Identification of glycidyl ethers in biological and environmental samples using LC-APCI-ms / ms

Abstract

Isopropyl glycidyl ether (IPGE) is a glycidyl ether used as a reactive diluent in epoxy resin production, but its toxicokinetics are poorly understood. To address this gap, a sensitive LC– MS / MS method was developed and validated for quantifying IPGE in rat plasma using tert- butyl glycidyl ether as an internal standard. An in-source derivatization based on the Meerwein reaction was applied, markedly improving sensitivity and achieving a lower limit of quantification of 0.01 μg / mL. The method was applied to rat plasma samples from a single-dose oral toxicity study, revealing dose-dependent superproportional systemic exposure. Also, seven IPGE metabolites were tentatively identified.

Description

[0001] Identification of glycidyl ethers in biological and environmental samples using LC-APCI-MS / MS

[0002] Isopropyl glycidyl ether (IPGE) is the member of the large glycidyl ether family frequently used as e.g., reactive diluent during the epoxy resin manufacturing process. Although the toxicity induced by this type of chemicals has been investigated in many studies of different aspects (acute, subchronic, genotoxic, reproduction, etc.), there is still little known about their toxicokinetics. To gain information about the attainable systemic concentration, a liquid chromatography - tandem mass spectrometry (LC-MS / MS) method was developed and validated for the quantification of IPGE in rat plasma using its structural analogue tert-butyl glycidyl ether as the internal standard. Two types of atmospheric pressure ionization techniques have been utilized; however, the protonated molecule ion could be observed in neither ionization mode. First, the ammonium adduct form was used for fragmentation albeit this MRM transition proved to be not sensitive enough for real study sample analysis. In order to achieve the desired sensitivity, Meerwein reaction was applied as an in-source derivatization tool to generate a product by using the ethylnitrilium ion formed from the eluent acetonitrile. This gas- phase reaction enabled us to build up a method with a substantial sensitivity increase (LLOQ of 0.01 pg / mL) compared to that one obtained with ammonium adduct. After method validation, real study samples from a single-dose oral toxicity study were analyzed to evaluate blood plasma concentration of IPGE at three dose levels. Dose-dependent superproportional systemic exposure was observed in the studied dose range (1000 - 2000 mg / kg). Additionally, seven metabolites of IPGE were tentatively identified in rat plasma: 3 -isopropoxy -2-hydroxy- 1-propanol (Ml), sulfate-conjugate of IPGE (M3), glucuronide-conjugate of IPGE (M4), 3- isopropoxy-2-hydroxypropionic acid (M5), O-isopropyl-A-acetylserine (M6), <9-(2-hydroxy- isopropyl )-M acetylserine (M7), and glutathione-conjugate of IPGE (Ml 1). Present work may pave the way to other methods that are able to quantify compounds similar to IPGE even in human plasma, which could provide valuable information to assist exposure assessment / biomonitoring in occupational health and safety studies.

[0003] Keywords

[0004] isopropyl glycidyl ether, LC-MS / MS, derivatization, rat plasma, toxicokinetics, Meerwein reaction, metabolite

[0005] IntroductionGlycidyl ethers, the commonly used materials in the chemical industry, contain at least one oxirane (epoxide) ring which can be actively taken into chemical reaction due to its strained structure. By the ring opening, it can form various types of materials since reaction partners can come from a wide variety of chemicals such as carboxylic acids, hydrogen cyanide, halogen acids, amines, aldehydes, etc.1Monofunctional glycidyl ethers and aliphatic glycidyl ethers are most frequently used as reactive diluents, viscosity reducers, flexibilizers, and adhesion promoters in the manufacture of epoxy resins which are generally prepared from phenol, such as bisphenol A and a reactive epoxide, epichlorohydrin. Glycidyl ethers can be modifiers for elastomers, fibers, and adhesives, additives in epoxy chemicals used for food contact products. Furthermore, they can function as reactive intermediates in electrical product coatings and stabilizers for chlorinated materials.2-4Epoxy resins are ubiquitous materials in all kinds of applications such as protective coatings, reinforced plastics, flooring, paving construction materials as well as bonding materials and adhesives. Thus, their production is needed on a very large scale. Consequently, occupational exposure to glycidyl ethers during the manufacturing procedure via inhalation, ingestion, and skin / eye contact can be significant. Thus, all information about their toxicological effects is relevant to the field of occupational safety and human health risk assessment.

[0006] Reactive epoxides are often suspected to cause toxic effects (i.e. idiosynchratic toxicity) through hapten formation because of their binding to nucleophilic macromolecules, such as DNA, and proteins. The reactivity, molecular weight, and solubility are all factors in the extent of adverse effect.

[0007] The toxicity of glycidyl ethers was studied as early as 1956 by Hine etal.5in different species (mice, rats and rabbits), and IPGE, / / -butyl glycidyl ether, and phenyl glycidyl ether were described as compounds exhibiting systemic toxicity following repeated exposure by inhalation. Furthermore, skin irritation and dermatitis were mentioned as potential ailments among workers with such materials. For IPGE, an LD50 value of 4200 mg / kg in rats was described in this study.

[0008] Epoxide hydrolase (EH) has been identified as the dominant enzyme responsible for the metabolism and detoxification of glycidyl ethers.1Eadsforth et al.6performed an in vivo metabolism study withl4C- / / -butyl glycidyl ether where urinary excretion was described as the main route for elimination. Three major metabolites have been identified; the hydrolytic opening of the epoxide ring occurs, followed by the conversion of the formed diol to 3 -butoxy -2-acetylaminopropionic acid, 3-butoxy-2-hydroxypropionic acid, and subsequent oxidative decarboxylation to butoxyacetic acid. Later on, Chen et al.7have identified all remainingmetabolites e.g. glutathione conjugates. Furthermore, species differences have been demonstrated with mice excreting higher amount of the administered dose in expired air as14CO2than rats.

[0009] Whilst there is a vast amount of literature available on the analytical techniques of bisphenol A

[0010] diglycidyl ethers (BADGE)8’9, the information about aliphatic glycidyl ethers are rather limited. Standardized gas chromatography methods have been included in the Occupational Safety and Health Administration (OSHA) Occupational Chemical Database for / / -butyl glycidyl ether, phenyl glycidyl ether, IPGE, and allyl glycidyl ether, under the method IDs 1616, 1619, 1620 and 2545, respectively.10A GC-FID method was described for monitoring phenyl glycidyl ether in workplace air by using a solvent desorption technique.11An application note describing an HPLC method for the analysis of allyl glycidyl ether has also been reported; however, the chromatographic details have been vaguely given.12

[0011] In our present study, IPGE (also known as 2-[(propan-2-yloxy)methyl]oxirane; 2-3-epoxypropyl isopropyl ether) was selected as a target compound from the class of aliphatic glycidyl ethers to study its toxicokinetic behavior. Besides the published data of acute toxicity and skin / eye irritation5, results of a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test according to OECD 42213are available in the Registration Dossiers of the European Chemicals Agency.14This study revealed reduced body weight gain, macro- and microscopic changes in the stomach, complete lack of fertility at the dose of 300 mg / kg body weight and above, and notably reduced fertility at the dose of 100 mg / kg bodyweight. However, there has been only an expert statement about the basic toxicokinetics derived from the chemical structure and available physicochemical and toxicological data of IPGE. Due to its hydrophilic nature with an estimated partition coefficient (log P) below 1 and its relatively low molecular weight (MW= 116.18), detection of IPGE is expected to be rather challenging in biological samples.14These properties might serve as an explanation for why there has been no bioanalytical method targeting blood plasma analysis of IPGE or any glycidyl ethers in the literature yet.

[0012] An interesting approach has been reported where glycidyl ethers were aimed to be quantified as hemoglobin adducts in mice.15The research presented a method that can estimate the potential of human exposure to glycidyl ethers. For identification of bisphenol A diglycidyl ether (BADGE), it has been essential to develop analytical methods, since its possible biotransformation to bisphenol A (BPA) has triggered a high alert on human health safety issues as an endocrine disruptor. The potential effects of occupational exposure to BADGE have beenevaluated by urinalysis of BPA and plasma hormone concentration measurements.16An LC-MS / MS for the identification of BADGE and its two hydrolytic metabolites in biological samples (blood and urine) has been presented for biomonitoring purposes in a general population.17Besides bioanalytical purposes, LC-MS / MS methods have been developed for identification of potential migrants from can coatings as a highly probable exposure source, as well.3A common point in these papers is that glycidyl ethers were detected as an adduct formed. It has been recognized that the protonated molecule ion was not produced directly in the ion source in high yield, but its ammonium adduct counterpart resulted in a much more abundant precursor ion for multiple-reaction monitoring (MRM).17-19

[0013] Since there are no papers available about bioanalytical methods on glycidyl ethers we turned our attention towards other molecules with epoxy moieties where the epoxy ring of the analyte can be exploited to construct a suitable method via derivatization reaction. Newman and Hammock screened derivatization experiments on epoxy lipids with nine thiol compounds.

[0014] 2,3,5,6-Tetrafluorobenzenethiol proved to be an optimal derivatizing agent for GC-ELMS measurements of a, P -di substituted epoxy fatty acids.20Bai et al. elaborated on two interesting techniques to create sensitive methods for compounds with epoxy rings (identified as pharmaceutical genotoxic impurities): a derivatization reaction with dimethylamine and coordination ion-spray spectrometry in order to circumvent multiple reaction products. Not only ammonium but lithium, sodium, and potassium adducts were investigated, concluding the latest one as the optimum.21Another interesting work from this research group described a gas- phase derivatization strategy via the Meerwein reaction. To conduct a reaction between epoxides and ethylnitrilium ions, acetonitrile was used as the mobile phase; it served as in situ derivatization reagent, and atmospheric pressure chemical ionization (APCI) was the preference over the other two ionization techniques (ESI and APPI) due to its superior sensitivity and robustness.22, 23An epoxy-containing drug candidate (5R)-hydroxytriptolide was quantified in human plasma using benzylamine as a derivatization reagent to enhance ESI-MS detection after the liquid-liquid extraction.24Sun et al. developed and validated an LC-MS / MS method for the determination of l,2:5,6-dianhydrogalactitol, a hexitol epoxide in plasma and tissue homogenates. Its highly polar nature and the lack of chromophores inspired the research group to use sodium diethyldithiocarbamate as a derivatization reagent.25Glowka et al. have developed a bioanalytical method for the quantification of the biologically active epoxy- metabolite formed from the pro-drug, treosulfan. 3 -Nitrobenzenesulfonic acid has been used as a selective derivatization reagent reacting only with the epoxide group and not with the hydroxyl group. Coupling a chromophore to the molecule, an RP-HPLC-UV method was developed forbioanalysis in human plasma.26

[0015] In this paper, we present a sensitive, validated LC-MS / MS method for the bioanalysis of IPGE in rat plasma. In situ in-source derivatization of the analyte and the selected structural analogue IS (tert-butyl glycidyl ether) by adapting the gas-phase Meerwein reaction provided a substantial increase in sensitivity. Toxicokinetic information about IPGE in an acute singledose oral study has been acquired for the first time demonstrating the applicability of the method presented.

[0016] Materials and Methods

[0017] Standards and reagents

[0018] IPGE (98%), tert-butyl glycidyl ether (99%), neopentyl glycol diglycidyl ether (technical grade), trifluoroacetic acid >99.0% were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, 2-propanol, formic acid, acetic acid, ammonium formate and ammonium acetate were LC-MS grade and were purchased from VWR International (Leuven, Belgium). HPLC gradient methanol was purchased from Carlo Erba Reagents S. A. S. (Vai de Reuil, France). Ultrapure water was prepared in-house by using an ELGA Veolia PURELAB Chorus water purification system (High Wycombe, UK). Blank rat plasma was prepared at Toxi-Coop Toxicological Research Center with the anticoagulant K3-EDTA and stored in a freezer (at -20 °C ±5 °C).

[0019] Calibration and quality control (QC) solutions and samples

[0020] Two IPGE stock solutions of 1 mg / mL for the calibrator and quality control (QC) solutions and one tert-butyl glycidyl ether (IS) stock solution of 1 mg / mL were prepared in methanol. Working solutions were prepared in methanol immediately after the stock solutions by serial dilution from the stock solutions to achieve 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 pg / mL (calibrator) and 0.05, 0.15, 15, 40 pg / mL (QC) spiking solutions of the analyte, and 1 pg / mL for the IS in acetonitrile. The solutions stored in a freezer (at -20 °C ±5 °C) were stable for 49 days.

[0021] Calibrator samples were prepared at the following 10 nominal concentration levels: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 pg / mL, and QC samples were prepared at the following 4 concentration levels: 0.01, 0.03, 3 and 8 pg / mL. Calibration and QC samples were prepared by spiking 50 pL of blank rat plasma with 10 pL working solution, and 150 pL solution of ISin acetonitrile.

[0022] Sample preparation

[0023] The plasma samples were thawed at room temperature. An aliquot of 50 pL of rat plasma sample was transferred into a 1.5-mL microcentrifuge tube, and then 10 pL of methanol and 150 pL of 1 pg / mL IS working solution in acetonitrile were added. Then the samples were mixed by vortex (Velp Scientifica, Usmate Velate, Italy) and were centrifuged (Heraeus Biofuge Stratos, Osterode. Germany) at room temperature for 10 minutes with 6596*g. An aliquot of 100 pL of the supernatant was pipetted into autoinjector vials and kept at 10 °C in the autosampler until bioanalysis.

[0024] LC-MS / MS parameters

[0025] A Shimadzu NexeraX2 system (Kyoto, Japan) containing two LC-30AD pumps, a DGU-20ASR degassing unit, a CTO-20AC column oven, a SIL-30AC autosampler with a CBM-20A communication bus module coupled to a Shimadzu LCMS-8060 triple quadrupole tandem mass spectrometer (Kyoto, Japan) equipped with a Peak Scientific Genius 1051 nitrogen / air generator (Inchinnan, UK) was used for the bioanalytical measurements. The injection volume was 10 pL. The chromatograph was equipped with a YMC-Triart Cl 8 column (3pm, 2.1x75 mm) with a YMC-Triart C18 (3pm, 2.1x10 mm) guard column (Kyoto, Japan) thermostated at 30 °C. The temperature of the sample tray was set to 10°C. Gradient elution was applied using the mobile phases A: 0.5% formic acid in ultrapure water and B: 0.5% formic acid in acetonitrile, at a flow rate of 0.4 mL / min. The injector washing solvent was 2-propanol. The initial eluent composition was 10% B for 0.5 min; then a linear gradient was applied to 90% B until 3.5 min post-injection, held at 90% B for 0.5 min, and decreased to 10% B over the next 0.5 min. That composition (10% B) was kept for 0.5 min for re-equilibration of the system prior to the next injection. The total run time was 5 minutes, and there was a 2-minute waiting time between injections. The mass spectrometric parameter settings were the following: MRM detection mode, atmospheric pressure chemical ionization in the positive mode, interface temperature 350 °C, desolvation line temperature 300 °C, interface voltage 4 kV, heat block temperature 300 °C, nebulizing gas flow 3 L / min, drying gas flow 5 L / min. The analyte was detected at the transitions of mass-to-charge ratio (m z) 157.90 —> 116.10 (quantifier), 157.90 74.10, 157.90 57.15 with 100 ms of dwell time and with collision energy of-14.0 V, -

[0026] 15.0 V, -19.0 V, respectively. The IS was at the transitions of (m / z) 172.00 57.25, 172.0074.15, 172.00 116.10 (quantifier) with 100 ms of dwell time and with collision energy of- 14.0 V, -15.0 V, -19.0 V, respectively. Method parameters are summarized in the Supporting Information in Table S3.

[0027] LC-MS / MS parameters for metabolite identification

[0028] During the metabolite identification some of the settings were changed. The injection volume was either 1 or 10 pL. Gradient elution was applied using the mobile phases A: 0.1% formic acid in ultrapure water and B: 0.1% formic acid in acetonitrile, at a flow rate of 0.4 mL / min. The initial eluent composition was 0% B for 0.5 min; then a linear gradient was applied to 100% B until 10 min post-injection, held at 90% B for 0.5 min, and decreased to 0% B over the next 1 min. That composition (10% B) was kept for 0.5 min for re-equilibration of the system prior to the next injection. The total run time was 12 minutes, and there was a 2-minute waiting time between injections. The mass spectrometric parameter settings were the following: single ion monitoring (SIM) detection mode, electrospray ionization in the positive and negative modes, interface temperature 400 °C, desolvation line temperature 200 °C, interface voltage 4 kV, heat block temperature 300 °C, nebulizing gas flow 3 L / min, drying gas flow 3 L / min. The analyte was detected as Meerwein reaction product at the mass-to-charge ratio (m z) 158 with 100 ms of dwell time. The metabolites were detected at the mass-to-charge ratio (m z) 135, 151, 231, 309, 149, 190, 206, 165, 119, 296, 424, 280 respectively with 100 ms of dwell time.

[0029] Method validation

[0030] The bioanalytical method was validated according to the ICH M10 guideline.27Selectivity was studied with six individual blank plasma samples collected from six different rats (three male lots and three female lots). Linearity was assessed by measuring calibrator samples in the 0.01-10 pg / mL concentration range. The analyte / IS peak area ratio was plotted as a function of the nominal concentration of IPGE and a linear calibration curve was fitted using the least squares method with 1 / x2weighing. Intra-day and inter-day accuracy and precision were evaluated at four QC concentration levels in six replicates on three different days. Carry-over was studied in system suitability tests: the highest calibrator sample was injected five times followed by one matrix blank sample and then the lowest calibrator sample. The accuracy and precision for each individual matrix lot were evaluated. The plasma stability was assessed by studying the following conditions: short-term room temperature stability, long-term stability in an ultra-low temperature freezer (-75°C ± 10 °C), three freeze-thaw cycles, processed sample stability in the autosampler, and reinjection reproducibility. Short-term room temperature working solution stability was studied by preparing plasma samples with working solutions without benchtopstorage and with solutions stored at room temperature for 4 hours. Dilution integrity was tested. Long-term stock and working solution stability were checked by preparing plasma samples with freshly prepared working solutions and with solutions kept in a freezer for 49 days. The processed samples were stable in the autosampler for at least 2 days.

[0031] Toxicokinetic study samples

[0032] A toxicological study with IPGE with supplementary toxicokinetic sampling was conducted by a single oral treatment with gavage (vehicle: sunflower oil; treatment volume 5 mL / kg) in HamWIST of Wistar origin rats (T oxi-Coop Toxicological Research Center). Three dosing groups (low dose of 1000 mg / kg, medium dose of 1500 mg / kg, and high dose of 2000 mg / kg) with nine animal s / sex / dosegroup were included in the study. The dose of 1000 mg / kg did not result in any toxic symptoms or any clinical signs in a preliminary study, therefore this dose has been selected as the low dose in the present study. The high dose of 2000 mg / kg is generally considered as the limit dose for an acute oral toxicity study, so this had been selected accordingly in this study, too.28The medium dose has been selected as an interpolation between the low and high doses. Due to the unknown elimination of the test item, nine time points as pre-dose and 0.5, 1, 2, 4, 6, 8, 12, and 24 h post-dose were scheduled for blood sampling. Sparse sampling was applied, three animals / sex were sampled at each time-point. The number of animals used throughout the experiments is shown in Table S6. We made efforts to minimize the number of animals while maximizing the number of samples, in accordance with the 3Rs principles. Blood samples were taken from the retro-orbital venous plexus under Isofluran CP® anesthesia. Animals were sampled at two or three different time points (within 0-24 hr) and were euthanized by exsanguination after over-anesthesia at the termination of sampling. The sampling volume did not exceed 1 mL / animal / day according to Diehl et al.29Blood samples were drawn into tubes containing Ka-EDTA as an anticoagulant (spray-dried MiniCollect 0.5 mL, Greiner Bio-One GmbH, Kremsmunster, Austria) and were centrifuged at 2000 / for 15 min. The plasma samples were immediately snap-frozen on dry ice and then placed in an ultralow temperature freezer (-75°C ± 10 °C) until further analysis.

[0033] Mean plasma concentration vs. time curves were subjected to non-compartmental analysis with the sparse sampling linear trapezoidal and linear / log interpolation calculation method by the Phoenix WinNonlin PK / TK software version 8.1 (Certara L.P., USA). A list of the reported toxicokinetic parameters can be found in the Supporting Information.

[0034] The animal study was reviewed and permitted by the Institutional Animal Care and Use Committee and was conducted according to the National Research Council Guide for the Careand Use of Laboratory Animals and in compliance with the principles of the Hungarian Act 2011 CL VIII (modification of Hungarian Act 1998 XXVIII) and Government Decree 40 / 2013 regulating animal protection.

[0035] Results

[0036] Optimization of LC and MS / MS conditions

[0037] The present work aimed to develop an LC-MS / MS method to support the toxicokinetic characterization of IPGE in rat. First, an LC-ESLMS / MS method was elaborated and validated using the ammonium adduct of the analyte for fragmentation. Method parameters and results are summarized in Table SI and Table S2 in the Supporting Information.

[0038] A pilot animal study with a single oral dose of 1000 mg / kg body weight was conducted to generate plasma samples, and the bioanalysis revealed that the LC-ESI-MS / MS method was not fit-for-purpose; the sensitivity was not sufficient (with an LLOQ of 5 pg / mL) for the toxicokinetic analysis. None of the plasma samples exhibited quantifiable concentration of IPGE; therefore, a derivatization approach was used to enhance the sensitivity of mass spectrometric detection retaining the same liquid chromatographic conditions used in the first method. Method parameters and results are summarized in Table S3 and Table S4 in the Supporting Information.

[0039] During method optimization the signal intensity of the derivatization product was studied with the ionization sources available. Two ion sources, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were tested, with various eluent modifiers, formic acid, acetic acid, and trifluoroacetic acid added to the eluents at various concentrations (Figure 1). APCI source provided the best result in terms of sensitivity using formic acid as an eluent additive. The injection volume of 10 pL was an optimal compromise between peak shape symmetry and sensitivity. The LC-APCI-MS / MS method ion source optimization process summary can be found in the Supporting Information (Tables S5 and Figure SI).

[0040] Figure 1. Signal intensity of the analyte (TIC = total ion chromatogram, representing the sum of the three MRM transitions [157.90 116.10, 157.90 74.10, and 157.90 57.15]) with various eluent compositions using ESI or APCI source

[0041] Method validation

[0042] LC-MS / MS methods with both ESI and APCI modes were validated (comprehensive tables presented for the validation results in the Tables S2 and S4, in the Supporting Information).The validation of the method with substantial sensitivity improvement was performed when the relevant concentration range for study sample analysis had been determined.

[0043] The selectivity criteria were fulfilled in the bioanalytical method validation. There was no interference observed above the 20% of the LLOQ sample in the studied six individual rat blank plasma samples. In Figure 2 A and B, representative chromatograms acquired at the MRM signals of the analyte and the IS are presented.

[0044] Figure 2. A) Representative MRM chromatograms of the analyte [157.90 — 116.10] and IS [172.00 —> 116.10] in LLOQ sample: analyte (solid), IS (dashed). B) Representative MRM chromatograms of the analyte [157.90 —> 116.10] with red pointing arrows to the peaks: matrix blank (black), IS blank (orange), LLOQ sample (green) and a real study sample (blue), inset: MRM chromatogram of the corresponding IS [172.00 — 116.10] in the matrix blank sample as proof of selectivity for IS (black)

[0045] As IPGE and the IS Zc / V-butyl glycidyl ether possess the same quantifier product ion, the crosstalk phenomenon did not occur by the evaluation of the blank sample with IS and the highest calibrator sample prepared without IS.

[0046] Good linearity in the concentration range of 0.01-10 pg / mL was observed with a correlation coefficient (r) not lower than 0.995. The intra-day (n=6) and inter-day (n=18) accuracy and precision met the acceptance criteria (mean accuracy: ±15%, ±20% at the LLOQ, precision < 15 CV%, 20 CV% at the LLOQ) (Table 1).

[0047] Table 1. Accuracy and precision of IPGE measurement in rat plasma

[0048] The highest precision (12.3 % and 10.3%) and accuracy (+13.7 % and -13.0 %) for the individual QC samples of 8 pg / mL and 0.03 pg / mL concentration levels were acceptable, respectively. Consequently, the matrix effect did not affect the method. The result of the recovery test was determined in two ways: peak area (106 % for 8 pg / mL, 94.6 % for 0.03 pg / mL) and peak ratio (111 % for 8 pg / mL, 114 % for 0.03 pg / mL). Performing a precipitation step with acetonitrile during sample preparation did not adversely affect the recovery. Recovery evaluation was done following the FDA’s Bioanalytical Method Validation (2018) guideline.

[0049] 30The peak area of the analyte and IS obtained for the processed low and high QC samples were compared to the peak area of the analyte and IS obtained for processed blank samples post-spiked after extraction with the corresponding spiking solutions prepared in sextuplicate expressed as peak area ratio percentage. The possibility of fivefold, fifteenfold and hundredfold dilution with blank plasma was justified. The spiked samples after three freeze-thaw cycles did not show degradation, and the percentage of the nominal concentrations were 96.6 % and 90.3 % for 8 pg / mL and 0.03 pg / mL, respectively (Table 2).Table 2. Stability results of IPGE in rat plasma

[0050] The spiked samples were stable for 4 hours at room temperature (88.9% 8 pg / mL, 95.7% for 0.03 pg / mL), but some degradation was observed after 24 hours at the same conditions (67.4% 8 pg / mL, 45.0% for 0.03 pg / mL). After the 26-day storage period in an ultra-low temperature freezer, the samples were 91.5% and 86.0% of the nominal at 8 pg / mL and 0.03 pg / mL concentration levels, respectively. The working solutions of the analyte proved to be stable for 4 hours at room temperature and also for 49 days stored in a freezer (-30 °C to -15 °C). The stock solution (1 mg / mL) of the analyte and the IS was proved to be stable for 49 days stored in a freezer (-30 °C to -15 °C). Table 3 summarizes stability data for the solutions under different storage conditions, over specific stability periods, at specified concentration ranges, showing deviations from freshly prepared counterparts in percentage values.

[0051] Table 3. Stability results of IPGE (analyte) and IS solutions

[0052] Application to toxicokinetic bioanalysis

[0053] After validation, the method was used for the bioanalysis of toxicokinetic samples in a main toxicity (conducted at three dose levels) study. Plasma samples were analyzed in three batches with duplicate QC samples at three concentration levels. None of the QC samples exceeded the accuracy value of ±14.8%. Inter-day (n=6) accuracy was +2.00%, -1.00%, and -5.00% and inter-day precision was 3.10%, 7.33%, and 10.3% at levels of QCH, QCM, and QCL, respectively. Incurred sample reanalysis (ISR) was performed on 32.2% of the samples collected from test-item-treated rats using the same plasma aliquot. 87.5% of the incurred samples met the acceptance criterion stipulated in the guideline confirming the reliability of the method (percent difference between initial and reanalysed results should be < ±20% for at least 2 / 3 of the repeats). All ISR data can be found in the Supporting Information in Table S7. IPGE was not detected in the pre-dose samples. The highest observed individual concentration of the analyte was 373 pg / mL (high-dose group), and the concentrations were below the LLOQ only in four samples of the last time-point in the low-dose group confirming the appropriate calibration range. No significant gender difference was observed in the examined toxicokinetic parameters; therefore, total (male and female data together) mean concentration profiles are presented in Figure 3 at the three investigated dose levels with each point representing the average value of a minimum of 3 and a maximum of 6 rats (at least one from each sex) with SEM (standard error of mean) values. For the 1000 and 1500 mg / kg doses, the mean of 3 female and 3 male samples is presented for each measurement point. In the case of the 2000 mg / kg dose, the sample size differs from the other two dose groups due to mortality (SupportingInformation, Table S8). Due to mortality at the high dose, the curves of the surviving and dead animals are presented separately in a different graph (Supporting Information, Figure S2). The calculated toxicokinetic parameters are given in Supporting Information Table S9. Rapid absorption was observed for IPGE with a Tmax of 0.5 hour (medium and high doses) and 1 hour (low dose). The elimination was moderately slow; the half-life was about 14-15 hours (1500 mg / kg and 2000 mg / kg doses) and about 9 hours (1000 mg / kg dose). Dose-dependent systemic exposure was seen with a distinct superproportional characteristics (Figure 4) that was explained by non-linear kinetics at the dose concentrations investigated. The differences in plasma concentrations between the low and high doses can be explained by the magnitude change in IPGE metabolism, and the non-linear kinetics are thought to be caused by the saturation of metabolizing enzymes which is likely to result in mortality at the highest dose level.

[0054] Figure 3: Plasma concentration rv. time curves in rats after oral administration of IPGE at three dose levels (A: 1000 mg / kg; B: 1500 mg / kg; C: 2000 mg / kg). The points represent the mean plasma concentrations of 3-6 animals, whereas the whiskers are for the SEM values. Inset figures show the terminal elimination phase magnified, open symbol indicates exclusion from the terminal elimination phase fit.

[0055] Figure 4. Dose normalized AUCiast values of IPGE in male and female rats at three dose levels

[0056] At the high dose, toxic effects, such as piloerection, closed eyes, incoordination, decreased / depressed motor activity, and hypotonicity, were observed in rats. Even mortality occurred in 8 cases after the maximum plasma concentration which was rather surprising since the selected dose was far below the reported LD50 value (4200 mg / kg).5At the low and medium doses there were no symptoms for any toxicological adverse effects. (The list of toxicokinetic parameters can be found in the Supporting Information.) The large SEM values can be explained by the pronounced inter-individual variability among the experimental animals. The ADME (absorption, distribution, metabolism and excretion) process might be largely different from rats to rats (depending on how distantly it exceeded the linear kinetics range), especially considering the highest dose (2000 mg / kg) triggering mortality for some animals but not for all of them. This diverse susceptibility to the administered test item is manifested by the various plasma concentration values and resulted in large SEM values.Metabolite Identification

[0057] Further experiments aimed at identifying metabolites formed from IPGE. The potential metabolites were predicted based on two references cited in the Introduction.6’7Under acidic conditions with 12-minute long gradient elution, employing the ESI ion source (both in positive and negative modes), and utilizing the single ion monitoring mode, the precursor ions of several metabolites were detected. Among the monitored precursor ions, sulfate and glucuronide conjugates were detected in negative ESI, while the others were detected in positive ESI mode. The chemical structures of the 12 potential IPGE metabolites are depicted in the Supporting Information (Table S10). For metabolite searching, we used the plasma samples stored for several months from toxicokinetic studies, and additionally we also treated one male and one female rat with a dose of 1500 mg / kg to obtain fresh samples. The representative chromatogram of the 4-hour sampling time point for the female (no difference observed between sexes) rat freshly treated is shown in Figure 5. Seven of these possible metabolites are visible on the chromatogram: 3 -isopropoxy-2-hydroxy-l -propanol (Ml), sulfate-conjugate of IPGE (M3), glucuronide-conjugate of IPGE (M4), 3-isopropoxy-2-hydroxypropionic acid (M5), O-isopropyl-A-acetyl serine (M6), O-(hydroxy-isopropyl)-7V-acetyl serine (M7), and glutathione-conjugate of IPGE (Mil). The retention times are 2.6, 3.3, 2.8, 2.7, 3.2, 2.9, and 3.0 minutes, respectively. The retention time of the analyte under these conditions is 3.6 minutes. During the measurements, we set the precursor ion for each possible metabolite but only displayed those seven that gave a distinct peak. The IPGE metabolites were absent in matrix blank samples.

[0058] Figure 5. Representative SIM chromatograms of the possible metabolites

[0059] [1-1-10 pL injection volume] A) O-isopropyl-A-acetylserine (M6) 190 m / z (blue), glucuronide-conjugate of IPGE (M4) 309 m / z (green), O-(hydroxy-isopropyl)-A-acetyl serine (M7) 206 m / z (orange); B) 3-isopropoxy-2-hydroxypropionic acid (M5) 149 m / z (black), glutathione-conjugate of IPGE (Ml 1) 424 m / z (brown), sulfate-conjugate of IPGE (M3) 213 m / z (red); C) 3 -isopropoxy-2-hydroxy-l -propanol (Ml) 135 m / z (magenta)

[0060] Discussion

[0061] This study presents a novel LC-MS / MS method developed and validated for the bioanalysis of plasma samples collected in a toxicological study to gain information about the systemic exposure of IPGE administered to rats. As a consequence of the REACH-regulation coming into effect in the European Union, demand for toxicological studies of a wide variety of chemicals has tremendously increased in the past decade.31OECD guidelines for toxicological studies (e.g. OECD 474 or OECD 443) call for the need for toxicokinetic sampling, andemphasize the importance of plasma bioanalysis to prove the systemic exposure.32, 33Hence, to equip our analytical toolbox for the compounds of the widely used industrial chemical glycidyl ether family the need is essential.

[0062] The method development was started for the quantification of IPGE by utilizing the LC-ESI-MS / MS technique. At the very beginning of the method optimization, we faced that the protonated molecule ion of the analyte was not produced in the ion source at all. Ammonium buffers as commonly used eluent modifiers offer the formation of ammonium adducts in the ion source, which can provide a valuable ionization alternative in mass spectrometry. Using ammonium acetate or ammonium formate buffer in the mobile phase, the corresponding adducts for the glycidyl ethers were recognized in the mass spectrum; therefore, MRM transitions with the ammonium adduct precursor ions could be constructed. The diglycidyl analogue of IPGE, namely neopentyl glycol diglycidyl ether, was selected as the internal standard.

[0063] Due to the hydrophilic character of IPGE, the stationary phase of YMC-Triart Cl 8, which bears excellent retention stability even with 100% aqueous mobile phase, was selected. Nevertheless, such an extreme eluent composition was not necessary to reach sufficient retention of IPGE. Ammonium acetate over ammonium formate in the mobile phase was proved to be a better source for abundant formation of ammonium adduct. The measurements with matrix-matched calibration compared to plasma-free samples suggested no substantial matrix effect.

[0064] The method provided a moderate LLOQ value of 5 pg / mL in rat plasma.

[0065] Since there was no information on the toxicokinetics of the glycidyl ether analyte, it was anticipated that the moderate LLOQ concentration might be sufficient for sample analysis in a single-dose toxicity study. IPGE was expected to display no substantial distribution to the organ tissues, and to mostly remain in the bloodstream, reaching rather high plasma concentrations because of its hydrophilic character. The analysis of plasma samples collected in the pilot toxicokinetic study (single dose of 1000 mg / kg p.o.) highlighted that this method using ESI source was unable to give relevant information about the systemic exposure of IPGE. No valid concentration results could be acquired at any time-points. Hence, it was essential to improve the bioanalytical method with high sensitivity performance characteristics. Solutions for better ionization efficiency was required to create precursor ions abundantly for subsequent fragmentation. A plausible direction was to target the rigid epoxy ring in the molecule, which was expected to be easily forced into some chemical reaction. Several derivatization reagents, such as benzylamine and 3 -nitrobenzenesulfonic acid can provide feasible options for a much more detectable molecule variant. An approach that did not require tedious multiple derivatization steps prior to the analysis and yielded a reaction product in the analyticalinstrument itself with high sensitivity, was desired. Gas-phase derivatization via the Meerwein reaction21’22was used with acetonitrile serving as the derivatization reagent per se, which was ionized into ethylnitrilium ions, and reacted with the epoxy group moiety of the analyte glycidyl ether (Figure 6). Fragmentation of the analyte (Figure S3) and Full scan spectrum and product ion scan spectra of the analyte (Figure S4) can be found in the Supporting Information.

[0066] Figure 6. Reaction scheme of the Meerwein reaction taking place in the ion source

[0067] After selecting APCI as the optimal ion source by comparing signal intensities with different eluents, it was optimized for the corresponding Meerwein reaction products of the analyte and the IS in terms of gas and temperature settings.

[0068] Because of the accuracy and precision requirements, the need for another internal standard emerged. The neopentyl glycol diglycidyl ether, internal standard of the first method was not an optimal one due to its inability to compensate for the analyte signal throughout the analytical run. The yield of the Meerwein reaction with the diglycidyl component most probably differed from that of a molecule with a single epoxy ring; therefore a monoglycidyl ether analogue, tertbutyl glycidyl ether was selected with which provided sufficient signal stability for the quantification.

[0069] The transformation of the mass spectrometric conditions by replacing the ion source ESI with APCI combined with an in-source derivatization approach resulted in a several hundred-fold sensitivity increase. After establishing a promising, highly sensitive LC-MS / MS method, namely with an LLOQ of 10 ng / mL, the samples of the pilot study were reanalyzed to gain information on whether the improved method could provide relevant toxicokinetic data. A full toxicokinetic curve was outlined even from the concentration results of the samples stored in an ultra-low temperature freezer for 123 days, and justified that the improved LC-MS / MS method was able to hit the relevant concentration range and to be adapted for subsequent toxicokinetic analyses in rat at three dose levels.

[0070] Although there is no bioanalytical method for IPGE accessible in the literature, methods analyzing compounds with epoxy moiety was using derivatization served as a starting point. Some methods targeting epoxy derivatization used liquid-liquid extraction techniques from relatively large volume of plasma samples (500 pL) with dichloromethane or diethyl ether as extraction solvents.24, 26The additional sample preparation step and solvent application may be considered to be disadvantages of these methods. Glowka etal. presented a LC-MS / MS method for the pharmacologically active monoepoxide metabolite of the prodrug treosulfane with moderate sensitivity (LLOQ of 0.93 pM equal to 170 ng / mL); however, the use of Amicon Ultra Centrifugal Filter vials provided a less cost-effective way for a facile sample preparationcomparing to our simple protein precipitation method.34

[0071] IPGE bears epoxy ring similarly to bisphenol A diglycidyl ether (BADGE), often used in epoxy resins, whereas its derivative bisphenol A (BP A) contains phenolic rings similarly to BADGE. Both BADGE and BPA have been studied extensively due to their potential health impacts as endocrine disruptors. One must also note that IPGE lacks the phenolic rings present in BADGE and BPA; however, analytical methods available for aliphatic glycidyl ethers like IPGE are rather limited. Understanding chemical differences betwee BADGE, BPA and IPGE is crucial for assessing their potential health and environmental impacts and for developing effective analytical approaches to monitor their presence in various matrices. Various bioanalytical approaches targeting the abovementioned aromatic compounds. Cambien et al. focused on chlorinated derivatives of BPA in human plasma,17, 35and the solid-phase extraction (SPE) technique was the only technique achieved a lower limit of quantification as low as 0.1 ng / mL in plasma sample (500 pL) with a 13 -minute acquisition runtime using water and methanol eluents. Chang et al. studied BADGE and its metabolites in human urine and plasma, and liquidliquid extraction using hexane and ethyl acetate solvents was applied to achieve an LLOQ value of 0.05 ng / mL for BADGE in 200 pL of plasma.

[0072] The successful implementation of in-source derivatization addresses several challenges encountered in the analysis of IPGE, including its low ionization efficiency and possible susceptibility to matrix interference. By incorporating a derivatization reagent directly into the ionization source, we achieved efficient conversion of IPGE into more readily detectable derivatives ionizable by APCI source; thereby, enhancing sensitivity and retaining sample preparation requirements on the minimum with low sample volume need and organic solvent consumption. In terms of matrix effect considerations, APCI is the ion source of choice, if the analyte permits, as this is considered to less susceptible to matrix effect than ESI.36Based on the toxicokinetic data and experimental observations, it is worth mentioning that our work is in concordance with Terry and Hays’ recommendations emphasizing that incorporation of toxicokinetic analysis into in vivo toxicity studies can give evidence that the toxicity at high doses observed in animals does not mean potential risk to humans where the worst-case exposure is expected to be much lower.37

[0073] Based on the articles by Chen et al. and Eadsforth et al. , with the assistance of further metabolite search measurements, we were able to identify seven potential metabolites [3 -isopropoxy -2-hydroxy-1 -propanol (Ml), sulfate-conjugate of IPGE (M3), glucuronide-conjugate of IPGE(M4), 3-isopropoxy-2-hydroxypropionic acid (M5), O-isopropyl-A-acetylserine (M6), (9-(hydroxy-isopropyl)-TV-acetyl serine (M7), and glutathione-conjugate of IPGE (Mil)].6, 7The proposed metabolic pathways of IPGE in rats can be seen in Figure 7. (Figure 7. Proposed metabolic pathways of IPGE in rats.)

[0074] Conclusions

[0075] Glycidyl ethers are commonly used chemicals in the epoxy resin industry, and exposure assessment / biomonitoring has been getting more and more reasonable attention nowadays. An LC-MS / MS method was developed and validated for the quantification of IPGE in rat plasma for the first time. An in-source derivatization approach was applied in order to achieve adequate sensitivity. The selected precursor ion was the gas-phase Meerwein reaction product between the target epoxide and the ethylnitrilium ion produced from the eluent acetonitrile in the APCI source. Thus, several hundred-fold sensitivity improvement was achieved compared to that observed in the ESI mode using ammonium adduct as a precursor ion. This finding made it possible to develop a facile sample preparation protocol with protein precipitation without the demand for a multi-step derivatization / preconcentration process.

[0076] Using the novel bioanalytical method in a single-dose oral toxicity study in the rat at three dose levels (1000 mg / kg, 1500 mg / kg and 2000 mg / kg) IPGE showed fast oral absorption and moderately slow elimination in rats, whereas its systemic exposure displayed dose-dependence with distinct super-proportionality without substantial gender difference. Furthermore, seven IPGE metabolites [3-isopropoxy-2-hydroxy-l-propanol (Ml), sulfate- conjugate of IPGE (M3), glucuronide-conjugate of IPGE (M4), 3-isopropoxy-2-hydroxypropionic acid (M5), O-isopropyl-A-acetylserine (M6), < -(hydroxy-isopropyl)-A-acetyl serine (M7), and glutathione-conjugate of IPGE (Ml 1)] were tentatively identified in rat plasma.

[0077] The present method may provide a starting point for the development of bioanalytical methods by other counterparts from the class of glycidyl ethers to acquire valuable information not only in the field of toxicology, but also in occupational health and safety studies.

[0078] Associated content

[0079] Supporting Information

[0080] The Supporting Information is available free of charge at the webpage of Chemical Research in Toxicology online.

[0081] LC-MS / MS method parameters;

[0082] bioanalytical method validation results;

[0083] LC-APCI-MS / MS method ion source optimization process summary;the number of animals throughout the experiments;

[0084] incurred sample reanalysis data;

[0085] the number of samples throughout the experiments in the dose group of 2000 mg / kg; calculated toxicokinetic parameters;

[0086] descriptive list of toxicokinetic parameters;

[0087] fragmentation of the analyte;

[0088] full scan spectrum and product ion scan spectra of the analyte;

[0089] names and formulas of the analyte and the possible metabolites

[0090] Notes

[0091] The authors declare no competing financial interest.

[0092] Funding

[0093] This work was supported by the grants of 2018-1.2. l-NKP-2018-00005 and TKP2021-EGA-31 (National Research, Development and Innovation Fund of Hungary). Project no. 2020-1.1.2-PIACI-KFI-2020-00021 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the 2020.1.1 ,2-PIACI-KFI funding scheme. Supported by the 2024-2.1.2-EKOP-KDP New National Excellence Program of the Ministry for Culture and Innovation, from the source of the National Research, Development and Innovation Fund. Acknowledgments

[0094] The authors express their warm thanks to Agnes Szeker, Maria Nemeth (bioanalytics), and Timea Csorge, Marcell Madar (sample collection, animal experiments) for their excellent technical assistance.References

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Claims

CLAIMS1. A method for the identification and quantification of glycidyl ethers in biological and environmental samples, comprising:o the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for the sensitive detection of isopropyl glycidyl ether (IPGE) in biological matrices, using its structural analogue tertbutyl glycidyl ether as an internal standard,o the application of atmospheric pressure ionization (APCI) techniques with an insource derivatization reaction employing the Meerwein reaction to generate a product by reacting IPGE with the ethylnitrilium ion formed from acetonitrile, o achieving a lower limit of quantification (LLOQ) of 0.01 pg / mL in plasma samples,o using the validated method to analyze real study samples from a single-dose oral toxicity study to evaluate blood plasma concentrations of IPGE at multiple dose levels (three), observing dose-dependent superproportional systemic exposure in the range of 1000-2000 mg / kg;o identifying seven possible IPGE metabolites in rat plasma.

2. The method of claim 1, wherein the detection and quantification were performed specifically in plasma samples, while it could also be adapted for use in other biological matrices, such as serum or other body fluids, through similar derivatization and analytical protocols.

3. The method of claim 1, could be applied for:o occupational health biomonitoring to assess exposure to glycidyl ethers, including isopropyl glycidyl ether, during industrial applications,o toxicokinetic studies to evaluate absorption, distribution, metabolism, and excretion (ADME) characteristics of IPGE and its possible metabolites in preclinical or clinical settings.

4. The method of claim 1, is characterized by its simplified sample preparation protocol involving protein precipitation with acetonitrile, reducing the complexity and resource requirements compared to traditional methods.S-l

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