Expert systems for method development and validation in HPLC

ESCA, Expert Systems Applied to Chemical Analysis, started its research in March 1987, with the aim of building prototype expert systems for HPLC method development. Results of this research have been published as the work has progressed. The project is now completed and this paper summarises some of the overall project conclusions. Seven different expert systems have been built which tackle problems throughout the process of method development, four stand-alone systems and three integrated systems. The object of ESCA was to evaluate the applicability of expert system technology to analytical chemistry and not all the systems were built for commercial uses. Many of the systems tackle problems specific to one or more of the partners and thus may not be useful outside this environment. However, the results of the work are still pertinent to analysts wishing to build their own systems. These results are described, however, the emphasis of the paper is on those systems developed for method validation.Method validation for HPLC is a complex task which requires many characteristics of the method to be tested, e.g. accuracy, precision, etc. The expert systems built within ESCA concern the validation of precision. Two systems were developed for repeatability testing and ruggedness testing. The method validation process can be divided into several discrete stages, these include: (1) The selection of the method feature to test, for instance which factors can influence the ruggedness of a method. (2) The definition of a test procedure, for instance an efficient statistical design. (3) The execution of experiments and the interpretation of results. (4) A diagnosis of any observed problem. This paper describes these two systems in some detail and summarises some of the results obtained from their evaluation. It concludes that expert systems can be useful in solving analytical problems and the integration of several expert systems can provide extremely powerful tools for the analyst.     

Method validation approach on the basis of a quadratic regression model

A method validation approach that bases on a quadratic regression model in which two types of error are incorporated is presented and applied to an experimental data set. The validation approach enables the determination of analytical performance characteristics referred to in Commission Decision 2002/657/EC (i.e., repeatability, within-laboratory reproducibility, decision limit, detection capability).     

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.     

Assessment of reproducibility and uncertainty of food microbiology methods: statistical approach of a multi-site laboratory

The validation of food microbiology methods and assessment of measurement uncertainty are required for laboratories operating EN ISO/IEC 17025 accreditation systems. This paper aims to describes the statistical approach adopted by a multi-site laboratory to satisfy these requirements and to routinely check the performance of the methods. A validation protocol was performed, reflecting a great variability of experimental conditions, represented by the period of time during which determinations were made, the different laboratories concerned, the large number of technicians involved, the differences in the level of contamination of the matrix analyzed and the operating conditions (equipment, reagents, culture media, environmental conditions, etc.). Despite the very high variability of the experimental conditions, the values of repeatability and reproducibility obtained for the methods were lower than those stated in the respective regulations. In addition, the top-down approach adopted in this study is to be considered effective overtime and allows to randomize all variable factors, including the effects of sensitivity and specificity, in order to simulate the conditions of real reproducibility. A specific Excel spreadsheet was also developed for the routine expression of analytical results. This spreadsheet represents a very useful tool for operators to calculate and routinely check the results, as well as to assess the uncertainty of measurement. This particular quality assessment system is enabling the multi-site laboratory to implement the accuracy, reliability and comparability of the analytical results and to ensure the analytical control of data output.     

Validation of a digestion system using a digester block/cold finger system for the determination of lead in vegetable foods by electrothermal atomic absorption spectrometry

This paper presents the validation of a system for sample digestion using a digester block/cold finger to determine the lead content in vegetables by electrothermal atomic absorption spectrometry (ETAAS). After mineralization, lead contents were determined by ETAAS using a calibration curve based on aqueous standards prepared in 2.60 M nitric acid solutions containing 5 microg ammonium phosphate as chemical modifier. A pyrolysis temperature of 900 degrees C and atomization temperature of 2000 degrees C were used. This method allowed the determination of lead with a characteristic mass of 35 pg; LOD and LOQ of 0.6 and 2 nglg, respectively, were found. The precision was investigated in terms of reproducibility and repeatability. Reproducibility was estimated by analysis of nine different portions of a certified reference material (CRM) of spinach leaves, and the repeatability was determined through the analysis of nine aliquots of the same solution. The reproducibility and repeatability were found to be 4.27 and 2.94% RSD, respectively. The accuracy was confirmed by analysis of whole meal flour, spinach leaves, and orchard leaves CRMs, all furnished by the National Institute of Standards and Technology. Lead contents were measured using the newly developed technique in 11 different potato samples. The lead contents ranged from 12.80 to 69.27 ng/g, with an average value of 28.59 ng/g. These values were in agreement with data reported in the literature.     

Method validation for the analysis of pesticide residues in grain by thin-layer chromatography

Method validation is an important requirement in the practice of pesticide residue analysis and is the process of verifying that a method is fit for its purpose. To make a correct decision on the validity of the method, the following method performance parameters have to be taken into consideration: scope, specificity, limit of detection, limit of quantification, linear range, accuracy, precision, repeatability, reproducibility, recovery, ruggedness and robustness. The goal of this study was to validate previously adapted thin-layer chromatographic methods for the pesticide residue analysis in grain. Confirmation of validation parameters for some compounds was also performed by gas chromatographic analysis.     

Fuzzy logic-based procedures for GMO analysis

Key to sound validation studies is the formalization and harmonization of procedures for design of experiment and interpretation of results. International guidelines (ISO 5725, ENGL) are available for the validation of GMO detection methods, and ad-hoc validation statistics (e.g. per cent bias, repeatability and reproducibility) are used for in-house and inter-laboratory testing and decision-making. Acceptability criteria have been set but not every situation can be covered by a preset rule; the interpretation of results in validation largely depends on expert judgement being a matter of professional judgment and expertise. Fuzzy logic-based techniques may be used to summarize the information obtained by independent validation statistics and are helpful in such respect. A comprehensive indicator of method performance permits direct comparison between methods and facilitates the evaluation of multiple, yet contradictory statistics. The European Union Reference Laboratory for GM Food and Feed has already proposed the fuzzy principle in the context of method validation. Other studies have also proved its applicability in other areas of GMO analysis, but the application has been limited hitherto. In this article, we review the fuzzy logic principle and its potential to support the continuous progress of GMO science and routine laboratory analyses.     

A direct RP-HPLC method for the determination of furanic aldehydes and acids in honey

In this study 5-hydroxymethyl-2-furaldehyde (HMF), 2-furaldehyde, 3-furaldehyde, 2-furoic acid and 3-furoic acid are contemporarily determined in honey using a swift and direct RP-HPLC approach. The validation protocol was performed in terms of detection and quantification limits, precision (by repeatability and reproducibility), linearity and accuracy (by recovery tests); the acceptability of the precision and accuracy results was positively verified using Horwitz's model and AOAC guidelines, respectively. The method was tested on 18 honey samples of different ages, and botanical and geographical origin. HMF and 2-furaldehyde correlated highly with the age of the samples, whereas no correlation was observed with regards to 2-furaldehyde and 2-furoic acid. Hypotheses relating to the formation of minority furanic compounds are also proposed.     

Online fully automated SPE‐HPLC‐MS/MS determination of ceftiofur in bovine milk samples employing a silica‐anchored ionic liquid as sorbent

Solid‐phase extraction coupled online with high performance liquid chromatography and tandem mass spectrometry was successfully applied to determine low concentrations of ceftiofur antibiotic in bovine milk samples. A silica‐anchored ionic liquid was applied as sorbent material to be used as extraction phase in the proposed online system. The material was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. In order to improve the system reproducibility, the following experimental parameters were optimized: organic solvent percentage, time and sample loading flow rate. Subsequently, the method was validated presenting satisfactory results as adequate selectivity, good linearity and correlation coefficient higher than 0.98. The limit of detection and quantification were 0.1 and 0.7 μg/L, respectively. The precision of the methodology was evaluated as repeatability and intermediate precision, with relative standard deviation values lower than 15%. The accuracy of the method ranged from 72.8 to 137% and the minimum and maximum recovery values were 73.4 and 111.3%, respectively. After the validation, seven milk samples were analyzed and although ceftiofur was not detected in any of them the method was demonstrated to be efficient when applied to the analysis of milk samples fortified with the pollutant of interest.     

Development of an improved method for the determination of polycyclic aromatic hydrocarbons in mainstream tobacco smoke

A gas chromatograph/mass-selective detection (GC/MS) method has been developed and validated for the quantitation of 16 polycyclic aromatic hydrocarbons found in mainstream tobacco smoke condensate. The utilization of two types of solid-phase extraction media combined with capillary column technology removed matrix interferences, afforded a significant reduction in analysis run time, and increased accuracy. Also, the addition of a chilled impinger was used to trap semi-volatile polycyclic aromatic hydrocarbons and to provide more accurate data. This was done without sacrificing the repeatability, reproducibility, and precision obtained in previously published methods. The development and validation studies discussed in this paper resulted in an improved, robust analytical method capable of increasing laboratory capacity and reducing sample reporting time.     

Expert system for precision testing in validation of liquid chromatographic methods

After a liquid chromatographic method has been developed, it must be validated to establish its limitations in daily use. Method validation is becoming increasingly important as stricter rules are applied by regulatory authorities. Precision testing is a vital step in this validation; both intralaboratory testing and interlaboratory testing are needed. In an intralaboratory test, repeatability and ruggedness tests are usually done. Expert systems are available for both tests. Here they are integrated to form an intralaboratory precision-testing expert system; special integration architecture is described. Important features of the integrated system are a supervisor containing planning knowledge about the tests and a common data structure containing all the objects necessary for an expert system in this area.     

测量方法精密度共同试验测量数据的统计分析

以钼蓝分光光度法测定石灰石、白云石中二氧化硅分析方法标准的精密度共同试验测量数据为例,按GB/T 6379.2—2004的统计方法进行统计,最终确定了分析方法的重复性限r和再现性限R与含量(水平)m的函数关系式。对统计中数据的处理、回归方程的图示确定、分析方法精密度的表示等问题进行了讨论。以含量分段表示重复性限和再现性限,在分析实践中更为实用。对共同试验和数据处理中要注意的问题进行了深入讨论。     

Analysis of estrogens in environmental matrices

Since the increase in interest in the presence of estrogens in the environment, several publications have been published. This article aims first to review the state-of-the-art analytical methods used to measure these compounds in environmental matrices, and second to scrutinize how these methods and their performances were validated. Our review will cover the methodologies used for quantification of total estrogens (i.e. free and conjugated estrogens) and verification of sample stability. The fundamental point for a good method validation is the use of a real matrix to estimate linearity, precision (including repeatability and reproducibility), specificity and, especially, sensitivity and recovery.     

Nonlinear Regression Using the “Big Three” Spreadsheets

Nonlinear regression using the spreadsheet Excel has appeared in the literature in several places. This paper extends the use of this technique to two other popular spreadsheets, Lotus 123 and Quattro Pro. Associated files, which can be downloaded, are included. These are the spreadsheet templates and ScreenCam movies that illustrate the use of equation solvers for minimization in these spreadsheets.     

Method validation and qualification of instruments for atomic spectrometry

This work describes the analytical procedures for atomic absorption and inductively coupled plasma (ICP) techniques that have to be used in order to obtain a license to sell drugs in the USA. The qualification of atomic absorption spectrometers and ICP instruments is described. The method validation characteristics, e.g., accuracy, precision, linearity, range, detection limits, and quantification are discussed. The time involved and the quality of documentation are pointed out. The consequences for laboratory personnel and operating costs are also discussed.     

In-house validation of a liquid chromatography tandem mass spectrometry method for the analysis of lipophilic marine toxins in shellfish using matrix-matched calibration

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 μg kg^-1 for OA, AZA1 and PTX2 and 1,000 μg kg^-1 for YTX. For SPX1, 400 μg kg^-1 was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 μg kg^-1 OA equivalents and 30 μg kg^-1 AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests.     

Multi-method for the determination of antibiotics of different substance groups in milk and validation in accordance with Commission Decision 2002/657/EC

The presented method is able to analyse 47 substances of the antibiotic groups tetracyclines, quinolones, macrolides, sulfonamides, diamino-pyrimidine derivatives and lincosamides simultaneously in a single analytical run. Applying an in-house validation concept, the validation of the multi-method was successfully accomplished with a low number of experiments. Each substance was validated at least at the concentrations 0.5, 1.0 and 1.5 MRL (maximum residue limit), or respectively, at concentrations as low as possible for substances without MRL. The calculated relevant validation parameters, e.g. the decision limit CCα, the detection capability CCβ, the repeatability, the within-laboratory reproducibility and the recovery, are in an acceptable range and in compliance with the requirements of Commission Decision 2002/657/EC. A proficiency test and the implementation of the method by other laboratories were performed successfully.     

A simplified approach to the estimation of analytical measurement uncertainty

The present study summarizes the measurement uncertainty estimations carried out in Nestlé Research Center since 2002. These estimations cover a wide range of analyses of commercial and regulatory interests. In a first part, this study shows that method validation data (repeatability, trueness and intermediate reproducibility) can be used to provide a good estimation of measurement uncertainty.In a second part, measurement uncertainty is compared to collaborative trials data. These data can be used for measurement uncertainty estimation as far as the in-house validation performances are comparable to the method validation performances obtained in the collaborative trial.Based on these two main observations, the aim of this study is to easily estimate the measurement uncertainty using validation data.     

Validation of a multi-residue method for the determination of several antibiotic groups in honey by LC-MS/MS

The presented multi-method was developed for the confirmation of 37 antibiotic substances from the six antibiotic groups: macrolides, lincosamides, quinolones, tetracyclines, pleuromutilines and diamino-pyrimidine derivatives. All substances were analysed simultaneously in a single analytical run with the same procedure, including an extraction with buffer, a clean-up by solid-phase extraction, and the measurement by liquid chromatography tandem mass spectrometry in ESI+ mode. The method was validated on the basis of an in-house validation concept with factorial design by combination of seven factors to check the robustness in a concentration range of 5-50 μg kg(-1). The honeys used were of different types with regard to colour and origin. The values calculated for the validation parameters-decision limit CCα (range, 7.5-12.9 μg kg(-1)), detection capability CCβ (range, 9.4-19.9 μg kg(-1)), within-laboratory reproducibility RSD(wR) (<20% except for tulathromycin with 23.5% and tylvalosin with 21.4 %), repeatability RSD(r) (<20% except for tylvalosin with 21.1%), and recovery (range, 92-106%)-were acceptable and in agreement with the criteria of Commission Decision 2002/657/EC. The validation results showed that the method was applicable for the residue analysis of antibiotics in honey to substances with and without recommended concentrations, although some changes had been tested during validation to determine the robustness of the method.     

Validation of a reversed phase high performance thin layer chromatographic-densitometric method for secoisolariciresinol diglucoside determination in flaxseed

The validation of a HPTLC-densitometric method for the determination of secoisolariciresinol diglucoside (SDG) in flaxseed was performed improving the reproducibility of a previously reported HPTLC densitometric procedure by the use of fully wettable reversed phase plates (silica gel 60 RP18W F(254S), 10cmx10cm) with MeOH:HCOOH 0.1% (40:60, v/v) mobile phase. The analysis required only the alkaline hydrolysis in aqueous medium of undefatted samples and densitometry at 282nm of HPTLC runs. The method was validated following the protocol proposed by the Société Francaise des Sciences et Techniques Pharmaceutiques (SFSTP) giving rise to a dependable and high throughput procedure well suited to routine application. SDG was quantified in the range of 321-1071ng with RSD of repeatability and intermediate precision not exceeding 3.61% and accuracy inside the acceptance limits. Flaxseed of five cultivars of different origin was elected as test-bed.