Development and validation of a rapid method for the detection of latrunculol A in plasma

Latrunculol A is a recently discovered 6,7-dihydroxy analog of the potent actin inhibitor latrunculin A. Latrunculol A has exhibited greater cytotoxicity than latrunculin A against both murine and human colon tumor cell lines in vitro. Currently, there are no reports regarding the bioavailability of latrunculol A in vivo. This study was undertaken as a prelude to pharmacokinetic assessments and it is the first work where bioavailability of latrunculol A was studied. In the present work, a simple plasma preparation and a rapid HPLC method have been developed. Mouse plasma containing latrunculol A was first treated by acetonitrile and then centrifuged at 14,000 rpm at 4 °C for 25 min. The supernatant was injected in an HPLC system comprising a Waters Symmetry NH₂ column, a mobile phase of acetonitrile/water (95/5, v/v), a flow rate of 1.0 mL/min, at 220 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 9 ng/mL, and a good precision with a coefficient variation of 1.65, 1.86, and 1.26% for 20, 400, and 800 ng/mL, respectively. With this simple method, excellent separation and sensitivity of latrunculol A are achieved, thus allowing a rapid analysis of the plasma samples for absorption, distribution, and metabolism studies.     

Sequential injection analysis with electrochemical detection as a tool for economic and rapid evaluation of total antioxidant capacity

This work presents a new flow-based coupled electrochemical technique for evaluation of “total antioxidant capacity (TAC)”. A sequential injection (SI) with amperometric detection was applied to the TAC analysis of commercial instant ginger infusion beverages using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Besides having chromogenic properties, the ABTS reagent behaves as an electroactive species at the glassy carbon electrode in phosphate buffer pH 7.0, the decrease of the cathodic current signal of the ABTS⁺ radical after reaction with antioxidants can be monitored. The SI system, furnished with an in-house electrochemical detection cell (ECD), was optimized with respect to the applied potential, sample and reagent volume, and flow rate to the detector. Gallic acid was used as the standard antioxidant and the capacity was reported as gallic acid equivalent (GAE) unit. TAC measurements of ginger infusions at the optimum condition were performed using the proposed technique and also with the classical batch spectrophotometric ABTS assay. TAC values obtained from our method and the standard method are in good agreement (r² = 0.956). The SI-amperometric technique provided satisfactory precision (4.11% RSD) with rapid sample throughput (40 samples h⁻¹). Also using this method, the consumption of the expensive ABTS reagent was greatly reduced.     

Cobalamin as an analytical tool for analysis of oxirane metabolites of 1,3-butadiene: development and validation of the method

The reduced form of vitamin B12 [cob(I)alamin] is known to be a supernucleophile, with the ability to react 10(5) times faster than standard nucleophiles. Procedures have been developed where cob(I)alamin is used as an analytical tool for the trapping of electrophilically reactive compounds. In the present work, a sensitive and accurate method for determination of reactive metabolites produced in vitro has been developed and validated. Diepoxybutane (DEB), a metabolite of 1,3-butadiene, was used as a model compound. The intermediate precursor 1,2-epoxybutene (EB) was incubated in a mouse liver S9 metabolic system and the formation of DEB was studied. Samples were taken at different times from the incubation mixture and added to the cob(I)alamin. The alkyl-cobalamins (alkyl-Cbl) formed were directly analysed by a miniaturized LC-MS/MS method and column switching. The assay was linear over the concentration range of 1.5-500 microM with acceptable precision and accuracy.     

Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC₅₀) of cephalexin and cefadroxil were obtained at 1.5 ng mL⁻¹; IC₅₀ of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL⁻¹, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL⁻¹). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL⁻¹ in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.     

Development and single laboratory validation of an optical biosensor assay for tetrodotoxin detection as a tool to combat emerging risks in European seafood

Tetrodotoxin (TTX) is a potent neurotoxin emerging in European waters due to increasing ocean temperatures. Its detection in seafood is currently performed as a consequence of using the Association of Analytical Communities (AOAC) mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins, but TTX is not monitored routinely in Europe. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. An AOAC-accredited high-performance liquid chromatography (HPLC) method has now been accepted by the European Union as a first action screening method for PSP toxins to replace the MBA. However, this AOAC HPLC method is not capable of detecting TTX, so this potent toxin would be undetected; thereby, a separate method of analysis is required. Surface plasmon resonance (SPR) optical biosensor technology has been proven as a potential alternative screening method to detect PSP toxins in seafood. The addition of a similar SPR inhibition assay for TTX would complement the PSP assay in removing the MBA. The present report describes the development and single laboratory validation in accordance with AOAC and IUPAC guidelines of an SPR method to be used as a rapid screening tool to detect TTX in the sea snail Charonia lampas lampas, a species which has been implicated in 2008 in the first case of human TTX poisoning in Europe. As no current regulatory limits are set for TTX in Europe, single laboratory validation was undertaken using those for PSP toxins at 800 μg/kg. The decision limit (CCα) was 100 μg/kg, with the detection capability (CCβ) found to be ≤200 μg/kg. Repeatability and reproducibility were assessed at 200, 400, and 800 μg/kg and showed relative standard deviations of 8.3, 3.8, and 5.4 % and 7.8, 8.3, and 3.7 % for both parameters at each level, respectively. At these three respective levels, the recovery of the assay was 112, 98, and 99 %.     

Simultaneous determination of adenosine and its metabolites by capillary electrophoresis as a rapid monitoring tool for 5'-nucleotidase activity

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of micro-molar adenosine, hypoxanthine and inosine in enzyme assays without using radioactive labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. Under the optimal condition, the good separation with high efficiency was achieved in 6 min. Using deoxyadenylate as an internal standard, the linear range of the method was 5-200 microM, and the concentration limits of detection of adenosine, hypoxanthine and inosine were 2.2, 3.6 and 1.4 microM, respectively. Application of the proposed method was demonstrated by the activity assay of 5'-nucleotidase from Hep G2 cells.     

Development and validation of a rapid and efficient method for simultaneous determination of methylxanthines and their metabolites in urine using monolithic HPLC columns

A new rapid, sensitive and validated HPLC method has been developed for the determination of methylxanthines and their metabolites in asthmatic patients. The method was initiated by using spiked urine samples on a silica monolithic column as a novel packing material. The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer/methanol (87.5:12.5 v/v), at a flow rate 1 mL/min. Detection was set at 274 nm. The LOQ for all the compounds ranged from 14 to 41 ng/mL. Excellent linearity was achieved over the studied range of concentration with correlation coefficients 0.9991–0.9998 (n = 6). The developed method was validated by precision and accuracy with RSD <2.55%. On extraction of the drugs and metabolites from the urine samples high recoveries were achieved ranging from 82.06 to 98.34% w/w on RP18 cartridges and methanol/chloroform (20:80 v/v) as the extraction solvent. This method has advantages over other methods using conventional C18 packings.     

Development and validation of a rapid and sensitive HPLC method for the quantification of 5‐fluorocytosine and its metabolites

To study the intracellular metabolism of the prodrug 5‐fluorocytosine (5FC), we developed a novel reverse‐phase high‐performance liquid chromatography method to simultaneously detect 5FC and its four major anabolic metabolites: 5‐fluorouracil, 5‐fluorouridine, 5‐fluorouridine‐monophosphate and 5‐fluoro‐2′deoxyuridine‐5′‐monophosphate. Separation of each compound was accomplished under isocratic conditions using a C₁₈ column and mobile phase of formic acid–water (1 : 99 v/v). The method was validated for both accuracy and reproducibility in cell culture media. Additionally, metabolites were assessed for stability at ambient temperatures and following freeze–thaw cycles. Calibration curves were linear over a range of 1–200 μg/mL. Limit of quantification for four of the five compounds was 1 μg/mL in cell culture media (RSD < 11%). This method was successfully used to monitor intracellular conversion of 5FC to its metabolic products over a 24h period. Copyright © 2009 John Wiley & Sons, Ltd.     

Enzyme amplification as detection tool in continuous-flow systems. I. Development of an enzyme-amplified biochemical detection system coupled on-line to flow-injection analysis.

The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplified biochemical detection (EA-BCD) system is described. The aim of this study is the development of a detection system able to detect biotin-containing compounds at low concentration levels. The detection system is based on the interaction of biotin with enzyme-labeled affinity proteins. Biotin possesses a high affinity to both streptavidin and anti-biotin Fab fragments, which are both tested. Several biotin derivatives are available with different reactive probes, which can be used to label analytes of interest. Therefore, biotin acts as a universal probe for the enzyme-amplified biochemical detection. Alkaline phosphatase (AP) was used as enzyme label. Several parameters, such as substrate type and concentration, concentration of enzyme-labeled affinity protein, reaction time and reaction temperature were examined. Biotin aminocaproic acid was used as a model compound. In addition to biotin aminocaproic hydrazide, other biotinylation reagents were also examined. With fluorescence detection of the enzyme-generated product, a mass detection limit of 1 fmol was achieved.     

Elimination profiles of flurbiprofen and its metabolites in equine urine for doping analysis

Flurbiprofen and its main acidic metabolites were detected in equine urine after a single-dose administration of 500 mg flurbiprofen to two 2.5–3.5-years-old mares, in order to be used in equine doping control routine analysis. The urine levels of the parent drug were determined using GC/MS. Five acidic metabolites were found in the urine. The structure of the proposed metabolites was confirmed by HRMS accurate mass measurements. The highest flurbiprofen concentration was 204 μg ml⁻¹ at 1–3 h post administration. Flurbiprofen could be detected for 24–37 h in urine using the standard screening procedure. All metabolites were present 25 h post administration, while 4′-hydroxyflurbiprofen could be traced for more than 48 h and it is regarded as the long-term metabolite of flurbiprofen in horse.     

Development of a new reference material for the validation of molecular detection methods for microbiological analysis

The standard methodology used for the detection of bacteria in environmental samples and food is primarily based on bacterial growth on specific culture media and confirmation by biochemical and/or immunological tests of all presumptive colonies. However, this methodology presents a number of drawbacks, such as low sensitivity and specificity, and the long time needed to obtain results. For this reason, the implementation of molecular methods in diagnostic laboratories has increased over the past several years. Nevertheless, most of these newer methods have not been included in current legislation, and, in most of cases, they have not yet been normalized. In this sense, the availability of appropriate reference materials (RMs) can help to overcome these deficiencies. The aim of this study was to develop and validate, following ISO Guide 34, a new RM, in a tablet format, for the quantification of Legionella pneumophila and Salmonella spp. by quantitative PCR (qPCR). This new RM can be used as a work reference sample in internal quality control, in the organization of proficiency testing schemes (PTS), as well as for the validation of molecular methods based on qPCR.     

Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BG) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BG, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BG which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes.     

Polarography as a tool in organic analysis. III

Summary Rapid indirect polarographic and visual micro-methods have been developed for the determination of azo, azo and nitro, or azo and nitroso groups in the same solution. First, the azo group is reduced with titanium(III) at p_H 1.7 and then the nitro group at p_H 5.5. The excess of reductant is determined polarographically. Mixtures containing nitroso and azo groups are analysed by indirect polarography by reducing the nitroso group first in 7N acid and then the azo group at p_H 5.

---

Zusammenfassung Indirekte polarographische und visuelle Mikromethoden zur Bestimmung von Azogruppen, Azo- und Nitrogruppen, oder Azo- und Nitrosogruppen in derselben Lösung wurden ausgearbeitet. Zuerst wird die Azogruppe bei p_H 1,7 mit Titan(III), dann die Nitrogruppe bei p_H 5,5 reduziert. Der Überschuß des Reduktionsmittels wird polarographisch bestimmt. Gemische mit Nitroso-und Azogruppen lassen sich indirekt polarographisch analysieren, indem man zuerst die Nitrogruppe in 7-n Säure und dann die Azogruppe bei p_H 5 reduziert.

     

Metabolomics analysis of human plasma metabolites reveals the age- and sex-specific associations

Abstract

Objectives: The objective of this study was the investigation of age- and sex-associations in a set of blood plasma metabolites in healthy male and female subjects.

Methods: A comparison study design with male and female subjects of various ages was used. Metabolic profiling was performed using electrospray ionization tandem mass spectrometry that yielded 186 metabolite concentrations for each study participant. The key age-related metabolites were identified using an integrative analysis of absolute concentrations, metabolite ratios and the differential correlation of pairwise metabolite concentrations. All of the age-associated metabolites were adjusted prior to the analysis to account for differences in Body Mass Index (BMI).

Results: A total of 236 plasma samples from 140 female and 96 male subjects aged 20 to 82 years-old were collected and analyzed in the study. 13 and 14 age-associated metabolites (|r|?>?0.33 and p < 6.6×10^?5), 438 and 337 age-associated metabolite ratios (|r|?>?0.37 and p < 3.5×10^?6), and 5 and 10 core metabolites were discovered in the female and male groups, respectively. 80% of the metabolites displaying associations with age belonged to sphingolipids and phosphatidylcholines, and the two sexes shared less than 50% of the age-associated metabolites.

Conclusion: The study found that changes in metabolite concentrations, metabolite ratios and differential correlations were age and sex-specific.     

Determination of nifursol metabolites in poultry muscle and liver tissue. Development and validation of a confirmatory method

A method is described for the identification and quantitative determination of 3,5-dinitrosalicylic acid hydrazide (DSH), the marker residue of nifursol metabolites in poultry (turkey, broiler) muscle and liver tissue. The method is based on the acid-catalysed hydrolysis of tissue-bound metabolites to free DSH and in situ derivatisation with 2-nitrobenzaldehyde to the corresponding nitrophenyl derivative NPDSH. A structural analogue of DSH, 4-hydroxy-3,5-dinitrobenzoic acid hydrazide (HBH) was synthesised to serve as an internal standard. The analytes were isolated from the matrix by liquid-liquid extraction with ethyl acetate. Determination was performed by LC-MS/MS with negative electrospray ionisation. The [M - H](+) ions of NPDSH and NPHBH at m/z 374 were fragmented by collision induced dissociation (CID) producing transition ions at m/z 182, 183 and 226. The transition ions at m/z 182 and 226 were selected for monitoring of NPDSH while the transition ion at m/z 183 was selected for NPHBH. The method has been validated according to the EU criteria of Commission Decision 2002/657/EC at 0.5, 1.0 and 1.5 microg kg(-1) in muscle and liver tissue. A decision limit (CC(alpha)) was obtained of 0.04 and 0.025 microg kg(-1) in muscle and liver, respectively. Similarly a detection capability (CC(beta)) was obtained of 0.10 and 0.05 microg kg(-1) in muscle and liver, respectively. The introduction of HBH as an internal standard did not lead to a significant improvement of the quantitative performance of the method. In fact for liver better performance characteristics were obtained when the IS was not taken into account. Nevertheless, as a qualitative marker for recovery, HBH could still be very useful in the analysis of unknown samples.     

Development and validation of rapid disequilibrium enzyme-linked immunosorbent assays for the detection of Methyl Yellow and Rhodamine B dyes in foods

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of two illegal synthetic dyes: Methyl Yellow (MY) and Rhodamine B (RB) in food. Polyclonal antibodies were raised against synthesised immunogens and employed in unique direct disequilibrium ELISAs. The time of the assays was only twenty minutes (five minutes for each incubation step with sample and enzyme conjugate and ten minutes with enzyme substrate). The IC(50) for MY was in the range 1.4-4.2 ng mL(-1) and for RB 0.1-0.5 ng mL(-1). A simple sample preparation method was developed for the analysis of a range of sauces. In the case of spices a dispersive solid phase extraction was applied to purify the extracts. The testing of twenty samples took approximately one and a half hours (including sample preparation and analysis). Both assays were validated according to the Commission Decision 2002/657/EC criteria for use in sauces and spices. The detection capability for MY in sauces and spices was determined to be less than 15 ng g(-1) and 50 ng g(-1), respectively and for RB, 10 ng g(-1) for both types of food samples. The precision of the developed assays was determined in a repeatability study. The intra- and inter-assay coefficients of variation were less than 25% for both tests and matrix types. The simplicity and performance of both assays indicate that they will be very reliable screening methods for the detection of the illegal dyes MY and RB in a range of food products.     

Development and validation of a rapid high-performance liquid chromatography method for the quantification of exenatide

The objective was to develop a simple HPLC method to quantify exenatide—a 39 amino acid residue incretin mimetic used in diabetes therapy. To date, only non‐validated, sometimes incomplete, gradient methods have been reported in the literature. Isocratic separation was achieved using a C₄ column and a mixed solvent system, A–B–C (48:45:7, v/v/v; pH* 5.2), where A represents KH₂PO₄ (pH 4.5; 0.1 m ) and MeCN (60:40, v/v), B corresponds to NaClO₄·H₂O (pH 6.0; 0.2 m ) and MeCN (60:40, v/v), and C is water. Exenatide eluted at 3.64 min and the total run time was 6 min. The method was specific and the response was accurate, precise and linear from 0.75 to 25 µg/mL. It was used to quantify exenatide transport across intact and laser‐porated porcine skin in vitro as a function of laser fluence [0 (i.e. intact skin), 9 and 15 J/cm², respectively]. Although no permeation was observed using intact skin, cumulative exenatide permeation after 8 h through laser porated skin was 9.6 ± 6.5 and 12.4 ± 6.4 µg/cm² at fluences of 9 and 15 J/cm², respectively. This is the first validated isocratic method for exenatide quantification and it may be of use in quality control analysis and with other biological matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Polarography as a tool in organic analysis

Summary Rapid indirect polarographic and visual micromethods have been developed for the determination of the quinone and azoxy groups by reduction with titanium(III) at pH values of 3 and 5.5, respectively. The excess of reductant is first determined polarographically followed by visual back-titration of the same solution with iron(III) using thiocyanate as indicator a drop of neutral red is added in case of the quinone function. Although the results obtained by the two finishes are of approximately the same order of accuracy (±0.46%), yet the polarographic finish proved to be of more general applicability particularly for quinones that give rise, after reduction, to a variety of colours on adding the iron(III) solution.

---

Anwendung der Polarographie in der organischen AnalyseIV. Bestimmung von Chinon- und Azoxygruppen durch Reduktion mit Titan(III)

Zusammenfassung Zur schnellen Bestimmung von Chinon- und Azoxygruppen durch Reduktion mit Titan(III) bei pH 3 bzw. 5,5 werden indirekte polarographische und visuelle Mikromethoden beschrieben. Der Überschuß an Reduktionsmittel wird zunächst polarographisch und anschließend volumetrisch durch Rücktitration mit Eisen(III)-lösung bestimmt, wobei Thiocyanat (bei der Chinongruppe noch zusätzlich Neutralrot) als Indicator dient. Beide Ergebnisse weisen etwa dieselbe Genauigkeit auf (±0,46%); doch ist die polarographische Methode allgemeiner anwendbar, besonders bei Chinonen, die nach der Reduktion auf Zusatz Eisen(III) verschiedenartige Färbungen hervorrufen.

---

---

Part III: Mikrochim. Acta 1969, 44.     

Polarography as a tool in organic analysis

Summary A polarographic micromethod has been developed for the analysis of mono-, di-, tri- and polynitro compounds, standard titanium(III) sulphate solution being used as a reducing agent. The cathodic part of the Kalousek cell has been modified to serve as a reaction vessel. The results agree favourably with those obtained by visual back-titration of excess of titanium(III) with iron(III), thiocyanate being used as indicator, and the relative error is ± 0.5%. This polarographic finish proved to be of general applicability particularly with nitro compounds that give rise to coloured reduction products.

---

Zusammenfassung Eine polarographische Mikromethode zur Analyse von Mono-, Di-, Tri- und Polynitroverbindungen mit Titan(III)-sulfatlösung als Reduktionsmittel wurde ausgearbeitet. Der Kathodenteil der Kalousek-Zelle wurde als Reaktionsgefäß umgestaltet. Die Analysenresultate stimmen sehr gut mit jenen überein, die man durch visuelle Rücktitration des überschüssigen Titan(III) mit Eisen(III) gegen Thiocyanat als Indikator erhält. Der relative Fehler beträgt ± 0,5%. Dieses polarographische Verfahren dürfte allgemein für Nitroverbindungen anwendbar sein, aus denen sich gefärbte Reduktions-produkte bilden können.