Development and validation of a gas chromatographic method for analysis of fosfomycin in chicken muscle samples

Summary A gas chromatographic method for the determination of residues of Fosfomycin in chicken muscle samples has been developed. Muscle samples were homogenised with TRIS buffer, containing phenylphosphonic acid (as internal standard) and Fosfomycin using a tissue homogenizer. Afterwards, the samples were ultrafiltered and the ultrafiltrate was evaporated to dryness. A silylation reagent for derivatization was used in order to reconstitute the residue. The linear concentration range of application was 10–150 μgg⁻¹, with a detection and quantitation limit of 3.11 and 10 μgg⁻¹, respectively. The method was efficient with a mean recovery of 87.83% from spiked muscle. The results obtained show that gas chromatography is a useful method for the determination of Fosfomycin residues in chicken muscle samples.     

Bufferized solvent extraction and HPLC fluorometric detection method for sarafloxacin in pig and chicken muscles

In this study, a method for the detection of sarafloxacin in pig and chicken muscles was developed using HPLC‐FLD as a regulatory residue technique. Good extraction efficiency was achieved using a mixture of 1% orthophosphoric acid–0.2 m MgCl₂ in water and acetonitrile as an extraction solvent, and n‐hexane partitioning and centrifugation for cleanup was used in the absence of dehydration. Specificity, linearity, detection and quantification limits, recovery, accuracy and precision were all validated, and all results were sufficient for the SARA regulatory residue method in pig and chicken muscles. The method developed and described herein was not only simple but also reliable, and was applied to market samples to determine their residue contents. Copyright © 2010 John Wiley & Sons, Ltd.     

Toward an international standard for PCR-based detection of foodborne Escherichia coli O157: validation of the PCR-based method in a multicenter interlaboratory trial

The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries. Twelve cattle swab samples, spiked at 4 levels (0, 1-10, 10-100, and 100-1000 colony-forming units, in triplicate) with E. coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR. The results were reported as positive when the correct amplicons were present after gel electrophoresis. The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2%. The diagnostic specificity was 100%. The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels. The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels. The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method. Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an intenational PCR standard.     

General method validation guidelines for pharmaceutical samples

Method validation guidelines, which provide an organizational structure for the design and evaluation of a validation procedure, are presented for a wide range of pharmaceutical applications. The validation guidelines are based on the analyte concentration/sample matrix combination to which the method will be applied. These guidelines include the selection of appropriate validation parameters, design considerations for evaluation, and a discussion of acceptance guidelines for the determination of acceptable method performance. A set of tables is included which illustrates the selection and testing procedure and tailors the entire validation process to the specific characteristics of the determination to be made.     

Reveal Salmonella enteritidis test for detection of Salmonella enteritidis in shell eggs and environmental samples

Reveal Salmonella Enteritidis (SE) is a lateral flow-based immunodiagnostic assay used for rapid detection of Salmonella enterica serovar Enteritidis from pooled shell eggs and environmental samples. This assay uses highly specific antibodies to accurately detect S. Enteritidis. Studies were conducted to compare the performance of this test against reference procedures for detection of S. Enteritidis from both pooled shell eggs and environmental samples. Pooled shell eggs were inoculated with low levels ofS. Enteritidis and were enriched according to the procedure prescribed by the U.S. Food and Drug Administration. Uninoculated samples were included in each trial. Reveal SE exhibited 100% sensitivity and 100% specificity in comparison to the reference method in all trials. An abbreviated 48 h/(no hold) enrichment procedure was also developed and validated for detection ofS. Enteritidis from pooled shell egg samples. This shortened enrichment procedure can be used in conjunction with the Reveal SE test and offers a significant enrichment time savings of 96 h. Chi-square analysis revealed that there was no significant difference between the abbreviated Reveal method and the reference procedure for detection ofS. Enteritidis from pooled shell egg samples. Out of 245 natural drag swabs screened internally, only three samples tested Reveal SE positive and were confirmed by the reference procedure, resulting in 100% sensitivity and 100% specificity. An external laboratory screened 147 poultry house environmental samples and obtained 35 Reveal SE confirmed positives for Reveal SE sensitivity of 100% and specificity of 90%. Inoculation trials with drag swabs resulted in 96% sensitivity and 100% specificity. Thus, these data demonstrate that Reveal SE is a highly sensitive and specific assay for the detection of S. Enteritidis from both pooled shell eggs and environmental samples.     

CE method for analyzing Salmonella typhimurium in water samples

Salmonella typhimurium is commonly described as a food‐borne pathogen. However, natural and drinking water are known to be important sources for the transmission of this pathogen in developing and developed countries. The standard method to determine Salmonella is laborious and many false positives are detected. To solve this, the present work was focused on the development of a capillary zone electrophoresis method coupled to ultraviolet detection for determination of Salmonella typhimurium in water (mineral and tap water). Separations were performed in less than 11 minutes using 4.5 mM Tris (hydroxymethyl)‐aminomethane, 4.5 mM boric acid and 0.1 mM ethylene diamine tetraacetate (pH 8.4) with 0.1% v/v poly ethylene oxide as separation buffer. The precision of the method was evaluated in terms of repeatability obtaining a relative standard deviation of 10.5%. Using the proposed method Salmonella typhimurium could be separated from other bacteria that could be present in water such as Escherichia coli. Finally, the proposed methodology was applied to determine Salmonella typhimurium in tap and mineral water.     

GC-MS method development and validation for anabolic steroids in feed samples

A GC-MS method for the determination of AAS used as growth promoting agents using SIM in piglet feed samples has been developed and validated, using testosterone as internal standard. The formation of volatile steroid derivatives was carried out by derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide. The optimum separation was achieved using a Zebron ZB-5 column under a gradient temperature elution, allowing the separation of steroids in 18 min. The required sample treatment process was discussed. A leaching using ACN, saponification using a binary NaOH/MgCl2 solution, and LLE using ethyl acetate were finally selected. Method validation has been carried out according to the Commission Decision 2002/657/EC criteria established for quantitative confirmatory methods. The extraction efficiencies, CCalpha and CCbeta for these compounds were in the ranges 78-98%, 10-21 and 18-35 mug/kg, respectively. The repeatability and the within-laboratory reproducibility at 1, 1.5, and 2 CCbeta concentration levels were smaller than 8.2, 7.5, and 5.8% and 12.2, 9.5, and 7.5%, respectively. Accuracy was in the 99-103% range. The robustness was evaluated using the Youden robustness test. The proposed method was applied to the analysis of steroids spiked in different kinds of animal feed samples with satisfactory results.     

Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance Tested Method 960801

Reveal Salmonella 2.0 is an improved version of the original Reveal Salmonella lateral flow immunoassay and is applicable to the detection of Salmonella enterica serogroups A-E in a variety of food and environmental samples. A Performance Tested Method validation study was conducted to compare performance of the Reveal 2.0 method with that of the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, raw shrimp, a ready-to-eat meal product, dry pet food, ice cream, spinach, cantaloupe, peanut butter, stainless steel surface, and sprout irrigation water. In a total of 17 trials performed internally and four trials performed in an independent laboratory, there were no statistically significant differences in performance of the Reveal 2.0 and reference culture procedures as determined by Chi-square analysis, with the exception of one trial with stainless steel surface and one trial with sprout irrigation water where there were significantly more positive results by the Reveal 2.0 method. Considering all data generated in testing food samples using enrichment procedures specifically designed for the Reveal method, overall sensitivity of the Reveal method relative to the reference culture methods was 99%. In testing environmental samples, sensitivity of the Reveal method relative to the reference culture method was 164%. For select foods, use of the Reveal test in conjunction with reference method enrichment resulted in overall sensitivity of 92%. There were no unconfirmed positive results on uninoculated control samples in any trials for specificity of 100%. In inclusivity testing, 102 different Salmonella serovars belonging to serogroups A-E were tested and 99 were consistently positive in the Reveal test. In exclusivity testing of 33 strains of non-salmonellae representing 14 genera, 32 were negative when tested with Reveal following nonselective enrichment, and the remaining strain was found to be substantially inhibited by the enrichment media used with the Reveal method. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device development time.     

Development and validation of an analytical method for detection of estrogens in water

An analytical procedure enabling routine analysis of four environmental estrogens at concentrations below 1 ng L^–1 in estuarine water samples has been developed and validated. The method includes extraction of water samples using solid-phase extraction discs and detection by gas chromatography (GC) with tandem mass spectrometry (MS–MS) in electron-impact (EI) mode. The targeted estrogens included 17- and 17-estradiol (aE2, bE2), estrone (E1), and 17-ethinylestradiol (EE2), all known environmental endocrine disruptors. Method performance characteristics, for example trueness, recovery, calibration, precision, accuracy, limit of quantification (LOQ), and the stability of the compounds are presented for each of the selected estrogens. Application of the procedure to water samples from the Scheldt estuary (Belgium – The Netherlands), a polluted estuary with reported incidences of environmental endocrine disruption, revealed that E1 was detected most frequently at concentrations up to 7 ng L^–1. aE2 was detected once only and concentrations of bE2 and EE2 were below the LOQ.Presented at the 9th FECS Conference on Chemistry and the Environment, Bordeaux, France, 29 August–1 September 2004     

PCR-based detection in a micro-fabricated platform

We present a novel, on-chip system for the electrokinetic capture of bacterial cells and their identification using the polymerase chain reaction (PCR). The system comprises a glass-silicon platform with a set of micro-channels, -chambers, and -electrodes. A platinum thin film resistor, placed in the proximity of the chambers, is used for temperature monitoring. The whole chip assembly is mounted on a Printed Circuit Board (PCB) and wire-bonded to it. The PCB has an embedded heater that is utilized for PCR thermal cycle and is controlled by a Lab-View program. Similar to our previous work, one set of electrodes on the chip inside the bigger chamber (0.6 microl volume) is used for diverting bacterial cells from a flowing stream into to a smaller chamber (0.4 nl volume). A second set of interdigitated electrodes (in smaller chamber) is used to actively trap and concentrate the bacterial cells using dielectrophoresis (DEP). In the presence of the DEP force, with the cells still entrapped in the micro-chamber, PCR mix is injected into the chamber. Subsequently, PCR amplification with SYBR Green detection is used for genetic identification of Listeria monocytogenes V7 cells. The increase in fluorescence is recorded with a photomultiplier tube module mounted over an epifluorescence microscope. This integrated micro-system is capable of genetic amplification and identification of as few as 60 cells of L. monocytogenes V7 in less than 90 min, in 600 nl volume collected from a sample of 10(4) cfu ml(-1). Specificity trials using various concentrations of L. monocytogenes V7, Listeria innocua F4248, and Escherichia coli O157:H7 were carried out successfully using two different primer sets specific for a regulatory gene of L. monocytogenes, prfA and 16S rRNA primer specific for the Listeria spp., and no cross-reactivity was observed.     

QbD-driven development and validation of HPLC method for determination of Bisphenol A and Bis-sulphone in environmental samples

ABSTRACT

A large number of hazardous chemicals have entered the environment due to the rapid growth of urbanisation and industrial development and are exerting harmful effects on wildlife as well as on human health. Plastic materials are one of the most leading causes for this contamination which are widely used in daily activities of human beings, i.e. disposal purpose, food packaging, bottles, containers, cups, grocery bags, etc. These materials contain Bisphenol A (BPA) and Bis-sulphone (BIS) which have been recognised as potential endocrine disruptors. In the present study, a selective and reliable high-performance liquid chromatography (HPLC) based method was developed with the mobile phase of 10 mM ammonium acetate buffer-acetonitrile (58:42 v/v, pH: 5) using quality by design (QbD) approach and the method was validated for the simultaneous assessment of BPA and BIS. The method was observed with a good linearity range of 50–500 ng/mL with an r² value of 0.998 and 0.999 for BPA and BIS, respectively. The developed and validated method was applied for the estimation of endocrine-disrupting chemicals in sewage water and soil samples. The results showed a considerable amount of BPA and BIS in the samples. This preliminary data explored the presence of BPA and BIS in these environmental samples that give the primary awareness of the effluence of BPA and BIS in the environment.     

Development and method validation for testosterone and epitestosterone in human urine samples by liquid chromatography applications

An isocratic high-performance liquid chromatographic method for the determination of testosterone (T) and epitestosterone (ET) in human urine using liquid-liquid or solid-phase extraction (SPE) is developed and validated. The optimum separation is achieved using a Hypersil C(18) column, water-acetonitrile (57:43, v/v) as the mobile phase and UV-absorbance detection at 245 nm. The recoveries obtained for T and ET in liquid-liquid and SPE demonstrate that these procedures are interchangeable. Quantitation limits for T and ET are 8.6 and 5.4 ng/mL using solvent extraction and 7.3 and 5.7 ng/mL using SPE, respectively. The proposed method is used to evaluate the urinary T, ET, and the T/ET ratio for a healthy male population using liquid-liquid extraction, and the T and ET excretion profile for nine healthy men using SPE.     

Development of a method for the detection of beta-lactamases in milk samples

With the rapid growth of the dairy industry and the establishment of strict antimicrobial residue limits in the People's Republic of China's (PRC) milk supply, a beta-lactamase product known as "antimicrobial destroyer" was introduced into dairy production without regulatory review. We developed a method for detecting this product in milk samples based on a modified cylinder plate method. The presence of beta-lactamase is defined as a difference between the inhibitory zones of the test samples (supplemented with 25 microg/mL sulbactam plus 0.5 microg/mL penicillin G) and control samples (supplemented only with 0.5 microg/mL penicillin G) > or = 3 mm. Using this method, 77 individually packaged milk samples were randomly collected from 5 retail stores in 3 cities over a 4-month period (May to August 2006). Of the 77 samples, 49 were found to be beta-lactamase-positive. In 2 undiluted milk samples showing extremely high beta-lactamase activity, 25 microg/mL sulbactam could not inhibit penicillin G activity. Because there is a lack of safety data on beta-lactamases in milk products, these data indicated a potentially serious safety concern for the dairy industry in the PRC.     

Use of the Eurachem guide on method validation for determination of uranium in environmental samples

Three analytical methods for determination of uranium in environmental samples by a fluorescence technique have been validated and compared in accordance with the Eurachem Guide on method validation. The first method depends on uranium separation from iron using the mercury anode technique; in the other two methods uranium is separated from iron on an anion exchange column by use of either a solution of hydrochloric acid containing ascorbic acid and hydrazine hydrate or a dilute sulfuric acid solution. Detection limits, repeatability, reproducibility, and recovery coefficient were the main validation characteristics. The results showed that better statistical values can be achieved by using the third method. Control charts for in-house control samples and international intercomparison samples have also shown that the third method is more statistically stable with time. In addition, uncertainties of measurement were estimated and compared for the three methods. It was found that the Eurachem Guide and comparison of quality statistical validation data can be good tools for selection of the appropriate method for an application.     

Development and validation of a method for analyzing polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in sewage sludge samples

Summary A method has been developed for the analysis of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) in sewage sludge samples. It was found that PCDD/F are best Soxhlet extracted from the matrix with toluene for 24h, after having tested other solvents (dichloromethane and hexane/acetone 41/59) and other extraction times. Several clean up steps (sulfuric acid, multilayer silica and Florisl columns) and concentration are required prior to analysis of the extract by high resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS). The complete procedure has been validated and the accuracy and precision data (repeatability and reproducibility) are given. The method is linear in the range studied and the limit of detection ranges between 0.2 and 2.2 pg g⁻¹ of dry matter for the 2,3,7,8-substituted congeners. Moreover, the suitability of the method has been checked in an international interlaboratory comparison. The successful application of this method to several samples from Catalan and Dutch urban wastewater treatment plants was demonstrated.     

An EOX method for the detection of halogenated hydrocarbons in soil samples

Analytical and Bioanalytical Chemistry -     

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.     

Development and validation of an SPE HG-AAS method for determination of inorganic arsenic in samples of marine origin

The present paper describes a novel method for the quantitative determination of inorganic arsenic (iAs) in food and feed of marine origin. The samples were subjected to microwave-assisted extraction using diluted hydrochloric acid and hydrogen peroxide, which solubilised the analytes and oxidised arsenite (As(III)) to arsenate (As(V)). Subsequently, a pH buffering of the sample extract at pH 6 enabled selective elution of As(V) from a strong anion exchange solid-phase extraction (SPE) cartridge. Hydride generation atomic absorption spectrometry (HG-AAS) was applied to quantify the concentration of iAs (sum of As(III) and As(V)) as the total arsenic (As) in the SPE eluate. The results of the in-house validation showed that mean recoveries of 101-104% were achieved for samples spiked with iAs at 0.5, 1.0 and 1.5 mg·kg(-1), respectively. The limit of detection was 0.08 mg kg(-1), and the repeatability (RSD(r)) and intra-laboratory reproducibility (RSD(IR)) were less than 8% and 13%, respectively, for samples containing 0.2 to 1.5 mg kg(-1) iAs. The trueness of the SPE HG-AAS method was verified by confirming results obtained by parallel analysis using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry. It was demonstrated that the two sets of results were not significantly different (P < 0.05). The SPE HG-AAS method was applied to 20 marine food and feed samples, and concentrations of up to 0.14 mg kg(-1) of iAs were detected.     

Instrumental validation in capillary electrophoresis and checkpoints for method validation

Capillary electrophoresis (CE) is increasingly being used in regulated and testing environments which demand validation. The design, development and production of CE instrumentation should be governed by qualifications which ensure the quality of the finished product. The vendor should therefore provide guidelines and procedures which assist the user in ensuring the adequate operation of the instrumentation and especially in designing installation qualification (IQ) and operational qualification/performance verification (OQ/PV) procedures. OQ/PV should test those functions of an instrument which directly affect the CE analysis, i.e. voltage, temperature, injection precision and detector function. In validation of CE methods care should be taken that those aspects which directly affect the precision of peak parameters are appreciated. The relationship between CE instrumentation, chemistry and validation parameters is discussed and guidelines are presented for definition of a CE method for submission to regulatory authorities.