, and working solution for the internal standards (WIS) (100 ng mL−1 ) were prepared by diluting appropriate volume of analytical standard mixture solution in ACN/H2 O (20:80, v/v), and stored at 4 ◦ C.
2.3. Sample collection and preservation Wild northern pikes (n = 49; length = 342–930 mm) were collected, using a beach seine, at two distinct sites located upstream and downstream of a municipal wastewater discharge in the St. Lawrence River, Montréal, QC, Canada in May and June of 2014, 2015 and 2016. Pike were euthanized in a 250 mg L−1 solution of clove oil. Protocols were approved by Environment and Climate Change Canada’s animal care committee working under the Canadian council on animal care. Blood was drawn from the dorsal artery in EDTA tubes, and kept on ice until centrifugation (14,000 rpm, 10 min) in the laboratory to obtain plasma. Plasma was stored at −80 ◦ C until chemical analysis.
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model CHROMA-358884; No. of Pages 8
ARTICLE IN PRESS S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
3
Table 1 Physicochemical properties of selected pharmaceuticals. Drugs
Chemical Formula
Molecular Mass g/mol
Exact Mass g/mol
pKa 1
pKa 2
Log Kow (logP)
Diclofenac Sulfamethoxazole Trimethoprim Ibuprofen 2-OH-Ibuprofen Naproxen Salbutamol
C14 H11 Cl2 NO2 C10 H11 N3 O3 S C14 H18 N4 O3 C13 H18 O2 C13 H18 O3 C14 H14 O3 C13 H21 NO3
296.15 253.28 290.32 206.28 222.28 230.26 239.31
295.0167 253.0521 290.1379 206.1307 222.1256 230.0943 239.1521
COOH = 4.15 Ar-NH2 = 5.7 N = 7.12 COOH = 4.91 COOH = 4.63 COOH = 4.15 NH = 9.4
NH = 1 SO2-NH = 1.6 NH2 = 17.33
4.26 0.79 1.28 3.84
2.4. Sample extraction optimization In the present study, two SPE cartridges were tested on a Vac Master Sample Processing Station from International Sor® ® bent Technology purchased from Biotage (Charlotte, NC) for the extraction of the seven selected analytes using 100 uL of ® fish plasma. First, the Waters Oasis PRiME HLB SPE cartridge, which requires no conditioning step, was tested. These cartridges contain 60 mg of Oasis PRiME HLB water-wettable polymeric sorbent. Sample pH was adjusted by adding 300 L of 1% formic acid to 100 L of plasma sample and 15 L of internal standard solution at 100 ng mL−1 . Sample load flow rate was about 2–4 mL min−1 . After sample loading, cartridges were washed with 1 mL of MeOH/H2 O (5:95) solution. Residual H2 O was removed from the cartridges using a manifold at maximum vacuum −0.5 bar. Analytes were eluted with 2 × 0.5 mL of ACN/IPA/DCM (80:10:10, ® v/v/v). Second, the Waters Oasis MCX mixed-mode cartridge (reverse phase/strong cation-exchange) which contains 30 mg of water-wettable polymeric sorbent with a particle size of 30 m was tested. SPE was first conditioned with 1 mL of MeOH followed by 1 mL of DI-H2 O. Sample pH was adjusted by adding 100 uL of a solution of 10 mM citric acid (pH 2) to 100 uL of plasma sample and 15 L of internal standard solution at 100 ng mL−1 . Sample load flow rate was about 2–4 mL min−1 . After sample loading, cartridges were washed with 1 mL of MeOH/H2 O (5:95) solution. Residual H2 O was removed from the cartridges using the manifold at maximum vacuum −0.5 bar. Analytes were eluted with 1 mL of ACN/IPA/DCM/MTBE (70:10:10:10, v/v/v/v) followed by 1 mL of ACN/IPA/DCM/MTBE/NH4OH (68:9:9:9:5, v/v/v/v/v). SPE extracts were collected in 10 mL centrifuge tubes and evaporated to dryness under a gentle stream of nitrogen, in a dry bath set to 50 ◦ C. Evaporated extracts were then reconstituted to 100 L with MeOH/H2 O/NH4 OH (5:90:5, v/v/v), vortexed for 10 s and transferred to 2-mL glass vials for LC-QqQMS analysis. 2.5. Identification and quantification of target analytes using LC-QqQMS Three chromatographic columns were tested for the separation of compounds, two HILIC columns and a C18 column. Mass spectrometry sensitivity was evaluated for each drug with various mobile phase pH and solvent. Optimized MS parameters are presented in Table 2. To gain sensitivity in ESI, HILIC chromatography was tested, with a gradient of ACN/MeOH mixture, at different pHs. The follow® ing columns were evaluated: YMC Triart Diol-HILIC 150 × 3 mm, ® 3 m, and Waters XBridge Amide HILIC 580 × 2.1 mm, 3.5 m. ® Waters CORTECS C18 + Column, 150 × 2.1 mm, 2.7 m was also tested with a gradient of ACN/H2 O and MeOH/H2 O at different pH values. This column contains a positively charged surface that allows better peak shape for basic compounds. Since the study investigates both acid and basic compound, this column has a better potential than conventional C18 . Optimum separation was achieved ® with Waters CORTECS C18 + Column using a gradient with sol-
OH = −2.7 OH = 10.12
2.99 0.34
vent (A) 5 mM ammonium acetate buffer at pH 5.5 in MeOH/H2 O (5:95, v/v) and solvent (B) 5 mM ammonium acetate buffer at pH 5.5 in MeOH/H2 O (95:5, v/v). Analysis of samples was done using a 1200 Series liquid chromatographer coupled to an electrospray triple-quadrupole mass spectrometer 6410 both manufactured by Agilent (Santa Clara, CA). The injection volume was 10 L, the flow rate was 400 L min−1 and the column was set at 40 ◦ C. The binary LC solvent gradient was: (%B): 0 min (5%), 2 min (5%), 6 min (60%), 9 min (60%), 10 min (80%), 11 min (80%), 12 min (5%), 14 min (5%). Ionization was performed with an electrospray ionization (ESI) source in positive mode. Source parameters were the following: gas temperature 350 ◦ C; drying gas flow 7 L min−1 , fragmentor 160 V; skimmer 60 V and capillary voltage 3000 V. LC–MS raw data obtained from LC-QqQMS analysis of plasma samples were analysed with MassHunter quantitative software manufactured by Agilent (Santa Clara, CA). Two MRM transitions were selected for each analyte, precursor ion and two fragments ions, the first with highest signal for quantification and a second one for qualitative identification confirmation. 2.6. Validation The method was first developed with commercially available wild salmon plasma, fished far from urban effluent, in order to make a complete validation in the matrix. Wild salmon is also less fat than culture salmon, which is more similar to pike. Validation was also performed in a wild salmon plasma matrix, spiked with a maximum of 5% of spiked solution at various concentrations which solvent was ACN/H2 O (20:80, v/v), in order to minimize protein plasma precipitation and solvent effects on the solubility of drugs in plasma. Quantification was done with standard curves for each analyte based on relative responses of the peak area ratio to their respective internal standard versus the corresponding concentration ratio. Validation parameters were determined according to FDA guidance for bioanalytical method validation [16]. 2.6.1. Linearity and limit of quantification Linearity was evaluated with a 5 point standard curve from 1 to 100 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and from 2 to 200 ng mL−1 for IBU and 2-OH-IBU with fixed concentration of their respective labeled internal standard. Linear regression was chosen in such a way that the correlation coefficients were ≥0.995 for all analytes. Lower limit of quantification (LLOQ) and higher limit of quantification (HLOQ) were evaluated on triplicate respectively at the lowest and highest point of the standard curve. The relative standard deviation (RSD) should not exceed 20% for LLOQ and 15% for HLOQ for the precision. The mean value should not exceed 20% of the theoretical value for the LLOQ and 15% for HLOQ for the accuracy. 2.6.2. Precision and accuracy Precision and accuracy were evaluated with triplicates of quality controls using plasma spiked at 3 different concentrations (lowmid-high) within the standard curves, namely 3, 30 and 60 ng mL−1
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model
ARTICLE IN PRESS
CHROMA-358884; No. of Pages 8
S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
4 Table 2 MS parameters for all pharmaceutical residues. Drugs
RT (min)
Quant/Qual
Prec Ion
Prod Ion
Frag (V)
CE (V)
Polarity
Sulfamethoxazole
2.77
SMX-13 C6 Salbutamol
2.77 4.64
Albuterol-d3 2-OH-ibuprofen + NH4+
4.64 5.25
2-OH-Ibuprofen-d6 Trimethoprim
5.25 6.97
Trimethoprim-d3 Naproxen
6.97 7.44
Naproxen-d3 Diclofenac
7.44 8.55
Diclofenac-13 C6 Ibuprofen + NH4+
8.55 8.88
Ibuprofen-d3 + NH4+
8.88
Quan Qual Quan Quan Qual Quan Quan Qual Quan Quan Qual Quan Quan Qual Quan Quan Qual Quan Quan Qual Quan
254.2 254.2 260.1 240.2 240.2 243.2 240.2 240.2 246.1 291.1 291.1 294.2 231.1 231.1 234.1 296.1 296.1 302.1 224.2 224.2 227.2
108.1 156 114 148 165.9 151.1 163 205.1 211.1 230.2 261.2 230.1 170.1 185.1 188.1 215.1 250.1 221.0 161.1 207.1 164.0
106 106 106 96 96 96 70 70 70 149 149 149 70 70 70 90 90 90 65 65 65
13 13 13 9 9 9 9 9 7 15 15 15 5 5 5 5 5 5 0 0 0
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
Abbreviations: Quan: Quantitative ion; Qual: Qualitative ion; Prec Ion: Precursor ion; Prod Ion: Product Ion; Frag : Fragmentation voltage; CE : collision energy.
for DCF, NAP, SMX, TRIM and SAL and at 6, 60 and 120 ng mL−1 for IBU and 2-OH-IBU. The RSD of the mean for each concentration should not exceed 15% for the precision. The mean value should not exceed 15% of the theoretical value for the accuracy. 2.6.3. Recovery/extraction efficiency/matrix effect The recovery, matrix effect and extraction efficiency were all evaluated in a same extraction experiment. Parameters were evaluated with triplicates of quality controls spiked at 3 different concentrations (low-mid-high) within the standard curve, namely 3, 30 and 60 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and 6, 60 and 120 ng mL−1 for IBU and 2-OH-IBU, before and after extraction on SPE. Reference solutions at the same concentrations are also used to calculate overall recovery. The recovery tests were calculated as follow: Extraction efficiency = QC spiked before extraction/QC spiked after extraction
Matrix effect = QC spiked after extraction/standard solution
Recovery = QC spiked before extraction/standard solution
3. Results and discussion 3.1. Extraction of analytes from fish plasma The methods currently published for the extraction of pharmaceuticals in fish plasma use polymeric adsorbent type Oasis HLB ® ® cartridges from Waters or Strata-X cartridges from Phenomenex because of their hydrophilic-lipophilic selectivity. This type of adsorbent was used for the analysis of DCF by ESI-LC–MS/MS [17], DCF, SMX and TRIM by ESI-LC–MS/MS [2], NAP, IBU and DCF by GC–MS [14], NAP and DCF by LC–MS/MS [18] and NAP, IBU ® and DCF by heated electrospray (HESI)-LC–MS/MS [19]. Waters Oasis MCX SPE also uses a mixed-mode type adsorbent, reversedphase/strong cation-exchange water-wettable polymer which is suitable for the purification of basic and neutral molecules. As an example, this adsorbent was used for DCF, NAP, IBU, 2-OH-IBU and SAL by LC–MS/MS [20]. However, none of these studies have covered the analyses of the present selected drugs representing a range of physico-chemical properties. In the present study, two SPE car-
tridges were tested for the extraction of selected the seven analytes using 100 uL of fish plasma. ® The Waters Oasis PRiME HLB SPE cartridge removes phospholipids and in turn, most of matrix effects in LC–MS/MS. In addition, the Oasis PRiME HLB SPE requires no conditioning. Different pH values at the loading step, and several washing and elution solvents were tested. This SPE cartridge is neutral but the pH at the load step was influencing the retention of drugs. At acidic pH, all ® drugs were retained except SAL. The Waters Oasis MCX mixedmode cartridge (reverse phase/strong cation-exchange) was also tested at different pH values during the loading step, and several different washing and elution solvents. Loading at pH 2 with 10 mM citric acid was successful for all drugs with an optimal recovery. The elution solvent was optimized for the recovery and to reduce matrix effect of each drug. IBU, 2-OH-IBU and SAL were retained by hydrophilic and lipophilic interactions and eluted with a solvent mixture, while DCF, SMX, NAP and TRIM were retained by ionic exchange interactions with SO3 − contained in the MCX and required the addition of a strong base, ammonium hydroxide, to be eluted. A solvent mixture was developed to achieve optimal recovery for all drugs, since with ACN or MeOH alone, or in combination, the recovery for SAL, DCF and SMX was not optimal. The addition of 10% isopropanol (IPA) increased the recovery of SAL, the addition of 10% methylene chloride (DCM) increased the recovery of DCF and the addition of 10% methyl tert-butyl ether (MTBE) increased the recovery of SMX. IBU, 2-OH-IBU and SAL were eluted with a mixture of ACN/IPA/DCM/MTBE (70:10:10:10, v/v/v/v), while DCF, SMX, NAP and TRIM were eluted with a mixture of ACN/IPA/DCM/MTBE/NH4 OH (68:9:9:9:5, v/v/v/v/v).
3.2. Chromatography and mass spectrometry Separation and retention of analytes were not possible with an HILIC chromatography because of the limited retention of compounds such as DCF, SMX, and IBU. The separation of analytes with ® good retention was efficiently performed using a Waters CORTECS C18 + column (150 × 2.1 mm, 2.7 m, 90 Å) (Fig. 1). This column containing a positively charged surface allowed better peak shape for basic compounds such as SAL and TRIM. A gradient composed of methanol and ammonium acetate buffer at pH 5.5 allowed the separation of all compounds in a run time of 10 min plus an equilibration time of 4 min. Maximum sensitivity in MS was achieved by select-
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model CHROMA-358884; No. of Pages 8
ARTICLE IN PRESS S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
5
Fig. 1. MRM chromatogram corresponding to the analysis of salmon plasma spiked with all analytes at 100 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and at 200 ng mL−1 for IBU and 2-OH-IBU.
ing collision energy providing best signal of product ions used for quantification. A second ion product was chosen to confirm the identity of the analytes. 3.3. Method validation parameters Quantification was done with relative responses based on the peak area ratio of analytes to their respective internal standard versus the corresponding concentration ratio. All validation results follow FDA guidelines for bioanalysis [16], except for matrix effect and QC’s below LLOQ. Matrix effects were higher than values permitted in FDA guidelines, but since internal standard are isotopically labeled drugs, they are corrected efficiently. All validation results are given in Table 3. 3.3.1. Linearity/LLOQ/HLOQ Linear range was evaluated with 5 calibration points from 1 to 100 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and from 2 to 200 ng mL−1 for IBU and 2-OH-IBU. Standard curves were linear (R2 > 0.995) for all drugs. The LLOQ was evaluated on triplicates and valued at 1 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and 2 ng mL−1 for IBU and 2-OH-IBU and was reproducible for all drugs with precision from 0.3% to 9% and an accuracy from 99% to 110%. Other studies have also evaluated LOQ (or MQL as Method Quantification Limit) as 10 times the signal to noise ratio (s/n) of target analytes. MQL of naproxen (2.3 pg/L), ibuprofen (2.1 pg/L) and diclofenac (1.6 pg/L) have been evaluated by [19] in 20 L of zebrafish plasma. LOQ of multiple drugs [14] have been evaluated in rainbow trout plasma exposed to undiluted effluent from three different stations. The reported LOQs varied from 0.05 to 5 ng/mL for 0.5 mL of extracted plasma. LOQ of carbamazepine, bisoprolol, diclofenac, ibuprofen and naproxen have also been evaluated by [21] and ranged from 0.86 to 20 ng/mL without clarifying single values, in 300 L of rainbow trout plasma. LLOQ results from the present work are thus lower than previously published studies considering the volume of plasma used, and results have been
proven to be precise, accurate and reproducible. The higher limit of quantification (HLOQ) was evaluated on triplicates and valued at 100 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and 200 ng mL−1 for IBU and 2-OH-IBU with a precision from 0.2% to 2.3% and an accuracy from 99% to 106%.
3.3.2. Precision/accuracy Precision results ranged between 3% to 11% while accuracy values ranged between 94% to 105% for all drugs. Since results from pike samples were all below LLOQ, additional QC’s were evaluated at 0.1, 0.3, and 0.5 ng mL−1 for DCF, NAP, SMX, TRIM and SAL and 1 ng mL−1 for IBU and 2-OH-IBU. Precision results ranged between 4% to 27% while accuracy values ranged between 84% to 147% for all drugs at determined concentrations, except IBU which was not detectable below LLOQ.
3.3.3. Recovery/matrix effect/extraction efficiency The recovery values were between 94 to 111% for all drugs. Matrix effect values ranged from 15% to 73% of signal suppression and extraction efficiency values ranged from 57 to 89%. Results demonstrated that the extraction efficiency did not take into account the matrix effect and signal suppression generated by matrix effect was reflected in the extraction efficiency. The recovery took into account both effects and confirmed that quantification by ratio with internal standard was effective to counterbalance the matrix effect.
3.3.4. Selectivity The selectivity was evaluated on triplicate blank salmon plasma. The matrix blanks contained no peak of all drugs except 4% of LLOQ for DCF and 8% of LLOQ for TRIM, which is below the value of 20% accepted by FDA guidelines.
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
G Model
ARTICLE IN PRESS
CHROMA-358884; No. of Pages 8
S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
6 Table 3 Validation results of all pharmaceuticals residues. Validation Results
IBU
2-OH-IBU
DCF
NAP
SMX
SAL
TRIM
Linearity (Concentration) Linearity R2 LLOQ concentration (ng/mL) LLOQ (Precision; n = 3) LLOQ (Accuracy; n = 3) HLOQ concentration (ng/mL) HLOQ (Precision; n = 3) HLOQ (Accuracy; n = 3) Selectivity (% of LLOQ; n = 3) Precision (3 QC low-mid-high; n = 3) Accuracy (3 QC low-mid-high; n = 3) Extraction efficacity (3 QC low-mid-high) Matrix effect (3 QC low-mid-high) Recovery (3 QC low-mid-high; n = 3) QC below LLOQ (ng/mL) Precision (n = 3) Accuracy (n = 3)
2–200 ng/mL >0.996 2 7.5% 103% 200 0.6% 106% 0% <11% 102–105% 82–87% 122% ± 6% 98–107% 1 NA NA
2–200 ng/mL >0.997 2 3.9% 100% 200 2.3% 102% 0% <6% 94–103% 79–89% 115% ± 4% 94–100% 1 20% 104%
1–100 ng/mL >0.998 1 2.8% 109% 100 0.9% 100% 4% <5% 97–100% 79–84% 128% ± 3.9% 101–104% 0.3 11% 84%
1–100 ng/mL >0.999 1 4.8% 107% 100 1.3% 100% 0% <4% 98–101% 82–84% 126% ± 0.6% 104–105% 0.3 11% 137%
1–100 ng/mL >0.999 1 2.0% 99% 100 1.2% 100% 0% <4% 96–101% 72–74% 146% ± 0.5% 104–111% 0.5 6% 147%
1–100 ng/mL >0.999 1 8.8% 110% 100 0.2% 100% 0% <3% 99–102% 57–62% 173% ± 6.7% 100–104% 0.3 4% 137%
1–100 ng/mL >0.999 1 0.3% 105% 100 0.2% 99% 8% <3% 99–102% 76–78% 130% ± 1.9% 100% 0.1 27% 133%
Abbreviation: LLOQ: Lower Limit of Quantification; HLOQ: Higher Limit of Quantification; QC: Quality Control at low. mid and high concentration.
3.4. Application to environmental samples Samples of wild pike plasma (n = 49), collected over a three-year period downstream and upstream of a wastewater discharge in the St. Lawrence River, were analyzed in duplicate for IBU, 2-OH-IBU, DCF, SMX, SAL, NAP, and TRIM, and results are shown in Table 4. The detected levels for all samples were below the limit of quantification (LLOQ) validated in the method. However, chromatographic peaks were present for DCF, NAP, TRIM and SAL in several samples. Quantification of these samples would be an extrapolation of the linearity of the quantification curve. Three additional QCs were therefore extracted at concentrations below LLOQ (i.e., 0.5, 0.3 and 0.1 ng mL−1 ) to determine the precision and accuracy for each drug. The results (Table 3) showed that the precision was variable and the accuracy was lower below the LLOQ but allow the interpretation of results obtained in fish plasma. Several results presented in Table 4 have values below the concentrations used for QC evaluated below LLOQ, but duplicate relative standard deviation (RSD) values were presented in order to give an overall picture for a maximum of specimens. The selected pharmaceuticals were detected in St. Lawrence pike, however no pattern of high contamination could be observed through the years or among individuals and sites. When extrapolating concentrations, NAP was found in fish collected at the downstream site in 2014 with a RSD of 43% and in 2016 fish from the upstream and downstream sites with RSD between 5 and 31%. SAL was found in a single sample collected downstream of the effluent outfall in 2014 with a RSD of 20%. DIC and TRIM were detected in fish plasma samples in 2014 and only DIC was detected in collected samples downstream in 2016. None of the investigated drugs were detected in samples from 2015. Overall, there was no significant difference between the samples collected downstream and upstream of the effluent discharge point. Pike are known to be sedentary to these specific sites. Also, when comparing plasma concentrations to those measured in the surface water samples collected by [22] in the St. Lawrence River downstream of the same effluent, levels for NAP (approx. 4 ng L−1 ), 2-OH-IBU (approx. 100 ng L−1 ) and IBU (approx. 25 ng L−1 ) as well as SMX (approx. 12 ng L−1 ), and TRIM (approx. <10 ng L−1 ) [6] were less concentrated in fish than in water. These results may indicate that the high flow of the river which causes a major dilution of the effluent could limit the exposure of fish to the pharmaceuticals and explain the lack of differences in pharmaceutical concentrations found in fish samples upstream and downstream of the effluent discharge point.
The ionization of the drugs could not solely explain the differences between the results for DIC, NAP, SMX, IBU, and 2-OH-IBU, which are negatively ionized and SAL which is positively ionized in the effluent plume at a stable pH value of 8.1. The chosen drugs were thus all ionized under environmental conditions except TRIM, which was predominantly neutral. Several studies investigated the absorption and the metabolism of pharmaceuticals in fish in comparison to humans [11,13,23] and differences in cytochrome (CYP) activities were observed. The habitat and feeding ecology can also influence the absorption and metabolism of pharmaceuticals in fish, and consequently, drug clearance. The plasma samples from wild pike were little contaminated with the investigated drugs, but the method still allowed record of signal for lower concentrations. Values previously published by [2,9] were indeed much higher in other rivers influenced by the relatively high flow of urban effluents which were less diluted by the river flow. The present developed method could be easily utilized in more contaminated environments, such as rivers in Texas [2] and Japan [9], to characterize the plasma concentrations of validated drugs and possibly several other pharmaceuticals with further validation. This developed method can indeed allow the detection of very low concentrations of pharmaceuticals of environmental interest in a relevant biological matrix. 4. Conclusion An analytical method based on SPE and LC-QqQMS was developed to identify and analyze DCF, IBU, 2-OH-IBU, NAP, SMX, SAL, and TRIM in plasma of wild pikes exposed in their natural habitat to municipal wastewater. The advantages of this method were the simultaneous analysis of six polar drugs and a common metabolite (2-OH-IBU) (log Kow = 0.79–4.26) in a small volume of 100 L of fish plasma with low LLOQ. Simple SPE extraction and chromatography with an ESI source in LC–MS/MS were successfully developed. Method sensitivity has been determined with a LLOQ of 1 ng mL−1 for DCF, NAP, SMX, SAL and TRIM and a LLOQ of 2 ng mL−1 for IBU and 2-OH-IBU in reproducibly manner with high precision and accuracy. Moreover, the analysis of plasma represents a less-invasive approach to collect small biological samples and monitor pharmaceuticals in fish. Thus, the developed method allowed the quantification of low concentrations of pharmaceuticals in fish plasma with better LLOQ than those reported in previous published studies, with high precision, accuracy and recovery. Therefore this method allowed for quantification of the selected drugs in plasma of wild pike chronically exposed to low levels of environmental contamination. This method could easily be used in more contam-
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
TRIM
NAP
Site
ID
Concentration
RSD
Concentration
RSD
Concentration
2014
IB
1 3 5 9 10 1 2 3 4 5 6 7 8 9 10
0.29 0.30 0.48 ND 0.14 0.16 0.15 0.42 0.15 ND ND ND ND ND ND
7% 32% 16%
0.13 0.13 0.14 0.14 0.12 0.17 0.13 0.13 ND ND ND ND ND ND 0.03
11% 17% 4% 14% 3% 18% 21% 19%
ND ND ND ND ND ND ND ND ND ND ND ND ND 0.10 ND
4 6 8 9 10 12 13 14 3 7 9 10 11 12 13 14 15
ND ND ND ND ND ND ND ND ND 0.20 ND ND ND ND 0.30 ND ND
0.12 0.10 0.12 0.11 0.09 0.13 0.10 ND 0.03 ND ND ND 0.04 ND 0.05 ND ND
3% 2% 40% 10% 41% 24% 3% – 6%
IV
2016
IB
IV
33% 29% 5% 11% 4%
36%
– – 23%
39%
26% 39%
0.20 ND 0.21 0.23 ND 0.18 0.19 0.21 ND 0.19 ND 0.18 0.18 0.20 0.16 0.15 0.22
RSD
43% 17% – 20% 11% 6% 8% 9% 5% 15% 22% 13% 7% 15% 31%
Concentration ND ND ND ND ND ND 0.09 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
SMX RSD
20%
Concentration
2-OH-IBU RSD
Concentration
IBU RSD
Concentration
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
RSD
Abbreviations: RSD: relative standard deviation; ND: Not detected. Concentrations in italics were below the LLOQ but the RSD was still acceptable. Site: IB: Îles de Boucherville, Upstream; IV: Îlet Vert, Downstream. a Data in 2015 were all ND.
ARTICLE IN PRESS
Year
SAL
G Model
CHROMA-358884; No. of Pages 8
DCF
S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055
Table 4 Mean concentrations (ng/mL) and RSD (%) of selected pharmaceuticals in plasma of northern pike.a
7
G Model CHROMA-358884; No. of Pages 8
ARTICLE IN PRESS S. Poirier Larabie et al. / J. Chromatogr. A xxx (2017) xxx–xxx
8
inated environments for the characterization of validated drugs in fish plasma and potentially other drugs with further validation. Furthermore, the chromatographic and mass spectrometry conditions developed could be further used to analyze these drugs in other fish tissues and allow a better understanding of the overall body distribution of pharmaceuticals in these vertebrates.
[10]
[11]
Acknowledgments
[12]
We are grateful to Mélanie Douville, Martin Pilote, Julie Reinling, Guillaume Cottin, and Alexandre Bernier-Graveline for their help in the field. This work was supported by the St. Lawrence Action Plan.
[13]
[14]
References [1] L. Viglino, K. Aboulfadl, A.D. Mahvelat, M. Prévost, S. Sauvé, On-line solid phase extraction and liquid chromatography/tandem mass spectrometry to quantify pharmaceuticals, pesticides and some metabolites in wastewaters, drinking, and surface waters, J. Environ. Monit. JEM 10 (2008) 482–489. [2] W.C. Scott, B. Du, S.P. Haddad, C.S. Breed, G.N. Saari, M. Kelly, L. Broach, C.K. Chambliss, B.W. Brooks, Predicted and observed therapeutic dose exceedances of ionizable pharmaceuticals in fish plasma from urban coastal systems, Environ. Toxicol. Chem. 35 (2016) 983. [3] D. Fatta-Kassinos, S. Meric, A. Nikolaou, Pharmaceutical residues in environmental waters and wastewater: current state of knowledge and future research, Anal. Bioanal. Chem. 399 (2011) 251–275. [4] F.O. Agunbiade, B. Moodley, Occurrence and distribution pattern of acidic pharmaceuticals in surface water, wastewater, and sediment of the Msunduzi River, Kwazulu-Natal, South Africa, Environ. Toxicol. Chem. 35 (2016) 36–46. [5] C.L. Chitescu, G. Kaklamanos, A.I. Nicolau, A.A.M. Stolker, High sensitive multiresidue analysis of pharmaceuticals and antifungals in surface water using U-HPLC-Q-Exactive Orbitrap HRMS. Application to the Danube river basin on the Romanian territory, Sci. Total Environ. 532 (2015) 501–511. [6] C. Robert, Bolduc, Anouk, DeBlois, Christian, Résultats du suivi des produits pharmaceutiques et de soins personnels ainsi que des hormones dans des eaux usées, de l’eau de surface et de l’eau potable au Québec, MDDEFPQ, Québec, 2009, pp. 115. [7] V. Christen, S. Hickmann, B. Rechenberg, K. Fent, Highly active human pharmaceuticals in aquatic systems: a concept for their identification based on their mode of action, Aquat. Toxicol. 96 (2010) 167–181. [8] A.J. Ramirez, R.A. Brain, S. Usenko, M.A. Mottaleb, J.G. O’Donnell, L.L. Stahl, J.B. Wathen, B.D. Snyder, J.L. Pitt, P. Perez-Hurtado, L.L. Dobbins, B.W. Brooks, C.K. Chambliss, Occurrence of pharmaceuticals and personal care products in fish: results of a national pilot study in the united states, Environ. Toxicol. Chem. 28 (2009) 2587–2597. [9] R. Tanoue, K. Nomiyama, H. Nakamura, J.-W. Kim, T. Isobe, R. Shinohara, T. Kunisue, S. Tanabe, Uptake and tissue distribution of pharmaceuticals and
[15]
[16] [17]
[18]
[19]
[20]
[21]
[22]
[23]
personal care products in wild fish from treated-wastewater-impacted streams, Environ. Sci. Technol. 49 (2015) 11649–11658. T.W. Valenti Jr., P. Perez-Hurtado, C.K. Chambliss, B.W. Brooks, Aquatic toxicity of sertraline to Pimephales promelas at environmentally relevant surface water pH, Environ. Toxicol. Chem. 12 (2009) 2685–2694. K.A. Connors, B. Du, P.N. Fitzsimmons, A.D. Hoffman, C.K. Chambliss, J.W. Nichols, B.W. Brooks, Comparative pharmaceutical metabolism by rainbow trout (Oncorhynchus mykiss) liver S9 fractions, Environ. Toxicol. Chem. 32 (2013) 1810–1818. J.N. Brown, N. Paxéus, L. Förlin, D.G.J. Larsson, Variations in bioconcentration of human pharmaceuticals from sewage effluents into fish blood plasma, Environ. Toxicol. Pharmacol. 24 (2007) 267–274. T.H. Hutchinson, J.C. Madden, V. Naidoo, C.H. Walker, Comparative metabolism as a key driver of wildlife species sensitivity to human and veterinary pharmaceuticals, Philos. Trans. R. Soc. B Biol. Sci. 369 (2014). J. Fick, R.H. Lindberg, J. Parkkonen, B. Arvidsson, M. Tysklind, D.G.J. Larsson, Therapeutic levels of levonorgestrel detected in blood plasma of fish: results from screening rainbow trout exposed to treated sewage effluents, Environ. Sci. Technol. 44 (2010) 2661–2666. C. Gagnon, A. Lajeunesse, Low removal of acidic and hydrophilic pharmaceutical products by various types of municipal wastewater treatment plants, J. Xenobiotics 2 (2012) 13–17. Guidance for Industry Bioanalytical Method Validation, U.S. Department of Health and Human Services Food and Drug Administration, 2001. B. Du, S.P. Haddad, A. Luek, W.C. Scott, G.N. Saari, L.A. Kristofco, K.A. Connors, C. Rash, J.B. Rasmussen, C.K. Chambliss, B.W. Brooks, Bioaccumulation and trophic dilution of human pharmaceuticals across trophic positions of an effluent-dependent wadeable stream, Philos. Trans. R. Soc. Lond. B Biol. Sci. 369 (2014). F. Cuklev, E. Kristiansson, J. Fick, N. Asker, L. Förlin, D.G.J. Larsson, Diclofenac in fish: blood plasma levels similar to human therapeutic levels affect global hepatic gene expression, Environ. Toxicol. Chem. 30 (2011) 2126–2134. F. Chen, Z. Gong, B.C. Kelly, Rapid analysis of pharmaceuticals and personal care products in fish plasma micro-aliquots using liquid chromatography tandem mass spectrometry, J. Chromatogr. A 1383 (2015) 104–111. Y. Aminot, Etude de l’impact des effluents urbains sur la qualité des eaux de la Garonne estuarienne: application aux composés pharmaceutiques et aux filtres UV, UMR EPOC, Universite Bordeaux 1, Bordeaux, 2013. M. Lahti, J.-M. Brozinski, A. Jylhä, L. Kronberg, A. Oikari, Uptake from water, biotransformation, and biliary excretion of pharmaceuticals by rainbow trout, Environ. Toxicol. Chem. 30 (2011) 1403–1411. A. Lajeunesse, C. Gagnon, Determination of acidic pharmaceutical products and carbamazepine in roughly primary-treated wastewater by solid-phase extraction and gas chromatography–tandem mass spectrometry, Int. J. Environ. Anal. Chem. 87 (2007) 565–578. E.M. Smith, F.I. Iftikar, S. Higgins, A. Irshad, R. Jandoc, M. Lee, J.Y. Wilson, In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes, Aquat. Toxicol. 109 (2012) 259–266.
Please cite this article in press as: S. Poirier Larabie, et al., Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.09.055