A multi-residue chiral liquid chromatography coupled with tandem mass spectrometry method for analysis of antifungal agents and their metabolites in aqueous environmental matrices

The presence and fate of antifungal agents in the environment have hardly been investigated. This is despite the increased usage of antifungal agents and higher prevalence of antifungal resistance. Stereochemistry of antifungal agents has been largely overlooked due to lack of analytical methods enabling studies at the enantiomeric level. This paper introduces a new analytical method for combined separation of achiral and chiral antifungal agents and their metabolites with the utilization of chiral chromatography coupled with triple quadrupole tandem mass spectrometry to enable comprehensive pro ﬁ ling of wide-ranging antifungal agents and their metabolites in environmental matrices. The method showed very good linearity and range ( r 2 > 0.997), method accuracy (61 – 143%) and precision (3 – 31%) as well as low (ng L (cid:1) 1 ) MQLs for most analytes. The method was applied in selected environmental samples. The following analytes were quanti ﬁ ed: ﬂ uconazole, terbina ﬁ ne, N -desmethyl-carboxyterbina ﬁ ne, tebuconazole, epoxiconazole, propiconazole and N -deacetyl ketoconazole. They were predominantly present in the aqueous environment (as opposed to wastewater) with sources linked with animal and plant protection rather than usage in humans. Interestingly, chiral fungicides quanti ﬁ ed in river water were enriched with one enantiomer. This might have consequences in terms of their ecological e ﬀ ects which warrants further study.


Introduction
Antifungal agents are widely used as pharmaceuticals, in household products and in agriculture, which has an impact on the environment. The global reporting of fungal diseases has increased signicantly in recent years because of an increasing population leading to a rise in the use of antifungal drugs. 1 Generally, there are 3 classes of antifungal agents used in medicines. These are azoles, polyenes, and allylamines. 2 Azole antifungal agents can also be used in an anti-dandruff shampoo 3 and for material preservation in paints, plastics, sealants, wall adhesives, binders, papers, or polymerised materials, such as leather, rubber, and paper. 4 As a result, antifungal agents, especially azoles, have emerged as a new group of pollutants in the environment and a risk to human health due to unintentional (non-clinical) exposure. 5,6 Furthermore, fungicides are commonly used on fruits and vegetables because fungal diseases are a major threat to crop production. The use of fungicides improves crop yield, quality and shelf-life. In the European Union (EU), fungicide sales constitute more than 40% of the total pesticide sales. In wine-growing regions, fungicides may account for more than 90% of all pesticide applications. Moreover, the trend of fungicide use is predicted to rise because of climate change, development of antifungal resistance, and invasive fungal species. 7 Antifungal agents are found in surface waters and wastewater at up to mg L À1 levels. 8 Although antifungal drugs and fungicides are determined at relatively low levels in the environment, there are effects of antifungal agents on the aquatic environment, humans, and animals, especially antifungal resistance, that require immediate attention. The impact of antifungal agents on the aquatic environment has been widely reported. These include effects on the survival, growth, molting, and reproduction of invertebrates. The growth rates of plants and mortality of sh were also the result of contamination with antifungal agents. 8,9 Moreover, azole agents were linked with the decrease in the formation of estradiol and testosterone in humans. 10 Worldwide emergence of resistance to antifungal drugs has been reported. The use of antifungal agents for the treatment of fungal diseases in animals, humans and plants can lead to the development of antifungal resistance. 1 Resistance in Candida spp. to triazole antifungal pharmaceuticals has increased in patients, including patients with AIDS, because triazole agents were used widely for prophylaxis and treatment. 11 In addition, azole-resistance in Aspergillus fumigatus has been found in Western European countries as well as in the Asia-Pacic due to the use of fungicides in agriculture to treat cereal crops and wheat. Thus, the risk of endocrine effects was considered in farmers and greenhouse workers from preparing azole spray mixtures. 4 An important overlooked phenomenon characteristic of many antifungal agents is their chirality. Enantiomers of the same drug have different biological properties 12 leading to enantiomer-dependent effects on human metabolism, as well as occurrence in and biological effects on the environment. [13][14][15] However, despite several papers published on the enantiomerdependent fate and effects of several pharmaceuticals, the role of stereochemistry of most antifungal agents in the context of their fate and effect remains unknown. One of the reasons for this is the lack of available sensitive and selective analytical methods that can differentiate between enantiomers of the same pharmaceutical. Though several chiral methods have been developed to analyse chiral pharmaceuticals in the environment, high-performance liquid chromatography (HPLC) is the most commonly used technique. Chiral drugs are present in the environment at trace levels and in very complex matrices. Therefore, HPLC tandem mass spectrometry with triple quadrupole (QqQ) needs to be used for sensitive targeted identication and quantication. High resolution mass spectrometry such as QTOF can also be used for retrospective analysis and suspect screening, albeit with usually lower sensitivity. There are many factors which inuence chiral recognition. These include the type of chiral selector, as well as mobile phase composition. HPLC-MS/MS has been applied in the analysis of enantiomers of antifungal agents in human serum using albumin (HSA), a1-acid glycoprotein (AGP), cellulose, and amylose columns. The occurrence of antifungal agents and their enantiomers was reported in raw wastewater, sludge, soil, and fruit samples. [16][17][18][19][20][21][22][23][24] Although the presence of antifungal agents in the environment has become a major clinical and public health problem, 1 only a few reports have been published on the investigations of antifungal agents in China, 16 Germany, 25 Switzerland, 8 Ireland, 26 Belgium, 27 Spain 28 and UK. 29 Additionally, there is a lack of research in metabolism and transformation of chiral and achiral antifungal agents in the environment. Thus, this paper's objective is to introduce a new analytical method for combined separation of achiral and chiral antifungal agents and their metabolites with the utilization of chiral chromatography coupled with triple quadrupole tandem mass spectrometry to enable comprehensive proling of wide-ranging antifungal agents and their metabolites in environmental matrices.

Sample collection and preparation
River water, wastewater effluent and inuent samples were collected in South West England in PTFE bottles as 24 h ow proportional composite samples (inuent and effluent wastewater) or grab samples (river water) and placed in a cool box with ice during the transport from the site of sampling to the laboratory. Once in the laboratory, and aer adjustment to pH 7 and addition of internal standards (to give the following concentrations: 1 ng mL À1 in wastewater and 0.5 ng mL À1 in river water or 100 ng mL À1 in SPE extracts), samples were subject to ltration and solid-phase extraction (SPE) as described below.

Solid phase extraction
SPE was carried out using Oasis HLB cartridges (60 mg, Waters, UK). The SPE protocol is discussed in detail elsewhere. 32 Briey aer ltration through a GF/F lter (0.7 mm), 100 mL of river water or 50 mL wastewater was loaded into Oasis HLB cartridges (at 3 mL min À1 ) and pre-conditioned with 2 mL of MeOH and 2 mL of H 2 O (at 1 mL min À1 ). The cartridges, aer drying under vacuum for 30 min, were then eluted with 4 mL MeOH at 1 mL min À1 . The obtained eluate was subject to evaporation under nitrogen using a TurboVap evaporator (40 C, N 2 , <5 psi) and reconstituted with 500 mL mobile phase (NH 4 OAC/MeOH 1 : 99).  ionisation source (ESI) in positive mode with an optimised capillary voltage of 3 kV, source temperature of 350 C, desolvation temperature of 350 C and desolvation gas ow of 650 L h À1 . Nitrogen, supplied by a high purity nitrogen generator (Peak Scientic, UK), was used as a nebulising and desolvation gas. Argon (99.999%) was used as a collision gas. The system was controlled using MassLynx 4.1 soware (Waters, UK). The data processing soware was TargetLynx (Waters, Manchester, UK).

Chiral liquid chromatography-mass spectrometry (cLC-MS/MS)
2.5 SPE-cLC-MS/MS performance 2.5.1. cLC-MS/MS performance. The instrument linearity and concentration range were assessed using a 21-point calibration curve with a concentration range of 0 to 1000 ng mL À1 . Internal standards were used at 100 ng mL À1 . All calibration standard solutions were run in triplicate. Standard stock solutions were prepared in methanol, acetonitrile and DMSO at 1 mg mL À1 . Mixed working solutions containing all analytes were prepared from stock solutions at different concentration levels by dilution with the mobile phase.
The instrumental limit of detection (IDL) and the instrumental limit of quantication (IQL) were measured from the calibration curve as the lowest measured concentration with an average peak signal to noise ratio (S/N) greater than or equal to 3 (S/N $ 3) across three repeat injections. The IQL was determined as the lowest measured concentration with an average S/ N $ 10 across three repeat injections.
The enantiomeric fraction (EF) was calculated from the concentration of the rst-(E 1 ) and the second-eluted enantiomer (E 2 ) of chiral compounds from eqn (1). The EF provided the relative concentration of enantiomers of chiral compounds as follows: EF equals 1 or 0 in the case of an enantiomerically pure compound, and 0.5 in the case of a racemate. 33 The resolution of enantiomeric pairs (R s ) was calculated from the retention times of the rst-(t 1 ) and the second-eluted enantiomer (t 2 ) and the widths of the responses at the baseline (w 1 , w 2 ) on the basis of the following equation: 33 Instrument accuracy and precision were calculated from eqn (3) and (4). Standard solutions were spiked in the mobile phase at 10, 100 and 500 ng mL À1 . The accuracy and precision were determined by replicate measurements of the same concentrations (three times) within one day (intra-day) (n ¼ 3) and over different three day periods (inter-day) (n ¼ 9) where x is the theoretical concentration and x 1-3 is the concentration measured in each sample. 33 Intra-day accuracy ð%Þ ¼ x 2.5.2. SPE-cLC-MS/MS performance. Relative recovery was calculated by comparison of analyte concentrations in river water or wastewater (analyte conc. x ) to analyte concentrations in the mobile phase (analyte conc. QC x ). The concentration of analyte in the blank river water and wastewater samples (analyte conc. 0 ) was subtracted from the measured concentration, to account for analyte already present in the matrix (eqn (5)). Recoveries were determined in triplicate at three different  Table 3 Instrumental performance data for selected antifungals and metabolites in the mobile phase concentrations, and then averaged. The analyte was spiked into the sample matrix with the internal standard, before ltration and SPE as described above. 33 Overall relative recovery ð%Þ ¼ Analyte conc: x À analyte conc: 0 Analyte conc: QC x Â 100 The matrix effect (ME) was calculated by comparing the concentrations of the post-spiked sample (analyte conc. ME,x ) minus analyte concentrations in the blank (analyte conc. 0 ) to analyte concentrations in the mobile phase (analyte conc. QC x ) at the following concentration levels (eqn (6)). 33 Matrix effect ð%Þ ¼ Analyte conc: ME;x À analyte conc: ME;0 Analyte conc: QC x À 1 Â 100 In environmental samples, the method detection limit (MDL) was calculated using the following equation: 34 In the same way, the method quantication limit (MQL) in the environmental samples was calculated as follows: 34 Rec is the relative recovery of the analyte in the matrix, that is the average of the recoveries obtained at three different concentrations considering the internal standard, and CF is the concentration factor.
Method accuracy (MD) was calculated (eqn (9)) to determine how close the measured concentration (analyte conc. x 1 -x 3 ) was to spiked concentrations (x) and method precision (MP) was used to measure how similar the measured concentration values were to each other (eqn (10)). The concentration of the analyte in the blank river water and wastewater samples (analyte conc. 0 ) x 1 -x 3 was subtracted from the measured concentration. The standard deviation of analyte concentration is denoted by s.

Liquid chromatography-tandem mass spectrometry
Antifungal compounds and their metabolites were analysed using cLC-MS/MS in ESI+ mode. Optimised multiple reaction monitoring (MRM) transitions are presented in Table 2. Seventeen compounds were separated using a chiral CHIRALCEL® OZ-RH column ( Table 4 Method performance data for antifungal agents and metabolites results of 2 chiral center compounds (ketoconazole, ketoconazole metabolite, epoxiconazole and propiconazole) provided 2 peaks because chemical compounds in this research study are a racemic mixture of 2 enantiomers. Other racemic compounds (tebuconazole, hydroxy-tebuconazole and prothioconazoledesthio) could not be separated and are reported as the sum of two enantiomers. The method provided very good separation and peak shapes for achiral compounds.
Inter-day and intra-day instrument precision were studied at three different concentrations, 10, 100 and 1000 ng mL À1 . As can be seen in Table 3, intra-day and inter-day instrumental precision was <15% for all compounds. Moreover, the method is characterized by high accuracy between 88 and 115% for most compounds.
The EF provided the relative ratio of enantiomers of chiral compounds. As can be seen from Table 3, EFs of econazole, epoxiconazole, miconazole, ketoconazole and its metabolite, propiconazole and prothioconazole are within 0.49-0.55 at low, medium and high concentration levels. The resolutions of enantiomers are between 0.54 and 1.87. Very good method sensitivity was achieved with IDLs ranging from 0.001 to 11.6 ng mL À1 and IQLs ranging from 0.004 to 38.6 ng mL À1 .

SPE-cLC-MS/MS performance
The SPE methodology utilized a hydrophilic lipophilic balanced (HLB) copolymer as the extraction phase. SPE recoveries and matrix effects were calculated using eqn (3) and (4), respectively. As can be seen from Fig. 2-4, the SPE recoveries and matrix effects of antifungal agents are on average 98%. The recoveries of ketoconazole, miconazole, terbinane, N-desmethyl-carboxyterbinane and voriconazole of river water, inuent and effluent samples were between 80 and 119% with deviation from 100% linked with matrix effects. Lower apparent recoveries of epoxiconazole, uconazole, hydroxyte-buconazole, propiconazole, pothioconazole, and prothioconazole-desthio in the inuent are due to ion suppression as shown by the high negative percentage of matrix effects in Fig. 2-4. Table 4 shows method performance parameters. MDLs and MQLs were calculated from eqn (5) and (6), respectively. MQLs for liquid matrices ranged from 1.9 ng L À1 for naine in surface water, to 30362.5 ng L À1 for the metabolite of ketoconazole in the effluent. The MDLs and MQLs of most analytes are low enough to measure in the environment. 8,16,26,27,29,[35][36][37][38][39][40][41] EFs are within 0.46-0.64. The resolutions of enantiomeric pairs are between 0.51 and 2.04 in river water, effluent and inuent. Most Table 5 Average concentrations of antifungal agents and metabolites found in several matrices

Application to environmental matrices
The new multi-residue analytical method was applied to determine the concentration of antifungal drugs and plant fungicides in river water, inuent and effluent samples collected in South West England ( Table 5). The fungicide tebuconazole was found at the following concentrations: 252.4 AE 70.2, 927.5 AE 2.4 and 115.1 AE 37.6 ng L À1 in river water, effluent and inuent, respectively. It is worth noting that its concentrations were higher in river water than wastewater inuent indicating other than communal sources of this fungicide in the aqueous environment. Interestingly, effluent concentrations are the highest, which warrants further study regarding transformation of tebuconazole during wastewater treatment. Indeed, tebuconazole is primarily used on crops. Its metabolite, hydroxytebuconazole, was quantied only in the river water at 228.9 AE 54.8 ng L À1 conrming its usage and environmental transformation. Terbinane (used in both human and animal treatment) was also determined in river water (50.2 AE 6.5 ng L À1 ) at higher concentrations than in wastewater inuent (30.5 AE 2.4 ng L À1 ). Its metabolite, N-desmethyl-carboxyterbinane, was identied only in river water at <MDL indicating other than communal sources of this contaminant.
Fluconazole was present at <MQL and 101.0 AE 35.6 ng L À1 in river water and effluent, respectively. Epoxiconazole enantiomers (with primary usage on crops) were quantied only in river water with signicant predominance of the E 1 enantiomer: 67.3 AE 26.5 ng L À1 and 13.2 AE 4.4 ng L À1 for E 1 and E 2 , respectively. Propiconazole enantiomers (with primary usage on crops) were also quantied only in river water at concentrations of 32.2 AE 2.0 ng L À1 and 41.3 AE 0.9 ng L À1 for E 1 and E 2 enantiomers, respectively. However, only one enantiomer of deacetylketoconazole was determined in effluent wastewater at a concentration of 218.21 AE 38.62 ng L À1 . In summary, the results of this study indicate predominance of antifungal agents in the aqueous environment with sources linked with animal and plant protection rather than usage in humans. Interestingly, chiral fungicides quantied in the river water were enriched with one enantiomer. This might have consequences in terms of their ecological effects which warrants further study.

Conclusions
A new multiresidue method utilizing chiral chromatography (with a chiral CHIRALCEL® OZ-RH column) and triple quadrupole tandem mass spectrometry was developed for sensitive and selective enantiomer-dependent analysis of fungicides and their metabolites in aqueous matrices such as river water and wastewater. The method showed very good linearity and range (r 2 > 0.997), method accuracy (61-143%) and precision (3-31%) as well as low MQLs (1.9-30362.5 ng L À1 ). The method was applied in selected environmental samples. The following analytes were quantied: uconazole, terbinane, N-desmethyl-carboxyterbinane, tebuconazole, hydroxy-tebuconazole, epoxiconazole, propiconazole and N-deacetyl ketoconazole. They were predominantly present in the aqueous environment (as opposed to wastewater) with sources linked with animal and plant protection rather than usage in humans. Interestingly, chiral fungicides quantied in the river water were enriched with one enantiomer. This might have consequences in terms of their ecological effects which warrants further study, also focussed on identication of individual enantiomers.

Conflicts of interest
The authors declare no conicts of interest.