Development and validation of LC methods with visible detection using pre-column derivatization and mass detection for the assay of voglibose

Two sensitive and selective liquid chromatographic methods were developed for the assay of voglibose (VB) and validated as per International Conference on Harmonization (ICH) guidelines. First method is based on the pre-column derivatization of VB followed by visible detection (LC-VD) and second method involves mass spectrometric detection (LC-MS). In LC-VD method, VB was derivatized with sodium metaperiodate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH). The derivatized color product of VB (DCPVB) was run through Novapak C18 (300 × 3.9 mm, 4 μm) column using the mobile phase containing buffer (0.01 M mixture of sodium di hydrogen orthophosphate and disodium hydrogen orthophosphate, pH 6.0) and acetonitrile in 35:65 v/v ratio. The eluted DCPVB was monitored at 667 nm. The fixation of optimum conditions in LC-VD method is described. DCPVB structure was confirmed by mass spectral analysis. In LC-MS method, VB was passed through Venusil XBPPH (150 × 4.6 mm, 5 μm) column using a 95:5 v/v mixture of 0.01% formic acid and methanol as mobile phase. The assay concentrations of VB in pure form and in tablets for LC-VD and LC-MS methods are 25 and 5 ng ml⁻¹, respectively.     

Protein immunoassay methods for detection of biotech crops: applications,limitations, and practical considerations

Immunoassay methods are available for detection and quantitation of proteins expressed by most biotechnology-derived crops in commercial production. The 2 most common test formats are enzyme-linked immunosorbent assay (ELISA) and immunochromatographic (lateral flow) strip tests. Two ELISA methods, one for Roundup Ready soybeans and one for MON810 CrylAb corn, were the subject of large international collaborative studies and were demonstrated to quantitatively determine the concentrations of biotech crops in samples of ground grain. Quantitative ELISA methods are also useful for analysis of processed fractions of agricultural commodities such as soybean toasted meal or corn flour. Both strip tests and ELISAs for biotech crops are currently being used on a large scale in the United States to manage the sale and distribution of grain. In these applications, tests are used to determine if the concentration of biotech grain is above or below specified threshold limits. Using existing U.S. Department of Agriculture sampling techniques, the reliability of the threshold determination is expressed in terms of statistical confidence rather than analytical precision. Combining the use of protein immunoassays with Identity Preservation systems provides an effective means of characterizing the raw and processed agricultural inputs to the food production system in a way that allows food producers to comply with labeling laws.     

Increased efficacy for in-house validation of real-time PCR GMO detection methods

To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSD_r and RSD_R) were calculated. The results showed that not only the PCR reaction but also the factors ‘DNA isolation’ and ‘PCR day’ are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods.     

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.     

Premalbrancheamide: synthesis, isotopic labeling, biosynthetic incorporation, and detection in cultures of Malbranchea aurantiaca

An advanced metabolite, named premalbrancheamide, involved in the biosynthesis of malbrancheamide (1) and malbrancheamide B (2) has been synthesized in double (13)C-labeled form and was incorporated into the indole alkaloid 2 by Malbranchea aurantiaca. In addition, premalbrancheamide has been detected as a natural metabolite in cultures of M. aurantiaca. The biosynthetic implications of these experiments are discussed.     

On-chip integrated hydrolysis, fluorescent labeling, and electrophoretic separation utilized for acetylcholinesterase assay

A sensitive on-chip acetylcholinesterase (AChE) assay that serves as a basis for the development of a fully integrated on-chip AChE-inhibitor detection assay is presented. The sequential steps required for the on-chip analysis process were integrated into a microchip. Transport and mixing of the reagents occurred by a combination of electroosmosis and electrophoresis using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was determined by on-chip reaction of thiocholine with eosinmaleimide, and the resulting thioether was electrophoretically separated and detected by laser-induced fluorescence (LIF). Enzyme-substrate mixing and reaction by confluent flow of reagents was compared with electrophoretically mediated microanalysis (EMMA), based on injection of an enzyme plug, and the utilization of differences in electrophoretic mobility as a driving force for efficient mixing and reaction. Both methods yielded similar results, however the EMMA-plug technique is preferable. The EMMA-plug technique was optimized for length and pushing time of enzyme plug, length of dyes mixture plug, acetylthiocholine concentration, and detector location. Detection of O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and paraoxon, two AChE inhibitors, was demonstrated by off-chip mixing of the inhibitor and AChE, followed by the on-chip AChE assay. Limit of detection of VX for 5.5 min incubation and of paraoxon for 8 min incubation was 4 × 10⁻¹⁰ and 4 × 10⁻⁷ M, respectively. Utilization of the AChE microchip assay for inhibition kinetics was demonstrated also by evaluation of the inhibitor-enzyme bimolecular reaction constant (k_i). The evaluated k_i values for VX and paraoxon for AChE from the electric eel were 3.5 × 10⁷ and 1.7 × 10⁵ M⁻¹ min⁻¹, respectively, conforming well to reported values obtained by bulk methods.     

Analytical methods for detection of gluten in food--method developments in support of food labeling legislation

The current essential therapy of celiac disease is a strict adherence to a gluten-free diet. Besides food products that are naturally gluten-free, "very low gluten" and "gluten-free" bakery products have become available. The availability of immunochemical and other analytical methods to determine gluten markers in foods is of utmost importance to ensure the well being of gluten-sensitive individuals. The aim of this review was to evaluate if currently available methodologies are suitable to meet the requirements of food labeling standards for individual gluten source declaration, in order to achieve policy objectives. Codex Alimentarius and European Union (EU) legislation and gluten detection methodologies applicable at present have been summarized and compared. In 2009, the European Commission issued Regulation No. 41/2009 concerning the composition and labeling of foodstuffs suitable for people intolerant to gluten. This review constitutes a basis to investigate the possibility to develop a proteomic-based method for the specific detection of gluten-containing cereals in food products, especially at or around the limits specified in EU legislation.     

EZ Gluten for the qualitative detection of gluten in foods, beverages, and environmental surfaces

The EZ Gluten assay is a rapid immunochromatographic screening method for qualitative detection of gluten in raw and cooked foods and beverages and on environmental surfaces. This AOAC Performance Tested Method study evaluated the EZ Gluten assay as an effective method for the detection of gluten in four selected matrixes: rice flour, cooked dough, beer, and dog food. In addition, the method was evaluated for its effectiveness in detecting gluten contamination of > or =1 microg/2 in.2 (25 cm2) stainless steel surface area. The EZ Gluten demonstrated 100% specificity [probability of detection (POD) 0.00, confidence interval (CI) 0.00-0.01] and 99% sensitivity (POD 0.99, CI 0.97-0.995) at the 10 ppm level for all four matrixes, and 100% specificity (POD 0.00, CI 0.00-0.11) and sensitivity (POD 1.00, CI 0.886-1.00) at the 1 microg level on the stainless steel surface. Independent laboratory testing confirmed the internal validation results in one matrix and on the stainless steel surface. Lot-to-lot, stability, and robustness studies provided evidence that the EZ Gluten is a rugged, consistent method for the detection of gluten at levels as low as 10 ppm.     

Development and validation of an HPLC-UV detection assay for the determination of rufinamide in human plasma and saliva

The development of a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of the new antiepileptic drug rufinamide (RFN) in human plasma and saliva is reported. Samples (250 μl) are alkalinized with ammonium hydroxide (pH 9.25) and extracted with dichloromethane using metoclopramide as internal standard. Separation is achieved with a Spherisorb silica column (250 × 4.6 mm i.d., 5 μm) at 30 °C using as mobile phase a solution of methanol/dichloromethane/n-hexane 10/25/65 (vol/vol/vol) mixed with 6 ml ammonium hydroxide. The instrument used was a Shimadzu LC-10Av chromatograph and flow rate was 1.5 ml min^-1, with a LaChrom L-7400 UV detector set at 230 nm. Calibration curves are linear [r ² = 0.998 ± 0.002 for plasma (n = 10) and r ² = 0.999 ± 0.001 for saliva (n = 9)] over the range of 0.25–20.0 μg ml^-1, with a limit of quantification at 0.25 μg ml^-1. Precision and accuracy are within current acceptability standards. The assay is suitable for pharmacokinetic studies in humans and for therapeutic drug monitoring.     

Development, validation and data quality assurance of screening methods: a case study

Despite the growing importance of qualitative screening tests in routine laboratories involved in the EU official control, their validation is not as deeply explained in Commission Decision 2002/657/EC as the validation of quantitative confirmatory methods. At the same time, the issue of quality assurance of screening assays defining internal quality control (IQC) procedures as required by accreditation bodies is undoubtedly less developed in this analytical field. As an example the present study describes the development, the validation and the IQC implemented for a commercial enzyme linked immunosorbent assay (ELISA) able to detect 17-α-19-nortestosterone (α-NT) and 17-β-19-nortestosterone (β-NT) isomers in bullock urine. In order to select a suitable sample treatment, two SPE purification protocols were preliminary compared. The chosen method was therefore fully validated determining the mandatory parameters required by Commission Decision 2002/657/EC: specificity, detection capability and robustness. An in-depth discussion was carried out illustrating the possible validation approaches and their implications especially in the assessment of the key performance characteristic: detection capability. Finally, the control charts implemented for continuous method verification during analyses of real samples were reported.     

Decision protocol for checking robustness with previous outlier detection in the validation of analytical methods

A general procedure based on variation of experimental design for checking robustness in the validation of analytical methods is presented. This procedure, which is easy to apply, consists in estimating the main total effects, in detecting outliers, checking the curvature and in determining the main side effects. Two methodologies based on the analysis of a) the residuals from the reduced model and b) the replicates from the reconstructed design were employed for the detection of outliers. In further studies, general experimental design principles were applied using two- and three-level factorial designs. In some cases, a dummy variable was introduced in order not to modify the structure of the designs utilized. Received: 30 October 1998 / Revised: 19 April 1999 / Accepted: 6 May 1999     

Decision protocol for checking robustness with previous outlier detection in the validation of analytical methods

A general procedure based on variation of experimental design for checking robustness in the validation of analytical methods is presented. This procedure, which is easy to apply, consists in estimating the main total effects, in detecting outliers, checking the curvature and in determining the main side effects. Two methodologies based on the analysis of a) the residuals from the reduced model and b) the replicates from the reconstructed design were employed for the detection of outliers. In further studies, general experimental design principles were applied using two- and three-level factorial designs. In some cases, a dummy variable was introduced in order not to modify the structure of the designs utilized.     

Design of a reversible biotin analog and applications in protein labeling, detection, and isolation

To expand the applicability of the biotin-(strept)avidin system, a biotin analog with reversible binding under non-denaturing conditions has been designed, and its applications in protein labeling, detection, and isolation have been evaluated.     

Comparison of three electrochemical end-point detection methods to assay potassium dichromate by coulometric titration

Coulometric titrations with three electrochemical end-point detection methods were performed to assay potassium dichromate as a standard for oxidation–reduction titration. The assay as an oxidizing agent was carried out with ferrous ions produced by electrolytically reducing ferric ions. Three end-point detection methods were employed and compared with each other: constant potential amperometry, potentiometry, and constant voltage biamperometry (a dead-stop method). The last one was found to provide high accuracy in the coulometric titration of potassium dichromate. Solution form samples were also measured to confirm the possible existence of chromium(III) in potassium dichromate by both coulometric titration and ion-exchange chromatography with inductively coupled plasma time-of-flight mass spectrometry.     

Xenoestrogens: mechanisms of action and detection methods

Estrogenic compounds exert pleiotropic effects in wildlife and humans, and endogenous estrogens, like 17-estradiol, regulate growth and development of their target tissues. Environmental, industrial, or naturally occurring chemicals that possess estrogenic and/or antiestrogenic activities are termed xenoestrogens and may interfere with endocrine systems. These xenoestrogens are therefore defined as endocrine-active or endocrine-disrupting compounds. The estrogen receptor (ER) is the major regulatory unit within the estrogen-signaling pathway and the molecular mechanisms of estrogen and ER actions are described briefly. Based on the mechanism of ER action, in vitro test systems are described that can be employed for screening but also for the elucidation of mechanisms of action of (anti)estrogenic compounds. How screening assays and mechanistic studies can aid in human risk assessment for potential endocrine-active compounds is discussed also.     

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.     

Quantification of dexamethasone and corticosterone in rat biofluids and fetal tissue using highly sensitive analytical methods: assay validation and application to a pharmacokinetic study

A sensitive, specific, accurate and precise LC/MS/MS method was developed for the simultaneous measurement of dexamethasone and corticosterone in rat plasma. The method was extended to dexamethasone analysis in rat plasma ultrafiltrate and fetal tissues. Samples were processed using SPE involving Oasis HLB cartridges, which offered complete extraction recovery for the analytes. Samples were subsequently analyzed using LC/MS/MS. A structurally related corticosteroid, prednisolone, was used as the internal standard. Using a 500 microL plasma sample, limits of quantification of 0.2 and 2.0 ng/mL were achievable for dexamethasone and corticosterone. This level of sensitivity allowed characterization of maternal/fetal dexamethasone profiles after administration of multiple doses of dexamethasone sodium phosphate to rats. However, this sensitivity was not satisfactory for corticosterone during pharmacokinetic studies involving dexamethasone due to its strong adrenosuppressive effect. This led us to investigate the suitability of a commercially available radioimmunoassay kit, which through extensive testing and minor modifications was found to offer extremely sensitive, specific, accurate and precise analysis of corticosterone. Knowledge of the steroid profiles captured using these highly sensitive analytical tools may potentially help in the optimization of corticosteroid therapy during pregnancy.     

Rapid and subnanomolar assay of recombinant human erythropoietin by capillary electrophoresis using NanoOrange precolumn labeling and laser‐induced fluorescence detection

Because of less functionally critical carbohydrate sectors that contributed to the stability, efforts have been made to quantify intact recombinant human erythropoietin. A simple, rapid capillary electrophoresis with laser‐induced fluorescence method for the assay of recombinant human erythropoietin was developed, with a limit of detection of intact recombinant human erythropoietin at subnanomolar concentration (up to 10 ng/mL or 3 × 10^?10 M), which is among the lowest reported. High sensitivity was accomplished by precolumn derivatization with the noncovalent dye NanoOrange. Capillary electrophoresis separation and reaction conditions were carefully manipulated for avoiding microheterogeneity of glycoforms and inhomogeneity of multiple labeling products. The fluorescence signal was linear over the range of 10 ng/mL–10 μg/mL, corresponding to the detection requirement of recombinant human erythropoietin in biofluids and pharmaceutical samples, as demonstrated by a real sample analysis. Although the salt in reaction mixtures showed a detrimental effect on the fluorescence of the derivatives, this method could tolerate a certain amount of salt, extending its application in biofluid analysis. In addition, zero‐order fluorescence emission kinetics was obtained, indicating that the rapid decay of recombinant human erythropoietin was derived from a self‐quenching effect.