Biomarkers of Organophosphate and Polybrominated Diphenyl Ether (PBDE) Flame Retardants of American Workers and Associations with Inhalation and Dermal Exposures.

This study evaluated workers' exposures to flame retardants, including polybrominated diphenyl ethers (PBDEs), organophosphate esters (OPEs), and other brominated flame retardants (BFRs), in various industries. The study aimed to characterize OPE metabolite urinary concentrations and PBDE serum concentrations among workers from different industries, compare these concentrations between industries and the general population, and evaluate the likely route of exposure (dermal or inhalation). The results showed that workers from chemical manufacturing had significantly higher (p <0.05) urinary concentrations of OPE metabolites compared to other industries. Spray polyurethane foam workers had significantly higher (p <0.05) urinary concentrations of bis(1-chloro-2-propyl) phosphate (BCPP) compared to other industries. Electronic scrap workers had higher serum concentrations of certain PBDE congeners compared to the general population. Correlations were observed between hand wipe samples and air samples containing specific flame-retardant parent chemicals and urinary metabolite concentrations for some industries, suggesting both dermal absorption and inhalation as primary routes of exposure for OPEs. Overall, this study provides insights into occupational exposure to flame retardants in different industries and highlights the need for further research on emerging flame retardants and exposure reduction interventions.

Assessment of triphenyl phosphate (TPhP) exposure to nail salon workers by air, hand wipe, and urine analysis.

Triphenyl phosphate (TPP or TPhP) is commonly used as an additive plasticizer or organophosphate flame retardant (OPFR) in consumer products including nail polish. We evaluated exposure to TPhP from 12 nail salon technicians working at four nail salons located in California over a period of two work days. Bulk samples of 15 nail polishes and other nail products were collected. Study participants also provided two personal air samples, two hand wipe samples (pre- and post-shift on day two), and two urine samples (pre-shift day one and post-shift day two). The geometric mean (GM) of TPhP air sampling concentrations was 7.39 ng/m3. Post-shift TPhP hand wipe concentrations (GM 1.35 µg/sample) were significantly higher (p = 0.024) than pre-shift hand wipe concentrations (GM 0.29 µg/sample). Diphenyl phosphate (DPP or DPhP), a urinary metabolite of TPhP used in this study as a biomarker of exposure, was detected in all post-shift urine samples and 75% of urine pre-shift samples. DPhP post-shift concentrations (GM 1.35 µg/g creatinine) were significantly higher than pre-shift concentrations (GM 0.84 µg/g creatinine; p = 0.012). In addition, DPhP post-shift concentrations were correlated with TPhP post-shift hand wipe concentrations, suggesting dermal contact may be a relevant exposure pathway for nail salon workers.

Maternal urinary concentrations of organophosphate ester metabolites: associations with gestational weight gain, early life anthropometry, and infant eating behaviors among mothers-infant pairs in Rhode Island.

BACKGROUND: Organophosphate esters (OPEs)-used as flame retardants and plasticizers-are associated with adverse pregnancy outcomes such as reduced fecundity and live births and increased preterm delivery. OPEs may interfere with growth and metabolism via endocrine-disruption, but few studies have investigated endocrine-related outcomes. The objective of this pilot study (n = 56 mother-infant pairs) was to evaluate associations of OPEs with gestational weight gain (GWG), gestational age at delivery, infant anthropometry, and infant feeding behaviors. METHODS: We quantified OPE metabolites (bis-2-chloroethyl phosphate [BCEP], bis (1,3-dichloro-2-propyl) phosphate [BDCPP], diphenyl phosphate [DPHP]) in pooled maternal spot urine collected throughout pregnancy (~ 12, 28, and 35 weeks' gestation). We obtained maternal sociodemographic characteristics from questionnaires administered at enrollment and perinatal characteristics from medical record abstraction. Trained research assistants measured infant weight, length, head and abdominal circumferences, and skinfold thicknesses at birth and 6 weeks postpartum. Mothers reported infant feeding behavior via the Baby Eating Behavior Questionnaire (BEBQ). Using multiple linear regression, we assessed associations of log2-transformed maternal urinary OPE metabolites with GWG, gestational age at delivery, infant anthropometry at birth, weekly growth rate, and BEBQ scores at 6 weeks postpartum. We used linear mixed effects (LME) models to analyze overall infant anthropometry during the first 6 weeks of life. Additionally, we considered effect modification by infant sex. RESULTS: We observed weak positive associations between all OPE metabolites and GWG. In LME models, BDCPP was associated with increased infant length (ß = 0.44 cm, 95%CI = 0.01, 0.87) and weight in males (ß = 0.14 kg, 95%CI = 0.03, 0.24). BDCPP was also associated with increased food responsiveness (ß = 0.23, 95%CI = 0.06, 0.40). DPHP was inversely associated with infant abdominal circumference (ß = - 0.50 cm, 95%CI = - 0.86, - 0.14) and female weight (ß = - 0.19 kg, 95%CI = - 0.36, - 0.02), but positively associated with weekly growth in iliac skinfold thickness (ß = 0.10 mm/wk., 95%CI = 0.02, 0.19). Further, DPHP was weakly associated with increased feeding speed. BCEP was associated with greater infant thigh skinfold thickness (ß = 0.34 mm, 95%CI = 0.16, 0.52) and subscapular skinfold thickness in males (ß = 0.14 mm, 95%CI = 0.002, 0.28). CONCLUSIONS: Collectively, these findings suggest that select OPEs may affect infant anthropometry and feeding behavior, with the most compelling evidence for BDCPP and DPHP.

Quantification of 16 urinary biomarkers of exposure to flame retardants, plasticizers, and organophosphate insecticides for biomonitoring studies.

Chlorinated alkyl and non-chlorinated aryl organophosphate flame retardants (OPFRs) and some brominated flame retardants (FR) were introduced as replacements for polybrominated diphenyl ethers (PBDEs) after PBDEs phase-out in 2004 and 2013. Organophosphorous (OP) insecticides are mainly used in agricultural settings since the Food Quality Protection Act of 1996 phased-out most residential uses of OP insecticides in the United States. Urinary metabolites of FRs and OPs are known exposure biomarkers to FRs and OP insecticides, respectively. For large population-based studies, concurrent quantification of these metabolites using a small urine volume is desirable, but until now was not possible. We developed an analytical approach to quantify in 0.2 mL urine 10 FRs and six OP insecticide metabolites: diphenyl phosphate, bis(1,3-dichloro-2-propyl) phosphate, bis(1-chloro-2-propyl) phosphate, bis(2-chloroethyl) phosphate, dicresyl phosphates, dibutyl phosphate, dibenzyl phosphate, 2,3,4,5-tetrabromobenzoic acid, 2-((isopropyl)phenyl)phenyl phosphate, 4-((tert-butyl)phenyl)phenyl phosphate, dimethyl phosphate, diethyl phosphate, dimethyl thiophosphate, dimethyl dithiophosphate, diethyl thiophosphate, and diethyl dithiophosphate. The method relies on enzymatic deconjugation, automated off-line solid phase extraction, high-performance liquid chromatography, and isotope dilution tandem mass spectrometry. Detection limits ranged from 0.05 to 0.5 ng mL-1, accuracy from 89 to 118%, and imprecision was <10%. . This method is the first to quantify simultaneously trace levels of 16 biomarkers of FRs and OP insecticides in only four drops of urine. We confirmed the method suitability for use in large epidemiological studies to assess background and occupational exposures to these classes of environmental pollutants by analyzing 303 samples collected from the general population and a group of firefighters. FR metabolite and DAPs concentrations in the general population group were lower than in the firefighters group, and within the ranges reported in the U.S. general population and other non-occupationally exposed populations.

Assessment of spray polyurethane foam worker exposure to organophosphate flame retardants through measures in air, hand wipes, and urine.

Tris(1-chloro-2-propyl) phosphate (TCPP, also referenced as TCIPP), a flame retardant used in spray polyurethane foam insulation, increases cell toxicity and affects fetal development. Spray polyurethane foam workers have the potential to be exposed to TCPP during application. In this study, we determined exposure to TCPP and concentrations of the urinary biomarker bis(1-chloro-2-propyl) phosphate (BCPP) among 29 spray polyurethane foam workers over 2 work days. Work was conducted at residential or commercial facilities using both open-cell (low density) and closed-cell (high density) foam. Study participants provided two personal air samples (Day 1 and Day 2), two hand wipe samples (Pre-shift Day 2 and Post-shift Day 2), and two spot urine samples (Pre-shift Day 1 and Post-shift Day 2). Bulk samples of cured spray foam were also analyzed. Sprayers were found to have significantly higher TCPP geometric mean (GM) concentration in personal air samples (87.1 µg/m3), compared to helpers (30.2 µg/m3; p = 0.025). A statistically significant difference was observed between TCPP pre- and post-shift hand wipe GM concentrations (p = 0.004). Specifically, TCPP GM concentration in post-shift hand wipe samples of helpers (106,000 ng/sample) was significantly greater than pre-shift (27,300 ng/sample; p < 0.001). The GM concentration of the urinary biomarker BCPP (23.8 µg/g creatinine) was notably higher than the adult male general population (0.159 µg/g creatinine, p < 0.001). Urinary BCPP GM concentration increased significantly from Pre-shift Day 1 to Post-shift Day 2 for sprayers (p = 0.013) and helpers (p = 0.009). Among bulk samples, cured open-cell foam had a TCPP GM concentration of 9.23% by weight while closed-cell foam was 1.68%. Overall, post-shift BCPP urine concentrations were observed to be associated with TCPP air and hand wipe concentrations, as well as job position (sprayer vs. helper). Spray polyurethane foam workers should wear personal protective equipment including air-supplied respirators, coveralls, and gloves during application.

Exposure to organophosphate flame retardant chemicals in the U.S. general population: Data from the 2013-2014 National Health and Nutrition Examination Survey.

BACKGROUND: Use of organophosphate flame retardants (OPFRs) including tris(1,3-dichloro-2-propyl) phosphate, triphenyl phosphate, tris(1-chloro-2-propyl) phosphate, and tris-2-chloroethyl phosphate, in consumer products is on the rise because of the recent phase out of polybrominated diphenyl ether (PBDE) flame retardants. Some of these chemicals are also used as plasticizers or lubricants in many consumer products. OBJECTIVES: To assess human exposure to these chlorinated and non-chlorinated organophosphates, and non-PBDE brominated chemicals in a representative sample of the U.S. general population 6years and older from the 2013-2014 National Health and Nutrition Examination Survey (NHANES). METHODS: We used solid-phase extraction coupled to isotope dilution high-performance liquid chromatography-tandem mass spectrometry after enzymatic hydrolysis of conjugates to analyze 2666 NHANES urine samples for nine biomarkers: diphenyl phosphate (DPHP), bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), bis-(1-chloro-2-propyl) phosphate (BCIPP), bis-2-chloroethyl phosphate (BCEP), di-n-butyl phosphate (DNBP), di-p-cresylphosphate (DpCP), di-o-cresylphosphate (DoCP), dibenzyl phosphate (DBzP), and 2,3,4,5-tetrabromobenzoic acid (TBBA). We calculated the geometric mean (GM) and distribution percentiles for the urinary concentrations (both in micrograms per liter [µg/L] and in micrograms per gram of creatinine). We only calculated GMs for analytes with an overall weighted frequency of detection >60%. For those analytes, we also a) determined weighted Pearson correlations among the log10-transformed concentrations, and b) used regression models to evaluate associations of various demographic parameters with urinary concentrations of these biomarkers. RESULTS: We detected BDCIPP and DPHP in approximately 92% of study participants, BCEP in 89%, DNBP in 81%, and BCIPP in 61%. By contrast, we detected the other biomarkers much less frequently: DpCP (13%), DoCP (0.1%), TBBA (5%), and did not detect DBzP in any of the participants. Concentration ranges were highest for DPHP (<0.16-193µg/L), BDCIPP (<0.11-169µg/L), and BCEP (<0.08-110µg/L). Regardless of race/ethnicity, 6-11year old children had significantly higher BCEP adjusted GMs than other age groups. Females had significantly higher DPHP and BDCIPP adjusted GM than males, and were more likely than males to have DPHP concentrations above the 95th percentile (odds ratio=3.61; 95% confidence interval, 2.01-6.48). CONCLUSIONS: Our results confirm findings from previous studies suggesting human exposure to OPFRs, and demonstrate, for the first time, widespread exposure to several OPFRs among a representative sample of the U.S. general population 6years of age and older. The observed differences in concentrations of certain OPFRs biomarkers by race/ethnicity, in children compared to other age groups, and in females compared to males may reflect differences in lifestyle and exposure patterns. These NHANES data can be used to stablish a nationally representative baseline of exposures to OPFRs and when combined with future 2-year survey data, to evaluate exposure trends.

Variability and predictors of urinary concentrations of organophosphate flame retardant metabolites among pregnant women in Rhode Island.

BACKGROUND: Organophospate flame retardants (PFRs) are chemicals of emerging concern due to restrictions on polybrominated diphenyl ether flame retardant formulations. We describe the occurrence, variability, and predictors of urinary metabolites of PFRs among pregnant women. METHODS: In 2014-2015, 59 women from Providence, RI provided up to 3 spot urine samples during pregnancy (~12, 28, and 35 weeks' gestation). We created a pooled urine sample per woman and measured nine relevant metabolites in individual and pooled samples. We used linear mixed models to calculate intraclass correlation coefficients (ICCs) across the 3 measurements and to assess sociodemographic and dietary predictors of PFRs. RESULTS: The median (IQR) of bis-2-chloroethyl phosphate (BCEP), bis(1,3-dichloro-2-propyl) phosphate (BDCPP), and diphenyl phosphate (DPhP), the metabolites most frequently detected, from pooled samples were: 0.31 µg/L (0.17-0.54), 1.18 µg/L (0.64-2.19), 0.93 µg/L (0.72-1.97), respectively. We observed fair to good reproducibility for BCEP (ICC = 0.50), BDCPP (ICC = 0.60), and DPhP (ICC = 0.43), and excellent agreement between the urinary flame retardant metabolite concentrations averaged across pregnancy versus pooled urine sample concentrations for BCEP (ICC = 0.95), BDCPP (ICC = 0.89), and DPhP (ICC = 0.93). Adjusting for pertinent sociodemographic factors and gestational week of urine collection, each 1 kg increase in pre-pregnancy weight was associated with greater BCEP (1.1%; 95% CI: 0.1, 2.1), BDCPP (1.5%; 95% CI: 0.3, 2.7), and DPhP (0.5%; 95% CI: 0.0, 1.1). Dietary factors were generally not associated with urinary flame retardant metabolites. CONCLUSIONS: Urinary concentrations of BCEP, BDCPP, and DPhP were frequently detected among women in this pilot study and had fair reproducibility across pregnancy. Body size may be an important predictor of urinary flame retardant metabolite concentrations.

Quantification of three chlorinated dialkyl phosphates, diphenyl phosphate, 2,3,4,5-tetrabromobenzoic acid, and four other organophosphates in human urine by solid phase extraction-high performance liquid chromatography-tandem mass spectrometry.

Polybrominated diphenyl ethers (PBDEs), produced as flame retardants worldwide, have been phased-out in many countries, and chlorinated and non-chlorinated organophosphates and non-PBDE brominated formulations (e.g., Firemaster 550 (FM550)) have entered the consumers' market. Recent studies show that components of organophosphate esters and FM550 are frequently detected in many products common to human environments. Therefore, urinary metabolites of these compounds can be used as human exposure biomarkers. We developed a method to quantify nine compounds in 0.4 mL urine: diphenyl phosphate (DPhP), bis(1,3-dichloro-2-propyl) phosphate (BDCPP), bis-(1-chloro-2-propyl) phosphate, bis-2-chloroethyl phosphate, di-p-cresylphosphate, di-o-cresylphosphate (DoCP), di-n-butyl phosphate, dibenzyl phosphate (DBzP), and 2,3,4,5-tetrabromobenzoic acid. The method relies on an enzymatic hydrolysis of urinary conjugates of the target analytes, automated off-line solid phase extraction, reversed phase high performance liquid chromatography separation, and isotope dilution-electrospray ionization tandem mass spectrometry detection. The method is high-throughput (96 samples/day) with detection limits ranging from 0.05 to 0.16 ng mL-1. Spiked recoveries were 90-113 %, and interday imprecision was 2-8 %. We assessed the suitability of the method by analyzing urine samples collected from a convenience sample of adults (n = 76) and from a group of firefighters (n = 146). DPhP (median, 0.89; range, 0.26-5.6 ng mL-1) and BDCPP (median, 0.69; range, 0.31-6.8 ng mL-1) were detected in all of the non-occupationally exposed adult samples and all of the firefighter samples (DPhP [median, 2.9; range, 0.24-28 ng mL-1], BDCPP [median, 3.4; range, 0.30-44 ng mL-1]); DBzP and DoCP were not detected in any samples.

Two-dimensional high performance liquid chromatography separation and tandem mass spectrometry detection of atrazine and its metabolic and hydrolysis products in urine.

Atrazine [6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine] is the most widely used herbicide in the United States. In recent years, there has been controversy about atrazine's potential endocrine/reproductive and neurological adverse effects in wildlife and humans. The controversy triggered several environmental and epidemiologic studies, and it generated needs for sensitive and selective analytical methods for the quantification of atrazine, atrazine metabolites, and degradation or hydrolysis products. We developed a two-dimensional high performance liquid chromatography (2D-HPLC) method with isotope dilution tandem mass spectrometry detection to measure atrazine in urine, along with 11 atrazine metabolites and hydrolysis products, including 6-chloro (Cl), 6-mercapto (Mer) and 6-hydroxy (OH) derivatives, and their desethyl, desisopropyl and diamino atrazine analogs (DEA, DIA and DAA, respectively). The 2D-HPLC system incorporated strong cation exchange and reversed phase separation modes. This versatile approach can be used for the quantitative determination of all 12 compounds in experimental animals for toxicological studies. The method requires only 10 µL of urine, and the limits of detection (LODs) range from 10 to 50 µg/L. The method can also be applied to assess atrazine exposure in occupational settings by measurement of 6-Cl and 6-Mer analogs, which requires only 100 µL of urine with LODs of 1-5 µg/L. Finally, in combination with automated off-line solid phase extraction before 2D-HPLC, the method can also be applied in non-occupational environmental exposure studies for the determination of -6-Cl and 6-Mer metabolites, using 500 µL of urine and LODs of 0.1-0.5 µg/L.

High-throughput sample preparation for the quantitation of acephate, methamidophos, omethoate, dimethoate, ethylenethiourea, and propylenethiourea in human urine using 96-well-plate automated extraction and high-performance liquid chromatography-tandem mass spectrometry.

Acephate, methamidophos, o-methoate, and dimethoate are organophosphorus pesticides, and ethylenethiouria and propylenethiourea are two metabolites from the bisdithiocarbamate fungicide family. They are some of the most widely used pesticides and fungicides in agriculture both domestically and abroad. The existing high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the measurement of these compounds in human urine was improved by using a 96-well plate format sample preparation; the use of HPLC-MS/MS was comparable with a concentration range of 0.125 to 50 ng/ml. Deuterium-labeled acephate, ethylenethiouria, and methamidophos were used as internal standards. The sample preparation procedure, in the 96-well format with a 0.8-ml urine sample size, uses lyophilization of samples, followed by extraction with dichloromethane. The analytes were chromatographed on a Zorbax SB-C3 (4.6 × 150 mm, 5.0-µm) column with gradient elution by using 0.1% formic acid in aqueous solution (solvent A) and 0.1% formic acid in methanol (solvent B) mobile phase at a flow rate of 1 ml/min. Quantitative analysis was performed by atmospheric pressure chemical ionization source in positive ion mode using multiple-reaction monitoring of the precursor-to-product ion pairs for the analytes on a TSQ Quantum Ultra HPLC-MS/MS. Repeated analyses of urine samples spiked with high (15 ng/ml), medium (5 ng/ml), and low (1 ng/ml) concentrations of the analytes gave relative SDs of <13%. The limits of detection were in the range of 0.004-0.01 ng/ml. The method also has high accuracy, high precision, and excellent extraction recovery. Furthermore, the improved sample preparation method decreased the cost and labor required while effectively doubling the analytic throughput with minimal matrix effect.

Measurement of ethyl methanesulfonate in human plasma and breast milk samples using high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Ethyl methanesulfonate (EMS) is a mesylate ester, which is known to be a potent mutagen, teratogen, and possibly carcinogen. Mesylate esters have been found in pharmaceuticals as contaminants formed during the manufacturing process and may potentially pose an exposure hazard to humans. We have developed and validated a method for detection of trace amounts (ng/ml levels) of EMS in human plasma and breast milk. The samples were extracted by matrix solid-phase dispersion with ethyl acetate using Hydromatrix and the ASE 200 Accelerated Solvent Extractor. The extracts were separated by high-performance liquid chromatography (HPLC) using a HILIC column. The detection was performed with a triple quadrupole mass spectrometer (TSQ Quantum Ultra, Thermo Electron Corporation) using atmospheric pressure chemical ionization in negative-ion mode and multiple reaction monitoring. The use of a surrogate internal standard in combination with HPLC-MS/MS provided a high degree of accuracy and precision. The extraction efficiency was greater than 70%. Repeated analyses of plasma and breast milk samples spiked with high (100 ng/ml), medium (50 ng/ml) and low (5 ng/ml) concentrations of the analytes gave relative standard deviations of less than 12%. The limits of detection were in the range of 0.5-0.9 ng/ml for both matrices.

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography-tandem mass spectrometry.

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.

Guanine radical reaction processes: a computational description of proton transfer in X-irradiated 9-ethylguanine single crystals.

Computational methods based on DFT procedures have been used to investigate proton-transfer processes in irradiated 9-ethylguanine crystals. Previous experimental results from X-irradiation and study of this system at 10 K found significant concentrations of two main products, R1, formed by N7-hydrogenation of the purine ring, and R2, the primary one-electron oxidation product (Jayatilaka, N.; Nelson, W. H. J. Phys. Chem. B 2007, 111, 7887). The objective of this work is to describe the processes leading to these products using computational methods that take into account molecular packing and bulk dielectric properties. The basic concept is that a proton will transfer following ionization if the net electronic energy of the system, consisting of the donor plus the acceptor plus any intervening molecules, becomes lower. Three approaches were used to investigate this concept, two based on energies computed for single molecules and one based on energies computed for two-molecule clusters arranged as in the crystals. The results are that the methods successfully predict the observed behavior, that it is energetically favorable on one-electron reduction for proton H1 to transfer from a neutral molecule to N7 of the neighbor, forming the N7-hydrogenated product, and that there is virtually no energy advantage for a proton to transfer upon one-electron oxidation. The results also support the proposal that the C8 H-addition radical, found only upon irradiation at 300 K, was the product of intramolecular transfer of the H7 proton to C8 in a process apparently requiring sufficient thermal energy for activation. Finally, the computations predict hyperfine couplings and tensors in very good agreement with those from experiment, thereby providing additional evidence for the success of the computations in describing the experimental observations.

Structure of radicals from X-irradiated guanine derivatives. 2. An experimental and computational study of 9-ethylguanine single crystals.

An EPR (electron paramagnetic resonance) and ENDOR (electron-nuclear double resonance) study of 9-ethylguanine crystals X-irradiated at 10 K detected evidence for three radical forms. Radical R1, characterized by three proton and two nitrogen hyperfine interactions, was identified as the product of net hydrogenation at N7 of the guanine unit. R1, which evidently formed by protonation of the primary electron addition product, exhibited an unusually distorted structure leading to net positive isotropic components of the alpha-coupling to the hydrogen attached to C8 of the guanine unit. Radical R2, characterized by two nitrogen and three proton hyperfine couplings, was identified as the primary electron loss product, *G+. Distinguishing between *G+ and its N1-deprotonated product is difficult because their couplings are so similar, and density functional theory (DFT) calculations were indispensable for doing so. The results for R2 provide the most complete ENDOR characterization of *G+ presented so far. Radical R3 exhibited a narrow EPR pattern but could not be identified. The identification of radicals R1 and R2 was supported by DFT calculations using the B3LYP/6-311+G(2df,p)//6-31+G(d,p) approach. Radical R4, detected after irradiation of the crystals at room temperature, was identified as the well-known product of net hydrogenation at C8 of the guanine component. Spectra from the room temperature irradiation contained evidence for R5, an additional radical that could not be identified. Radical concentrations from the low temperature irradiation were estimated as follows: R1, 20%; R2, 65%; R3, 15%.

Structure of radicals from X-irradiated guanine derivatives: an experimental and computational study of sodium guanosine dihydrate single crystals.

In sodium guanosine dihydrate single crystals, the guanine moiety is deprotonated at N1 due to growth from high-pH (>12) solutions. Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) studies of crystals X-irradiated at 10 K detected evidence for three radical forms. Radical R1, characterized by two proton and two nitrogen hyperfine interactions, was identified as the product of net hydrogenation at N7 of the N1-deprotonated guanine unit. R1 exhibited an unusually distorted structure leading to net positive isotropic components of the hydrogen alpha-couplings. Radical R2, characterized by one proton and one nitrogen hyperfine coupling, was identified as the primary electron-loss product. This product is equivalent to that of deprotonation at N1 by the guanine cation and represents the first ENDOR characterization of that product. Radical R3, characterized by a single hydrogen hyperfine coupling, was identified as the product of net dehydrogenation at C1' of the ribose moiety. The identification of radicals R1-R3 was supported by density functional theory (DFT) calculations on several possible structures using the B3LYP/6-311G(2df,p)//6-31G(d,p) approach. Radical R4, detected after warming the crystals to room temperature, was identified as the well-known product of net hydrogenation of C8 of the (N1-deprotonated) guanine component. Radical R1, evidently formed by protonation of the primary electron addition product, was present as roughly 60% of the total radicals detected at 10 K. Radical R2 was present as roughly 27% of the total yield, and the concentration of R3 contributed the remaining 13%. R3 is evidently the product of one-electron oxidation followed by deprotonation; thus, the balance of oxidation and reduction products is approximately equal within experimental uncertainty.