Determination of Benzotriazoles and Benzothiazoles in Human Urine

Article pubs.acs.org/ac

Determination of Benzotriazoles and Benzothiazoles in Human Urine by Liquid Chromatography-Tandem Mass Spectrometry Alexandros G. Asimakopoulos,†,‡ Anna A. Bletsou,†,‡ Qian Wu,† Nikolaos S. Thomaidis,‡ and Kurunthachalam Kannan*,† †

Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Albany, New York, United States ‡ Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis Zografou, Athens, Greece S Supporting Information *

ABSTRACT: Benzotriazole (BTR) and benzothiazole (BTH) derivatives are used in a wide variety of industrial and consumer products and have been reported to occur in the environment. Owing to a lack of analytical methods, human exposure to BTR and BTH is still unknown. In this study, a liquid chromatography-electrospray ionization tandem mass spectrometry (LC− ESI(+)MS/MS) method was developed for simultaneous determination of five 1,2,3-benzotriazoles and five 1,3-benzothiazoles in human urine. The target benzotriazoles were 1H-benzotriazole, 1-hydroxy-benzotriazole, tolyltriazole, xylyltriazole (or 5,6-dimethyl-1H-benzotriazole), and 5-chloro-benzotriazole, and the target benzothiazoles were benzothiazole, 2-hydroxy-benzothiazole, 2methylthio-benzothiazole, 2-amino-benzothiazole, and 2-thiocyanomethylthiobenzothiazole. Urine specimens were enzymatically deconjugated with βglucuronidase and extracted by a solid-phase extraction (SPE) procedure for the measurement of total concentrations (i.e., free + conjugated forms) of BTRs and BTHs. Additionally, a liquid−liquid extraction (LLE) method was developed for comparison of extraction efficiencies between SPE and LLE. The limits of detection (LODs) ranged from 0.07 (2-amino-benzothiazole) to 4.0 ng/mL (benzothiazole) for the SPE method and from 0.04 (tolyltriazole) to 6.4 ng/mL (benzothiazole) for the LLE method. A total of 100 urine specimens, collected from Athens, Greece, were analyzed by enzymatic deconjugation and SPE. Benzothiazole and tolyltriazole were found frequently, and their concentrations were on the order of a few ng/mL. To our knowledge, this is the first study on the occurrence of 10 BTR and BTH compounds in human urine.

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in plastics, and antifogging agents.4,7,8,10,11,13−16 BTRs can be found in pigments, dishwasher detergents, and deicing/antiicing fluids.7,9−11,15 1H-BTR is an important chemical intermediate in the production of dyes, pharmaceuticals, and fungicides.14 BTHs also are used as corrosion inhibitors and as herbicides, algicides, slimicides (in paper and pulp industry), fungicides (in lumber and leather industry), and photosensitizers; BTHs are constituents of azo dyes.17−23 Additionally, the applications of BTHs in deicing/anti-icing fluids, drugs, food flavors, and rubber production have been documented.17−23 Novel BTH derivatives are synthesized and evaluated as enzyme inhibitors.24,25 In addition to anthropogenic sources, BTHs can be derived from natural sources. For instance, BTH and 2-SH-BTH are known constituents of tea leaves and cranberries, respectively.23,26 Detection of BTH in human atherosclerotic aortas at a concentration of 10 ng/g was reported in 1985.27 The presence of BTH in tobacco

ompounds that belong to the classes of 1,2,3benzotriazoles (BTRs, substances that contain 1,2,3benzotriazole skeleton) and 1,3-benzothiazoles (BTHs, substances that contain 1,3-benzothiazole skeleton) are used in a variety of industrial and consumer products and are highproduction volume chemicals.1−3 BTRs include 1H-benzotriazole (1H-BTR), 1-hydroxy-benzotriazole (1-OH-BTR), tolyltriazole (TTR, a mixture of isomers of 4-methyl-1Hbenzotriazole [4-Me-1H-BTR] and 5-methyl-1H-benzotriazole [5-Me-1H-BTR]), xylyltriazole (XTR or 5,6-dimethyl-1Hbenzotriazole [5,6-diMe-1H-BTR]), and 5-chloro-1H-benzotriazole (5-Cl-1H-BTR).4−11 The commonly known BTHs are benzothiazole (BTH), 2-hydroxy-benzothiazole (2-OH-BTH), 2-methylthio-benzothiazole (2-Me-S-BTH), 2-amino-benzothiazole (2-amino-BTH), 2-mercaptobenzothiazole (2-SHBTH), and 2-thiocyanomethylthio-benzothiazole (2SCNMeS-BTH).9,12 BTRs are categorized as “high production volume chemicals” with an annual production of over 9000 tons in the U.S. and a much greater production worldwide.13 BTRs have been used as flame and corrosion inhibitors, ultraviolet (UV) light stabilizers © XXXX American Chemical Society

Received: November 9, 2012 Accepted: December 4, 2012

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smoke28,29 and use of BTH in plastics were suggested as sources of this compound in humans.27 Little is known, however, about human exposure and metabolism of BTR and BTH derivatives. 1H-BTR and TTR have been shown to be hazardous to plants.4,14 1H-BTR is mutagenic in bacteria cell systems (Salmonella, Escherichia coli),14 and TTR is toxic to microorganisms.16 Recently, the estrogenic potential of 1H-BTR was reported in marine fish.30 The Dutch Expert Committee on Occupational Standards classified 1H-BTR as a suspected human carcinogen.14 BTRs and BTHs have been detected in environmental media.7,9,12,18,20,31−40 Gas chromatography (GC) and liquid chromatography (LC) techniques, coupled with mass or tandem mass (MS or MS/MS) spectrometric detection, have been used in the analysis of BTRs and BTHs. Asimakopoulos et al.40 developed a multicompound LC-ESI(+)MS/MS method for simultaneous determination of four BTRs and four BTHs in wastewater and sludge. To the best of our knowledge, however, a method for analysis of BTR and BTH derivatives in human specimens is not available. The polarity and low molecular weight of BTRs/BTHs and their coelution with other matrix components preclude accurate determination in complex biological samples. Further, GC/MS methods have been shown to overestimate the concentrations of BTRs and BTHs in wastewaters.20 In this study, an LC-ESI(+)MS/MS method was developed and applied for the quantification of the 10 aforementioned BTRs and BTHs in human urine. For optimal chromatographic separation, a C18 column that provided a balanced retention of polar and hydrophobic molecules was used.40 Two sample preparation protocols were developed and validated. The first method was based on solid-phase extraction (SPE) that determined total concentration of target analytes in urine after enzymatic deconjugation with β-glucuronidase enzyme (from Helix pomatia). This method was applied in the analysis of BTRs and BTHs in 100 urine specimens collected from Athens, Greece. The performance of the SPE method was evaluated by the extraction of samples with and without enzymatic deconjugation. Further, two different SPE cartridges were tested for their extraction efficacy. The SPE method was compared with a liquid−liquid extraction (LLE) method. Both SPE (with and without deconjugation) and LLE methods were applied in the analysis of the selected urine specimens, and the results were compared.

d5; 100%) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.). BTR-d4 and ATR-d5 were used as internal standards (ISs).9 Formic acid (98.2%), creatinine (99%), and βglucuronidase from Helix pomatia (145 700 units/mL βglucuronidase; 887 units/mL sulfatase) also were purchased from Sigma-Aldrich. Creatinine-d3 (99%) was purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). SPE cartridges, Strata-X-CW RP 6 cc/200 mg (Phenomenex, Bellefonte, PA, U.S.) with 32 μm average particle diameter, 87 Å average pore diameter, and 806 m2/g specific surface area, and Oasis HLB 6 cc/200 mg (Waters, Milford, MA, U.S.) with 30 μm average particle diameter, 82 Å average pore diameter, and 823 m2/g specific surface area, were compared. Standard stock solutions (1000 μg/mL) of all target analytes, except for 2-SCNMeS-BTH, were prepared in methanol (MeOH). 2-SCNMeS-BTH stock solution (100 μg/mL) was prepared in acetonitrile (ACN). Two sets of standard stock solutions (10 μg/mL), one for each IS, were prepared in MeOH. All standard stock solutions were stored at −20 °C for up to 3 months. The calibration standards were prepared from stock solutions through serial dilutions with MeOH/ACN (1:1 v/v). The stock solutions of creatinine and creatinine-d3 were prepared at 1 mg/mL in Milli-Q water. For the development and validation of analytical methods, pooled urine samples (total volume of 100 mL) obtained from 14 individuals (7 male and 7 female donors) were used. Sample Collection. A total of 100 volunteers, from Athens, Greece, provided urine specimens during March and April 2012. Some donors (25%) were employees or students at the University of Athens, and the rest were from three urology centers in Athens. Samples were collected from 50 males and 50 females who ranged in age from 2.5 to 87 years. All urine samples were collected in 15 mL polypropylene conical tubes and stored at −20 °C until analysis. The age range of male and female donors is shown in Figures S1 and S2, Supporting Information, respectively. Instrumentation. The chromatographic separation was carried out using an Agilent 1100 Series HPLC system (Agilent Technologies Inc., Santa Clara, CA, U.S.). Identification and quantification was performed with an Applied Biosystems API 2000 electrospray triple quadrupole mass spectrometer (ESI− MS/MS; Applied Biosystems, Foster City, CA, U.S.). A Zorbax SB-Aq (150 mm × 2.1 mm, 3.5 μm) column serially connected to a Javelin guard column (Betasil C18, 20 mm × 2.1 mm, 5 μm) and a 24-port SPE vacuum manifold from Mallinckrodt Baker (Phillipsburg, NJ, U.S.) were used. An Eppendorf Centrifuge 5804 (Hamburg, Germany) was used for centrifugation, a MultiVap 118 Nitrogen Evaporator (Organomation Associates Inc., West Berlin, MA, U.S.) was used for evaporation of solvents; a Corning Pinnacle 540 pH meter (Corning Inc., Corning, NY, U.S.) was used for pH measurements, and a Lab Companion SI-300 (Jeio Tech Co., Des Plaines, IL, U.S.) incubator shaker was used for incubation of samples. Conical autosampler vials (1.5 mL) with an assembled screw cap (with PTFE/silicone septum) and vial inserts (200 μL) were obtained from National Scientific (Rockwood, MI, U.S.). Polypropylene conical tubes (BD Falcon, NJ, U.S.) were used during sample preparation. Sample Preparation by SPE. Two milliliters of urine were transferred into a 50 mL polypropylene tube. All blanks and samples were spiked with a known amount of ISs before extraction. Matrix-spiked samples were fortified with a known amount of ISs and an appropriate amount of target analytes

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EXPERIMENTAL SECTION Chemicals and Reagents. Ammonium acetate (98%), hydrochloric acid (HCl, 37%), ammonium hydroxide (NH4OH, 29.5% assayed as NH3), and all organic solvents (analytical grade) used in the experiments were purchased from Mallinckrodt Baker (Phillipsburg, NJ, U.S.). Milli-Q water was purified by an ultrapure water system (Barnstead International, Dubuque, IA, U.S.). Analytical standards of 1H-BTR (99%), BTH (97%), and 2OH-BTH (98%) were purchased from Alfa Aesar GmbH & Co KG (Karlsruhe, Germany). 2-SCNMeS-BTH (100%) was purchased from AccuStandard (New Haven, CT, U.S.). 5Me-1H-BTR (98%), 5,6-diMe-1H-BTR hydrate (99%), and 2amino-BTH (97%) were purchased from Acros Organics (Morris Plains, NJ, U.S.). 1-OH-BTR hydrate (≥98%), 2-MeS-BTH (97%), 4-Me-1H-BTR (≥90%), 5-Cl-1H-BTR (99%), 1H-benzotriazole-d4 (BTR-d4; 100%), and atrazine-d5 (ATRB

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Table 1. Tandem MS Parameters for the Analysis of Benzotriazoles (BTRs) and Benzothiazoles (BTHs) precursor ions [M + H]+ (m/z)

product ions (m/z)

declustering potential (V)

entrance potential (V)

collision cell entrance potential (V)

collision energy (V)

1H-BTR

120.0

1-OH-BTR

136.0

TTR

134.2

XTR

148.0

5-Cl-1H-BTR

154.0

BTH

136.0

2-OH-BTH

152.0

2-Me-S-BTH

182.0

2-Amino-BTH

151.0

2-SCNMeS-BTH

180.0

BTR-d4 ATR-d5

124.2 221.0

65.2 92.2a 64.1 91.2a 79.2 77.3a 93.3 91.3a 99.3 73.2a 109.2 65.2a 80.3 65.0a 167.3 109.2a 124.1 109.1a 136.3 109.2a 69.1 179.1

60 45 40 40 50 50 40 40 35 30 60 60 60 60 50 50 60 60 45 45 60 40

5 5 8 8 8 8 5 5 8 2 8 8 10 8 10 10 6 6 4 4 5 5

21.8 21.8 22.1 22.1 22.1 22.1 22.3 22.3 22.4 22.4 22.1 22.1 22.4 22.4 22.9 22.9 22.3 22.3 22.9 22.9 21.9 23.6

35 25 40 45 35 25 25 35 35 50 40 45 40 45 30 50 35 35 25 40 35 20

target analytes and ISs

a

Confirmation ion.

prior to the extraction of samples (referred to as pre-extraction matrix spikes). The samples were digested with 4 mL of 1 M ammonium acetate that contained 291.4 units of βglucuronidase (0.77 g of ammonium acetate dissolved in 10 mL of Milli-Q water with 20 μL of β-glucuronidase solution) at 37 °C for 24 h in the incubator shaker. Thereafter, the samples were diluted with Milli-Q water to approximately 20 mL, and the pH was adjusted to 3.00 ± 0.10, with HCl and/or ammonium hydroxide and vortex mixed. Isolation of target analytes from urine samples was accomplished with the use of Oasis HLB 6 cc/200 mg cartridges. The cartridges were conditioned by 10 mL of MeOH and equilibrated with 10 mL of acidified Milli-Q water (adjusted to pH 3.00 ± 0.10 with HCl and/or ammonium hydroxide). Then, the samples were passed through the cartridge, and the cartridges were dried under a vacuum for 15 min. The cartridges were washed with 2 × 5 mL of acidified Milli-Q water/MeOH (95:5% v/v) (adjusted to pH 3.00 ± 0.10) and then dried under vacuum. The target analytes were eluted with 10 mL of mixture of MeOH/ACN (1:1 v/v). The solvent was evaporated to near-dryness under a gentle stream of nitrogen, reconstituted with a 200 μL mixture of MeOH/ACN (1:1 v/v), vortex-mixed, and transferred into an autosampler vial for LC-MS/MS analysis. For the determination of recoveries and matrix effects, ISs and target analytes were spiked into final extracts (referred to as post-extraction matrix spikes). Another set of procedures that involved SPE, without enzymatic deconjugation, was used for the comparison of concentrations of target analytes determined with enzymatic deconjugation, as discussed above. Sample Preparation by LLE. A volume of 600 μL of urine was transferred into a 15 mL polypropylene tube. After spiking the ISs, pH was adjusted to 3.00 ± 0.10, and 6 mL of ACN/ dichloromethane (DCM) (1:1 v/v) was added. Samples were vortex-mixed and then sonicated for 45 min. The mixture was left overnight in a freezer at −20 °C for the separation of the

two layers of solvents. The organic layer at the bottom (5.5 mL) was collected, and 7 mL of MeOH was added as a keeper solvent. The solution was evaporated to near-dryness under a gentle stream of nitrogen, reconstituted with 150 μL of MeOH/ACN (1:1 v/v), and transferred into an autosampler vial for HPLC-MS/MS analysis. Analysis of Creatinine. An aliquot of urine (10 μL) was diluted with Milli-Q water (∼160-fold), and 800 ng of creatinine-d3 was added. Creatinine was analyzed by LC− ESI(+)MS/MS, and the multiple reaction monitoring (MRM) transitions monitored were m/z 114 > 44 for creatinine and m/ z 117 > 47 for creatinine-d3.41,42 LC-MS/MS Analysis. Chromatographic separation was carried out using a gradient elution of ACN and Milli-Q grade water (acidified with 0.1% v/v formic acid) as a binary mobile phase at a flow rate of 250 μL/min. The gradient elution started with 10% (v/v) ACN and increased linearly to 40% ACN in 4.5 min and then to 100% ACN in 11.5 min, which was held for 6.1 min (until 17.6 min) and reverted to 10% ACN at 18 min and re-equilibrated for 7.1 min (from 18.0 to 25.1 min) at 10% ACN for a total run time of 25.1 min. The tandem MS system was operated in the positive ion multiple reaction monitoring (MRM) mode. The compound-specific MS/MS parameters are shown in Table 1. Nitrogen was used as both a curtain and collision gas. All tandem MS parameters were optimized by infusion of an individual compound into the mass spectrometer through a flow-injection system. The electrospray ionization voltage was set at +4.7 kV. The curtain gas flow rate was set at 10 psi; the collision gas flow rate was set at 4 psi, and the source heater was set at 550 °C. The nebulizer gas was set at 69 psi, whereas the heater gas was set at 68 psi. The data acquisition was set at 195 ms for scan speed and 0.70 FWHM for resolving power. The final composition of all standard solutions and sample extract C

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Table 2. Percent Recoveries (RSD%) of 1H-BTR, 1-OH-BTR, TTR, XTR, 5-Cl-1H-BTR, 2-Me-S-BTH, 2-amino-BTH, and 2SCNMeS-BTH at a fortification level of 100 ng, BTH and 2-OH-BTH at 500 ng, ATR-d5 at 10 ng, and BTR-d4 at 50 ng (N = 6) analyte 1H-BTR 1-OH-BTR TTR XTR 5-Cl-1H-BTR BTH 2-OH-BTH 2-Me-S-BTH 2-Amino-BTH 2-SCNMeSBTH BTR-d4 ATR-d5 a

absolute recovery Oasis HLB/Strata X-CWa

relative recovery (to BTR-d4) Oasis HLB/Strata X-CWa

relative recovery (to ATR-d5) Oasis HLB/Strata X-CWa

110 (4.8)/76 (3.1) 117 (8.5)/113 (19.4) 93.6 (5.8)/147 (3.7) 98.4 (5.0)/121 (10.2) 93.5 (8.0)/139 (4.5) 4.7 (11.4)/29.0 (38.6) 98.0 (7.4)/135 (7.8) 17.3 (11.9)/58.3 (12.6) 90.4 (6.8)/11.5 (12.6) 98.7 (13.1)/119 (10.8)

94.3 (5.1)/97.2 (9.3) 101 (8.2)/87.1 (25.7) 80.3 (4.6)/112 (7.7) 84.3 (4.3)/93.3 (14.3) 79.8 (3.6)/105 (10.3) 4.0 (4.8)/22.0 (38.3) 84.3 (5.8)/103 (11.0) 14.8 (10.7)/44.5 (16.9) 76.8 (4.9)/9.0 (16.9) 90.9 (15.4)/95.8 (13.3)

128 (9.2)/136 (12.9) 136 (9.7)/100 (16.2) 109 (10.0)/129 (13.1) 115 (9.9)/107 (16.0) 109 (10.3)/125 (11.0) 5.5 (10.6)/25.9 (47.3) 114 (10.2)/118 (17.2) 19.5 (8.0)/52.0 (17.7) 102 (6.5)/10.4 (17.7) 117 (18.0)/96 (10.7)

117 (8.9)/131 (6.4) 86.5 (10.4)/105 (9.4)

All compounds were eluted during the washing step with organic solvent.

was MeOH/ACN 1:1 (v/v), and 10 μL was injected for analysis. Data Analysis. Data were acquired with the Analyst 1.4.1 software package (Applied Biosystems). Statistical treatment was performed with STATGRAPHICS Centurion XV software package (Stat Point, Inc., Version 2002) and Excel (Microsoft, 2010). Concentrations below the lower limit of quantification (LLOQ) were substituted with a value equal to LLOQ divided by the square root of 2, for the calculation of geometric mean (GM).

provided adequate compensation for variations in the extraction percentages of BTRs and BTHs as the recoveries of BTR-d4 were proportional to the recoveries of target analytes. The absolute recoveries of BTH and 2-Me-S-BTH through OASIS HLB were 5% and 17%, respectively; the corresponding recoveries through STRATA-X CW were 29% and 58%. Although the recoveries (absolute and relative) of BTH and 2Me-S-BTH were higher through STRATA-X CW than through OASIS HLB, the RSD values were higher (13% to 47%) in the former than in the latter. 2-Amino-BTH was retained strongly by STRATA-X CW, which is explained by the strong interaction of −NH2 group with the carboxylic acid moiety present in the sorbent. The recovery of 2-amino-BTH through STRATA-X CW cartridge was low (11−12%). The matrix effect (ME%) was calculated by comparing the instrumental response of the matrix matched standard, spiked (i.e., post-extraction matrix spike) into the final extract prior to instrumental analysis, with that of the external calibration standard prepared in solvent (Figure S4, Supporting Information). Urine extracts analyzed by both types of SPE cartridges produced matrix suppression ranging from 4.5% to 88.8%; however, the suppression was more significant for BTH, 2-Me-S-BTH, and 2-SCNMeS-BTH from STRATA-X CW. The recoveries of BTH and 2-Me-S-BTH through Oasis HLB cartridges were low but were significantly improved after enzymatic deconjugation. The absolute recovery of BTH increased from 5% to 20%, and the recovery of 2-Me-S-BTH increased from 17% to 33%. The observed improvement in recovery was due to the change in the ionic strength of the urine matrix after the addition of 1 M ammonium acetate in the deconjugation step. Changes in ionic strength can denature biomolecules (particularly proteins) and consequently alter the interactions of target analytes with the sample matrix and the SPE sorbent. BTHs are structurally similar to naturally occurring purines as they can interact with charged biomolecules (i.e., proteins and phospholipids).44 The interaction of thiol compounds (like 2-Me-S-BTH and BTH) with different biomolecules has been reported earlier.25,44−46 Liquid−Liquid Extraction. Although the SPE method did not yield optimal recoveries for BTH and 2-Me-S-BTH, the recoveries of these compounds increased to >70% by LLE. We evaluated various solvents for their ability to extract all target analytes while sustaining less background noise. The nonpolar

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RESULTS AND DISCUSSION Solid-Phase Extraction. SPE cartridges have been evaluated for extraction and purification of BTRs and BTHs in environmental samples.9,15,40 In this study, OASIS HLB and STRATA-X CW were evaluated for extraction and isolation of BTRs and BTHs from urine matrix. Both cartridges have the ability to present hydrophobic, π−π, and hydrophilic intermolecular forces to target analytes. STRATA-X CW, however, is designed to increase the retention of basic compounds by strong cationic exchange through its deprotonated carboxylic acid moieties. BTRs are weak bases, whereas BTHs present both acidic and basic properties, depending on the functional group that is attached to the ring.5,43 The recovery of target analytes from the urine matrix was examined in six replicate analyses (by fortification of the urine matrix with standards prior to extraction) without enzymatic deconjugation. A fortification level of 100 ng for each target analyte, except for BTH and 2-OH-BTH (500 ng), was used. BTR-d4 and ATR-d5 were spiked at 50 and 10 ng, respectively. Samples were extracted by passage through OASIS HLB, as described above, and through STRATA-X CW, as shown in Figure S3, Supporting Information. Absolute and relative recoveries of target analytes are shown in Table 2. The recoveries of target analytes and the relative standard deviations (RSD%, N = 6) suggest more optimal performance of OASIS HLB in comparison with STRATA-X CW. The absolute recoveries for 7 of 10 target analytes through STRATA-X CW were >76%, whereas the recoveries were >90% for 8 of 10 target analytes through OASIS HLB. 1HBTR, TTR, XTR, 5-Cl-1H-BTR, 2-OH-BTH, and 2-SCNMeSBTH were successfully extracted and isolated from the human urine matrix with either SPE sorbent (Table 2). The IS BTR-d4 D

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Figure 1. XIC and MRM chromatograms of spiked urine sample at a fortification level of 200 ng for BTH and 2-OH-BTH and 40 ng for the rest of the target analytes and extracted and purified by SPE method (MRM transitions depicted).

solvents, methyl-t-butyl ether (MTBE), DCM, and ethylacetate (EtAC), introduced a high background noise for BTHs analysis. We then evaluated mixtures of each of the above solvents at 1:1 (v/v) ratio with more polar solvents, ACN, MeOH, and

acetone (AC). Solvent mixtures with MeOH yielded more impure extracts than the rest, whereas solvent mixtures with AC yielded low recoveries (in most cases 60% for the LLE method). Ionization suppression was less for 2-Me-S-BTH and 2-SCNMe-S-BTH (