In vitro and in vivo studies of triacetone triperoxide (TATP) metabolism in humans

Optima HPLC grade methanol, Optima HPLC grade water, Optima HPLC grade acetonitrile, American Chemical Society (ACS) grade acetone, ACS grade methanol, ACS grade pentane, hydrochloric acid, ammonium acetate, dipotassium phosphate, monopotassium phosphate, magnesium chloride (MgCl₂) and reduced glutathione (GSH) were purchased from Fisher Chemical (Fair Lawn, NJ, USA); NADPH, 1-aminobenzotriazole, methimazole, 1-naphthol and hydroxyacetone from Acros Organics (Morris Plain, NJ, USA); UDPGA, saccharolactone and 2,4-dichlorophenoxyacetic acid from Sigma-Aldrich (St. Louis, MO, USA); bupropion, benzydamine and alamethicin from Alfa Aesar (Ward Hill, MA, USA); oxcarbazepine from European Pharmacopoeia Reference Standard (Strasbourg, France); ticlopidine from Tokyo Chemical Industry (Tokyo, Japan); hydroxybupropion from Cerilliant Corporation (Round Rock, Texas, USA); deuterated acetone (acetone-d₆) from Cambridge Isotope Labs (Cambridge, MA, USA); hydrogen peroxide (50%) from Univar (Redmond, WA, USA); HLM, rat liver microsomes (RLM), DLM and human lung microsomes (HLungM) from Sekisui XenoTech (Kansas City, KS, USA); human recombinant CYP (rCYP) bactosomes expressed in Escherichia coli (E. coli) from Cypex (Dundee, Scotland); human recombinant flavin monooxygenase (rFMO) supersomes and human recombinant UGT (rUGT) supersomes expressed in insect cells from Corning (Woburn, MA, USA).

TATP was synthesized following the literature methods using hydrochloric acid as the catalyst [1]. TATP was purified by recrystallization, first with methanol/water (80:20, v/v) and then with pentane. TATP-d₁₈ was synthesized as above using acetone-d₆. TATP-OH was synthesized as above using hydrogen peroxide (50 wt%)/acetone/hydroxyacetone (2:1:1, v/v/v) [15]. TATP-OH was purified using a CombiFlash RF + system with an attached PurIon S MS system (Teledyne Isco, Lincoln, NE, USA), followed by two cycles of drying and reconstituting in solvent to sublime away the TATP. Separation was performed using a C-18 cartridge combined with a liquid chromatograph flow of 18 mL/min with 10% methanol (A) and 90% aqueous 10 mM ammonium acetate (B) for 1 min, before ramping to 35%A/65%B over 1 min, followed by another ramp to 95%A/5%B over the next 1 min, holding for 2 min, before a 30 s transition to initial conditions, with a hold of 2 min [15].

Metabolite identification was performed by high‐performance liquid chromatography coupled to Thermo Scientific Exactive or Thermo Scientific LTQ Orbitrap XL high-resolution mass spectrometers (HPLC–HRMS) (Thermo Fisher Scientific, Waltham, MA, USA). A CTC Analytics PAL autosampler (CTC Analytics, Zwingen, Switzerland) was used for LC injections, solvent delivery was performed using a Thermo Scientific Accela 1200 quaternary pump, and data collection/analysis was done using Xcalibur software (Thermo Scientific, version 2.1).

Metabolite quantification was performed by high‐performance liquid chromatography coupled to AB Sciex Q-Trap 5500 triple quadrupole mass spectrometer (HPLC–MS/MS) (AB Sciex, Toronto, Canada). A CTC Analytics PAL autosampler was used for LC injections, solvent delivery was performed using a Thermo Scientific Accela 1200 quaternary pump and data collection/analysis was done with Analyst software (AB Sciex, version 1.6.2).

The HPLC method for all TATP derivatives was as follows: sample of 40 µL (Exactive and LTQ Orbitrap XL) or 20 µL (Q-Trap 5500) in acetonitrile/water (50:50, v/v) was injected into LC flow at 250 µL/min of 10%A/90%B for introduction onto a Thermo Syncronis C18 column (50 × 2.1 mm i.d., particle size 5 µm). Initial conditions were held for 1 min before ramping to 35%A/65%B over 1 min, followed by another ramp to 95%A/5%B over the next 1 min. This ratio was held for 2 min before reverting to initial conditions over 30 s, which was held for additional 2 min. The Exactive MS tune conditions for atmospheric pressure chemical ionization (APCI) in positive mode were as follows: N₂ sheath gas flow rate, 30 arbitrary units (AU); N₂ auxiliary gas flow rate, 30 AU; discharge current, 6 µA; capillary temperature, 220 ℃; capillary voltage, 25 V; tube lens voltage, 40 V; skimmer voltage, 14 V; and vaporizer temperature, 220 ℃. The Exactive MS tune conditions for electrospray ionization (ESI) in negative mode were as follows: N₂ sheath gas flow rate, 30 AU; N₂ auxiliary gas flow rate, 15 AU; spray voltage, − 3.4 kV; capillary temperature, 275 ℃; capillary voltage, − 35 V; tube lens voltage, − 150 V; and skimmer voltage, − 22 V. The LTQ Orbitrap XL MS tune conditions for ESI−, used for TATP-O-glucuronide verification, were as follows: N₂ sheath gas flow rate, 30 AU; N₂ auxiliary gas flow rate, 15 AU; spray voltage, − 4 kV; capillary temperature, 275 °C; capillary voltage, − 15 V; and tube lens voltage, − 84 V. The single-reaction monitoring settings were as follows: m/z 413.13 with isolation width m/z 1.7, activated by higher-energy collision dissociation at 35 eV. The Q-trap 5500 MS tune and multiple reaction monitoring (MRM) conditions are shown in Table 1. TATP and TATP-OH quantification was done as the area ratio to TATP-d₁₈ (internal standard, IS) using a standard curve ranging 10–20,000 ng/mL and 10–500 (Fig. S1) or 500–8,000 ng/mL, respectively. TATP-O-glucuronide relative quantification was done as the area ratio to 2,4-dichlorophenoxyacetic acid (IS).

The HPLC method for bupropion, hydroxybupropion, benzydamine, benzydamine N-oxide, and oxcarbazepine (IS) was as follow: sample of 10 μL in acetonitrile/water (50:50, v/v) was injected into LC flow at 250 μL/min with 30%A/70%B for introduction onto a Thermo Scientific Acclaim Polar Advantage II C18 column (50 × 2.1 mm i.d., particle size 3 µm). Initial conditions were held for 1 min before instant increase to 95%A/5%B, held for 2.5 min, and then reversed to initial conditions over 30 s, with a hold of 1 min for the bupropion, hydroxybupropion and oxcarbazepine method or with a hold of 3 min for the benzydamine, benzydamine N-oxide and oxcarbazepine method.

All incubations were performed in triplicate in a Thermo Scientific Digital Heating Shaking Drybath set to body temperature 37 ℃ and 800 rpm. An incubation mixture containing phosphate buffer (pH 7.4), MgCl₂ [17], and NADPH (CYP cofactor [11]) was prepared so that at a final volume of 1 mL, their concentrations were 10, 2 and 1 mM, respectively. When the incubation times were relatively long (greater than 15 min), it was thought necessary to use closed vessels to avoid loss of the volatile TATP or TATP-OH; as a result, prior to incubation, oxygen gas was bubbled through the buffer to ensure ample oxygen availability [15]. To this mixture, microsomes or recombinant enzymes were added and equilibrated for 3 min before the reaction was initiated by adding the substrate. Substrates included TATP in acetonitrile, TATP-OH in methanol, and bupropion and benzydamine in water. Organic solvents can disrupt metabolism, but the catalytic activity of most CYP enzymes is unaffected by less than 1% acetonitrile or methanol [18]. At the end point, an aliquot was transferred to a vial containing equal volume of ice-cold acetonitrile and immediately vortex-mixed to quench the reaction. The sample was centrifuged for 5 min at 14,000 rpm, and the supernatant was analyzed by LC–MS.

Metabolite identification studies used microsomes (HLM, RLM and DLM) at protein concentrations of 1 mg/mL in the incubation mixture. The substrate, TATP, TATP-OH, or TATP-d₁₈ (10 µg/mL) was allowed to incubate for several min before MS analysis. Negative controls consisted of the incubation mixture excluding either microsomes or NADPH. Positive control used 100 µM bupropion, a probe substrate for CYP2B6 [19‐21].

Phase II TATP metabolism was examined by two studies. Metabolism by glutathione S-transferase (GST) was probed by equilibrating 5 mM GSH (GST cofactor [11]) for 5 min in the incubation mixture including HLM before the substrate (TATP) was added. Ticlopidine (10 µM) was the substrate for the GST positive control (Fig. S2) [22]. To examine metabolism by UGT, HLM, buffer, and alamethicin (50 µg/mL in methanol/water) were equilibrated cold for 15 min, before saccharolactone (1 mg/mL, β-glucuronidase inhibitor [23]), MgCl₂, and NADPH were added, and the mixture was warmed to 37 ℃ and shaken at 800 rpm. After 3 min equilibration, the substrate (TATP or TATP-OH) was added, and in 2 min, the reaction was started by the addition of 5.5 mM UDPGA (UGT cofactor [11]) [24, 25]. Positive control used 100 µM 1-naphthol, an UGT1A6 substrate (Fig. S3) [26, 27]. Alamethicin was employed to replace membrane transporters, in allowing UGT (located in the endoplasmic reticulum lumen) easy access to the UDPGA cofactor [11].

TATP was incubated as described in the previous section with various enzyme inhibitors in HLM (1 mg/mL). TATP-OH formation was first monitored and benchmarked against incubations without inhibitors. Chemical inhibitors, such as 1-aminobenzotriazole (1 mM) [28, 29], methimazole (500 µM) [30, 31], or ticlopidine (100 µM) [32, 33], were pre-equilibrated in the incubation mixture for 30 min prior to the addition of the substrate (100 µM, TATP or controls). TATP was also tested as a possible CYP2B6 inhibitor; TATP or bupropion was pre-equilibrated in the incubation mixture before starting the reaction with a known CYP2B6 substrate (bupropion) or TATP, respectively. FMO inhibition by heat was also tested [11, 34]. In that experiment, HLM was mixed with buffer and preheated at 37 or 45 ℃ for 5 min. After an hour, cooling on ice, the incubation procedure was resumed. Bupropion, a CYP2B6 substrate [19‐21], and benzydamine, an FMO substrate [34], were used as positive and negative control substrates to assess CYP, FMO and CYP2B6 inhibition. Samples not preincubated with chemical inhibitors nor heated to 45 ℃ were used as 100% TATP-OH formation. Inhibition studies were quenched after 15 min incubation.

Recombinant CYP and FMO enzymes were employed to identify the isoform responsible for the NADPH-dependent metabolism. Human bactosomes expressed in E. coli were used for CYP isoform identification; CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 (100 pmol CYP/mL) were tested. Human supersomes expressed in insect cells were used for FMO isoform identification; FMO1, FMO3 and FMO5 (100 µg protein/mL) were examined. Both TATP and TATP-OH were tested as the substrate (10 µg/mL) in the incubation mixture with the recombinant enzymes. Negative control incubations were done in E. coli control or insect cell control. Positive control incubations were done in HLM (200 pmol CYP/mL), which contains all CYP and FMO enzymes. Recombinant enzymes studies were quenched after 10 min incubation.

Recombinant UGT enzymes were used to identify the isoform responsible for phase II metabolism. Human supersomes expressed in insect cells were used for UGT isoform identification; UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 were examined. The incubation mixture was similar to the glucuronidation incubation previously described, except that saccharolactone was not added [35]; 500 µg protein/mL was used; and the substrate was 10 µg/mL TATP-OH. Negative control incubations were done in insect cell control. Positive control incubations were done in HLM (1 mg protein/mL), which contained all UGT enzymes. Glucuronidation with recombinant enzymes was quenched after 2 h incubation.

Kinetics experiments were done to determine the affinity of the enzyme CYP2B6 to the substrate TATP. The human CYP2B6 bactosomes used contained human CYP2B6 and human CYP reductase coexpressed in E. coli, supplemented with purified human cytochrome b₅. Cytochrome P450 reductase is responsible for the transfer of electrons from NADPH to CYP, a task sometimes extended to cytochrome b₅ [11]. The incubation mixture (1 mL) contained 10 mM phosphate buffer (pH 7.4), 2 mM MgCl₂, 50 nM rCYP2B6, 1 mM NADPH, and various concentrations of 0.1 to 20 µM TATP. The reaction was initiated by adding TATP after a 3 min pre-equilibration and stopped at different end points (up to 5 min) to determine rate of TATP hydroxylation. The rate of TATP hydroxylation in lungs was also investigated by incubating TATP (100 µM) in the incubation mixture containing 1 mg/mL HLM or HLungM, instead of rCYP2B6, for up to 10 min.

TATP-OH metabolism by CYP2B6 was evaluated by incubating 10 µg/mL TATP-OH in CYP2B6 bactosomes according to the above procedure, with or without NADPH, except that the buffer was preoxygenated so that the incubation could be performed in a closed vessel. Aliquots were removed and quenched at different time points, up to 30 min. TATP-OH depletion by HLM was determined using the same procedure, except that 1 µM TATP-OH and up to 60 min reaction times were used.

When TATP was incubated in HLM, TATP was depleted, and one observable product, TATP-OH, was formed over time (Fig. 1). Metabolism of TATP in HLM consists of hydroxylation at one methyl group with the peroxide bonds and nine-membered ring structure preserved (Fig. 2) [15]. Opsenica and Solaja [12] reported various monohydroxylated and dihydroxylated products during microsomal incubations with cyclohexylidene and steroidal mixed tetraoxanes where the peroxide bond was also preserved. A TATP-OH standard (Fig. S4) was chemically synthesized to confirm the metabolite by retention time and mass-to-charge ratio (m/z). TATP-OH, identified as [TATP-OH + NH₄]⁺ (m/z 256.1391) by accurate mass spectrometry, increased in incubation samples as time progressed. Attempts to confirm the hydroxylated metabolite with the deuterated substrate were unsuccessful due to the persistence of a contaminant with the same mass that the metabolite would have had, even in samples where TATP-d₁₈ was not used as the substrate. As previously observed, TATP incubations in different species (dogs and rats) yielded the same metabolite, TATP-OH (Fig. S5) [15]. Other suspected metabolites, including the dihydroxy-species and additional oxidation of the TATP-OH to the aldehyde and carboxylic acid were not observed. Small polar molecules, such as acetone and hydrogen peroxide, the synthetic reagents of TATP, could not be chromatographically separated or are below the lower mass filter limit.

TATP was investigated for phase II metabolism routes of glutathione and glucuronide conjugation. Incubation in HLM with GSH produced no detectable glutathione metabolite conjugates, indicating that TATP is most likely not a substrate for microsomal GSTs (Fig. S6). When TATP was incubated with UDPGA, the TATP-OH glucuronic acid metabolite (TATP-O-glucuronide) was observed (Fig. 2). The m/z 432.1712 for [TATP-O-glucuronide + NH₄]⁺ was observed at very low levels after 2 and 3 h of incubation. When the sample was dried and reconstituted in low volume, the intensity of [TATP-O-glucuronide + NH₄]⁺ increased, but TATP and TATP-OH were evaporated along with the solvent. TATP and TATP-OH are volatile, limiting their use in quantification experiments since sample concentration is not feasible [5]. However, TATP-O-glucuronide is a non-volatile TATP derivative that is amenable to sample preparation. Formation of TATP-O-glucuronide over time was monitored, using concentrated samples, as an intensity increase of m/z 432.1712 (Fig. S7). Even though TATP and TATP-OH generally form ammonia adducts under positive ion APCI, the glucuronide favors negative ion mode ESI. The m/z 413.1301 for [TATP-O-glucuronide − H]⁻ was easily seen at 1–3 h without sample concentration. The common fragments of glucuronic acid, m/z 175, 113, and 85, were observed in the fragmentation pattern of m/z 413.1301, confirming the presence of a glucuronic acid conjugate (Fig. 3) [36]. TATP-O-glucuronide was not observed in negative controls without UDPGA or NADPH, indicating that TATP-OH must be formed and then be further metabolized into TATP-O-glucuronide. Glucuronide conjugates are highly polar compounds that are easily eliminated by the kidney, suggesting that TATP-O-glucuronide would likely progress via urinary excretion [11].

TATP was only metabolized into TATP-OH in HLM in the presence of NADPH, indicating that the metabolism is NADPH-dependent. The predominant microsomal enzymes that require NADPH for activity are CYP and FMO [11].

The usual roles of CYP are hydroxylation of an aliphatic or aromatic carbons, epoxidation of double bonds, heteroatom oxygenation or dealkylation, oxidative group transfer, cleavage of esters and dehydrogenation reactions [11]. 1-Aminobenzotriazole is considered a general mechanism-based inhibitor of CYPs, initiated by metabolism into benzyne, which irreversibly reacts with the CYP heme [28, 29]. When CYP activity was inhibited by 1-aminobenzotriazole, TATP-OH formation was also inhibited, with only 3.6% formed (Table 2), suggesting that CYP is involved in the hydroxylation of TATP. To support this evidence and to narrow down the CYP isoform catalyzing TATP hydroxylation, TATP was incubated with rCYPs (Fig. 4, Table S1). The CYPs selected for testing are responsible for the metabolism of 89% of common xenobiotics [37]. TATP-OH was not observed when TATP was incubated with CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. TATP hydroxylation was performed exclusively by CYP2B6, with 5.6 ± 0.3 µM TATP-OH produced in 10 min. CYP2B6 has been found to metabolize endoperoxides by hydroxylation, as observed for TATP [13, 14]. HLM, which contains CYP2B6, also exhibited TATP hydroxylation (1.5 ± 0.1 µM).

Since DLM studies indicated that TATP is metabolized by CYP2B11 [15], it was not surprising that another CYP2B subfamily enzyme, CYP2B6, metabolizes TATP in humans. CYP2B6 metabolism of TATP was further investigated by incubating TATP with ticlopidine, a mechanism-based inhibitor of CYP2B6 [32, 33]. When CYP2B6 activity was inhibited by ticlopidine, TATP-OH formation was also inhibited by 95% (Table 2), further supporting the primary involvement of CYP2B6 in the metabolism of TATP. Bupropion hydroxylation is catalyzed by CYP2B6; therefore, it was chosen as a positive control for CYP and CYP2B6 inhibition tests [28]. Hydroxybupropion formation was inhibited by 77 and 90% when incubated with 1-aminobenzotriazole and ticlopidine, respectively (Table 2).

FMO catalyzes oxygenation of nucleophilic heteroatoms, such as nitrogen, sulfur, phosphorous and selenium [34, 38]. Although FMO involvement in the hydroxylation of the TATP methyl group was unlikely, it seemed prudent to examine this enzyme class. Addition of methimazole, an FMO competitive inhibitor [30, 31], caused a decrease in TATP-OH formation by 66% (Table 2), but this is not necessarily direct inhibition of TATP metabolism by FMO since methimazole has been reported to reduce CYP2B6 activity by up to 80% [34, 39]. While this result was inconclusive about the FMO contributions to TATP metabolism, it could be considered further support for the role of CYP2B6. TATP was incubated with available rFMOs: FMO1, FMO3 and FMO5 (Fig. 4, Table S1). FMO1 is expressed in adults in the kidneys, and it should not contribute to the liver metabolism of TATP [34]; indeed, none appeared to be involved as no TATP-OH was produced (positive control, Fig. S8). Since FMO is inactivated by heat [11, 34], TATP was incubated in HLM pre-heated to 45 ℃ for 5 min. Table 2 compares the formation of TATP-OH at 37 ℃ to that at 45 ℃ and to the N-oxidation of the positive control, benzydamine. Although, there was a decrease in TATP hydroxylation, the inhibitory effect was not as significant as compared to the decrease in benzydamine N-oxidation, which is catalyzed by FMO.

TATP-OH appears to be metabolized by HLM in an NADPH-dependent manner; therefore, TATP-OH was incubated for 10 min with recombinant enzymes (CYP and FMO) to determine which isoform is responsible for this secondary phase I metabolism (Table 3). Although TATP-OH is not nearly as volatile as TATP, some TATP-OH was lost in all incubations (i.e., 37 ℃); however, notable depletion (by 40%) was observed only with CYP2B6. TATP-OH depletion in CYP2B6 was faster in the presence of NADPH than in its absence, supporting metabolism by CYP2B6 (Fig. S9). Unfortunately, no subsequent metabolite was identified.

To identify which isoform is responsible for TATP glucuronidation, TATP-OH was incubated with the most clinically relevant rUGTs (Fig. 5, Table S2) [40]. Glucuronidation was not observed with UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9. TATP-O-glucuronide was formed only in UGT2B7 incubations with 0.05 ± 0.03 area count relative to IS produced after 2 h of incubation. HLM, which contains UGT2B7, also displayed TATP-O-glucuronide (0.26 ± 0.02 area count relative to IS). Relative quantification of TATP-O-glucuronide was done by area ratio to the IS because a TATP-O-glucuronide standard is not available. Endoperoxide glucuronidation by UGT2B7 has been reported, in which urine analysis of patients treated with artesunate, an artemisinin derivative, found dihydroartemisinin-glucuronide to be the principal metabolite excreted [16].

Rate of TATP hydroxylation by CYP2B6 was evaluated by plotting concentration of TATP-OH formed over time. The initial rate of TATP hydroxylation at various TATP concentrations was used to estimate enzyme kinetics using the Michaelis–Menten model: \(v\, = \,V_{{\max}} \, \times \,\left[ S \right]/\left( {K_{{\text{m}}} \, + \,\left[ S \right]} \right)\). Here, [S] is the substrate (TATP) concentration, V_max is the maximum formation rate, K_m is the substrate concentration at half of V_max, and k_cat is the turnover rate of an enzyme–substrate complex to product and enzyme [41]. The kinetic constants were obtained using nonlinear regression analysis on GraphPad Prism software (version 8.2.1). The Michaelis–Menten evaluation for TATP hydroxylation by CYP2B6 (Fig. 6) yielded K_m of 1.4 µM; V_max of 8.7 nmol/min/nmol CYP2B6; and k_cat of 174 min^− 1. Linearized models, such as Lineweaver–Burk (Fig. S10), Eadie–Hofstee (Fig. S11) and Hanes–Woolf (Fig. S12), give similar values.

The low K_m indicates TATP has a high affinity for CYP2B6 [42]. Table 2 shows that TATP inhibits bupropion hydroxylation by CYP2B6, with only 62% hydroxybupropion formation in 15 min. However, in the presence of bupropion, TATP-OH formation was enhanced, with 125% formed compared to the reaction uninhibited by bupropion (Table 2). CYP2B6 preference for TATP affects the metabolism of bupropion, but further testing is needed to establish the specific type of inhibition.

Using the Michaelis–Menten parameters, in vitro intrinsic clearance (\(Cl_{{{\text{int}}}} \, = \,V_{{\max}} /K_{{\text{m}}}\)) was calculated to be 6.13 mL/min/nmol CYP2B6 [11, 43, 44]. Scale-up of the Cl_int to yield intrinsic clearance on a per kilogram body weight was done using values of 0.088 nmol CYP2B6/mg microsomal protein, 45 mg microsomal protein/g liver wet weight and 20 g liver wet weight/kg human body weight [43]. Taking that into account, the scale-up Cl_int was calculated to be 485 mL/min/kg.

In vivo intrinsic clearance (Cl) is the ability of the liver to metabolize and remove a xenobiotic, assuming normal hepatic blood flow (Q = 21 mL/min/kg [43, 45]) and no protein binding [43]. Cl can be extrapolated using the well-stirred model excluding all protein binding as \(Cl\, = \,Q\, \times \,Cl_{{\text{int}}} /Q\, + \,Cl_{{\text{int}}}\) [43]. The in vivo intrinsic clearance of TATP was estimated as 20 mL/min/kg. Compared to common drugs, TATP has a moderate clearance [46].

TATP-OH kinetics were also investigated by substrate depletion. Substrate depletion was plotted as the natural log of substrate percent remaining over time (Fig. 7). Half-life (t_1/2) was calculated to be 16 min, as the natural log 2 divided by the negative slope of the substrate depletion plot. In vitro intrinsic clearance (Cl_int) can also be estimated using half-life as Cl_int = (0.693/t_1/2) × (incubation volume/mg microsomal protein) [47, 48]. The in vitro intrinsic clearance of TATP-OH was estimated as 0.042 mL/min/mg. Even though we identified two metabolic pathways for TATP-OH, it appears to be cleared slower than TATP.

Inhalation is the most probable pathway for systemic exposure since TATP is both volatile and lipophilic. With passive diffusion into the bloodstream being very possible, TATP metabolism in the lung was also investigated. TATP was incubated in lung and liver microsomes for comparison of metabolic rate. The results, shown in Table 4, indicated that TATP hydroxylation in the lungs was negligible. Though CYP2B6 gene and protein are expressed in the lungs, enzyme activity in lungs is minimal as compared to the liver, limiting TATP metabolism [49, 50]. This suggests that TATP is most likely distributed through the blood to the liver for metabolism. News reported that traces of TATP was found in the blood samples extracted from the 2016 Brussels suicide bombers [51]. This indicates the possibility of using blood tests as forensic evidence for TATP exposure.

Laboratory workers, who normally work with TATP on a daily basis, performing tasks like explosive sensitivity testing, were screened for TATP exposure. These laboratory workers volunteered to collect their urine before and after exposure to TATP vapor. Since the health effects of TATP exposure are unknown, to minimize any additional risks to these workers, this pilot study was performed in duplicates using only three volunteers to establish some reproducibility. TATP and TATP-OH were not observed in the urine of any of the workers. However, TATP-O-glucuronide was present in all urine samples collected 2 h after TATP exposure (Fig. 8). Two out of the three volunteers still showed TATP-O-glucuronide in the urine collected the next day (Table 5). TATP-O-glucuronide was identified in human urine samples as both [TATP-O-glucuronide − H]⁻ and [TATP-O-glucuronide + NH₄]⁺. The presence of TATP-O-glucuronide in the urine of all three volunteers is summarized in Table 5.