ARTICLE pubs.acs.org/est
Determination of Benzotriazole and Benzophenone UV Filters in Sediment and Sewage Sludge Zifeng Zhang,†,‡ Nanqi Ren,† Yi-Fan Li,†,§ Tatsuya Kunisue,‡ Dawen Gao,† and Kurunthachalam Kannan†,‡,* †
International Joint Research Center for Persistent Toxic Substances (IJRC-PTS), State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (HIT), Harbin 150090, People's Republic of China ‡ 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, Empire State Plaza, PO Box 509, Albany, New York 12201-0509, United States § Science and Technology Branch, Environment Canada, Toronto, Ontario, M3H 5T4 Canada ABSTRACT: Benzophenones and benzotriazoles are widely used as ultraviolet (UV) light filters and stabilizers in cosmetics, skin creams, and body lotions and as corrosion inhibitors in building materials, automobile components, and automotive antifreeze cooling systems. Benzophenones and benzotriazoles have been reported to occur in the environment. Some of these UV filters have been reported to possess significant estrogenic activity. Despite this, very few studies have examined their occurrence and profiles in the environment. In this work, we determined five benzophenone-type UV filters and two benzotriazole-type corrosion inhibitors, namely, 2-hydroxy-4-methoxybenzophenone (2OH-4MeO-BP), 2,4-dihydroxybenzophenone (2,4OH-BP), 2,20 -dihydroxy-4-methoxybenzophenone (2,20 OH4MeO-BP), 2,20 ,4,40 -tetrahydroxybenzophenone (2,20 ,4,40 OHBP), 4-hydroxybenzophenone (4OH-BP), 1H-benzotriazole (1H-BT), and 5-methyl-1H-benzotriazole (5Me-1H-BT), in sediment and sewage sludge samples, using liquidliquid extraction and liquid chromatography-tandem mass spectrometry (LCMS/MS). In addition, four benzotriazole-type UV stabilizers, namely, 2-(3-t-butyl-2-hydroxy-5-methylphenyl)-5-chlorobenzotriazole (UV-326), 2,4-di-t-butyl-6-(5-chloro-2H-benzotriazole-2yl) phenol (UV-327), 2-(2H-benzotriazole-2yl)-4,6-di-t-pentylphenol (UV-328), and 2-(5-t-butyl-2-hydroxyphenyl) benzotriazole (TBHPBT) were determined by gas chromatography (GC)-mass spectrometry (MS). The limits of quantitation (LOQ) were in the ranges of 0.060.33 ng g1 dry weight (dw) and 0.11.65 ng g1 dw for sediment and sludge samples, respectively. Recoveries of target compounds spiked into sample matrices and passed through the entire analytical procedure ranged from 70% to 116% (RSD: 3.3213.8%) and from 82% to 106% (RSD: 2.898.09%) for the compounds analyzed by LCMS/MS and GCMS, respectively. The methods were applied to the analysis of sediment samples from the Songhua, Saginaw, and Detroit Rivers; the sum concentrations of target compounds were 3.299.93, 5.8122.5, and 190389 ng g1 dw, respectively. Five sludge samples collected from five wastewater treatment plants in northeastern China contained the sum concentrations of target compounds in the range of 1046370 ng g1 dw. The concentration of UV-328 in sludge was the highest (mean: 1300 ng g1 dw) among the target compounds. To our knowledge, this is the first work to report the occurrence of 2OH-4MeO-BP, 2,4OH-BP, 2,20 OH-4MeO-BP, 2,20 ,4,40 OH-BP, and 4OH-BP in sediment and sludge samples.
’ INTRODUCTION A growing awareness of the risks associated with skin exposure to ultraviolet (UV) radiation over the past few decades has led to increased use of sunscreen agents, which can absorb the UV radiation and attenuate the negative effects; these chemicals are commonly referred to as UV filters. Benzophenone-type UV filters are not only contained in increasing amounts in sunscreen products 1,2 but also in many products of daily use, such as cosmetics, skin creams, body lotions, hair sprays, hair dyes, and shampoos. These chemicals enter the aquatic environment either directly, via wash off r 2011 American Chemical Society
from skin and clothes during water recreational activities, or indirectly, via discharge of sewage and swimming pool waters. Benzotriazole-type UV stabilizers are known to absorb a full spectrum of UV light, UV-A (320400 nm) and UV-B (280320 nm).3 Some benzotriazole-type UV stabilizers are Received: February 3, 2011 Accepted: March 30, 2011 Revised: March 18, 2011 Published: April 11, 2011 3909
dx.doi.org/10.1021/es2004057 | Environ. Sci. Technol. 2011, 45, 3909–3916
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Figure 1. Chemical structures of benzotriazole and benzophenone compounds analyzed (KOW, octanolwater partition coefficient).
used in a variety of plastic products, such as building materials, automobile components, wax, paint, adhesive agents, film, shoes, glasses, and some sports equipment, to prevent yellowing and degradation of the products. Benzotriazoles are high-production volume chemicals, applied in various industrial processes as corrosion inhibitors in deicing fluids for aircrafts and in automotive antifreeze cooling systems.46 A few investigations have indicated that some organic UV filters possess significant estrogenic effects in laboratory animals.7 In the U.S., 2-hydroxy-4-methoxybenzophenone (2OH-4MeO-BP), a benzophenone (BP) derivative, is widely used in a variety of cosmetic products as a UV filter.8 2OH-4MeO-BP has been shown to elicit weak estrogenic activity in vitro and in vivo 812 and antiandrogenic activity in vitro.12,13 2,4-Dihydroxybenzophenone (2,4OH-BP) and 2,20 -dihydroxy-4-methoxybenzophenone (2,20 OH-4MeO-BP), which are used in cosmetic products as sunscreen agents, are shown to be metabolized to 2OH4MeO-BP in rats and piglets.1417 Other benzophenone derivatives, such as 2,20 ,4,40 -tetrahydroxybenzophenone (2,20 ,4,40 OH-BP) and 4-hydroxybenzophenone (4OH-BP), have been shown to have higher estrogenic activities than that of 2OH-4MeOBP.911,15,18 Additionally, benzotriazole (BT) UV stabilizers and corrosion inhibitors, such as 2-(3-t-butyl-2-hydroxy-5-methylphenyl)5-chlorobenzotriazole (UV-326), 2,4-di-t-butyl-6-(5-chloro-2Hbenzotriazole-2-yl) phenol (UV-327), 2-(2H-benzotriazole-2yl)4,6-di-t-pentylphenol (UV-328), 2-(5-t-butyl-2-hydroxyphenyl) benzotriazole (TBHPBT), 1H-benzotriazole (1H-BT), and 5-methyl-1H-benzotriazole (5Me-1H-BT), are toxic to animals. The LD50 value of UV-327 was >2000 mg kg1 in rats 19 and >25 mg L1 in fish.20 A gender-related difference in the toxicity of UV-327, which was associated with alterations in sex hormones, was reported in neonatal rats.21 In addition, 1H-BT has been shown to elicit developmental effects in the marine invertebrate Ciona intestinalis.22 Some UV absorbers and corrosion inhibitors have been detected in some environmental samples 2333 as well as in
human urine samples.34,35 2OH-4MeO-BP, 2,4OH-BP, 2,20 OH4MeO-BP, 1H-BT, and 5Me-1H-BT have been determined in water, soil, sediment, and fish samples by gas chromatography (GC)-mass spectrometry (MS) techniques.2528,3032 Nevertheless, earlier studies found only certain BP and BT derivatives in environmental samples. In this study, we developed a method for the determination of five BP and six BT derivatives in sediment and sludge samples: 2OH-4MeO-BP, 2,4OH-BP, 2,20 OH4MeO-BP, 2,20 ,4,40 OH-BP, 4OH-BP, 1H-BT, and 5Me-1H-BT (Figure 1), with LCMS/MS and UV-326, UV-327, and UV328 and TBHPBT (Figure 1), using GCMS. The method was applied in the analysis of sediment collected from China and the U.S. and sewage sludge collected from China. To our knowledge, this is the first work to report the detection of UV filters, 2OH4MeO-BP, 2,4OH-BP, 2,20 OH-4MeO-BP, 2,20 ,4,40 OH-BP, and 4OH-BP, in sediment and sludge samples.
’ MATERIALS AND METHODS Chemicals and Reagents. 2OH-4MeO-BP, 2,4OH-BP, 2,20 OH-4MeO-BP, 2,20 ,4,40 OH-BP, UV-326, UV-328, d10-phenanthrene, and d16-bisphenol A (all with a purity of 9798%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1HBT, 5Me-1H-BT, TBHPBT, UV-327, and 4OH-BP (purity 9598%) were purchased from TCI America (Portland, OR, USA). Organic solvents used in this study were analytical grade and were purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA). Water for chromatographic purposes was purified by a Milli-Q system (Millipore, Billerica, MA, USA). Sample Collection. Surface sediment samples (SHR1-SHR6, 020 cm) were collected in 2009 from the Songhua River (downstream of 5 large cities located along the river), which is the largest tributary of the Heilong River in northeastern China. Surface sediment samples were also collected from the Saginaw River (SR1, SR2, and SR3; downstream of Saginaw City, mouth of the river, and Shelter Island) (in 2002) and the Detroit River 3910
dx.doi.org/10.1021/es2004057 |Environ. Sci. Technol. 2011, 45, 3909–3916
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Table 1. Elemental Composition and Ions Monitored for Two Benzotriazole and Five Benzophenone Derivatives Analyzed by LCMS/MS and for Four Benzotriazole Derivatives Analyzed by GCMS target compounds
CAS No.
elemental composition
molecular weight
retention time (min)
MS/MS or SIM ions (m/z)
1H-BT
95147
C6H5N3
119.12
9.56
118/50
5Me-1H-BT 2,20 ,4,40 OH-BP
136856 131555
C7H7N3 C13H10O5
133.15 246.22
10.06 10.37
132/64 245/91
4OH-BP
1137424
C13H10O2
198.22
10.99
197/92
d16-BPA
96210876
C15D16O2
244.38
11.29
241/142
2,4OH-BP
131566
C13H10O3
214.22
11.56
213/91
2,20 OH-4MeO-BP
131533
C14H12O4
244.25
11.90
243/93
2OH-4MeO-BP
131577
C14H12O3
228.25
12.77
227/211
d10-phenanthrene
1517222
C14D10
188.29
12.61
188
TBHPBT UV-326
3147760 3896115
C16H17N3O C17H18ClN3O
267.33 315.8
19.07 23.08
252, 267, 253 300, 315, 272
UV-328
25973551
C22H29N3O
351.49
24.58
322, 351, 336
UV-327
3864991
C20H24ClN3O
357.88
24.72
342, 344, 357
(DR1, DR2, and DR3) (in 1998) in Michigan, USA. Five sludge samples were collected from WWTPs serving five large cities located along the Songhua River: Jiamusi, Mudanjiang, Qiqihar, Changchun, and Jilin in Heilongjiang and Jilin Provinces, in July 2009. The surface sediment (020 cm) and sludge samples were collected and stored in aluminum containers, freeze-dried, and kept at 20 °C in darkness. Sediment and sludge samples taken from several spots (46 spots within 10 m) at a given sampling location were pooled to obtain a representative sample. Total organic carbon content (TOC) of sediment and sludge samples was determined by total organic carbon analyzer (TOC-VCPH, Shimadzu, Tokyo, Japan). Sample Preparation. Benzophenone UV absorbers (2OH4MeO-BP, 2,4OH-BP, 2,20 OH-4MeO-BP, 2,20 ,4,40 OH-BP, and 4OH-BP) and two benzotriazole corrosion inhibitors (1H-BT and 5Me-1H-BT) were determined in sediment and sludge samples with LC-MS/MS. Approximately 0.51 g of sediment or 0.050.1 g of sludge (freeze-dried and homogenized earlier) was taken in a 15-mL polypropylene tube, and 10 ng of BPA-d16 were spiked as a surrogate standard. After 1 h of equilibration, 5 mL of methanol were added, shaken for 30 min, and centrifuged at 4000 rpm for 4 min, and the solvent layer was then transferred into a new 15-mL polypropylene tube. The extraction was repeated twice, and the extracts were combined. The extract was evaporated under a gentle stream of N2 to approximately 0.5 mL, and 10 mL of Milli-Q water was added; after thorough mixing, extracts were passed through a solid phase extraction (SPE) cartridge (Oasis HLB 0.5 g/6 cc, Waters Corporation) and conditioned with 5 mL of methanol for 5 min. Five milliliters of Milli-Q water were then passed through the cartridge at a rate of approximately 1 mL min1. The sample extract was loaded onto the cartridge at approximately 1 mL min1, and the cartridge was washed with 5 mL of 5% methanol in water and dried under vacuum for 20 min. Target chemicals were eluted with 6 mL of 15% methanol in ethyl acetate at a flow rate of 1 mL min1. The extract was concentrated under a gentle stream of N2 to 1 mL for LCMS/MS analysis. Benzotriazole-type UV stabilizers (UV-326, 327, 328, and TBHPBT) were analyzed in sediment and sludge samples by use of a GCMS. Approximately 5 g sediment or 1 g sludge (freeze-dried and homogenized earlier) were taken in a 50-mL polypropylene tube, and 50 ng of d10-phenanthrene were spiked
as a surrogate standard. After 1 h of equilibration, 25 mL of a mixture of ethyl acetate and dichloromethane (1:1 v/v) were added, shaken for 30 min, and centrifuged at 4000 rpm for 4 min, and the solvent layer was transferred into a round-bottom flask. The extraction was repeated twice, and the extracts were combined. The extract was concentrated by rotary evaporation to 23 mL, and 5 mL of isooctane were added and evaporated under a gentle stream of N2, to approximately 1 mL. The extract was further purified by passage through a silica gel (0.5 g) packed column, which was eluted with 6 mL of ethyl ether/n-hexane (1:9 v/v). The eluate was concentrated under a gentle stream of N2 to 1 mL, for GCMS analysis. Instrumental Analysis. An API 2000 electrospray triple quadrupole mass spectrometer (ESIMS/MS; Applied Biosystems, Foster City, CA, USA) equipped with an Agilent 1100 Series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), consisting of a binary high-pressure pump and an automatic sampler, was used for the measurement of BP and BT derivatives. Ten microliters of the extract were injected onto a Thermo Betasil C18 (100 mm length 2.1 mm internal diameter, 5 μm particle diameter) chromatographic column serially connected with a guard column (20 2.1 mm, 5 μm; Thermo Electron Corporation, Bellefonte, PA, USA), at a flow rate of 300 μL min1. The mobile phase was methanol (solvent A), and deionized water (solvent B). The target compounds were separated by gradient elution starting with 15% A at 0 min, held for 1.5 min; increased to 80% A at 1.5 to 4.5 min, held for 1.5 min; increased to 90% A at 6.0 to 6.5 min, held for 1.5 min; increased to 99% A at 8.0 to 8.5 min, held for 10 min; and decreased to 15% A at 18.5 to 19.5 min, held for 5 min, with a total run time of 24.5 min. The negative ion multiple reaction monitoring (MRM) mode was used, and the MRM transitions monitored are given in Table 1. Nitrogen was used as both curtain and collision gas. The optimized MS/MS parameters for the target compounds are summarized in Table 2. A representative total ion chromatogram of a standard mixture, showing chromatographic resolution of the target compounds analyzed by LCMS/MS, is shown in Figure 2. Concentrations of UV-326, UV-327, UV-328, and TBHPBT were determined with an Agilent 6890 GC interfaced with an Agilent 5973 MSD (Agilent Technologies, Foster City, CA, USA). Chromatographic separation was accomplished by use of a 30-m 3911
dx.doi.org/10.1021/es2004057 |Environ. Sci. Technol. 2011, 45, 3909–3916
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Table 2. MS/MS Parameters Optimized for the Analysis of Two Benzotriazole and Five Benzophenone Derivatives in Sediment and Sludge Samplesa target compounds
CUR (psi)
CAD (psi)
IS (V)
TEM (°C)
GS1 (psi)
GS2 (psi)
DP (V)
FP (V)
EP (V)
CE (eV)
CXP (V)
1H-BT
10
9
4500
400
90
70
25
400
12
45
9
5Me-1H-BT 2,20 ,4,40 OH-BP
10 15
12 7
4500 4000
400 400
80 50
70 60
40 25
400 400
12 9
35 40
12 5
4OH-BP
10
7
4000
400
50
60
30
400
10
40
5
d16-BPA
20
8
4000
400
50
60
30
400
12
35
5 5
2,4OH-BP
10
7
4000
400
50
60
30
400
10
40
2,20 OH-4MeO-BP
20
7
4000
400
50
60
25
300
9
40
5
2OH-4MeO-BP
10
6
4500
400
50
60
25
400
10
30
7
a
CUR: Curtain gas; CAD: Collision gas; IS: Ion spray voltage; TEM: Temperature; GS1: Ion source gas 1; GS2: Ion source gas 2; DP: Declustering potential; FP: Focusing potential; EP: Entrance potential; CE: Collision energy; CXP: Collision cell exit potential.
to 270 at 5 °C min1 and held for 5 min; postrun time was 5 min at 300 °C. The MS was operated in an electron impact selected ion-monitoring (SIM) mode. The primary and secondary ions monitored for the identification and quantification and retention times of the target compounds are given in Table 1. Details regarding quality assurance and quality control protocols are given below.
’ RESULTS AND DISCUSSION Extraction and Purification of Samples for Analysis by LCMS/MS. The extraction and cleanup procedures for the
Figure 2. (a) Representative total ion chromatogram (TIC) of a standard mixture (5 ng mL1) injected (10 uL) into LCMS/MS; 1 = 1H-BT, 2 = 5Me-1H-BT, 3 = 2,20 ,4,40 OH-BP, 4 = 4OH-BP, 5 = d16BPA, 6 = 2,4OH-BP, 7 = 2,20 OH-4MeO-BP, 8 = 2OH-4MeO-BP. (b) TIC of sludge sample from Changchun WWTP.
ZB-5MS fused silica capillary column (0.25 mm inner diameter, 0.25 μm film thickness, Phenomenex, Torrance, CA, USA). Two microliters of the aliquot were injected in splitless mode at 250 °C. The column oven temperature was programmed from 80 °C for 1 min, increased to 200 °C at a rate of 10 °C min1, and
analysis of BP and BT derivatives in sediment and sludge were optimized by testing extraction efficiencies of various solvents. We tested extraction efficiencies of methanol, ethyl acetate, dichloromethane, and a 1:1 mixture of methanol/ethyl acetate. Methanol provided the greatest recoveries (g70% for sludge and g80% for sediment) for all target analytes. An earlier work reported the analysis of benzophenone-type UV filters in water and soil samples without purification of the extracts.31 Due to the complex nature of sediment and sludge samples, to minimize matrix effects in the LCMS/MS analysis, removal of matrix components was necessary. In this work, we developed a cleanup step by passing sample extracts through an Oasis HLB cartridge (500 mg/6 cc). Prior to analysis of the samples, we loaded selected sample extracts (0.050.1 g sludge sample from Changchun WWTP, ∼1 g sediment sample from Jilin city), spiked with 50 ng and 100 ng of mixed BP and BT standards, onto preconditioned cartridges; after washing the cartridge with 5 mL of 5% methanol in water, 100% ethyl acetate, 10%, 15%, and 20% methanol in ethyl acetate were used for the elution of target compounds. Six milliliters of 15% methanol in ethyl acetate showed good recoveries for all target compounds (g70% in sludge and g80% in sediments), except for 2OH-4MeO-BP (recovery 90%) for all target compounds. Detection Limit and Procedural Recoveries. The target analytes were quantified by an external calibration curve prepared at concentrations ranging from 0.05 to 100 ng mL1 for LCMS/MS (correlation coefficient = r g 0.997), and 0.5 to 100 ng mL1 for GCMS (r g 0.994). Instrumental limits of detection (LOD) were calculated from the signal-to-noise ratio (S/N > 3) of 3 for the pure standard solutions injected onto the column. Limits of quantification (LOQ) for the entire method were determined by the signal-to-noise ratio (S/N > 10) of 10 for the injected extracts. The LOD and LOQ values were determined based on the standard deviations of six replicate analyses, using the lowest calibration standard. The calculated LOD and LOQ values were, respectively, 0.041 and 0.14 ng mL1 for 2,20 OH-4MeO-BP, 2,4OH-BP, and 4OH-BP; 0.067 and 0.22 ng mL1 for 2OH-4MeO-BP, 2,20 ,4,40 OH-BP, 1H-BT, and 5Me-1H-BT; 0.1 and 0.3 ng mL1 for both UV-327 and TBHPBT; and 0.5 and 1.65 ng mL1 for both UV-326 and UV-328. Because 1 g sediment sample or 0.1 g sludge sample was extracted for the analysis by LCMS/MS, and 5 g sediment sample or 1 g sludge sample was extracted for the analysis by GCMS, the method LOD and LOQ values for sediment analyzed in this study were as follows: 0.041 and 0.14 ng g1 for 2,20 OH-4MeO-BP, 2,4OH-BP, and 4OH-BP; 0.067 and 0.22 ng g1 for 2OH-4MeO-BP, 2,20 ,4, 40 OH-BP, 1H-BT, and 5Me-1H-BT; 0.02 and 0.06 ng g1 for both UV-327 and TBHPBT; and 0.1 and 0.33 ng g1 for both UV-326 and UV-328; and the method LOD and LOQ values for sludge samples were 0.41 and 1.4 ng g1 for 2,20 -OH-4MeO-BP,
Figure 4. LCMS/MS chromatograms of target benzophenone and benzotriazole compounds in sediment and sludge sample.
2,4OH-BP, and 4OH-BP; 0.67 and 2.2 ng g1 for 2OH-4MeOBP, 2,20 ,4,40 OH-BP, 1H-BT, and 5Me-1H-BT; 0.1 and 0.3 ng g1 for both UV-327 and TBHPBT; and 0.5 and 1.65 ng g1 for both UV-326 and UV-328. Recoveries of target compounds spiked at 50- and 100-ng levels in sediment and sludge and passed through the entire analytical procedure (n = 6) ranged from 70% to 116%, with a relative standard deviation (RSD) of 3.3213.8% for the compounds analyzed by LCMS/MS, except for 2OH-4MeO-BP (mean =38.3%, SD ( 3.14%). The mean recoveries ranged from 82% to 106% (RSD: 2.898.09%) for the compounds analyzed by GCMS. 3913
dx.doi.org/10.1021/es2004057 |Environ. Sci. Technol. 2011, 45, 3909–3916
Environmental Science & Technology
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Table 3. Concentrations of Benzophenone and Benzotriazole Compounds in Sediment and Sludge Samples Analyzed (ng g 1 dw)a sediment compounds 1H-BT
5Me-1H-BT
values
Songhua River,
Saginaw and Detroit Rivers,
WWTPs from northeastern
China (6 sites)
U. S. (6 sites)
China (5 sites)
range
0.385
0.42433.4
17.2198
mean frequency
0.385 1/6
9.43 6/6
142 5/5
range
ND
mean frequency 2,20 ,4,40 OH-BP
range
ND
mean range mean
2,20 OH-4MeO-BP
range
ND
UV-326
UV-327
UV-328
TBHPBT
a
5/6
3/5
2.65
ND
0.3120.951 0.530
2.6610.1 5.74
3/6
4/5
0.2590.607
4.4191.6
mean
0.454
32.7
frequency
4/6
5/5
0.1330.796
ND
range
ND
mean 2OH-4MeO-BP
30.0104 71.5
1/6 ND
frequency 2,4OH-BP
1.59165 40.0
2.65
frequency 4OH-BP
sludge
0.424
frequency range
0.2720.545
4/6 0.7284.66
mean
0.380
2.34
12.8
frequency
6/6
6/6
5/5 23.3136
2.0513.3
range
1.712.01
5.88
mean
1.86
5.88
77.4
frequency
2/6
1/6
5/5
range
0.310
0.221.90
1.808.40
mean frequency
0.310 1/6
0.850 3/6
3.68 4/5
range
2.067.12
0.72224
40.65920
mean
3.81
116
1300
frequency
6/6
5/6
5/5
range
ND
ND
0.7301.18
mean
0.955
frequency
2/5
ND: not quantified (below the LOQ).
For each batch of 10 samples analyzed, a procedural blank, a matrix spike (20 ng g1 for UV-326, 327, 328, and TBHPBT analyzed by GCMS, and 10 ng g1 for 2OH-4MeO-BP, 2,4OH-BP, 2,20 OH-4MeO-BP, 2,20 ,4,40 OH-BP, 4OH-BP, 1H-BT, and 5Me-1H-BT analyzed by LCMS/MS), and a matrix spike duplicate were analyzed in sequence to check for contamination and for peak identification and quantification. There were no target compounds found in procedural blanks. The recoveries of target compounds from matrix spikes and matrix spike duplicate were 71.2% to 115% (RSD:
