Elsevier

Food Chemistry

Volume 190, 1 January 2016, Pages 529-536
Food Chemistry

Pharmaceuticals in grocery market fish fillets by gas chromatography–mass spectrometry

https://doi.org/10.1016/j.foodchem.2015.06.003Get rights and content

Highlights

  • A reproducible GC–SIM-MS method is used and validated for drugs analysis in fish.

  • Edible fish samples from the local grocery markets were evaluated.

  • Samples were homogenized, extracted, concentrated and examined.

  • DZP for the first time in edible fish fillets.

  • The concentrations were lower than the environmental fish.

Abstract

Occurrences of pharmaceuticals are evident in aquatic organisms. A reproducible gas chromatography–mass spectrometry (GC–MS) method using selected ion monitoring (SIM) has been used to determine the anti-histamine diphenhydramine (DPH), anti-anxiety diazepam (DZP), anti-seizure carbamazepine (CZP) drugs and their metabolites in grocery stores fish that were homogenized, extracted, pre-concentrated, cleaned up, and examined. Identifications of the compounds in extracts were obtained by comparing similar mass spectral features and retention properties with standards. Among nine frequently detected drugs, only DPH and DZP were observed and ranged from 0.61 to 6.21 and 1.99 to 16.57 ng/g, respectively, in fourteen fish species. These concentration values were lower than the environmental fish. Mean spike recoveries of analytes exceeded 75% with relative standard deviations (RSD) < 10%. The statistically-derived method detection limits (MDLs) for nine compounds ranged from 0.13 to 5.56 ng/g. Average surrogate recoveries were 80–85% with 4–9% RSD.

Introduction

Pharmaceuticals are a large group of chemical compounds that are being increasingly used in human and animal applications. Tons of these chemicals are produced annually worldwide (Christen et al., 2010, Santos et al., 2010). After consumption, these compounds are loaded or excreted into ecosystems via urine, feces or residues as either parent compounds or their metabolites. Wastewater treatment plants are not commonly designed to eliminate the drugs because they are non-regulated water contaminants (Daughton, 2004, Daughton and Ternes, 1999). Based on the process design of the treatment plants, the elimination rates of drugs range from <10% (e.g. atenolol and carbamazepine) to almost complete removal e.g. propranolol (Miege, Choubert, Ribeiro, Eusebe, & Coquery, 2009). As these compounds are continuously released into aquatic systems, the effluents from the wastewater treatment plants are considered as the main routes of human pharmaceuticals into the environment (Fent, Weston, & Caminada, 2006), reaching concentrations of ng/L to μg/L (Mompelat, Le Bot, & Thomas, 2009). The non-regulated pharmaceuticals and personal care products (PPCPs), as environmental contaminants, consistently exposed the aquatic organisms. Many people and researchers around the world were unaware that a new environmental health concern had emerged. Nowadays, regulatory authorities, health agencies, and professional organizations, all over the globe are greatly concerned and this drives research on the presence, occurrence, fate of the PPCPs and metabolites (Brodin et al., 2013, Kelly et al., 2007).

Pharmaceutical drugs are all around, resulting from the use in humans and livestock. Commercial and domestic use and discharge of these compounds into municipal sewage have contributed to their occurrences in the aquatic environment and organisms (Snyder, Westerhoff, Yoon, & Sedlak, 2003). Drugs and metabolites have been detected in aquatic and terrestrial organisms (Oost, Beyer, & Vermeulen, 2003), surface water (van der Ven et al., 2004, Wu et al., 2008, Wu et al., 2014), lake Michigan water and sediments (Blair, Crago, Hedman, & Klaper, 2013), municipal effluent (Gagne, Blaise, & Andre, 2006), sewage effluent (Osemwengie & Steinberg, 2001), marine sediments (Beretta, Britto, Tavares, da Silva, & Pletsch, 2014), fish-eating birds and fish (Tanoue et al., 2014), effluent-dominated river water fish (Ramirez et al., 2009), Pecan Creek fish (Mottaleb et al., 2009), German fish specimen Bank (Subedi et al., 2012), receiving marine waters and marine bivalves (McEneff, Barron, Kelleher, Paull, & Quinn, 2014). Recent studies have indicated that many of pharmaceuticals and metabolites are environmentally persistent, bioactive, and have potential for bioaccumulation (Gomez et al., 2012, Mottaleb et al., 2004, Valdes et al., 2014). Acute aquatic toxicities of drugs and metabolites were examined, by Kim et al., 2007 using marine bacteria (Vibrio fischeri), a freshwater invertebrate (Daphnia magna), and the Japanese medaka fish (Oryzias latipes). They demonstrated that Daphnia was the most susceptible among the tested organisms. Correa and Hoffmann (1999) studied the variation of response of the drugs d-amphetamine, sodium pentobarbital, diazepam, β-carboline, and saline into weak electric fish (Gymnotus carapo). They concluded a reduction of the degree of alertness by the barbiturate and a decrease in emotionality and/or stress by the benzodiazepine interfered with the novelty response. Brandao et al. (2013) evaluated biochemical and behavioral effects employing neuro-active anticonvulsant drugs (diazepam, carbamazepine, and phenytoin) on pumpkin-seed sunfish (Lepomis gibbosus). They illustrated the behavioral changes of sunfish through use of oxidative stress parameters, such as glutathione reductase, glutathione S-transferases, catalase and lipid peroxidation.

Pharmaceuticals are polar compounds commonly analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Beretta et al., 2014, Blair et al., 2013, Du et al., 2012, Du et al., 2014, McEneff et al., 2014, Ramirez et al., 2007, Ramirez et al., 2009, Tanoue et al., 2014, Valdes et al., 2014, van der Ven et al., 2004, Wu et al., 2008). Previously our research group reported the LC–MS/MS method using electrospray ionization (ESI) for analysis of 25 pharmaceuticals from environmental fish (Ramirez et al., 2009, Ramirez et al., 2007). The LC–MS/MS protocol allowed us to characterize the drugs with MDL of 0.05–6.68 ng/g, with exceptions of miconazole and ibuprofen levels, extraction recovery of 60–90%. This method also showed a strong positive matrix influence (up to 1111% for erythromycin) and or negative matrix effect (up to 95% for miconazole) using positive/negative ESI operation modes for the target compounds analysis. Recently Tanoue et al. (2014) demonstrated an isotope dilution LC–MS/MS method employing silica gel and solid phase extraction clean-up steps for determination of 17 polar PPCPs in biological samples. They reported MDL values ranging from 0.019 to 3.2 ng/g in fish liver/brain, and recoveries from of 48% to 88%, with strong matrix influences. On the other hand, Subedi, Mottaleb, Chambliss, and Usenko (2011) described GC–MS/MS approach for two pharmaceuticals (carbamazepine and diazepam) and 12 personal care products and showed similar method performance, but required a sample derivation step prior to analysis. The GC–MS/MS method provided 98% recovery and 18 ng/g MDL for carbamazepine, and 97% recovery with 3.7 ng/g MDL for diazepam.

Although LC–MS/MS method favors the analysis of polar pharmaceuticals and their metabolites from environmental matrices, the method requires about 50–150 times more volume of samples, or pure standards, per injection than GC–MS/MS analysis. Additionally, GC–MS tools are less expensive and have fewer maintenance costs compared to LC–MS. Furthermore, the prices of standards, surrogates and isotopic analog and their hazard maintenance, laboratory chemical safety and disposal costs are increasingly higher day by day. Thus, the cost effective GC–MS techniques were explored for the analysis of drugs and metabolites.

As the drugs and metabolites have been repeatedly reported as present in water and fish, the consumption of fish and water can lead to an ingestion of these compounds in humans and animals. Employing our previous experience (Foltz, Mottaleb, Meziani, & Islam, 2014), in this study, we developed and validated a gas chromatography–mass spectrometry method using the selected ion morning (GC–SIM-MS) mode. We extended the capability of this technique to analyze the frequently occurring three-parent drugs and six-metabolites, without derivatization, from edible fish fillets. Detailed protocols together with analytical method performance, sample characterization and extraction procedures are discussed. The method is applied to determine the concentration of the target compounds in 14-different varieties of fish fillets purchased from grocery markets in the Midwest region of the United States. As very limited information is available about edible fish fillets, therefore this report may generate more interest among scientists and regulatory authorities for screening other PPCPs in grocery store edible fish.

Section snippets

Solvents, chemicals and reference materials

Nine pharmaceutical drugs including their metabolites from different therapeutic classes were chosen, in this study, because three parent compounds; diazepam (DZP), diphenhydramine (DPH) and carbamazepine (CZP) were frequently detected in the environmental samples. Commercially available highest purity grade reference pharmaceuticals and metabolites standards, surrogates, the internal standard and solvents were purchased from local vendors. The reference pharmaceuticals standards included CZP,

Results and discussion

The GC–SIM-MS method was developed to exploit its applicability for frequent analysis of the detected nine pharmaceuticals and their metabolites in the edible fish fillets. Silica gel cleaned up extracts of 14-different species of edible fish, developed in salt- and fresh-water environments collected from several grocery stores of Maryville and Kansas City, Missouri were analyzed by the GC–SIM-MS method. The protocol was established and validated. Target analytes listed in Table 1 did not

Conclusion

The occurrences of antihistamine DPH and anti-anxiety DZP drugs have been confirmed by GC–SIM-MS method in edible fillets originating from 14-different species from the local grocery Stores. The concentrations of DPH and DZP were obtained as 0.61–6.21 and 1.99–16.57 ng/g, respectively, in fish that developed in fresh and salt-water, and were 2–10 times lower than the environmental fish samples. To our knowledge, the presence of DZP is reported for the first time in edible fish fillets collected

Acknowledgements

The authors acknowledge the help of Dr. Christian Daughton, U.S. EPA, Las Vegas, Nevada for initiating the pharmaceutical and personal care products analysis in the environment. Author (M.A. Mottaleb) would like to thank Dr. G. Wayne Sovocool, Retired Research Chemist, Environmental Sciences Division, U. S. EPA, Las Vegas, Nevada for editing and improving the quality of the manuscript. The authors also thank to the Research Committee, the Dean of the College and Arts and Sciences, and the CIE

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