Interactions of triazole terconazole with copper and zinc cations

Terconazole is one of the triazole derivatives with an antifungal effect. It is used locally to treat vaginal yeast infections. Terconazole could interact with copper cations in the vagina or uterus of women who use an intrauterine device releasing copper cations. Other triazole derivatives in complexes with copper cations differ in their properties like toxicity or mobility. Cu2+ can be reduced in the presence of triazoles, which can lead to a Fenton-like reaction and the production of free radicals. Thus, the Cu2+/Cu+ terconazole system can be potentially dangerous. Stoichiometry, fragmentation, and stability of forming complexes were studied with tandem mass spectrometry. The Cu2+/Cu+ terconazole system was measured within the pH range of 4.0–7.2, with a concentration ratio of CuCl2 and terconazole 1:5 to 20:1, with water and ethanol as solvents (1:1). The achieved results suggest that terconazole is partially oxidized in the Cu2+/terconazole system and that this oxidation depends on the pH of a solution. Apart from the oxidized and protonated terconazole, other detected ions and complexes were not significantly affected by pH. The detection of complexes containing Cu+ is alarming because this could correspond to the occurrence of a Fenton-like reaction.


Introduction
Triazole fungicides can inhibit the enzyme lanosterol 14α demethylase (CYP51) from the family of cytochromes P450 [1,2].This enzyme catalyzes the demethylation of lanosterol in the process of ergosterol synthesis [3], which is an important fungal lipid membrane sterol that has a similar function to cholesterol in the animal cell membrane.The gradual accumulation of lanosterol and disruption of the cell membrane permeability leads to the inhibition of cell growth [4].In animals, cholesterol is produced from lanosterol.It is primarily received from food, and the damage to the animal cell membrane plays a not-so-important role.However, the activities of triazoles have several side effects on the animal cell, which are discussed in [3].
Azoles, which contain an imidazole, triazole, or tetrazole ring, are bound in the active site of this enzyme as the sixth axial ligand to the iron atom in the heme.This results in a blocking of the active site and a coordination-covalent bond between the nitrogen and iron additionally prevents the reduction of the Fe 3+ atom [1,5].Triazole chemotherapeutics are less toxic than polyene antifungals causing perforation of the fungal cell wall [6].
The coordination-covalent bond between the nitrogen atom and the iron is not specific only to the mentioned demethylase, and inhibition can also occur with other enzymes in the CYP23 family.In addition to demethylase, triazoles inhibit in the same way, e.g., aromatase, which also contains heme in the active center and catalyzes the conversion of androgen precursors androstenedione, testosterone, and 16α-hydroxytestosterone into estrogenic substances estrone, estradiol and estriol [7,8].Because of this, some triazoles are used in the treatment of hormone-dependent breast cancer.They also inhibit the CYP3A4 enzyme, an important detoxification enzyme that is also linked to testosterone metabolism [9].Triazoles can be characterized as potential endocrine disruptors because they inhibit CYP450 enzymes involved in the metabolism of steroid hormones [10], and in addition, some triazoles act as nuclear receptor ligands [11].Grains treated with triazole fungicides have been shown to endanger the reproductive cycle of farm animals, as these dietary substances disrupt their steroid hormone levels [12].
Spraying tomatoes with triazoles reduces the content of chlorophylls a and b in leaves, slows the growth of leaves and stems, and causes oxidative stress.However, the interaction of triazoles with nutrient cations in the soil can also play an important role, and thus the limited intake of nutrients causes plant wilting [13].On the other hand, the results of some other experiments point to the protective role of triazoles in stress experiments with tomatoes.Toxicological studies of triazole fungicides on model organisms such as zebrafish (Brachydanio rerio) have demonstrated their immunotoxicity [14], reproductive toxicity [15], teratogenicity, cardiotoxicity, and neurotoxicity [16].In mixtures, some triazoles show a synergistic effect [14].
The coordination-covalent bond between the N-4 atom of the triazole molecule and transition metals, which is responsible for the binding of triazoles to the active center of several enzymes, enables the formation of stable complexes in the case of free metal ions [17,18].Depending on the structure of the triazole molecule, this bond can also be formed between the N-2 atom, possibly with π electrons of the benzene ring.The complex can be further stabilized, for example, by the interaction of the metal with oxygen from the deprotonated hydroxyl group [18,19].The study of such complexes can be performed using MS/MS (tandem mass spectrometry) combined with quantum mechanical calculations, due to which we can describe the structure of the complexes and, based on fragmentation reactions, calculate the so-called appearance energy (AE) of particular complexes [19].AE is the value of the lowest necessary energy that needs to be supplied to an atom or molecule for fragmentation leading to the formation of an ion.Thus it indicates the stability of the given molecule, in our case, of the complex.Metal complexes with triazoles formed in this way can have properties, such as toxicity or mobility, different from those of the triazoles themselves, which can be dangerous to humans and the environment [20,21].The interaction of triazoles was studied, among others, with copper ions.The copper ion in complexes with triazoles can occur in the oxidation state of Cu 2+ and Cu + .The oxidation state of Cu 2+ is stabilized in the complexes in two ways: Either the number of ligands (triazoles) is usually four or more, which sterically hinders the reduction of the copper atom, or the Cu 2+ state is stabilized by the negative charge of a ligand (e.g., deprotonated triazole) [19,22].On the one hand, copper is an environmental pollutant, but above all, it was previously used as a fungicide, especially in viticulture, which led to soil contamination in vineyards.A study from 2008 focused on soil analyzes from six Czech and Moravian vineyards demonstrated that the copper limit was exceeded compared to the current legislation in all investigated locations except one.Therefore, the application of triazole fungicides in such an environment can carry various ecotoxicological risks [23].One of the commonly used fungicides is penconazole, which exhibits a higher affinity to soil particles in complex with copper ions.In this way, these pollutants can be accumulated in the soil [20].In addition, the complex of penconazole with Cu 2+ ions significantly increases aromatase inhibition compared to penconazole or Cu 2+ ions alone [21].
One of the triazole fungicides is terconazole 1), which is used to treat vulvovaginal infections caused by yeasts of the genus Candida [24].Candidiasis affects up to 75% of women worldwide at least once in their life, and 50% of women experience a recurrence.Candidiasis is the second most common type of vaginal infection after the group of bacterial infections.However, approximately one in five women may not even know about the infection due to the asymptomatic course [23].
The advantage of terconazole is that the therapeutic doses applied are more effective than those of fluconazole [25].The drug is administered as a cream or suppository.Its application was approved by the US Food and Drug Administration in 1987.Its anatomical therapeutic chemical code is G01AG02.In addition, terconazole has trypanocidal effects and could therefore be used, for example, to treat Chagas disease, which is particularly widespread in Latin America [26].If its solubility would be increased (e.g., using surfactants), terconazole could be used to treat eye infections [27,28].The LD50 value (oral administration, rat) of terconazole varies between 849 and 1741 mg kg −1 .The biological half-life is 6.9 h [29].Terconazole was determined, for example, by micellar liquid chromatography in blood plasma [30] or by the LC/MS/MS method in the liver and muscle tissue of chickens [31].
Although terconazole contains two chiral centers, there are only two enantiomers of this substance, due to the cis configuration of the dichlorophenyl group and the hydrogen on the dioxolane.Their separation can be performed using enantioselective capillary electrophoresis [32,33].Terconazole is stable in basic and neutral matrices and does not undergo thermodegradation.On the contrary, it is unstable in an acidic aqueous matrice and under oxidative conditions under which terconazole can undergo N,N' oxidation.In an acidic environment, terconazole breaks down into at least four degradation products, which have been described using MS/MS and NMR methods [34,35].
This study clarifies the reactivity of terconazole in presence of copper and zinc cations.This research aimed to provide information on the behavior of terconazole (as a part of antimitotic vagina creams) in the presence of copper (e.g., from an intrauterine device).

Full ESI-MS spectrum of terconazole
The spectrum of terconazole in ethanol with water (50 μmol dm −3 ) was measured for 8 min.The m/z signal of protonated terconazole 532.2 exhibited the highest intensity (Table 1).The m/z signal 496.2 (Fig. 2) belongs to the [(Ter-Cl-H)+H] + fragment, in which a new bond between the aryl radical and the triazole occurs after the cleavage of chlorine [35].When comparing the simulated isotopic profile of this ion (Fig. 3) with the height of the third peak (m/z 498.2) in the measured isotopic profile (Fig. 2), it can be seen that the measured profile also contains the signal of protonated monochlorinated terconazole   In previously published studies, these dechlorinated ions were identified as degradation products of terconazole solution by UV radiation [35].Terconazole also formed a dimer that produced a m/z signal of 1062.4,indicating that one of the terconazole molecules occurred here in the oxidized form [Ter] •+ .
[Ter] •+ is a radical cation formed by donating one electron from the piperazine ring [36].
The described ions and their position in the spectrum are shown in Table 1.

Full ESI-MS spectrum of terconazole with CuCl 2
The addition of an aqueous solution of CuCl 2 to terconazole dissolved in ethanol to their final equal concentration 50 µmol dm −3 caused increased oxidation of the terconazole molecule, which is evident from the intensity of [Ter] •+ (Table 2), and also from the formation of several other complexes.It is clear from the isotopic profile of the first peak of m/z of 593.0 (Fig. 4) that it represents two overlapping signals, namely [Cu(Ter-H)] + (m/z 593) containing copper in the Cu 2+ oxidation state and [CuTer] + (m/z 594) containing copper in the Cu + oxidation state.The evaluated isotopic profile shows that the second peak (m/z 594) is higher than the m/z peak of 593, and, similarly, the m/z peak of 596 is higher than it should be according to the simulation of the isotopic profile [Cu(Ter-H)] + (Fig. 5).Therefore, the peak m/z of 594 corresponds to the peak of the complex [CuTer] + and at the same time to the peak [Cu(Ter-H)] + .The signal of the ion m/z of 593.0 had the lowest intensity among the monitored signals.However, based on the intensity ratio of the signal m/z of 593.0 and the noise, the quantification limit of S/N ≥ 10 was reached.The complexes [CuTerCl] + (m/z 629.1) (Fig. 4) and [CuTer] + (m/z 594.1) were selected also for MS/MS experiments.Terconazole did not form oligomers with a copper ion with a significant presence in the spectrum, as is the case with other triazoles in the presence of this metal.This molecule is larger than the molecules of these related substances.It may affect the stability of such complexes [19,22].The described ions are shown in Table 2.

Oxidation of terconazole in the presence of Cu 2+ ions
The measurement results showed that terconazole oxidation increases in the presence of Cu 2+ ions.Depending on the ligands present, the Cu 2+ ion is normally reduced under electrospray conditions [37,38].However, there is no direct oxidation of the ligand in the case of terconazole.One of the motivations of this research was to prove or disprove the existence of potentially dangerous free radicals in terconazole complexes with copper.This phenomenon was   described in [39] (and elsewhere) in the presence of nitrogen heterocycles, amine groups and oxygen in the ligand molecule.Although the terconazole molecule is "suitable" for this, no such complex has been observed.It can be confirmed that radicals are formed in the presence of copper ions [39], however, the presence of [Ter] •+ in equimolar ratio is very low (Table 2).To determine the dependence of the ratio of the intensities of the oxidized [Ter] •+ and the protonated form [Ter+H] + of terconazole on the concentration of Cu 2+ ions, we performed a series of seven measurements (in three repetitions) of a solution of terconazole (50 μmol dm −3 ) with increasing concentrations of added CuCl 2 (10, 20, 50, 100, 200, 500, and 1 000 μmol dm −3 ) (Fig. 6).From the ratio of signal intensities of [Ter] •+ and [Ter+H] + (Fig. 7), it can be seen that in samples of CuCl 2 and terconazole, prepared in ratios 1:1, 1:5, and 2:5, the ratios of the signal intensities of oxidized terconazole to protonated terconazole amounted to 1.6% on average and increase significantly up to 9.3%, 14.7%, and

Quantification of [Ter] •+ and [Ter+H] + signals
The occurrence of both forms of terconazole, [Ter] •+ and [Ter+H] + , was derived from the intensity of the peaks belonging to their lightest isotopes (m/z 531 and m/z 532).The intensity of the first peak of [Ter] •+ (m/z 531) can be read directly from the registered spectrum.However, the second peak of [Ter] •+ also contributes to the intensity of the first peak of [Ter+H] + (m/z 532).Thus, the oxidized form causes a change in the isotopic profile of [Ter+H] •+ and the peak m/z of 532 is a composite signal, as seen in the simulation of the isotopic profiles of both ions (Fig. SI1).

Terconazole in the presence of Zn ions
To clarify the role of Cu 2+ ions in the oxidation of terconazole, analogous experiments were performed with Zn 2+ ions.The same stoichiometric ratios as in the case of Cu 2+ ions were used.Nevertheless, their reduction does not occur in the ESI environment.The aim was therefore to clarify the possible effect of the ionic strength of the solution on the oxidation of terconazole, which is identical for both ions.Therefore, using the same procedure, three times repeated sequences of seven measurements were performed: terconazole (50 µmol dm −3 ) and Zn 2+ ions with concentrations of 10, 20, 50, 100, 200, 500, and 1000 µmol dm −3 .The signal of protonated terconazole [Ter+H] + and its oxidized form [Ter] o+ was monitored.The signal of oxidized terconazole was not higher than 1% in any concentration of ZnCl 2 (Fig. SI2).It follows that the terconazole oxidation is caused by the presence of Cu 2+ ions and not by the ionic strength of the solution from which the sample was sprayed.

Determination of the stability of precursors [Ter+H] + , [CuTer] + , and [CuTerCl] +
Based on the spectra of CuCl 2 and terconazole (see above, Figs. 4, 5), the ions [Ter+H] + , [CuTer] + , and [CuTerCl] + were selected for MS/MS experiments.However, as discussed above, the signal of the [CuTer] + ion (m/z 594) is also the second peak of the [Cu(Ter-H)] + ion, and therefore, the results may be biased and loaded with error.During the CID experiments, the precursor ions fragmented immediately into several product ions.Their structures are described in the following part.To determine precursor stability using Eq. ( 1), only fragments with relative intensity higher than 10% at the highest used NCE value were selected.Furthermore, the interpolated sigmoid functions of product ions with a value of the correlation coefficient r < 0.99 were not used for the calculation of AE.Therefore, their curves are not even shown in the SI (Figs.SI3-SI5).The particular unaveraged AE values of the precursor ions, calculated using all the listed product ions, are shown in Table 3.The ion [Ter+H] + (m/z 532.2) fragmented into four product ions with a relative intensity greater than 10% at the highest used NCE value: m/z of 490.1, 277.2, 235.1, and 219.1.Averaging the AE values calculated according to Eq. (1) using the indicated product ions, an AE value of 218.51 kJ mol −1 was calculated for this precursor ion (Fig. SI4).
The precursor ion [CuTerCl] + (m/z 629.1) fragmented into two product ions with higher relative intensity than 10% at the highest used NCE value, i.e., m/z of 614.1 and 495.1.Using the same procedure as in the previous two cases, the AE value of 163.52 kJ mol −1 was determined for this precursor ion.The stability of this complex is therefore 25.2% lower than the stability of the [Ter+H] + ion (Fig. SI5).

Structure of [CuTerCl] + fragments
The fragment m/z of 614.1 corresponds to the formation of a double bond and the loss of the CH 2 group from the piperazine ring (Fig. 9A).Based on proposed degradation mechanisms [34], in the first step, an unstable product is formed from terconazole by incorporation of an oxygen atom into the piperazine ring, from which the CH 3 OH molecule is subsequently cleaved, thus forming a fivemembered ring.As with the previous precursor ion, but without a double bond, a fragment with a signal of m/z of 495.1 was formed.It belongs to terconazole without a chlorine atom, in which the bond between the aryl radical and the triazole molecule was formed, and which is oxidized on the piperazine ring (Fig. 9B) [35,36].

Effect of pH on the formation of complexes
As mentioned in the theoretical part, the pH of the vaginal area ranges from 4 to 5, and the local application of triazole fungicides can lower the pH even slightly [40,41].The pH of the solution of terconazole (50 µmol dm −3 ) and CuCl 2 (500 µmol dm −3 ) was 7.2.Therefore, it was necessary to exclude the possible effect of a lower pH on the formation of complexes or the properties of terconazole.For these experiments, a CuCl 2 concentration of 500 µmol dm −3 was used, which was also used for the fragmentation experiments above.Simultaneously, the ratio of the oxidized terconazole to protonated terconazole was sufficiently high at this concentration, i.e., 14.7% (Fig. 8).As the presence of other ions would complicate the formation and determination of the studied complexes, it was not appropriate to use a buffer.Therefore, the pH was adjusted by adding formic acid.First, four solutions of terconazole (50 µmol dm −3 ) acidified with formic acid (concentrations 50, 70, 100, and 500 µmol dm −3 ) were prepared.Then five solutions of terconazole (50 µmol dm −3 ) and CuCl 2 (500 µmol dm −3 ), (acidified with formic acid) with a concentration of 50, 70, 100, 300, and 500 µmol dm −3 .The pH was measured in all solutions (Table SI1, Table SI2) and then ESI-MS measurements were performed.The relative intensity of the mentioned ions in the dependence on the apparent pH* value (Fig. SI 6,7) was calculated as a proportion of their intensities from the total sum of the intensities of the most present ions.
In the CuCl 2 and terconazole (10:1) spectra, the presence of particular ions was relatively stable at all pH values studied (Fig. SI7).The only exception was the oxidized form of terconazole [Ter] •+ (m/z 531.2), which exhibited the relative intensity of 8.36% at pH* 7.2 (this corresponds to the presence in the spectrum, not the ratio to protonated terconazole).On the contrary, at pH* from 4.0 to 5.4, its relative intensity amounted to approx.2.21% (on average).Therefore, the terconazole protonation caused by the presence of H + ions was more significant than its oxidation caused by the presence of Cu 2+ ions, even if the CuCl 2 concentration was ten times higher than the concentration of formic acid (the sample of pH* 5.4 contained 50 μmol dm −3 of formic acid and 500 μmol dm −3 of CuCl 2 ).In addition, it can be assumed that the discussed intrauterine bodies release a lower concentration of the Cu 2+ ion than was used for the above-mentioned experiments [42,43].Even in this case, previously published degradation products formed in an acidic environment were not detected in the investigated pH range [34].The signal of m/z 266.6 belongs to the [Ter+2H] 2+ complex and the signal of m/z 315.0 belongs to the [CuTerCl+H] 2+ complex.Unlike the complex [CuTerCl+H] 2+ (m/z 315.0), in the case of the [CuTer] 2+ complex (m/z 297.1) cannot be determined whether it contains copper in the oxidation state Cu 2+ or Cu + together with oxidized terconazole.Since the relative intensity of this ion does not increase even at pH 7.2 (as in the case of [Ter] •+ ), it can be assumed that the From the obtained results, it is evident that the presence of copper cations (not so much zinc) significantly increases the reactivity of terconazole.Particularly alarming is the formation of terconazole radicals, the concentrations of which are directly proportional to the concentration of copper cations.Low pH values (around 5) that are common in the vagina under physiological conditions and even lower pH values during vaginal infection (around 3) increase the risk of terconazole radicals even more.Furthermore, terconazole causes the reduction of Cu 2+ to Cu + , which is known for its high reactivity and ability to trigger Haber-Weiss reactions leading to the generation of free radicals.
Therefore, it is possible to conclude that concomitant use of a vaginal cream containing terconazole against fungal diseases and a contraceptive device containing copper cations may increase the risk of oxidative stress.

Conclusions
Terconazole formed under Cu 2+ ions presence [CuTer] + and [CuTerCl] + complexes, which were further studied by ESI-MS/MS.Unlike other triazole fungicides, terconazole did not form oligomers with copper ions with a significant presence in the spectrum (probably due to its higher molecular weight affecting the stability of such complexes).The stability, determined as the AE value, of the most abundant copper complex [CuTerCl] + was 25.2% lower than that of the most abundant ion [Ter+H] + .The [CuTer] + complex, which contains copper in the Cu + oxidation state, had a stability lower by 3.6%.The fragments resulting from the Fig. 9 Fragmentation diagram of the precursor ion [CuTerCl] + (m/z 629) [34][35][36] CID experiments were in good agreement with several previously published fragments of terconazole from degradation studies.At the same time, it was found that the increasing concentration of Cu 2+ increased the intensity of the signal of [CuTerCl] + ion and the oxidation of terconazole at neutral pH.Terconazole is oxidized by donating one electron from its piperazine ring, thus forming a radical cation [36].This oxidation was mainly caused by Cu 2+ ions and possible Fenton reaction products.It was not affected by the ionic strength of the solution because the experiment with zinc ions did not increase the oxidation of terconazole.Furthermore, it was shown that in the terconazole spectra with a pH in the range of 4.1 to 5.8, simulating the pH of the vaginal area, protonated terconazole had a higher occurrence than at pH 7.2.In the spectra of terconazole and CuCl 2 (1:10), the presence of monitored ions in the pH range of 4.0-7.2 was relatively consistent, except the oxidized form of terconazole [Ter] •+ , whose occurrence increased 3.8 times at pH 7.2.Thus, pH in the pH range from 4.0 to 5.4 did not affect the abundance of terconazole complexes with copper ions.pH value influenced the oxidation of terconazole only.The degradation products that arise at acidic pH, described in the theoretical part [34], were not detected in any of the investigated samples.Even the adjusted pH simulating the pH of the vaginal area did not cause the formation of these fragments.On the contrary, dechlorinated fragments, arising according to the published degradation studies in terconazole solution under the effect of UV radiation, were detected in both MS and MS/MS spectra.The described complexes of copper ions with terconazole should be subjected to toxicological studies since they could exhibit different behavior from terconazole alone.

Preparation of solutions
The terconazole solution was prepared by dissolving in ethanol, from which a stock solution of a concentration of 5 mmol dm −3 was prepared.This was stored at a temperature of − 18 °C.A solution of a concentration of 100 µmol dm −3 was further prepared from this stock solution, which was stored at the temperature of − 4 °C.CuCl 2 and ZnCl 2 were separately dissolved in deionized water and stock solutions of a concentration of 0.01 mol dm −3 were prepared.Then, solutions of both substances with concentrations of 1000, 100, and 10 µmol dm −3 were prepared by their dilutions.These solutions were stored at − 4 °C.In all samples, both mentioned solvents (water and ethanol) were always used in the 1:1 ratio to maintain the same measurement conditions.Terconazole dissolved in ethanol was therefore mixed with the same volume of an aqueous solution of the respective ions, or formic acid, or only with deionized water, just before introduction into the quartz capillary.

Measurement conditions
ESI-MS measurements were carried out in the positive mode in the m/z range from 50 to 2000.During collisioninduced dissociation (CID) experiments, the range was adjusted to 1/3 m/z of the precursor ion up to 1000 m/z.Spectra were measured for two minutes unless otherwise noted.The sample was fed using an automatic dispenser through a quartz capillary at a flow rate of 0.5 cm 3 h −1 from a Hamilton syringe.Nitrogen was used as the nebulizing gas, and helium was used as the collision gas.The auxiliary and shielding gas flow was 15 and 20 arbitrary units, the spray voltage was 4.5 kV, and the capillary temperature was 180 °C.The Capillary Voltage (CV) and Tube Lens Offset (TLO) parameters were optimized for each ion separately.The experiments were carried out in the gas phase according to the method published in Ref. [18].

MS/MS experiments and appearance energy determination
The stability of the terconazole ion and its complexes with copper (estimated from AE [44]) was determined using the fragmentation reactions.Fragmentation was performed using CID.During the fragmentation reactions, the concentration of terconazole was 50 µmol dm −3 , and the concentration of CuCl 2 was 500 µmol dm −3 .The MS/MS sequence lasted 36 min, and the value of the normalized collision energy (NCE) increased after two minutes gradually from 0, 5,10,13,15,18,20,22,24,26,28,30,32,35,38,40,45, up to 50%.The used NCE values were then converted for the selected precursor ions to the absolute value of AE in kJ mol −1 using a conversion factor according to the method published in Ref [44].using the following procedure.The obtained points of the relative intensity of the precursor and product ion depending on the increasing NCE were fitted with a sigmoid curve.A tangent intersecting the x-axis at the point with the appropriate NCE value was obtained by the first derivative of this sigmoid function for the product ion at the point where the curves of the precursor and product ion sigmoid functions intersect.This NCE value was subsequently multiplied by a conversion factor of 7.3, see Eq. (1).The determination of this factor was previously performed on the same MS device, under the same CID conditions using the model fragmentation of benzylpyridinium ions [44].Within such a calibration, neither mass effects nor differences in density of states seriously affect the energetics derived from the ion-trap experiments.
Equation (1) for calculating the appearance energy of the precursor ion for the mass spectrometer used [44]: