Steffen Uhlig

Assessment of detection methods in trace analysis by means of a statistically based in-house validation concept

A matrix-considering in-house validation concept for analytical methods is presented which takes into account the uncertainty due to matrix- and time-induced deviations. It is based on a variance component model for univariate quantitative measurement data that can be adapted to both screening and confirmation methods and to both zero-tolerance and threshold decisions. The model allows the calculation of critical concentrations for given α-errors and the calculation of the corresponding power function to evaluate the performance of an analytical method. The model is applied to a real-life validation experiment.

Multi-residue method for non-steroidal anti-inflammatory drugs in plasma using high-performance liquid chromatography–photodiode-array detection: Method description and comprehensive in-house validation

A multi-residue high-performance liquid chromatography (HPLC) method with photodiode-array detection is presented for the determination of 12 non-steroidal anti-inflammatory drugs (NSAIDs) in plasma. This method has been validated under the consideration of actual alpha- and beta-errors according to an in-house validation concept based on a fractional factorial experiment. A wide range of matrices and other influencing factors have been included in the validation experiment. In order to assess the methods performance the power curve, which demonstrates the detection power of the analytical method, was computed and CCalpha and CCbeta values were calculated.

A top-down in-house validation based approach for the investigation of the measurement uncertainty using fractional factorial experiments

The recently presented in-house validation concept developed on the basis of a variance component model is extended and optimised by basing it on a fractional factorial sampling scheme. The presented approach relies on a comprehensive mathematical model to assess simultaneously the in-house reproducibility and its components in addition to uncertainty-related performance characteristics like power functions and critical concentrations. The influence of individual factors and the corresponding uncertainties can be determined separately in a single experiment with optimum utilization of the generated data, in this way achieving a favourable ratio between costs and result.

Results of a European interlaboratory method validation study for the quantitative determination of lipophilic marine biotoxins in raw and cooked shellfish based on high-performance liquid chromatography–tandem mass spectrometry. Part I: collaborative study

A European interlaboratory collaborative study was conducted to validate a method for the quantitative determination of lipophilic marine biotoxins based on high-performance liquid chromatography–tandem mass spectrometry. During this study, the diarrhetic shellfish poisoning toxins okadaic acid, dinophysis toxin1 and 2 including their esters, the azaspiracids 1-3, pectenotoxin2, and the yessotoxins were investigated at concentration levels near the limit of quantification and near the legal limit. Naturally contaminated blue mussels, both raw and cooked and spiked extracts of clams and oysters were studied and results were obtained for 16 test samples from 16 laboratories representing eight different countries. This article summarizes the study outcome concerning validation key parameters like specificity, linearity, limit of detection, accuracy/recovery, and precision. Further, influences of cooking of mussels before homogenization or hydrolysis on method robustness have been evaluated.

Evaluation and validation of a novel Arxula adeninivorans estrogen screen (nAES) assay and its application in analysis of wastewater, seawater, brackish water and urine.

A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene (phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene (ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase (GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6-25 h assay period with detection and determination limits and EC(50) values for 17β-estradiol of 2.8 ng L(-1), 5.9 ng L(-1), 33.2 ng L(-1) (nAES-P) and 3.1 ng L(-1), 6.7 ng L(-1) and 39.4 ng L(-1) (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors.

Development and assessment of a novel Arxula adeninivorans androgen screen (A-YAS) assay and its application in analysis of cattle urine

The novel A-YAS assay for the detection of androgenic activity in liquid samples such as urine has been developed and assessed. The assay is based on transgenic Arxula adeninivorans yeast cells as the bio-component. The cells were engineered to co-express the human androgen receptor (hAR) gene and the inducible phytase reporter gene (phyK, derived from Klebsiella sp. ASR1), under the control of an Arxula derived glucoamylase (GAA) promoter, which had been modified by the insertion of hormone-responsive elements (HREs). The Arxula transformation/expression platform Xplor®2 was used to select stable mitotic resistance marker free transformants and the most suitable cells were characterized for performance as a sensor bio-component. The assay is easy-to-use, fast (6-25 h) and is currently the most sensitive yeast-based androgen screen with an EC50, limit of detection and of quantification values for 5α-dihydrotestosterone (DHT) of 277.1±53.0, 56.5±4.1 and 76.5±6.7 ng L(-1), respectively. Furthermore, the assay allows the determination of androgenic and anti-androgenic activity of various compounds such as naturally occurring androgens and estrogens, pharmaceuticals and biocides. The robustness of the A-YAS assay enables it to be used for analysis of complex samples such as urine. The results of the analysis of a number of cattle urine samples achieved by the A-YAS assay correlate well with GC-MS analysis of the same samples.

A new profile likelihood confidence interval for the mean probability of detection in collaborative studies of binary test methods

A confidence interval for the probability of detection across laboratories (LPOD) for qualitative methods, described in the AOAC INTERNATIONAL guidelines for validation of microbiological methods for food and environmental surfaces, is considered. It is demonstrated that under certain conditions, the observed confidence of this confidence interval can be rather low, so that statistical minimum requirements are not fulfilled. A new profile likelihood confidence interval based on a latent random laboratory effect approach is proposed. Observed confidence levels for this confidence interval demonstrate its applicability already for a small number of laboratories.

Carcinoembryonic antigen and cytokeratin-19 fragments for assessment of therapy response in non-small cell lung cancer: a systematic review and meta-analysis

Background:This meta-analysis evaluated whether pretherapy serum levels of carcinoembryonic antigen (CEA) and cytokeratin-19 fragments (CYFRA 21-1) are predictive of response to therapy in non-small cell lung cancer (NSCLC) and whether changes in these markers during vs pretherapy are indicative of response.Methods:Original peer-reviewed studies enrolling adults with untreated advanced NSCLC were identified using PubMed. Two reviewers independently extracted data from eligible studies and assessed study heterogeneity and the risk of study bias.Results:Fourteen studies were eligible; 11 had objective response as an end point and three evaluated clinical benefit (i.e., response and stable disease). Study bias was relatively low. Both markers showed comparable modest predictive value across studies, with baseline CYFRA 21-1 numerically better in predicting treatment benefit. A good performance in identifying objective response during treatment was seen (AUC 0.724 (95% CI 0.667–0.785) for CYFRA 21-1 and 0.728 (95% CI, 0.599–0.871) for CEA). A decline in CYFRA 21-1 levels during treatment was highly indicative for objective response (sensitivity 79.1% (95% CI 71.5–85.1)).Conclusions:Comprehensive analysis of study heterogeneity and bias provides a high level of evidence for the clinical utility of CEA and CYFRA 21-1 for the prediction and monitoring of response in NSCLC.

Collaborative trial validation of cry1Ab/Ac and Pubi-cry TaqMan-based real-time PCR assays for detection of DNA derived from genetically modified Bt plant products

Presence of genetic modifications in rice products originating from China and imported to the European Union market is detected since 2006. Neither these products from China nor any other genetically modified rice lines are approved as food or feed in the EU. The transgenic rice varieties identified contain genetic elements and constructs coding for insect-resistance genes from Bacillus thuringiensis (Bt) coding for insecticidal crystal (cry) proteins. In particular, DNA sequences coding for codon-optimised or fused cry1Ab/c genes and constructs driven by the maize ubiquitin promoter (P-ubiZM1) were identified. For improved screening and identification of genetic modifications present in Asian rice products, two TaqMan-based real-time PCR assays targeting codon-optimised cry1Ab/Ac and the Pubi-cry construct have been developed. These assays have been validated in an international collaborative trial with 17 participants from 10 countries. Based on a new mathematical–statistical model and an adjusted experimental set-up of the collaborative trial, a close examination of the limit of detection (LOD95%) and the probability of detection of the qualitative PCR assays was conducted. The evaluation of the method performance characteristics and results of the collaborative trial validation are presented.

Robust Estimation of Variance Components with High Breakdown Point in the 1-Way Random Effect Model

For the 1-way random effect model three new robust variance component estimators are proposed. Consistency and breakdown properties are derived. The use of the estimators for interlaboratory tests is demonstrated in a practical example.