A pentaplex reverse-transcription polymerase chain reaction (Pentaplex RT-PCR) in a single tube was developed for the simultaneous detection of the pome fruit viruses: Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV). This is the first report of the simultaneous detection of all four viruses and host mRNA as an internal specific control. Pentaplex RT-PCR was applied successfully throughout the year, using different plant organs (leaves or dormant buds). The sensitivity of detection by monoplex- and pentaplex RT-PCR assays was comparable. Different combinations of mixed infections of viruses were identified in samples of infected apple and pear trees from different geographical regions. The pentaplex RT-PCR assay developed was sensitive, simple, rapid, and reliable for simultaneous detection of the four viruses in extracts of leaves or dormant buds.
- MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Financing, Organized MeSH
- Plant Leaves virology MeSH
- Malus virology MeSH
- RNA, Messenger analysis MeSH
- Reverse Transcriptase Polymerase Chain Reaction methods instrumentation MeSH
- Reproducibility of Results MeSH
- RNA Viruses isolation & purification classification MeSH
- Plant Extracts analysis MeSH
- Plant Viruses isolation & purification classification MeSH
- Sensitivity and Specificity MeSH
A novel non-aqueous capillary electrophoresis - tandem mass spectrometry method for the simultaneous separation, identification and quantification of nine designer benzodiazepines (bentazepam, etizolam, deschloroetizolam, diclazepam, flubromazepam, flubromazolam, nimetazepam, phenazepam, and pyrazolam) was developed. A non-aqueous running electrolyte consisting of 25mM ammonium acetate with 100mM trifluoroacetic acid in acetonitrile was used. The separation was carried out using a semipermanent coated capillary (successive multiple ionic-polymer coating) with a strong anodic electroosmotic flow at a negative separation voltage within twelve minutes. Electrospray ionization with a triple quadrupole mass spectrometry was utilized for the identification and quantification of selected designer benzodiazepines in a positive ionization mode. The developed method was validated and applied on the analysis of spiked serum sample following a simple liquid-liquid extraction. The LODs of the designer benzodiazepines were between 1.5 and 15.0ngmL-1.
- MeSH
- Benzodiazepines blood MeSH
- Electrophoresis, Capillary MeSH
- Liquid-Liquid Extraction MeSH
- Hexadimethrine Bromide chemistry MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Humans MeSH
- Limit of Detection MeSH
- Designer Drugs analysis MeSH
- Tandem Mass Spectrometry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
An HPLC procedure for the determination of lamotrigine (LAM) simultaneously with other antiepileptic drugs, primidone (PD), phenobarbital (PB), phenytoin (DPH), carbamazepine (CMZ), and two active metabolites 2-phenyl-2-ethyl-malonamide (PEMA) and 10,11-dihydro-10,11-epoxycarbamazepine (EPO) was developed and validated. The method involves an ordinary RP system and a liquid-liquid extraction. The mobile phase consisting of water/ACN/methanol/triethylamine in the ratio 72:23:5:0.1 with pH 7.0 was selected as the best one after the assays testing both pH and triethylamine contents. UV detection was carried out at a wavelength of 220 nm and the whole analysis took 15 min. The method was linear in the range of 0.5-25 mg/L for PEMA and LAM; 1.25-25 mg/L for PD and CMZ; 0.625-12.5 mg/L for EPO; 1.5-60 mg/L for PB; and 1.25-50 mg/L for DPH, respectively. Within-day CV% and between-day CV% were within 10%. The developed HPLC method can be used for routine therapeutic drug monitoring both in children and adults.
This paper describes the development of a method for the simultaneous determination of ten anticoagulant rodenticides (coumafuryl, warfarin, pindone, coumatetralyl, coumachlor, difenacoum, bromadiolone, brodifacoum, chlorophacinone and flocoumafen) in the liver and kidney based on column-switching liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry. The simple sample preparation includes extraction with methanol. A C18 trapping column was used for online solid-phase extraction before analytical separation with the mobile phase comprising a mixture of 0.1% formic acid in water, methanol and acetonitrile. Chromatographic separation was achieved using a Thermo Hypersil ultra high-performance liquid chromatography (UHPLC) C18 column with the mobile phase consisting of 5 mM ammonium formate buffer (pH = 9) and methanol. The column-switching procedure ensured no matrix effects during electrospray ionization (ESI). Extraction recoveries ranged between 91 and 100% for liver and between 89 and 97% for kidney. The method showed good linearity up to 750 ng g(-1). The limit of detection ranged between 0.001 and 0.022 ng g(-1) for liver and between 0.001 and 0.028 ng g(-1) for kidney. The developed method was successfully used in several animal poisoning cases.
- MeSH
- Anticoagulants analysis MeSH
- Spectrometry, Mass, Electrospray Ionization methods MeSH
- Liver chemistry MeSH
- Kidney chemistry MeSH
- Limit of Detection MeSH
- Dogs MeSH
- Rodenticides analysis MeSH
- Sus scrofa MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Animals MeSH
- Check Tag
- Dogs MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Validation Study MeSH
A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.
- MeSH
- Acetyl Coenzyme A analysis MeSH
- Adenosine Triphosphate analysis MeSH
- Chemistry Techniques, Analytical methods standards MeSH
- Cytidine Triphosphate analysis MeSH
- Embryonic Stem Cells chemistry MeSH
- Guanosine Triphosphate analysis MeSH
- Humans MeSH
- Limit of Detection MeSH
- Paracoccus denitrificans chemistry MeSH
- Reproducibility of Results MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
This paper presents a method for the simultaneous determination of α-amanitin, β-amanitin and muscarine in human urine by solid-phase extraction (SPE) and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry. The method can be used for a diagnostics of mushroom poisonings. Different SPE cartridges were tested for sample preparation, namely hydrophilic modified reversed-phase (Oasis HLB) and polymeric weak cation phase (Strata X-CW). The latter gave better results and therefore it was chosen for the subsequent method optimization and partial validation. In the course of validation, limits of detection, linearity, intraday and interday precisions and recoveries were evaluated. The obtained LOD values of α-amanitin and β-amanitin were 1ng/mL and of muscarine 0.09ng/mL. The intraday and interday precisions of human urine spiked with α-amanitin (10ng/mL), β-amanitin (10ng/mL) and muscarine (1ng/mL) ranged from 6% to 10% and from 7% to 13%, respectively. The developed method was proved to be a relevant tool for the simultaneous determination of the studied mushroom toxins in human urine after mushroom poisoning.
- MeSH
- Amanitins urine MeSH
- Chromatography, Liquid methods MeSH
- Solid Phase Extraction MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Limit of Detection MeSH
- Adolescent MeSH
- Muscarine urine MeSH
- Mushroom Poisoning diagnosis urine MeSH
- Aged, 80 and over MeSH
- Forensic Toxicology MeSH
- Check Tag
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells.
- MeSH
- Chromatography, Ion Exchange MeSH
- Intracellular Space chemistry MeSH
- Hyaluronic Acid metabolism MeSH
- Linear Models MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Streptococcus equi chemistry metabolism MeSH
- Uridine Diphosphate Sugars analysis MeSH
- Publication type
- Journal Article MeSH
A sensitive method for GC-ECD simultaneous determination of nitrendipine and its pyridine metabolite M1 in human plasma is described. Felodipine was used as the internal standard. The plasma samples were extracted with toluene. One microlitre of the extract was injected onto the capillary column (polymethylsiloxane) and measured with electron-capture detector. The developed method showed to be linear over the range 0.25-70 for nitrendipine and 0.3-61 ng/ml for its metabolite M1 with an inter-day and intra-day precision in terms of R.S.D. lower than 8% except the concentrations near lowest limit of quantification (LLOQ) (<11% R.S.D.). The LLOQ for nitrendipine was 0.25 and 0.3 ng/ml for its metabolite, respectively. The analytical recovery was 94% for nitrendipine and 89% for its pyridine metabolite M1. This GC-ECD method was developed for being used in clinical pharmacokinetic studies.
- MeSH
- Time Factors MeSH
- Chromatography, Gas methods MeSH
- Felodipine chemistry blood MeSH
- Calibration MeSH
- Humans MeSH
- Molecular Structure MeSH
- Nifedipine chemistry blood metabolism MeSH
- Pyridines blood metabolism MeSH
- Reference Standards MeSH
- Sensitivity and Specificity MeSH
- Drug Stability MeSH
- Freezing MeSH
- Check Tag
- Humans MeSH
