Simple and rapid determination of norethindrone in human plasma by supported liquid extraction and ultra performance liquid chromatography with tandem mass spectrometry.

We report for the first time an ultra performance liquid chromatographic method with tandem mass spectrometric detection (UPLC/MS/MS) for the determination of norethindrone alone in human plasma over the concentration range of 50.0-25000 pg mL(-1) using a sample volume of 0.250 mL. Norethindrone and its internal standard (ISTD), norethindrone-(13)C(2), were extracted from human plasma by supported liquid extraction (SLE). After evaporation of the organic solvent, samples were reconstituted and analyzed on an UPLC/MS/MS system. The UPLC system used a Waters BEH C18 (100 mm × 2.1mm, 1.7 µm) column with mobile phase A of 0.05% formic acid in water:acetonitrile (65:35, v/v) and mobile phase B of 0.05% formic acid in methanol:acetonitrile (50:50, v/v). The flow rate was 0.500 mL min(-1). The method was fully validated. The inter-run accuracy and precision at the lower limit of quantitation (LLOQ), low, mid and high quality control (QC) concentration levels were 99.2-108.4% with a <8.1% CV (coefficient of variation), respectively. The validated method has been successfully applied to analysis of thousands of pharmacokinetic samples.

A rapid and robust liquid chromatography/tandem mass spectrometry method for simultaneous analysis of anti-tuberculosis drugs--ethambutol and pyrazinamide in human plasma.

Ethambutol and pyrazinamide are two first-line anti-tuberculosis drugs. Though they are normally combined for the treatment, their highly different polarity complicates simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of these two drugs in human plasma with decent peak shape and retention. Here we report a rapid and robust LC/MS/MS method for the simultaneous determination of these two drugs in human plasma. Human plasma samples, together with the isotopically labeled internal standards were extracted using protein precipitation, and then separated on a Chromolith SpeedROD RP-18e column and detected with mass spectrometry. The mobile phase is 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in methanol. Addition of trifluoroacetic acid in the mobile phases was found to be able to improve peak shape as well as to increase the retention of ethambutol, thus being able to analyze these two drugs at the same time with both drugs having decent peak shape and enough retention on a C18 column. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The standard curve range was 10.0-5000 ng/mL for ethambutol and 50.0-25,000 ng/mL for pyrazinamide using a plasma sample volume of 50.0 microL. This method has a very short run time of 3.8 min. The method has been fully validated, and <15% relative standard deviation was obtained for both analytes.

Electronic deoxyribonucleic acid (DNA) microarray detection of viable pathogenic Escherichia coli, Vibrio cholerae, and Salmonella typhi.

An electronic deoxyribonucleic acid (DNA) microarray technique was developed for detection and identification of viable Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella typhi. Four unique genes, the E. coli O157 lipopolysaccharide (LPS) gene (rfbE) and H7 flagellin gene (fliC), the V. cholerae O1 LPS gene (rfbE), and the S. typhi LPS gene (tyv), were chosen as the targets for detection. These targets were selectively amplified from mRNA of viable cells using reverse transcription polymerase chain reaction (RT-PCR) and detected using the electronic DNA microarray technique. Specific captures and reporters were designed and examined for selective detection and correct identification of the target pathogens. The technique was able to detect as few as 2-150 cells of E. coli O157:H7. The co-presence of six other common bacteria and a parasite at 10- and 1000-fold higher concentrations than the target E. coli O157:H7 did not interfere with the specific detection. Comparative analysis of live and heat-killed E. coli O157:H7 cells showed that the technique only responded to the viable cells and not to the dead cells. Thus, the integration of RT-PCR of specific mRNA with the electronic DNA microarray technique enables specific and sensitive detection of viable target cells. This technique is potentially useful for high throughput screening of multiple pathogenic bacteria in different samples.

An electronic DNA microarray technique for detection and differentiation of viable Campylobacter species.

An electronic oligonucleotide microarray technique was developed for detection and differentiation of the viable Campylobacter species, C. jejuni, C. coli, and C. lari. This development consisted of four major components: identification of single nucleotide polymorphisms (SNPs) within the hsp60 gene as species markers, design of fluorescently labelled SNP-based reporters, development of an electronic microarray detection, and application of the integrated technique to analysis of Campylobacter species in food samples. A unique capability of this technique is the specific detection of viable cells and not dead ones. This is achieved by using mRNA of the 60 kDa heat-shock protein as the viability marker. The identification of two unique SNPs closely located at positions 291 and 294 of the hsp60 gene enabled the differentiation of the three Campylobacter species. This technique was able to detect as few as two viable Campylobacter cells. The analysis of 19 blind Campylobacter samples showed 100% agreement with their identities obtained using pulsed-field gel electrophoresis. The analysis of six chicken samples revealed the presence of C. coli in one of the samples.

Reverse transcription-multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi.

BACKGROUND: Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi are pathogenic bacteria that can be found in contaminated water supplies throughout the world. No currently available assays can simultaneously detect and identify all three pathogens. Our aim was to develop a rapid and reliable technique for simultaneous detection of these pathogens. METHODS: Four unique genes were chosen as the targets of detection. Forward and reverse primers were designed to specifically amplify different sizes of these target genes: a 239-bp region of the E. coli O157 lipopolysaccharide (LPS) gene (rfbE); a 179-bp region of the H7 flagellin gene (fliC); a 419-bp region of the V. cholerae O1 LPS gene (rfbE); and a 329-bp region of Salmonella Typhi LPS gene (tyv). To ensure the detection of only viable replicating bacteria, RNA was extracted for analysis. After reverse transcription, cDNAs were simultaneously amplified in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. To characterize the assay we analyzed, in a blinded fashion, seven unknown RNA samples containing various combinations of total RNA from these bacteria as well as clinical isolates. RESULTS: All seven unknown RNA samples were correctly identified. The assay was able to detect and identify as few as 30 cells of E. coli O157:H7 and Salmonella Typhi in clinical isolates, and the presence of other bacteria did not interfere with the analysis. CONCLUSION: An assay combining reverse transcription with single-tube multiplex PCR was successfully developed and validated for simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.

Determination of arsenic metabolic complex excreted in human urine after administration of sodium 2,3-dimercapto-1-propane sulfonate.

Sodium 2,3-dimercapto-1-propane sulfonate (DMPS) has been used to treat acute arsenic poisoning. Presumably DMPS functions by chelating some arsenic species to increase the excretion of arsenic from the body. However, the excreted complex of DMPS with arsenic has not been detected. Here we describe a DMPS complex with monomethylarsonous acid (MMA(III)), a key trivalent arsenic in the arsenic methylation process, and show the presence of the DMPS-MMA(III) complex in human urine after the administration of DMPS. The DMPS-MMA(III) complex was characterized using electrospray tandem mass spectrometry and determined by using HPLC separation with hydride generation atomic fluorescence detection (HGAFD). The DMPS-MMA(III) complex did not form a volatile hydride with borohydride treatment. On-line digestion with 0.1 M sodium hydroxide following HPLC separation decomposed the DMPS-MMA(III) complex and allowed for the subsequent quantification by hydride generation atomic fluorescence. Arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), MMA(III), and DMPS-MMA(III) complex were analyzed in urine samples from human subjects collected after the ingestion of 300 mg of DMPS. The administration of DMPS resulted in a decrease of the DMA(V) concentration and an increase of the MMA(V) concentration excreted in the urine, confirming the previous results. The finding of the DMPS-MMA(III) complex in human urine after DMPS treatment provides an explanation for the inhibition of arsenic methylation by DMPS. Because MMA(III) is the substrate for the biomethylation of arsenic from MMA(V) to DMA(V), the formation of DMPS-MMA(III) complex would reduce the availability of MMA(III) for the subsequent biomethylation.

Arsenic speciation analysis.

Nearly two dozen arsenic species are present in the environmental and biological systems. Differences in their toxicity, biochemical and environmental behaviors require the determination of these individual arsenic species. Considerable analytical progresses have been made toward arsenic speciation analysis over the last decade. Hyphenated techniques involving a highly efficient separation and a highly sensitive detection have become the techniques of choice. Methods based on high-performance liquid chromatography separation with inductively coupled plasma mass spectrometry, hydride generation atomic spectrometry, and electrospray mass spectrometry detection have been shown most useful for arsenic speciation in environmental and biological matrices. These hyphenated techniques have resulted in the determination of new arsenic species, contributing to a better understanding of arsenic metabolism and biogeochemical cycling. Methods for extracting arsenic species from solid samples and for stabilizing arsenic species in solutions are required for obtaining reliable arsenic speciation information.

Arsenic speciation in urine from acute promyelocytic leukemia patients undergoing arsenic trioxide treatment.

Arsenic has been used successfully in clinical trials for treating acute promyelocytic leukemia (APL). Although sublethal doses of inorganic arsenic are used, little is known about the pharmacokinetics and metabolism of the high levels of arsenic in APL patients. To fill this important gap, this study describes the speciation of arsenic in urine from four APL patients treated with arsenic. Each patient was injected daily with an arsenite (As(III)) solution that contained 10 mg of As(2)O(3) precursor. Speciation analysis of the patient urine samples collected consecutively for 48 h, encompassing two intravenous injections of arsenic, revealed the presence of monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), and dimethylarsinic acid (DMA(V)). The intermediate methyl arsenic metabolites, MMA(III) and DMA(III), were detected in most urine samples from all of the patients when a preservative, diethyldithiocarbomate, was added to the urine samples to stabilize these trivalent arsenic species. The major arsenic species detected in the urine samples from the patients were As(III), MMA(V), and DMA(V), accounting for >95% of the total arsenic excreted. The relative proportions of As(III), As(V), MMA(V), and DMA(V) in urine samples collected 24 h after the injections of As(III) were 27.6 +/- 6.1, 2.8 +/- 2.0, 22.8 +/- 8.1, and 43.7 +/- 13.3%, respectively. The relatively lower fraction of the methylated arsenic species in these APL patients under arsenic treatment as compared with that from the general population exposed to much lower levels of arsenic suggests that the high levels of As(III) inhibit the methylation of arsenic (inhibits the formation of methyl arsenic metabolites). The arsenic species excreted into the urine accounted for 32-65% of the total arsenic injected. These results suggest that other pathways of excretion, such as through the bile, may play an important role in eliminating (removing) arsenic from the human body when challenged by high levels of As(III).

Interaction of trivalent arsenicals with metallothionein.

Arsenic is a human carcinogen, causing skin, bladder, and lung cancers. Although arsenic in drinking water affects millions of people worldwide, the mechanism(s) of action by which arsenic causes cancers is not known. Arsenic probably exerts some toxic effects by binding with proteins. However, few experimental data are available on arsenic-containing proteins in biological systems. This study reports on arsenic interaction with metallothionein and established binding stoichiometries between metallothionein and the recently discovered trivalent metabolites of arsenic metabolism. Size exclusion chromatography with inductively coupled plasma mass spectrometry analysis of reaction mixtures between trivalent arsenicals and metallothionein clearly demonstrated the formation of complexes of arsenic with metallothionein. Analysis of the complexes using electrospray quadrupole time-of-flight tandem mass spectrometry revealed the detailed binding stoichiometry between arsenic and the 20 Cys residues in the metallothionein molecule. Inorganic arsenite (As(III)) and its two trivalent methylation metabolites, monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), readily bind with metallothionein. Each metallothionein molecule could bind with up to six As(III), 10 MMA(III), and 20 DMA(III) molecules, consistent with the coordination chemistry of these arsenicals. The findings on arsenic interaction with proteins are useful for a better understanding of arsenic health effects.