Translesion synthesis across 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-γ-HMHP-dA) adducts by human and archebacterial DNA polymerases.

The 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-γ-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N(6)-propano group on 1,N(6)-γ-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N(6)-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) ß, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N(6)-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol ß. Single nucleotide incorporation experiments indicated that although hpol η, κ, and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N(6)-γ-HMHP-dA and detected large amounts of -1 and -2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N(6)-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.

Capillary HPLC-accurate mass MS/MS quantitation of N7-(2,3,4-trihydroxybut-1-yl)-guanine adducts of 1,3-butadiene in human leukocyte DNA.

1,3-Butadiene (BD) is a high volume industrial chemical commonly used in polymer and rubber production. It is also present in cigarette smoke, automobile exhaust, and urban air, leading to widespread exposure of human populations. Upon entering the body, BD is metabolized to electrophilic epoxides, 3,4-epoxy-1-butene (EB), diepoxybutane (DEB), and 3,4-epoxy-1,2-diol (EBD), which can alkylate DNA nucleobases. The most abundant BD epoxide, EBD, modifies the N7-guanine positions in DNA to form N7-(2, 3, 4-trihydroxybut-1-yl) guanine (N7-THBG) adducts, which can be useful as biomarkers of BD exposure and metabolic activation to DNA-reactive epoxides. In the present work, a capillary HPLC-high resolution ESI⁺-MS/MS (HPLC-ESI⁺-HRMS/MS) methodology was developed for accurate, sensitive, and reproducible quantification of N7-THBG in cell culture and in human white blood cells. In our approach, DNA is subjected to neutral thermal hydrolysis to release N7-guanine adducts from the DNA backbone, followed by ultrafiltration, solid-phase extraction, and isotope dilution HPLC-ESI⁺-HRMS/MS analysis on an Orbitrap Velos mass spectrometer. Following method validation, N7-THBG was quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of DEB and in DNA isolated from blood of smokers, nonsmokers, individuals participating in a smoking cessation program, and occupationally exposed workers. N7-THBG concentrations increased linearly from 31.4 ± 4.84 to 966.55 ± 128.05 adducts per 109 nucleotides in HT1080 cells treated with 1-100 µM DEB. N7-THBG amounts in leukocyte DNA of nonsmokers, smokers, and occupationally exposed workers were 7.08 ± 5.29, 8.20 ± 5.12, and 9.72 ± 3.80 adducts per 109 nucleotides, respectively, suggesting the presence of an endogenous or environmental source for this adduct. The availability of sensitive HPLC-ESI⁺-HRMS/MS methodology for BD-induced DNA adducts in humans will enable future population studies of interindividual and ethnic differences in BD bioactivation to DNA-reactive epoxides.

NanoHPLC-nanoESI(+)-MS/MS quantitation of bis-N7-guanine DNA-DNA cross-links in tissues of B6C3F1 mice exposed to subppm levels of 1,3-butadiene.

1,3-Butadiene (BD) is an important industrial chemical and a common environmental pollutant present in urban air. BD is classified as a human carcinogen based on epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its potent carcinogenicity in laboratory mice. A diepoxide metabolite of BD, 1,2,3,4-diepoxybutane (DEB), is considered the ultimate carcinogenic species of BD due to its ability to form genotoxic DNA-DNA cross-links. We have previously employed capillary HPLC-ESI(+)-MS/MS (liquid chromatography-electrospray ionization tandem mass spectrometry) methods to quantify DEB-induced DNA-DNA conjugates, e.g. 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD), 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and 1,N(6)-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-HMHP-dA), in tissues of laboratory mice exposed to 6.25-625 ppm BD (Goggin et al. Cancer Res. 2009, 69(6), 2479-2486). However, typical BD human exposure levels are 0.01 to 3.2 ppb in urban air and 1-2.0 ppm in an occupational setting, requiring greater detection sensitivity for these critical lesions. In the present study, a nanoHPLC-nanoESI(+)-MS/MS method was developed for ultrasensitive, accurate, and precise quantitation of bis-N7G-BD in tissues of laboratory mice treated with low ppm and subppm concentrations of BD. The LOD value of the new method is 0.5 fmol/100 µg DNA, and the LOQ is 1.0 fmol/100 µg DNA, making it possible to quantify bis-N7G-BD adducts present at concentrations of 3 per 10(9) nucleotides. Bis-N7G-BD adduct amounts in liver tissues of mice exposed to 0.5, 1.0, and 1.5 ppm BD for 2 weeks were 5.7 ± 3.3, 9.2 ± 1.5, and 18.6 ± 6.9 adducts per 10(9) nucleotides, respectively, suggesting that bis-N7G-BD adduct formation is more efficient under low exposure conditions. To our knowledge, this is the first quantitative analysis of DEB specific DNA adducts following low ppm and subppm exposure to BD.

Quantitation of DNA adducts by stable isotope dilution mass spectrometry.

Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations and potentially contributing to the development of cancer. Because of their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples, including immunoassay, HPLC, and ³²P-postlabeling, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adduct concentrations in biological samples are between 0.01-10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry, especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS, have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke.

Persistence and repair of bifunctional DNA adducts in tissues of laboratory animals exposed to 1,3-butadiene by inhalation.

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen. The mechanism of BD-mediated cancer is of significant interest because of the widespread exposure of humans to BD from cigarette smoke and urban air. BD is metabolically activated to 1,2,3,4-diepoxybutane (DEB), which is a highly genotoxic and mutagenic bis-alkylating agent believed to be the ultimate carcinogenic species of BD. We have previously identified several types of DEB-specific DNA adducts, including bis-N7-guanine cross-links (bis-N7-BD), N(6)-adenine-N7-guanine cross-links (N(6)A-N7G-BD), and 1,N(6)-dA exocyclic adducts. These lesions were detected in tissues of laboratory rodents exposed to BD by inhalation ( Goggin et al. (2009) Cancer Res. 69 , 2479 -2486 ). In the present work, persistence and repair of bifunctional DEB-DNA adducts in tissues of mice and rats exposed to BD by inhalation were investigated. The half-lives of the most abundant cross-links, bis-N7G-BD, in mouse liver, kidney, and lungs were 2.3-2.4 days, 4.6-5.7 days, and 4.9 days, respectively. The in vitro half-lives of bis-N7G-BD were 3.5 days (S,S isomer) and 4.0 days (meso isomer) due to their spontaneous depurination. In contrast, tissue concentrations of the minor DEB adducts, N7G-N1A-BD and 1,N(6)-HMHP-dA, remained essentially unchanged during the course of the experiment, with an estimated t(1/2) of 36-42 days. No differences were observed between DEB-DNA adduct levels in BD-treated wild type mice and the corresponding animals deficient in methyl purine glycosylase or the Xpa gene. Our results indicate that DEB-induced N7G-N1A-BD and 1,N(6)-HMHP-dA adducts persist in vivo, potentially contributing to mutations and cancer observed as a result of BD exposure.

Using measured cannabidiol and tetrahydrocannabinol metabolites in urine to differentiate marijuana use from consumption of commercial cannabidiol products.

CONTEXT: Detecting marijuana use is a component of most urine drug screens targeting a single Δ9-tetrahydrocannabinol metabolite. Recently, the non-intoxicating cannabinoid, cannabidiol (CBD), has gained popular acceptance for a myriad of reasons. Commercially available CBD products sold without purity regulations have become ubiquitous. Many products contain trace tetrahydrocannabinol. Long-term or high dose use of CBD products can result in tetrahydrocannabinol exposures, potentially producing a positive marijuana drug test. These results are not false positives since marijuana biomarkers are present, but inaccurately identify donors as marijuana users. Addressing this conundrum, we developed an assay discriminating marijuana use from the use of CBD contaminated with tetrahydrocannabinol. METHODS: Following the synthesis of a primary CBD metabolite, a LC-MS/MS assay was developed measuring the urinary metabolites tetrahydrocannabinol, 11-nor-carboxy-Δ9-tetrahydrocannabinol, CBD, and 7-carboxy-cannabidiol. The assay was utilized on 425 patients claiming CBD use, and sixteen samples from trusted users of commercial CBD products. RESULTS AND DISCUSSION: Clear data clusters enabled metabolic cut-points assignments. Forty-three percent of samples contained CBD metabolites in ten-fold excess to tetrahydrocannabinol metabolites which was then used as a set point to classify donors as CBD users. An excess of tetrahydrocannabinol metabolites classify donors as marijuana users. Additionally, urine samples were procured from donors personally known to use commercial CBD ad libitum, yet abstain from tetrahydrocannabinol. Results from trusted users substantiated the use of the resulting metabolic ratios despite 11-carboxy-tetrahydrocannabinol measured in 75% of these samples. CONCLUSION: A method has been developed and utilized to distinguish marijuana use from tetrahydrocannabinol exposure from contaminated CBD use.

Column switching HPLC-ESI(+)-MS/MS methods for quantitative analysis of exocyclic dA adducts in the DNA of laboratory animals exposed to 1,3-butadiene.

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen on the basis of epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its carcinogenicity in laboratory rats and mice. BD is metabolically activated to epoxide intermediates that can react with nucleophilic sites of cellular biomolecules. Among these, 1,2,3,4-diepoxybutane (DEB) is considered the ultimate carcinogenic species of BD due to its potent genotoxicity and mutagenicity attributed to the ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. DEB mutagenesis studies suggest that adducts formed at adenine bases may be critically important, as DEB induces large numbers of A --> T transversion mutations. We have recently identified two regioisomeric exocyclic DEB-dA adducts, 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-gamma-HMHP-dA) and 1,N(6)-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-alpha-HMHP-dA) ( Seneviratne et al. ( ( 2010 ) Chem. Res. Toxicol. 23 , 118 - 133 ), which were detected in DEB-treated calf thymus DNA and in tissues of BD-exposed laboratory animals. In the present work, we describe a column switching HPLC-ESI(+)-MS/MS methodology for the quantitative analysis of 1,N(6)-HMHP-dA isomers in the DNA of laboratory mice exposed to BD by inhalation. On the basis of their exocyclic structure, which prevents normal Watson-Crick base pairing, these adducts could be responsible for mutations at the A:T base pairs observed following exposure to DEB.

Exocyclic deoxyadenosine adducts of 1,2,3,4-diepoxybutane: synthesis, structural elucidation, and mechanistic studies.

1,2,3,4-Diepoxybutane (DEB) is considered the ultimate carcinogenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant present in urban air. Although it preferentially modifies guanine within DNA, DEB induces a large number of A --> T transversions, suggesting that it forms strongly mispairing lesions at adenine nucleobases. We now report the discovery of three potentially mispairing exocyclic adenine lesions of DEB: N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (compound 2), 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (compound 3), and 1,N(6)-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (compound 4). The structures and stereochemistry of the novel DEB-dA adducts were determined by a combination of UV and NMR spectroscopy, tandem mass spectrometry, and independent synthesis. We found that synthetic N(6)-(2-hydroxy-3,4-epoxybut-1-yl)-2'-deoxyadenosine (compound 1) representing the product of N(6)-adenine alkylation by DEB spontaneously cyclizes to form 3 under aqueous conditions or 2 under anhydrous conditions in the presence of an organic base. Compound 3 can be interconverted with 4 by a reversible unimolecular pericyclic reaction favoring 4 as a more thermodynamically stable product. Both 3 and 4 are present in double stranded DNA treated with DEB in vitro and in liver DNA of laboratory mice exposed to 1,3-butadiene by inhalation. We propose that in DNA under physiological conditions, DEB alkylates the N-1 position of adenine in DNA to form N1-(2-hydroxy-3,4-epoxybut-1-yl)-adenine adducts, which undergo an S(N)2-type intramolecular nucleophilic substitution and rearrangement to give 3 (minor) and 4 (major). Formation of exocyclic DEB-adenine lesions following exposure to 1,3-butadiene provides a possible mechanism of mutagenesis at the A:T base pairs.

Salt-Assisted Liquid-Liquid Extraction of Meconium for Analysis of Cocaine and Amphetamines by Liquid Chromatography-Tandem Mass Spectrometry.

Meconium, the first stool of a newborn, can be analyzed to identify prenatal exposure to drugs of abuse. Meconium accumulates in a fetus during the second and third trimesters of pregnancy providing a wide window of exposure. Identification of in utero drug exposure is essential for the diagnosis and treatment of infants for dependency/withdrawal caused from the exposure. However, testing of meconium samples is often cumbersome and time-consuming. Unlike liquid samples, meconium is a viscous, semisolid, tar-like substance that needs to be individually weighed prior to extraction. Additionally, the meconium matrix is not homogeneous and not easily mixed or extracted. A method for analyzing cocaine and metabolites as well as amphetamines in meconium utilizing ceramic homogenizers prior to salt-assisted liquid-liquid extraction and liquid chromatography tandem-mass spectrometry (LC-MS/MS) is presented.

Reduced urinary opioid levels from pain management patients associated with marijuana use.

Aim: Marijuana use has been postulated to modulate opioid use, dependence and withdrawal. Broad target drug testing results provide a unique perspective to identify any potential interaction between marijuana use and opioid use. Materials & methods: Using a dataset of approximately 800,000 urine drug test results collected from pain management patients of a time from of multiple years, creatinine corrected opioid levels were evaluated to determine if the presence of the primary marijuana marker 11-nor-carboxy-tetrahydrocannabinol (THC-COOH) was associated with statistical differences in excreted opioid concentrations. Results & conclusion: For each of the opioids investigated (codeine, morphine, hydrocodone, hydromorphone, oxycodone, oxymorphone, fentanyl and buprenorphine), marijuana use was associated with statistically significant lower urinary opiate levels than in samples without indicators of marijuana use.

Quantitative high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the adenine-guanine cross-links of 1,2,3,4-diepoxybutane in tissues of butadiene-exposed B6C3F1 mice.

1,3-Butadiene (BD) is an important industrial chemical used in the manufacture of rubber and plastics as well as an environmental pollutant present in automobile exhaust and cigarette smoke. It is classified as a known human carcinogen based on the epidemiological evidence in occupationally exposed workers and its ability to induce tumors in laboratory animals. BD is metabolically activated to several reactive species, including 1,2,3,4-diepoxybutane (DEB), which is hypothesized to be the ultimate carcinogenic species due to its bifunctional electrophilic nature and its ability to form DNA-DNA and DNA-protein cross-links. While 1,4- bis-(guan-7-yl)-2,3,-butanediol ( bis-N7G-BD) is the only type of DEB-specific DNA adduct previously quantified in vivo, four regioisomeric guanine-adenine (G-A) cross-links have been observed in vitro: 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), 1-(guan-7-yl)-4-(aden-3-yl)-2,3-butanediol (N7G-N3A-BD), 1-(guan-7-yl)-4-(aden-7-yl)-2,3-butanediol (N7G-N7A-BD), and 1-(guan-7-yl)-4-(aden-6-yl)-2,3-butanediol (N7G-N (6)A-BD) ( Park ( 2004) Chem. Res. Toxicol. 17, 1638- 1651 ). The goal of the present work was to develop an isotope dilution HPLC-positive mode electrospray ionization-tandem mass spectrometry (HPLC-ESI (+)-MS/MS) method for the quantitative analysis of G-A DEB cross-links in DNA extracted from BD-exposed laboratory animals. In our approach, G-A butanediol conjugates are released from the DNA backbone by thermal or mild acid hydrolysis. Following solid-phase extraction, samples are subjected to capillary HPLC-ESI (+)-MS/MS analysis with (15)N 3, (13)C 1-labeled internal standards. The detection limit of our current method is 0.6-1.5 adducts per 10 (8) normal nucleotides. The new method was validated by spiking G-A cross-link standards (10 fmol each) into control mouse DNA (0.1 mg), followed by sample processing and HPLC-ESI (+)-MS/MS analysis. The accuracy and precision were calculated as 105 +/- 17% for N7G-N3A-BD, 102 +/- 25% for N7G-N7A-BD, and 79 +/- 11% for N7G-N (6)A-BD. The regioisomeric G-A DEB adducts were formed in a concentration-dependent manner in DEB-treated calf thymus DNA, with N7G-N1A-BD found in the highest amounts. Under physiological conditions, N7G-N1A-BD underwent Dimroth rearrangement to N7G-N (6)A-BD ( t 1/2 = 114 h), while hydrolytic deamination of N7G-N1A-BD to the corresponding hypoxanthine lesion was insignificant. We found that for in vivo samples, a greater sensitivity could be achieved if N7G-N1A-BD adducts were converted to the corresponding N7G-N (6)A-BD lesions by forced Dimroth rearrangement. Liver DNA extracted from female B6C3F1 mice that underwent inhalation exposure to 625 ppm BD for 2 weeks contained 3.1 +/- 0.6 N7G-N1A-BD adducts per 10 (8) nucleotides ( n = 5) (quantified as N7G-N (6)A-BD following base-induced Dimroth rearrangement), while the amounts of N7G-N3A-BD and N7G-N7A-BD were below the detection limit of our method. None of the G-A cross-links was present in control animals. The formation of N7G-N1A-BD cross-links may contribute to the induction of AT base pair mutations following exposure to BD. Quantitative methods presented here may be used not only for studies of biological significance in animal models but potentially to predict risk associated with human exposure to BD.

Anodyne by Design; Measuring the Prevalence of Esoteric Designer Opioids in Pain Management Patients.

The recent increase in illicit opioids sold on the black market, cut into heroin and masqueraded as prescription pills prompts a significant public health concern. Most designer opioids possess unknown potencies and unknown pharmacokinetics and their unregulated, variable dosages lead to rashes of overdoses. Additionally, many of the designer opioids, especially the fentanyl analogs are significantly more potent than heroin. High-profile cases involving overdoses of U-47700 and carfentanil have been reported in the media; however, the true prevalence of these and other designer opioids is unknown. Independent LC-MS-MS screen and confirmation methods have been developed and validated to identify and quantify fentanyl, and 18 designer opioids and their metabolites; methods were then exercised on urine specimens from contract pain management clients. Assuming patients in a pain management program may have a higher probability to seek out self-medication, samples from pain management patients were investigated for designer opioids. Similarly, pain management patients identified as using heroin may be more likely to experiment with or be accidentally exposed to designer opioids, specimens screening positive for the heroin metabolite 6-acetylmorphine were specifically chosen for designer opioid screening. Within this small group of pain management and heroin-positive samples, nine designer opioids were detected at a total prevalence of 25%. When screening random pain management samples not positive for heroin, a considerably lower percentage of samples (<1%) were identified as positive for designer opioids. Furanyl fentanyl, fluorobutyryl fentanyl and acetylfentanyl were the most prevalent designer opioids detected in both test groups.

Identification of Unique Metabolites of the Designer Opioid Furanyl Fentanyl.

The illicit drug market has seen an increase in designer opioids, including fentanyl and methadone analogs, and other structurally unrelated opioid agonists. The designer opioid, furanyl fentanyl, is one of many fentanyl analogs clandestinely synthesized for recreational use and contributing to the fentanyl and opioid crisis. A method has been developed and validated for the analysis of furanyl fentanyl and furanyl norfentanyl in urine specimens from pain management programs. Approximately 10% of samples from a set of 500 presumptive heroin-positive urine specimens were found to contain furanyl fentanyl, with an average concentration of 33.8 ng/mL, and ranging from 0.26 to 390 ng/mL. Little to no furanyl norfentanyl was observed; therefore, the furanyl fentanyl specimens were further analyzed by untargeted high-resolution mass spectrometry to identify other metabolites. Multiple metabolites, including a dihydrodiol metabolite, 4-anilino-N-phenethyl-piperidine (4-ANPP) and a sulfate metabolite were identified. The aim of the presented study was to identify the major metabolite(s) of furanyl fentanyl and estimate their concentrations for the purpose of toxicological monitoring.

Catching Fakes: New Markers of Urine Sample Validity and Invalidity.

Urine drug testing is common for workplace drug testing, prescription management, emergency medicine and the criminal justice system. Unsurprisingly, with the significant consequences based upon the results of urine drug testing, a donor in need of concealing the contents of their sample is highly motivated to cheat the process. Procedures and safeguards ensuring sample validity are well known, and include measuring sample temperature at the time of collection, and laboratory measurements of creatinine, specific gravity and pH. Synthetic urine samples are available and are designed to deceive all aspects of urine drug testing, including validity testing. These samples are sophisticated enough to contain biological levels of creatinine, and are at a physiological pH and specific gravity. The goal of our research was to develop new procedures designed to distinguish authentic samples from masquerading synthetic samples. We aimed to identify substances in commercial synthetic urines not expected to be present in a biological sample distinguishing fake specimens. Additionally, we aimed to identify and employ endogenous compounds in addition to creatinine for identifying biological samples. We successfully identified two compounds present in synthetic urines that are not present in biological samples and use them as markers of invalidity. Four new endogenous markers for validity were successfully evaluated. Validity assessment was further aided by monitoring metabolites of nicotine and caffeine. When the method was applied to patient samples, 2% of samples were identified as inconsistent with natural urine samples, even though they met the current acceptance criteria for creatinine, pH and specific gravity.

Profiting from Probability; Combining Low and High Probability Isotopes as a Tool Extending the Dynamic Range of an Assay Measuring Amphetamine and Methamphetamine in Urine.

A wide range of concentrations are frequently observed when measuring drugs of abuse in urine toxicology samples; this is especially true for amphetamine and methamphetamine. Routine liquid chromatography-tandem mass spectrometry confirmatory methods commonly anchored at a 50 ng/mL lower limit of quantitation can span approximately a 100-fold concentration range before regions of non-linearity are reached deteriorating accurate quantitation and qualitative assessments. In our experience, approximately a quarter of amphetamine and methamphetamine positive samples are above a 5,000 ng/mL upper limit of quantitation and thus require reanalysis with dilution for accurate quantitative and acceptable qualitative results. We present here the development of an analytical method capable of accurately quantifying samples with concentrations spanning several orders of magnitude without the need for sample dilution and reanalysis. For each analyte the major isotopes were monitored for analysis through the lower concentration ranges (50-5,000 ng/mL), and the naturally occurring, low probability 13C2 isotopes were monitored for the analysis of the high concentration samples (5,000-100,000 ng/mL amphetamine and 5,000-200,000 ng/mL methamphetamine). The method simultaneously monitors transitions for the molecules containing only 12C and 13C2 isotopologues eliminating the need for re-extraction and reanalysis of high concentration samples.

Analysis of mitragynine and metabolites in human urine for detecting the use of the psychoactive plant kratom.

The leaves of the South Asian plant kratom are described as having stimulating effects at low doses, and opiate-like analgesic and euphoric effects at high doses. A long history of use and abuse has led to the classification of kratom as a controlled substance in its native Thailand and other South Asian countries. However, kratom is not controlled in the United States, and the ready availability of kratom has led to its emergence as an herbal drug of abuse. With the growing popularity of kratom, efficient procedures are needed to detect kratom use. In the current study, both ultra-high-performance liquid chromatography and high-performance liquid chromatography-tandem mass spectrometry methods have been developed and validated for monitoring the major alkaloids and metabolites found in urine following kratom use. The primary unique alkaloid mitragynine is quantified in human urine from 1.00-500.00 ng/mL using mitraphylline as an internal standard. In addition, two metabolites (5-desmethylmitragynine and 17-desmethyldihydromitragynine) and the related active, alkaloid 7-hydroxy-mitragynine, are simultaneously qualitatively monitored. The presence of analytes are confirmed by an information-dependent acquisition-enhanced product ion procedure generating full fragmentation data used to positively identify detected analytes. The validated method has been utilized for clinical and forensic analyses of urine for the detection of kratom use.

Analysis of total and transferrin-bound iron in human serum for pharmacokinetic studies of iron-sucrose formulations.

BACKGROUND: Patients with iron-deficiency anemia benefit from intravenous iron therapies. Development of these pharmaceutical agents requires pharmacokinetic studies monitoring levels of both the administered agent and transferrin-bound iron (TBI). Successful pharmacokinetic methods must discriminate iron species. RESULTS: Routine colorimetric procedures were used to reliably measure total iron and TBI following iron-sucrose administration. Iron was liberated from iron-sucrose allowing the determination of all circulating iron. Solid-phase sample processing allowed the measurement of TBI. Circulating iron-sucrose could then be calculated as the difference between total iron and TBI. CONCLUSION: A reproducible and robust spectrophotometric method was developed and validated for measuring total iron and TBI in human serum following iron-sucrose therapy.

1,3-Butadiene: Biomarkers and application to risk assessment.

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol). The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for major species differences in carcinogenicity. We have conducted extensive exposure-biomarker studies on mice, rats and humans. Using low exposures that range from current occupational levels to human exposures from tobacco smoke has provided evidence that mice are very different from humans, with mice forming ∼200 times more DEB than humans at exposures of 0.1-1.5ppm BD. While no gender differences have been noted in mice and rats for globin adducts or N-7 guanine adducts, female rats and mice had 2-3-fold higher Hprt mutations and DNA-DNA cross-links, suggesting a gender difference in DNA repair. Numerous molecular epidemiology studies have evaluated globin adducts and Hprt mutations, SCEs and chromosomal abnormalities. None of the blinded studies have shown evidence of human genotoxicity at current occupational exposures and studies of globin adducts have shown similar or lower formation of adducts in females than males. If one calculates the EB dose-equivalents for the three species, mice clearly differ from rats and humans, being ∼44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD.

Molecular dosimetry of 1,2,3,4-diepoxybutane-induced DNA-DNA cross-links in B6C3F1 mice and F344 rats exposed to 1,3-butadiene by inhalation.

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen based on epidemiologic studies in occupationally exposed workers and animal studies in laboratory rats and mice. BD is metabolically activated to three epoxides that can react with nucleophilic sites in biomolecules. Among these, 1,2,3,4-diepoxybutane (DEB) is considered the ultimate carcinogen due to its high genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links. Our laboratory has developed quantitative high-performance liquid chromatography-muESI(+)-tandem mass spectrometry methods for two DEB-specific DNA-DNA cross-links, 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) and 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD). This report describes molecular dosimetry analysis of these adducts in tissues of B6C3F1 mice and F344 rats exposed to a range of BD concentrations (0-625 ppm). Much higher (4- to 10-fold) levels of DEB-DNA cross-links were observed in mice compared with rats exposed to the same BD concentrations. In both species, bis-N7G-BD levels were 1.5- to 4-fold higher in the liver than in other tissues examined. Interestingly, tissues of female animals exposed to BD contained higher concentrations of bis-N7G-BD adducts than tissues of male animals, which is in accord with previously reported differences in tumor incidence. The molecular dosimetry data presented herein suggest that species and gender differences observed in BD-induced cancer are directly related to differences in the extent of BD metabolism to DEB. Furthermore, a rat model of sensitivity to BD may be more appropriate than a mouse model for assessing human risk associated with BD exposure, because rats and humans seem to be similar with respect to DEB formation.

Cdk2 plays a critical role in hepatocyte cell cycle progression and survival in the setting of cyclin D1 expression in vivo.

Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2(-/-) mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication and survival. Cdk2(-/-) hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2(-/-) liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2(-/-) hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2(-/-) mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.