Development of immunoaffinity solid phase microextraction probes for analysis of sub ng/mL concentrations of 7-aminoflunitrazepam in urine.

We report on the development of solid phase microextraction probes for drug analysis, prepared with antibodies specific for benzodiazepines covalently immobilized to the surface. In the technique, immobilized antibody probes are exposed to a sample containing the drug for 30 min. Extracted drugs are subsequently desorbed from the probes in 500 microL of methanolic desorption solution, which is dried, reconstituted in a small volume of injection solution and analysed by LC-MS/MS. The antibodies were characterized both before and after immobilization, to facilitate the rational selection of antibodies for such analyses. Polyclonal and monoclonal antibodies were compared as was the impact of affinity purification of the polyclonal antibody to isolate the drug-specific fraction. The probes were evaluated for utility in analyzing 7-aminoflunitrazepam at sub ng/mL concentrations in urine, which is expected to be found several days after a single oral dose of 2 mg of flunitrazepam. Such analyses are required in monitoring for abuse of this drug, both in terms of 'club drug' use and in cases of drug-facilitated sexual assault. In these cases drug concentrations in blood and urine are much lower than in chronic abuse cases and are difficult to analyse by conventional methods. The method developed has a limit of detection of 0.02 ng/mL, with accuracy ranging from 1% to 27% and precision (% R.S.D.) ranging from 2% to 10% between the lower and upper limits of quantitation for the analysis of 7-aminoflunitrazepam in urine. The dynamic range of the method is from 0.02 ng/mL, which is limited by the instrument sensitivity, to 0.5 ng/mL, which is approaching the capacity of the probes. This would allow for quantitative analysis of samples at concentrations below that measurable by many other methods for general benzodiazepines analysis from urine, and a highly selective screen for samples at higher concentrations. The method has similar limits of detection to the most sensitive literature methods specifically designed for such analysis but with the advantage of significantly simplified sample preparation. This simplification makes the technique more amenable for use by both professionals and non-professionals.

Salmonella typhimurium strains expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of aromatic amines.

Epidemiological studies have established the carcinogenic risk of occupational exposure to aromatic amines such as benzidine, beta-naphthylamine, and 4-aminobiphenyl. Metabolic activation of these chemicals to reactive, genotoxic electrophiles, via enzymatic N-oxidation and subsequent conjugation reactions, is necessary for their carcinogenic potential to be realized. Many aromatic amines are mutagenic in prokaryotic test systems, in the presence of exogenous mammalian activating enzymes such as those contained in hepatic 9000 x g supernatant. However, in the Ames (Salmonella typhimurium) assay, induction of mutations by aromatic amines and nitroarenes is also almost completely dependent upon the activity of the endogenous bacterial enzyme, N-acetyltransferase/O-acetyltransferase. The relevance of this assay to the prediction of the carcinogenic potential of aromatic amines in humans is thus restricted by the likelihood that the bacterial and human enzymes possess different substrate specificities. In this paper we report the construction and use of new tester strains of S. typhimurium that express high levels of functional human arylamine N-acetyltransferases, NAT1 and NAT2, retaining characteristic arylamine substrate specificities that are distinct from those of the bacterial enzyme. These new strains support the mutagenic activation of benzidine, 2-aminofluorene and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in the Ames test and may provide a new tool for evaluating the carcinogenic potential of aromatic amines.

Bioactivation of aromatic amines by recombinant human cytochrome P4501A2 expressed in Ames tester strain bacteria: a substitute for activation by mammalian tissue preparations.

The most widely used bioassay in genetic toxicology is the Ames test, which combines a bacterial mutagenicity assay (reversion of Salmonella typhimurium histidine-auxotrophic tester strains) with an exogenous bioactivation system (hepatic postmitochondrial supernatant or "S9"). The enzymatic activities of S9 prepared from the tissues of experimental animals are difficult to control. We show that the requirement for S9 can be obviated by the engineered expression of enzymes of bioactivation within the bacterial cell. With this strategy, reactive metabolites are produced inside the bacterial cell, proximate to the genetic target. Species boundaries can be crossed, and chimeric or mutant enzymes can be studied. We have constructed an Ames tester strain, expressing both aromatic amine N-acetyltransferase and human cytochrome P4501A2, which detects aromatic amine mutagenicity in the absence of S9.

Applications of solid-phase microextraction in food analysis.

Food analysis is important for the evaluation of the nutritional value and quality of fresh and processed products, and for monitoring food additives and other toxic contaminants. Sample preparation, such as extraction, concentration and isolation of analytes, greatly influences the reliable and accurate analysis of food. Solid-phase microextraction (SPME) is a new sample preparation technique using a fused-silica fiber that is coated on the outside with an appropriate stationary phase. Analyte in the sample is directly extracted to the fiber coating. The SPME technique can be used routinely in combination with gas chromatography (GC), GC-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC) or LC-MS. Furthermore, another SPME technique known as in-tube SPME has also been developed for combination with LC or LC-MS using an open tubular fused-silica capillary column as an SPME device instead of SPME fiber. These methods using SPME techniques save preparation time, solvent purchase and disposal costs, and can improve the detection limits. This review summarizes the SPME techniques for coupling with various analytical instruments and the applications of these techniques to food analysis.

Inhibition of benzo[a]pyrene dihydrodiol epoxide mutagenicity by synthetic analogues of ellagic acid.

Dibenzo[b, d]pyran-6-one, hydroxylated and methoxylated derivatives of this ring system, and some other analogues of the natural product ellagic acid have been synthesized and examined as inhibitors of benzo[a]pyrene dihydrodiol epoxide (BPDE) mutagenicity in Salmonella typhimurium strain TA100. Some of these new compounds have inhibitory effectiveness comparable to the natural product. On the basis of our results, we suggest qualitative rules for predicting inhibitory activity of ellagic acid analogues.

Simple and rapid determination of amphetamine, methamphetamine, and their methylenedioxy derivatives in urine by automated in-tube solid-phase microextraction coupled with liquid chromatography-electrospray ionization mass spectrometry.

A simple and rapid method for the determination of amphetamine, methamphetamine, and their 3,4-methylenedioxy derivatives in urine samples was developed using automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). In-tube SPME is an extraction technique for organic compounds in aqueous samples in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC-MS analyses of stimulants were initially performed by liquid injection onto an LC column to determine spectra. Five stimulants tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M+H]+ were observed for all stimulants. The stimulants were well separated with a Supelcosil LC-CN column using acetonitrile/50mM ammonium acetate (15:85) as a mobile phase. In order to optimize the extraction of stimulants, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 35 microL of sample in 50mM Tris-HCI (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary column. The stimulants extracted by the capillary were easily desorbed by mobile phase flow, and carryover of stimulants was not observed. Using in-tube SPME-LC-ESI-MS with selected ion monitoring, the calibration curves of stimulants were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9985 (n = 18) and detection limits (S/N = 3) of 0.38-0.82 ng/mL. This method was successfully applied to the analysis of human urine samples without interference peaks. The recoveries of stimulants spiked into urine samples were above 81%.

Method optimization for the analysis of amphetamines in urine by solid-phase microextraction.

Solid-phase microextraction is under investigation in many laboratories for its usefulness in the analysis of an ever widening variety of compounds. As new classes of compounds are investigated and new challenges arise, the methods are adapted to accommodate them. Polar semivolatiles are increasingly under study as analytical targets, and difficulties with small partition coefficients and long equilibration times have been identified. Amphetamine and methamphetamine were selected as semivolatiles exhibiting these limitations, and methods to optimize their analyses were investigated. Amphetamines are frequently monitored in very complex matrixes. Headspace methods minimize interactions between the sample and the fiber and have proven useful for these analyses. Several areas of experimental design were considered in the process of method optimization. These included matrix modification by heating, stirring, methanol content, addition of salt, and pH buffering. It was found that these amphetamines could be reliably analyzed using modified sample conditions, with excellent sensitivity, limits of detection, and method linearity. Clinical urine samples were successfully analyzed and gave clean chromatograms with no interfering peaks. Finally, the method developed was found to be useful for the analysis of narcotic analgesics. In the future, it is hoped that the method can be used to develop a general screen for a wide range of drugs of abuse.

Reevaluation of the effect of ellagic acid on N-methyl-N-nitrosourea DNA alkylation and mutagenicity.

N-Methyl-N-nitrosourea (MNU) is a reactive, mutagenic methylating agent. MNU methylates DNA at various sites, including guanine N7, guanine O6, and adenine N3. Dixit and Gold [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8039-8043] reported that ellagic acid, a phenolic natural product, inhibited the mutagenicity of MNU in Salmonella typhimurium strain TA 100, inhibited salmon sperm DNA alkylation by [3H]MNU, and also greatly reduced the ratio of guanine O6 to guanine N7 alkylation. We have examined the MNU-induced alkylation of calf thymus DNA and evaluated the effect of ellagic acid on this binding. Ellagic acid had only a slight effect on total alkylation and did not alter the ratio of methylation at guanine-O6 and -N7 positions. In further experiments, ellagic acid did not significantly inhibit MNU mutagenicity. These findings do not support the potential use of ellagic acid as an inhibitor of biological damage induced by nitrosoureas.

Re-evaluation of the effect of ellagic acid on dimethylnitrosamine mutagenicity.

Dimethylnitrosamine (DMN) is activated to mutagenic species in the Ames test (Salmonella typhimurium strain TA100) by hamster hepatic S9 preparation. This S9 activity is induced by administration of ethanol to the animals. The organic solvents dimethylsulphoxide (DMSO) and N-methyl-2-pyrrolidinone (MP) inhibit this mutagenicity, apparently because they inhibit DMN demethylase activity (assayed as formaldehyde production). Ellagic acid, dissolved in DMSO or MP, had no inhibitory effect on DMN mutagenicity, beyond the effect of the solvent vehicle.

Dimethylnitrosamine genotoxicity: does N-acetyltransferase activity play a role?

Dimethylnitrosamine (DMN) genotoxicity was evaluated in two test systems: induction of the umu operon (as measured by expression of a lacZ gene fusion) and mutagenicity (as measured by the Ames assay). Three Salmonella typhimurium strains were used; the strains differ in the level of expression of the enzyme acetyl CoA:arylamine N-acetyltransferase (NAT). We did not observe increased sensitivity in a strain with a plasmid-borne copy of the nat gene, and expressing very high levels of NAT activity. In contrast to a recent report, we conclude that NAT-dependent metabolic activation does not play a major role in the genotoxicity of this carcinogenic nitrosamine.

Automated in-tube solid-phase microextraction-liquid chromatography-electrospray ionization mass spectrometry for the determination of ranitidine.

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was evaluated for the determination of ranitidine. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary column by repeated aspirate/dispense steps. In order to optimize the extraction of ranitidine, several in-tube SPME parameters such as capillary column stationary phase, extraction pH and number and volume of aspirate/dispense steps were investigated. The optimum extraction conditions for ranitidine from aqueous samples were 10 aspirate/dispense steps of 30 microliters of sample in 25 mM Tris-HCl (pH 8.5) with an Omegawax 250 capillary column (60 cm x 0.25 mm I.D., 0.25 micron film thickness). The ranitidine extracted on the capillary column was easily desorbed with methanol, and then transported to the Supelcosil LC-CN column with the mobile phase methanol-2-propanol-5 M ammonium acetate (50:50:1). The ranitidine eluted from the column was determined by ESI-MS in selected ion monitoring mode. In-tube SPME followed by LC-ESI-MS was performed automatically using the HP 1100 autosampler. Each analysis required 16 min, and carryover of ranitidine in this system was below 1%. The calibration curve of ranitidine in the range of 5-1000 ng/ml was linear with a correlation coefficient of 0.9997 (n = 24), and a detection limit at a signal-to-noise ratio of three was ca. 1.4 ng/ml. The within-day and between-day variations in ranitidine analysis were 2.5 and 6.2% (n = 5), respectively. This method was also applied for the analyses of tablet and urine samples.

Automated in-tube solid-phase microextraction coupled with liquid chromatography/electrospray ionization mass spectrometry for the determination of beta-blockers and metabolites in urine and serum samples.

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was evaluated for the determination of beta-blockers in urine and serum samples. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC/MS analyses of beta-blockers were initially performed by liquid injection onto a LC column. Nine beta-blockers tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M + H]+ were observed for all beta-blockers. The beta-blockers were separated with a Hypersil BDS C18 column using acetonitrile/methanol/water/acetic acid (15:15:70:1) as a mobile phase. To optimize the extraction of beta-blockers, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 30 microL of sample in 100 mM Tris-HCl (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary (Supelco, Bellefonte, PA). The beta-blockers extracted by the capillary were easily desorbed by mobile-phase flow, and carryover of beta-blockers was not observed. Using in-tube SPME/LC/ESI-MS with selected ion monitoring, the calibration curves of beta-blockers were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9982 (n = 18) and detection limits (S/N = 3) of 0.1-1.2 ng/mL. This method was successfully applied to the analysis of biological samples without interference peaks. The recoveries of beta-blockers spiked into human urine and serum samples were above 84 and 71%, respectively. A serum sample from a patient administrated propranolol was analyzed using this method and both propranolol and its metabolites were detected.

Metabolic activation of heterocyclic aromatic amines catalyzed by human arylamine N-acetyltransferase isozymes (NAT1 and NAT2) expressed in Salmonella typhimurium.

Heterocyclic aromatic amines formed during the cooking of meat and meat-derived products can be activated to reactive metabolites which bind to DNA, induce mutations and cause tumors in animals. A principal route of metabolic activation is N-oxidation to hydroxylamines, and their subsequent activation by acetyltransferase-catalyzed O-acetylation. We have used mutagenicity assays to study O-acetylation of heterocyclic arylhydroxylamines by the two isozymes of human N-acetyltransferase, NAT1 and NAT2, expressed in Salmonella typhimurium. N-Acetylation was also examined, using an HPLC method. In addition, Salmonella strains with endogenous acetyltransferase and lacking this activating activity were used. Hydroxylamines of nine heterocyclic aromatic amines, IQ, isoIQ, MeIQ, MeIQx, NI, PhIP, Glu-P-1, Glu-P-2, and Trp-P-2 were generated in situ by rat liver S9 mix. The strains expressing human NAT1 and lacking acetyltransferase activity showing little or no ability to activate these substrates. The strains expressing human NAT2 and Salmonella acetyltransferase supported to different extents the activation of all the compounds except PhIP and Trp-P-2. N-Acetylation of IQ, MeIQx and PhIP was slow or not detectable. In conclusion, human NAT2 but not NAT1 can O-acetylate heterocyclic hydroxylamines. NAT2 probably plays a key role in the genotoxic effects of the above heterocyclic amines except for PhIP and Trp-P-2, which have NAT2-independent mutagenic activity.