Determination of arylphenoxypropionic herbicides in water by liquid chromatography-electrospray mass spectrometry

The study described here investigates the replicability of gender-specific risk profiles for gonorrhea based on an Alaskan sample compared to a U.S. national sample of drug users at risk for HIV infection. The Alaska sample (interviewed at a field station in Anchorage, Alaska; N=1,049) and the national sample (interviewed at 18 sites other than Alaska; N=17,619) consisted of cocaine smokers and injection drug users not in drug treatment. A history of gonorrhea infection was self-reported and coded as ever or never. The Anchorage and national risk profile for men included the following factors: (a) history of intranasal or parenteral cocaine use, (b) being black versus nonblack, (c) being older, (d) income from illegal activity, and (e) history of amphetamine use. The Anchorage and national risk profiles for women included the following factors: (a) trading sex for money, (b) being Native American versus non-Native American, and (c) trading sex for drugs. The Anchorage model for women included perceived homelessness as a factor, but it was not retained in the national model. The extent of the replicability of these models illustrates the generalizability of Alaskan findings to other U.S. drug-using populations. The authors also discuss the implications of these findings for disease prevention.     

Determination of antibiotics in different water compartments via liquid chromatography-electrospray tandem mass spectrometry

The mechanisms by which BCG exerts its antitumour activity remain unclear. Attachment of BCG to the bladder via FN has been shown to be an important step in initiating its antitumorigenic activity. The mechanism(s) by which BCG operates requires LAK cells, BCG-activated killer cells, T lymphocytes (CD4) helper cells and CD8 suppressor/cytotoxic cells) and monocytes. The optimal route of administration is intravesical. The efficacy of a BCG vaccine depends on the viability, dose and strain. Differences in efficacy and side-effects have not been shown between different strains. Low-dose regimens successfully protect from recurrences, with fewer side-effects. The initial schedule of BCG is a course of six instillations in 6 weeks; when the patient fails this course, two possibilities arise. The first is maintenance therapy; response rates improve but there is more local and systemic toxicity. The second is a further 6-week course, and this seems most useful in those with a sustained response to the initial treatment. The clinical response to BCG therapy can be monitored using cytokine measurements or p53 determinations. Toxicity remains a major problem in BCG treatment and triple antituberculosis combination therapy should be given for 3 months in those with severe systemic side-effects. The use of prophylactic isoniazid is not recommend to decrease side-effects. The clinical results of BCG have been good, with success rates of 58-100%, with a minimal follow-up of one year in prophylaxis. BCG seems superior to intravesical therapy, but at the cost of inducing more adverse effects. BCG is not indicated for low- and intermediate-risk patients, in whom chemotherapy is the first choice. BCG can also be used to eliminate tumour after an incomplete TUR, or in patients who are unfit for surgery, with a 60-70% success rate. The primary and best treatment for CIS is intravesical BCG; encouraging results have been reported, with success rate of 42-83% after a minimal follow-up of one year. Although currently BCG seems to be the choice for high-risk superficial TCC, many questions remain unanswered, especially about the mechanism(s) of action, the optimal dose and clinical schedule.     

High-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry for the analysis of 1,2,3,4-tetrahydro-beta-carboline derivatives

Zinc has been shown to have antioxidant properties and to exhibit inhibitory effects on apoptosis. In this work we investigated the effect of zinc on DNA integrity and on apoptosis of HaCaT keratinocytes. Cells were submitted to zinc deprivation by a diffusible zinc chelator, (N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine) (TPEN) or supplied with zinc chloride and submitted to UVB radiation. After cell exposure to TPEN for 2 h, strand breaks significantly impaired DNA resistance to alkaline denaturation. DNA strand breaks induced by a 6 h TPEN application were significantly prevented if zinc chloride was supplied together with the chelator. TPEN also generated, after 4-6 h of application, cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes), features of cell death by apoptosis. Moreover, UVB irradiation led to early DNA strand breaks and to an increase in cytoplasmic nucleosomes which was maximum 10 h after irradiation. These effects were prevented by the supply of zinc chloride (0.1 mM) in the culture medium. These results suggest that zinc ions interfere with the apoptosis process at an early stage, by decreasing DNA damage able to trigger apoptosis.     

Determination of total cholesterol in serum by liquid chromatography-isotope dilution mass spectrometry

We have developed a liquid chromatography-isotope dilution mass spectrometry procedure to quantify total cholesterol in serum. A particle-beam interface was used for coupling the liquid chromatograph and the mass spectrometer. After electron impact ionization the ions m/z = 386 and m/z = 389 were used for selective ion monitoring of cholesterol and the internal standard [25,26,27-(13)C]cholesterol. The sample preparation steps required for serum materials are alkaline hydrolysis and an extraction of the cholesterol into the cyclohexane phase. Imprecision for the determination of cholesterol in control materials is typically <1.0%. The deviation from the certified reference values was <0.75% for all control materials tested. A method comparison of the results obtained by this method with those obtained by gas chromatography-isotope dilution mass spectrometry for n = 28 pooled human sera derived from samples analyzed in our routine laboratory did not show differences >2.5%.     

Determination of serum creatinine by isotope dilution method using discharge-assisted thermospray liquid chromatography/mass spectrometry

A discharge-assisted thermospray (plasmaspray) liquid chromatography/mass spectrometric method for the determination of serum creatinine is described. The method incorporates stable isotope dilution using (D3)creatinine as an internal standard. Separation is performed in reversed-phase high-performance liquid chromatography using 0.01 M aqueous ammonium acetate as a flow solvent. Effluents are directly introduced to the mass spectrometer and [MH]+ ions are monitored during LC/MS using the selected ion monitoring method. Satisfactory agreement between the analytical result and the certified value of the serum sample of standard reference material and relative standard deviation ranging from 0.6% to 1.2% was obtained.     

Structural characterization of site-specific N-glycosylation of recombinant human factor VIII by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry

The present study addresses the site occupancy and the site-specific carbohydrate microheterogeneity of N-linked oligosaccharides in recombinant human factor VIII, expressed in Chinese hamster ovary cells. The four factor VIIIa polypeptides, formed upon incubation with human thrombin, were isolated and separately subjected to proteolysis with trypsin. These tryptic digests were analyzed by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Selected ion monitoring of diagnostic carbohydrate ions was utilized to identify glycopeptide-containing chromatographic peaks. Oligomannose and complex carbohydrates were detected at the glycosylation sites of the 50 and the 73 kDa polypeptides, while all the oligosaccharides identified on the B-domain were complex-type structures. Only the 43 kDa polypeptide was found nonglycosylated. These studies established a biantennary core-fucosylated carbohydrate as the major substituent, consistent with the conclusions of the analyses on the entire N-linked carbohydrate pool (Kumar, H. P. M.; Hague, C.; Haley, T.; Starr, C. M.; Besman, M. J.; Lundblad, R.; Baker, D. Biotechnol. Appl. Biochem. 1996, 24, 207-216.). In addition, this mass spectrometric investigation revealed the presence of a complex nonfucosylated oligosaccharide not reported previously for this glycoprotein.     

Characterization of carbohydrates using a combination of derivatization, high-performance liquid chromatography and mass spectrometry

Mice trisomic for the distal portion of MMU 16 (Ts65Dn) were examined for differences in jejunal function and plasma amino acids as compared to diploid controls. Eighteen control and 19 Ts65Dn mice were compared for whole-body and intestinal O2 consumption, jejunal glucose uptake, and plasma amino acid concentrations. Ts65Dn mice consumed less (P < 0.02) O2 per gram of fasted body weight. No significant differences were found in either active or passive glucose uptake. Oxygen consumption by jejunal tissue was not different between Ts65Dn and control mice. The apparent energetic efficiency of jejunal active glucose uptake (eta mol ATP expended/eta mol glucose uptake) was significantly higher (115.6 vs. 80.8; P < 0.05) in Ts65Dn mice. Histomorphometric analysis of jejunal mucosa showed that Ts65Dn mice had shorter villus height (P < 0.04) and decreased planar villus circumference (P = 0.05). No differences were found in total jejunal protein (microgram/g) or DNA (mg/g) concentrations. Significantly higher concentrations of plasma tyrosine, phenylalanine, valine, leucine, isoleucine, and citrulline (P < 0.05) were found in Ts65Dn mice. Lower plasma concentrations of hydroxyproline were detected in Ts65Dn mice (P < 0.05). These data suggest that Ts65Dn mice have anomalies in digestive function and amino acid metabolism as compared to normal, diploid controls.     

Identification of eicosanoids in the red algae, Gracilaria asiatica, using high-performance liquid chromatography and electrospray ionization mass spectrometry

The "gold standard" inflow cytometric DNA analysis of breast cancer uses fresh tumor cells simultaneously labeled for cytokeratin (CK) and DNA. We developed a 2-parameter CK-DNA flow assay suitable for archival, paraffin-embedded tissue (PT). Six anti-CK monoclonal antibodies were tested by immunocytochemistry and our assay for staining of nuclei extracted from PT breast cancers by combination pepsin-trypsin digestion. Clone CAM 5.2 was inadequate for PT nuclear suspensions, but a cocktail of 2 anti-CK clones (AE1/AE3 and KL-1) distinguished epithelial from nonepithelial nuclei in 2-parameter flow dot plots. We studied 82 routine PT breast tumors by our assay and used a univariate flow DNA histogram based on fresh biopsy tissue for comparison. Three histogram data quality indicators were improved. A trend toward higher S-phase fractions was found for DNA diploid PT tumors, although when inflammation was evident histologically, the increment in S-phase fraction with gating was often marked. CK gating identified PT tumors containing concurrent CK-positive DNA diploid and nondiploid populations (27 of 56 DNA nondiploid histograms). By excluding nonepithelial nuclei, 2-color CK-DNA flow methods may increase the accuracy of ploidy and S-phase fraction measurements. Our method appears superior to previous techniques using clone CAM 5.2 for labeling of archival breast cancers.     

Identification and determination of pirlimycin residue in bovine milk and liver by high-performance liquid chromatography-thermospray mass spectrometry

Determinative and confirmatory methods of analysis for pirlimycin (I) residue in bovine milk and liver have been developed based on HPLC-thermospray (TSP) MS. Milk sample preparation consisted of precipitating the mill proteins with acidified acetonitrile followed by a solvent partitioning with a mixture of n-butyl chloride and hexane extraction of I from the aqueous phase into methylene chloride (MC), and solid-phase extraction clean-up. For liver, samples (2 g) were extracted with 0.25% trifluoroacetic acid in acetonitrile. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, then extracted with MC. The MC was evaporated to dryness and the dried residue reconstituted in 2.0 ml of 0.1 M ammonium acetate for analysis. A chromatographically resolved stereoisomer of I with TSP-MS response characteristics identical to I was used as an internal standard (I.S.) for quantitative analysis based on the ratio of peak areas of I to I.S. in the protonated molecular-ion chromatogram at m/z 411.2. The method for milk was validated by the analysis of control milk samples spiked with I at concentrations from 0.05 to 0.8 micrograms/ml. The overall recovery of pirlimycin across this concentration range was 95.4% +/- 8.7%. The limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were validated to be 0.05 micrograms/ml and 0.10 micrograms/ml, respectively. The method for liver was validated by the analysis of control liver samples spiked with I at concentrations ranging from 0.025 to 1.0 micrograms/g. The overall recovery of pirlimycin was 97.6% +/- 5.1% in this concentration range. The validated limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were 0.025 micrograms/g and 0.10 micrograms/g, respectively. Four diagnostic ions for I were monitored for confirmation: the pseudo-molecular ions (M+H)+ at m/z 411.2 (35Cl) and m/z 413.2 (37Cl), and fragment ions at m/z 375.2 and 158.1. Confirmatory criteria were defined for these assays.     

Characterisation of TNF-alpha-related peptides by high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry

Ion-spray triple quadrupole mass spectrometry and high-performance liquid chromatography were used to investigate the products from the solid phase synthesis of (H)-Leu-Thr-Glu-Asn-(OH), a TNF-alpha active-site probe. The target sequence was assembled using tert.-butoxycarbonyl (Boc) chemistry in stepwise fashion from the C-terminal on an Boc-Asn-OCH2-Pam-copoly(styrene-divinylbenzene) resin [Pam = 4-(carboxamidomethyl)benzyl ester]. The crude product was deprotected and cleaved from the resin by HF-p-cresol treatment for 1 h at 0 degrees C. HPLC analysis at 214 nm indicated two late-eluting major products and an early-eluting product. Preparative HPLC demonstrated that the early-eluting product contained ca. 80% of the expected recovered sample mass. Each component was then directly analysed by mass spectrometry and tandem mass spectrometry. The early eluting peak was confirmed as the desired LTEN sequence. Synthesis of the same sequence using 9-fluorenyl methoxycarbonyl (Fmoc) chemistry gave an identical product and confirmed the above analysis. The most significant by-product was derived from arylation of the glutamyl group by the quencher p-cresol. The likely origins of the by-products are discussed.     

Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry

The activity of mechanoreceptors was studied in normal rats' hindfoot and compared with that of rhizotomised animals. Evoked nerve impulses were recorded from afferent fibres of the n.tibialis. A possible autonomous functioning of decentralised mechanoreceptors, even though in an altered fashion, was shown. The registered shifts comprised a decrease in sensitivity to mechanical stimulation and lower adaptive properties. The findings suggest that the underlying mechanism involves a probable lack of trophic interaction leading to interruption of a link between the CNS and the peripheral nervous system.     

Determination of low 13C-glutamine enrichments using gas chromatography-combustion-isotope ratio mass spectrometry

Glutamine is an essential fuel for tissues with high rates of cell replication, such as enterocytes and lymphocytes. Infusion of 13C-labeled glutamine tracers allows for measurement of the rates of production, utilization and oxidation of glutamine's carbon skeleton in vivo. The use of this tracer, however, has been limited by its high cost and/or the difficulty is measuring low enrichments in biological fluids using conventional gas chromatography-mass spectrometry (GC/MS) techniques. We have developed a method using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that allows for the determination of low 13C enrichments (down to 0.06 mol.% excess) with a precision of 2% or better, and a within-day and between-day variability better than 5%, in plasma free glutamine. The method was applied to measuring the incorporation of 13C in plasma glutamine over the course of infusion of 13C-labeled acetate in a human subject.     

Proteins of rat serum: I. Establishing a reference two-dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography-electrospray mass spectrometry

In the present investigation, we have identified 56 major spots, or spot rows, corresponding to 22 proteins, in the 2-DE pattern of adult male rats. This was done mainly by applying two complementary techniques, namely immunoblotting and high performance liquid chromatography-mass spectrometry (HPLC-MS) peptide mapping. Glycoproteins were characterized by affinity blotting with six lectins. We have also detailed how rat serum differs from human serum in two main respects: (i) relative abundance of individual proteins, which amounts in some cases to a complete absence in either sample, and (ii) varying molecular parameters for homologous proteins. It was thus possible to establish a first-generation reference map of rat serum proteins, which can be accessed through http://weber.u.washington.edu/ruedilab/aebersold++ +.html. We hope the present database will be a useful reference for the evaluation of changes in serum protein distribution in the course of pharmacological and toxicological studies. The recognition of species-specific proteins appears of special relevance in this respect.     

Identification of pesticides by liquid chromatography--particle beam mass spectrometry using electron ionization and chemical ionization

Liquid chromatography-mass spectrometry (LC-MS) with a particle beam (PB) interface is used to separate and identify a group of pesticides. The mass spectra obtained under the different ionization modes, electron ionization (EI) and positive and negative chemical ionization (PCI and NCI) are compared. The operating conditions under each mode, determined by studying the influence on the ion abundance of the ion source temperature of the EI mode, and the gas pressure and ion source temperature in the methane CI were optimized. EI was more sensitive than PCI and NCI and of the latter two modes, NCI gave higher responses, especially for organophosphorus compounds. When on-line solid-phase extraction-LC-PB-MS was applied to real samples, limits of detection in full scan mode were in the range of 0.5 and 10 micrograms l-1 for EI. The analysis of real samples by on-line solid-phase extraction-LC-PB-MS enabled EI detection of one of the pesticides studied and confirmation by PCI and NCI. The combined EI/CI information also enabled the detection of some non-target compounds.     

Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.     

Isolation of adenoviruses and reoviruses from avian species other than domestic fowl

Adenoviruses and reoviruses were isolated from pigeons and mallard ducks. In addition, adenoviruses were isolated from budgerigars and a bantam and a reovirus was isolated from a turkey. Primary identification of these viruses was by electron-microscope examination. It was further possible to assign the 4 adenoviruses to recognized fowl serotypes, and the reoviruses shared a common antigen with fowl reoviruses. These viruses were isolated from a variety of clinical conditions.     

Determination of medetomidine, atipamezole and midazolam in pig plasma by liquid chromatography-mass spectrometry

Medetomidine, atipamezole and midazolam in pig plasma were determined by liquid chromatography-mass spectrometry (LC-MS) with an atmospheric pressure chemical ionization interface system by the use of detomidine as an internal standard. The method was applied to studies of pharmacokinetic behaviour of these drugs.     

Length and base composition of PCR-amplified nucleic acids using mass measurements from electrospray ionization mass spectrometry

A generally applicable algorithm has been developed to allow base composition of polymerase chain reaction (PCR) products to be determined from mass spectrometrically measured molecular weights and the complementary nature of DNA. Mass measurements of arbitrary precision for single-stranded DNA species are compatible with an increasingly large number of possible base compositions as molecular weight increases. For example, the number of base compositions that are consistent with a molecular weight of 35,000 is approximately 6000, based on a mass measurement precision of 0.01%. However, given the low misincorporation rate of standard DNA polymerases, mass measurement of both of the complementary single strands produced in the PCR reduces the number of possibilities to less than 100 at 0.01% mass precision, and base composition is uniquely defined at 0.001% mass precision. Taking into account the low misincorporation rate of standard DNA polymerases and the fact that the final PCR product also contains primers of known sequence (generally 15-20-mer in size, which flank the targeted region), this reduces the number of possible base combinations to only approximately 3 at MW = 35,000. In addition, the number of base pairs (i.e., length of the DNA molecule) is uniquely defined. We show that the use of modified bases in PCR or post-PCR modification chemistry allows unique solutions for the base composition of the PCR product with only modest mass measurement precision.     

Characterization of olanzapine (LY170053) in human liver slices by liquid chromatography/tandem mass spectrometry

Olanzapine metabolism was investigated using incubation of olanzapine with human liver slices. The intent of the investigation was to identify olanzapine metabolites and determine if the human liver slice incubations could potentially produce quantities of the olanzapine glucuronides for future studies. Along with known Phase 1 olanzapine metabolites, N-desmethyl-, 2-hydroxymethyl-, and 4'-N-oxide-, a new hydroxylated species was detected. Detection of Phase 2 metabolites included known N-10-glucuronides, a quaternary glucuronide and a novel glucuronide conjugate. This investigation showed the feasibility of using human liver slices to produce sufficient quantities of olanzapine glucuronides for further studies.