Multi-laboratory validation of a Δ9-tetrahydrocannabinol LC-MS/MS test kit designed for quantifying THC and marijuana metabolites in blood.

Marijuana legalization has increased the demand for testing of Δ9-tetrahydrocannabinol (THC) and THC metabolites. The THC ToxBox® test kit (THC ToxBox®) is commercially available and supports high-throughput LC-MS/MS analytical methods designed to quantify low levels of THC and THC metabolites in blood. The purpose of this study is to determine if this new test kit meets the rigors of laboratory accreditation and produces equivalent results across six states- and locally-funded laboratories. Each laboratory followed internal method validation procedures established for their clinical (CLIA) or international (ISO17025) accreditation program. Test performance indicators included accuracy, precision, measurement of uncertainty, calibration models, reportable range, sensitivity, specificity, carryover, interference, ion suppression/enhancement and analyte stability. Analytes and interferents were resolved within the 6-min analytical runtime, and the 48-well plate pre-manufactured with calibrators, second source quality control material, and internal standards at precise concentrations allowed for simple and consistent sample preparation in less than one hour. Every laboratory successfully validated test kit procedures for forensic use. Differences in sensitivity were generally associated with the use of older equipment. Statistical analysis of results spanning reportable ranges show that laboratories with different instrument platforms produce equivalent results at levels sufficiently low enough to support per se limit testing of THC and THC metabolites (1-5 ng/mL). THC ToxBox® represents a viable option for state- and locally-funded laboratories charged with investigating impaired driving cases involving marijuana use.

Tamoxifen, soy, and lifestyle factors in Asian American women with breast cancer.

PURPOSE: Soy foods have been a staple in Asia for centuries but the consumption of this food in the West is recent. Intake of soy among women at high risk for or with breast cancer has become a public health concern because genistein, a major component of soy, has weak estrogenic effects on breast epithelium, and has been found to negate the benefit of tamoxifen in some animal and in vitro studies. PATIENTS AND METHODS: We conducted a cross-sectional study in Asian Americans with breast cancer who were tamoxifen users (n = 380) to investigate the association between soy intake and circulating levels of tamoxifen and its metabolites (N-desmethyl tamoxifen [N-DMT], 4-hydroxytamoxifen [4-OHT], and 4-hydroxy-N-desmethyl-tamoxifen [endoxifen]). RESULTS: Serum levels of tamoxifen or its metabolites were unrelated to self-reported intake of soy or serum levels of isoflavones. Blood levels of tamoxifen were 81% higher in postmenopausal women age 65 or older compared with premenopausal women age 45 or younger (P = .005); similar patterns of results were observed for the tamoxifen metabolites. Levels of N-DMT were 27% (P = .03) lower among women in the highest tertile of body mass index (BMI, > 24.4 kg/m2) compared with those in the lowest category (BMI 21.5). Women who used hypertensive medications had higher levels of tamoxifen (P = .02) and N-DMT (P = .04) compared with nonusers. CONCLUSION: We found no evidence that soy intake adversely affected levels of tamoxifen or its metabolites. However, age, menopausal status, BMI, and use of hypertensive medications significantly influenced circulating levels of tamoxifen and its metabolites in this population.

Transplacental drug transfer and frequency of Tk and Hprt lymphocyte mutants and peripheral blood micronuclei in mice treated transplacentally with zidovudine and lamivudine.

In previous studies, we have shown that zidovudine (3'-azido-3'-deoxythymidine; AZT), but not lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC], is genotoxic when administered to neonatal mice, and that 3TC when coadministered with AZT does not alter the responses observed with AZT alone (Von Tungeln et al. [2002] Carcinogenesis 23:1427-1432). We now have investigated the transplacental transfer of these drugs and the induction of mutants and micronuclei in the neonatal offspring. From gestational day 12 until parturition, female C57BL/6N and C57BL/6N/Tk(+/-) mice, which had been mated to male C3H/HeNMTV mice, were treated daily by gavage with AZT, 3TC, or a combination of AZT and 3TC. In both dams and fetuses, AZT was found at much higher levels than its metabolites, AZT 5'-glucuronide and 3'-azido-3'-deoxythymidine. In the neonates, AZT and the mixture of AZT and 3TC caused a decrease in the percentage of reticulocytes (RETs) and an increase in the percentage of micronucleated RETs and micronucleated normochromatic erythrocytes. When assessed 3 weeks after birth, AZT and the combination of AZT and 3TC increased the thymidine kinase (Tk) mutant frequency in male mice; at 5 weeks, 3TC increased the Tk mutant frequency in female mice. The increase in Tk mutants in mice treated with AZT and the mixture of AZT and 3TC was associated with loss of the wild-type (Tk(+)) allele (loss of heterozygosity; LOH) and a pattern of discontinuous LOH. These data indicate that AZT, 3TC, and the combination of AZT and 3TC are transplacental mutagens and that the increase in mutants resulting from AZT is due mainly to large-scale genetic alterations.

Quantification of tamoxifen and metabolites and soy isoflavones in human plasma using liquid chromatography with electrospray ionization tandem mass spectrometry.

Tamoxifen is an important estrogen receptor antagonist used successfully for the treatment and prevention of breast cancer. The use of complementary and alternative medicines is an increasingly popular means for patients to participate in their own health care, and soy products, which contain phytoestrogens, have been widely promoted for possible beneficial effects on menopausal symptoms. The possibility that soy isoflavones could reduce tamoxifen efficacy has been demonstrated in animal models of post-menopausal breast cancer, but the occurrence of such an effect in women has not been explored. This paper describes the development and validation of a sensitive method using solid-phase extraction and isotope dilution liquid chromatography/tandem mass spectrometry with multiple reaction monitoring for the concurrent analysis of the major soy isoflavones (genistein and daidzein), an important metabolite of daidzein (equol), tamoxifen, and its important metabolites (4-hydroxytamoxifen, N-desmethyltamoxifen, and 4-hydroxy-N-desmethyltamoxifen or "endoxifen") in the serum of rats and women. The limits of quantification achieved are sufficient to determine accurately and precisely the concentrations of all of these analytes in women consuming soy foods and/or therapeutic doses of tamoxifen at levels consistent with modulation of estrogen receptor-mediated functions. These procedures enable future investigations of the possible impact of diet on the outcome of breast cancer therapy.

Effect of arylformamidase (kynurenine formamidase) gene inactivation in mice on enzymatic activity, kynurenine pathway metabolites and phenotype.

The gene coding for arylformamidase (Afmid, also known as kynurenine formamidase) was inactivated in mice through the removal of a shared bidirectional promoter region regulating expression of the Afmid and thymidine kinase (Tk) genes. Afmid/Tk -deficient mice are known to develop sclerosis of glomeruli and to have an abnormal immune system. Afmid-catalyzed hydrolysis of N-formyl-kynurenine is a key step in tryptophan metabolism and biosynthesis of kynurenine-derived products including kynurenic acid, quinolinic acid, nicotinamide, NAD, and NADP. A disruption of these pathways is implicated in neurotoxicity and immunotoxicity. In wild-type (WT) mice, Afmid-specific activity (as measured by formyl-kynurenine hydrolysis) was 2-fold higher in the liver than in the kidney. Formyl-kynurenine hydrolysis was reduced by approximately 50% in mice heterozygous (HZ) for Afmid/Tk and almost completely eliminated in Afmid/Tk knockout (KO) mice. However, there was 13% residual formyl-kynurenine hydrolysis in the kidney of KO mice, suggesting the existence of a formamidase other than Afmid. Liver and kidney levels of nicotinamide plus NAD/NADP remained the same in WT, HZ and KO mice. Plasma concentrations of formyl-kynurenine, kynurenine, and kynurenic acid were elevated in KO mice (but not HZ mice) relative to WT mice, further suggesting that there must be enzymes other than Afmid (possibly in the kidney) capable of metabolizing formyl-kynurenine into kynurenine. Gradual kidney deterioration and subsequent failure in KO mice is consistent with high levels of tissue-specific Afmid expression in the kidney of WT but not KO mice. On this basis, the most significant function of the kynurenine pathway and Afmid in mice may be in eliminating toxic metabolites and to a lesser extent in providing intermediates for other processes.

Multiresidue confirmation of beta-agonists in bovine retina and liver using LC-ES/MS/MS.

Misuse of numerous beta-agonist drugs for their growth promoting effects in livestock production requires significant regulatory enforcement activities worldwide. The proof of illegal drug use needed for regulatory action usually requires the high degree of specificity derived from mass spectrometric analysis of suspect tissues and body fluids. In this paper, we describe a multiresidue screening method for confirmation of nine beta-agonist compounds in bovine liver and retina. A wide range of analyte structures was selected in order to demonstrate applicability to other chemically related beta-agonists for which standards are not currently available. The class-specific method, which is based on mixed mode cation exchange/reverse phase solid phase extraction, reverse phase gradient LC separation using a cyanopropyl-silica phase, and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode, yields high analyte recoveries at the target level of 1 ppb (ng/g). In addition, acquisition of multiple MRM transitions for each analyte permits simultaneous confirmation of beta-agonists at the level of 1 ppb in liver and retina by using intensity ratios between fragment ions and protonated molecules. Estimated values for the limit of quantification (LOQ) for individual beta-agonists were 0.08-0.3 ppb in liver and 0.02-0.5 in retina; the estimated limits of confirmation, using accepted criteria from international regulatory agencies, were 0.25-0.8 ppb in liver and 0.1-1 ppb in retina. This method should be useful in supporting regulatory enforcement programs that monitor beta-agonist misuse.

Photodecomposition of Pigment Yellow 74, a pigment used in tattoo inks.

Tattooing has become a popular recreational practice among younger adults over the past decade. Although some of the pigments used in tattooing have been described, very little is known concerning the toxicology, phototoxicology or photochemistry of these pigments. Seven yellow tattoo inks were obtained from commercial sources and their pigments extracted, identified and quantitatively analyzed. The monoazo compound Pigment Yellow 74 (PY74; CI 11741) was found to be the major pigment in several of the tattoo inks. Solutions of commercial PY74 in tetrahydrofuran (THF) were deoxygenated using argon gas, and the photochemical reaction products were determined after exposure to simulated solar light generated by a filtered 6.5 kW xenon arc lamp. Spectrophotometric and high-pressure liquid chromatography (HPLC) analyses indicated that PY74 photodecomposed to multiple products that were isolated using a combination of silica chromatography and reversed-phase HPLC. Three of the major photodecomposition products were identified by nuclear magnetic resonance and mass spectrometry as N-(2-methoxyphenyl)-3-oxobutanamide (o-acetoacetanisidide), 2-(hydroxyimine)-N-(2-methoxyphenyl)-3-oxobutanamide and N,N''-bis(2-methoxyphenyl)urea. These results demonstrate that PY74 is not photostable in THF and that photochemical lysis occurs at several sites in PY74 including the hydrazone and amide groups. The data also suggest that the use of PY74 in tattoo inks could potentially result in the formation of photolysis products, resulting in toxicity at the tattoo site after irradiation with sunlight or more intense light sources.

Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta.

Equol is a metabolite produced in vivo from the soy phytoestrogen daidzein by the action of gut microflora. It is known to be estrogenic, so human exposure to equol could have significant biological effects. Equol is a chiral molecule that can exist as the enantiomers R-equol and S-equol. To study the biological activity of racemic (+/-)-equol, as well as that of its pure enantiomers, we developed an efficient and convenient method to prepare (+/-)-equol from available isoflavanoid precursors. Furthermore, we optimized a method to separate the enantiomers of equol by chiral HPLC, and we studied for the first time, the activities of the enantiomers on the two estrogen receptors, ERalpha and ERbeta. In binding assays, S-equol has a high binding affinity, preferential for ERbeta (K(i)[ERbeta]=16 nM; beta/alpha=13 fold), that is comparable to that of genistein (K(i)[ERbeta]=6.7 nM; beta/alpha=16), whereas R-equol binds more weakly and with a preference for ERalpha (K(i)[ERalpha]=50 nM; beta/alpha=0.29). All equol isomers have higher affinity for both ERs than does the biosynthetic precursor daidzein. The availability and the in vitro characterization of the equol enantiomers should enable their biological effects to be studied in detail.

Liquid chromatographic-mass spectrometric determination of the metabolism and disposition of the anti-retroviral nucleoside analogs zidovudine and lamivudine in C57BL/6N and B6C3F1 mice.

Transmission of HIV from mother to infant can be effectively prevented by zidovudine (3'-azido-3'-deoxythymidine; AZT) alone or in combination with other anti-retroviral drugs; however, significant evidence for genotoxicity, including transplacental carcinogenicity in mice, has been reported for AZT. A method, based upon solid phase extraction (SPE) in the 96-well format, gradient liquid chromatography (LC), and electrospray mass spectrometry (MS), was developed and validated to measure serum concentrations in maternal C57BL/6N and fetal B6C3F1 mice of the nucleoside analogs AZT, lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), and several metabolites selected based on importance in detoxification and bioactivation reactions. After intravenous (i.v.) and oral dosing with either 400 mg/kg AZT or 200 mg/kg 3TC, pharmacokinetics were determined for AZT, AZT-5'-glucuronide, 3'-amino-3'-deoxythymidine (AMT), AZT-5'-phosphate, 3TC, and 3TC-5'-phosphate in serum of adult female mice. Pharmacokinetics were also determined in spleen for AZT-5'-phosphate and 3TC-5'-phosphate following i.v. dosing. In addition, a preliminary assessment was made of placental transfer of AZT and 3TC and the presence of metabolites in the fetal compartment. The method described provides a means to evaluate thoroughly metabolism and disposition of anti-retroviral nucleoside analogs in maternal and fetal mice for comprehensive studies of genotoxicity.

Riddelliine N-oxide is a phytochemical and mammalian metabolite with genotoxic activity that is comparable to the parent pyrrolizidine alkaloid riddelliine.

Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are naturally-formed genotoxic phytochemicals that are widely distributed throughout the world. Although, the quantities of PAs and PA N-oxides in plants are nearly equal, the biological and genotoxic activities of PA N-oxides have not been studied extensively. PA N-oxides are major metabolites of PAs and are generally regarded as detoxification products. However, in this study, we report that rat liver microsomes converted riddelliine N-oxide to the genotoxic 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) metabolite. Metabolism of riddelliine N-oxide by rat liver microsomes under hypoxic conditions (argon) generated predominantly the parent PA, riddelliine. The reduction of riddelliine N-oxide to riddelliine was diminished, when the metabolism of riddelliine N-oxide with rat liver microsomes was conducted aerobically. Rat liver microsomal incubations of riddelliine N-oxide in the presence of calf thymus DNA produced a set of DHP-derived DNA adducts as detected and quantified by 32P-postlabeling/HPLC. The same DHP-derived DNA adducts were also found in liver DNA of F344 rats fed riddelliine N-oxide or riddelliine. When rats received doses of 1.0 mg/kg riddelliine N-oxide for three consecutive days, the level of DNA adducts was 39.9 +/- 0.6 adducts/10(7) nucleotides, which was 2.6-fold less than that measured in rats treated with riddelliine at the same dose. We have previously shown that these DHP-derived DNA adducts are produced by chronic feeding of riddelliine and that the adduct levels correlated with liver tumor formation. Results presented in this paper indicate that riddelliine N-oxide, through its conversion to riddelliine, is also a potential genotoxic hepatocarcinogen.

Identification of DNA adducts derived from riddelliine, a carcinogenic pyrrolizidine alkaloid.

Riddelliine is a naturally occurring carcinogenic pyrrolizidine alkaloid that produces liver tumors in experimental animals. Riddelliine requires metabolic activation to dehydroriddelliine and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) to exert its toxicity. Previously, (32)P-postlabeling HPLC was used to detect a set of eight DHP-derived adduct peaks from DNA modified both in vitro and in vivo. Among these DHP-derived DNA adducts, two were identified as epimers of DHP-2'-deoxyguanosine 3'-monophosphate. In this study, the remaining adducts have been characterized as DHP-modified dinucleotides. A series of dinucleotides, TpGp, ApGp, TpCp, ApCp, TpAp, ApAp, TpTp, and ApTp, were obtained by enzymatic digestion of calf thymus DNA with micrococcal nuclease (MN) and spleen phosphodiesterase (SPD) followed by HPLC separation and structural identification by negative ion electrospray tandem mass spectrometry (ES/MS/MS). Incubation of individual dinucleotides with DHP produced DHP-modified dinucleotide adducts that were also characterized using LC-ES/MS/MS. A parallel analysis of the isolated DHP-modified dinucleotides using (32)P-postlabeling recapitulated the series of unidentified adduct peaks that we previously reported from DHP-modified calf thymus DNA in vitro and rat liver DNA in vivo. Intact calf thymus DNA was also reacted with DHP and then digested by MN/SPD under the same conditions. The adduct profile obtained from LC-ES/MS/MS analysis was similar to that observed from the isolated dinucleotides. Structural analysis using LC-ES/MS/MS showed that DHP bound covalently to both 3'- and 5'-guanine, -adenine, and -thymine bases (but not cytosine) of dinucleotides to produce two or more isomers of each DHP-dinucleotide adduct. By comparing adduct formation at dissimilar bases within individual dinucleotides, the relative reactivity of DHP with individual bases was determined to be guanine > adenine approximately thymine. Identification of the entire set of DHP-derived DNA adducts further validates the conclusion that riddelliine is a genotoxic carcinogen and enhances the applicability of these biomarkers for assessing carcinogenic risks from exposure to pyrrolizidine alkaloids.