Validation and qualification: the fitness for purpose of mass spectrometry-based analytical methods and analytical systems

The context of validation for mass spectrometry (MS)-based methods is critically analysed. The focus is on the fitness for purpose depending on the task of the method. Information is given on commonly accepted procedures for the implementation and acceptance of analytical methods as ‘confirmatory methods’ according to EU criteria, and strategies for measurement. Attention is paid to the problem of matrix effects in the case of liquid chromatography-mass spectrometry-based procedures, since according to recent guidelines for bioanalytical method validations, there is a need to evaluate matrix effects during development and validation of LC-MS methods “to ensure that precision, selectivity and sensitivity will not be compromised”. Beneficial aspects of the qualification process to ensure the suitability of the MS analytical system are also evaluated and discussed.     

Validation of thin layer and high performance thin layer chromatographic methods

Analytical validation is a key requirement to asses and to prove a method's reliability and suitability for an intended use. Planar chromatographic procedures are used in different applications ranging from simple screening tests to sophisticated instrumental quantitative assays of analytes in complex matrices. This paper intends to give guidance on how to adopt international accepted formal requirements and guidelines for validation of these different TLC/HPTLC procedures. In addition, some selected parameters for robustness testing and for on going quality assurance of analytical performance based on control charts are reported.     

Recent Trends in Validation of Bioanalytical Methods

Abstract

The assessment of the quality levels of pharmaceutical formulations is done through various validation parameters. Validation of these analytical test procedures is the process by which it is established that the test methods meet the requirements for the intended analytical application. The validation criterion also applies to bioanalytical methods used for non‐human pharmacology/toxicology studies and preclinical studies. Bioanalytical method validation includes all the procedures, which demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine, is reliable and reproducible for the intended use.     

Bioanalytical method validation and its implications for forensic and clinical toxicology – A review

The reliability of analytical data is very important to forensic and clinical toxicologists for the correct interpretation of toxicological findings. This makes (bio)analytical method validation an integral part of quality management and accreditation in analytical toxicology. Therefore, consensus should be reached in this field on the kind and extent of validation experiments as well as on acceptance criteria for validation parameters. In this review, the most important papers published on this topic since 1991 have been reviewed. Terminology, theoretical and practical aspects as well as implications for forensic and clinical toxicology of the following validation parameters are discussed: selectivity (specificity), calibration model (linearity), accuracy, precision, limits, stability, recovery and ruggedness (robustness). Received: 16 June 2002 Accepted: 12 July 2002 Part of this review was published in the communications of the International Association of Forensic Toxicologists (TIAFT; TIAFT Bulletin 32 (2002): 16–23) and of the Society for Forensic and Toxicologic Chemistry (GTFCH; Toxichem and Krimitech 68 (2001): 116-126). Correspondence to F. T. Peters     

International Guidelines for Bioanalytical Method Validation: A Comparison and Discussion on Current Scenario

US FDA released guidelines for bioanalytical method validation in 2001 and it became the basis for guidelines such as ANVISA and EMA. Even though there is a general agreement between these guidelines in terms of evaluation of validation parameters, significant diversity exists with respect to methodology employed. Present review compares and summarizes the regulatory guidelines issued by US FDA, ANVISA and EMA for bioanalytical method validation. This review also discusses evaluation of certain validation parameters such as matrix effect, incurred sample reanalysis, various stability aspects, effect of anticoagulant counter ions, specificity in the presence of concomitant medications, and identification of pharmacokinetic repeats wherein specific guidance and general consensus amongst scientific community does not exist.     

Practical uses of proficiency testing as valuable tools for validation and performance assessment in environmental analysis

Besides their role as an external quality control tool, PT results or samples could be used as an alternative to fulfil some of the quality assurance requirements such as analytical precision, uncertainty assessment, and internal quality control. This additional use of proficiency testing could help laboratories to reduce the financial impact of their quality assurance process. The purpose of this paper is to highlight some practical uses of PT results or samples in the environmental analytical field, which have been implemented at ISSeP (Institut Scientifique de Service Public), either for method validation or for internal quality control.Presented at the Eurachem PT Workshop September 2005, Portorož, Slovenia     

US FDA–validated TLC method with four greenness assessment evaluations for simultaneous determination of prednisolone and esomeprazole in spiked human plasma

Recently, prednisolone has been used in treating many medical conditions, such as autoimmune diseases and cancer. It is also prescribed to mitigate the respiratory complications caused by COVID-19 infection. It can cause some health complications, such as GIT ulcers, so it should be co-administered with proton-pump inhibitors, such as esomeprazole, to prevent the risk of ulcers. This work aims to develop an ecofriendly and sensitive TLC method for simultaneous determination of esomeprazole and prednisolone in their binary mixtures and spiked human plasma. Separation was performed using a mixture of ethyl acetate, methanol, and ammonia (9.5:0.5:0.1, v/v/v) as an eluting system with UV scanning at 245 nm. Dapoxetine was used as an internal standard to correct the variation during sampling. The resulting R_f values for plasma, esomeprazole, prednisolone, and dapoxetine were 0.03, 0.51, 0.72 and 0.85, respectively. Four greenness assessment tools—national environmental method index, eco-scale assessments, analytical greenness metric approach (AGREE), and green analytical procedure index (GAPI)—were used to evaluate the greenness characteristics of the proposed method to the environment, and the results were acceptable and satisfactory. Validation parameters were checked according to the US FDA guidelines to achieve the international requirements for bioanalytical method validation, and the results were within the accepted ranges.     

Current use of high-resolution mass spectrometry in drug screening relevant to clinical and forensic toxicology and doping control

Clinical and forensic toxicology and doping control deal with hundreds or thousands of drugs that may cause poisoning or are abused, are illicit, or are prohibited in sports. Rapid and reliable screening for all these compounds of different chemical and pharmaceutical nature, preferably in a single analytical method, is a substantial effort for analytical toxicologists. Combined chromatography–mass spectrometry techniques with standardised reference libraries have been most commonly used for the purpose. In the last ten years, the focus has shifted from gas chromatography–mass spectrometry to liquid chromatography–mass spectrometry, because of progress in instrument technology and partly because of the polarity and low volatility of many new relevant substances. High-resolution mass spectrometry (HRMS), which enables accurate mass measurement at high resolving power, has recently evolved to the stage that is rapidly causing a shift from unit-resolution, quadrupole-dominated instrumentation. The main HRMS techniques today are time-of-flight mass spectrometry and Orbitrap Fourier-transform mass spectrometry. Both techniques enable a range of different drug-screening strategies that essentially rely on measuring a compound’s or a fragment’s mass with sufficiently high accuracy that its elemental composition can be determined directly. Accurate mass and isotopic pattern acts as a filter for confirming the identity of a compound or even identification of an unknown. High mass resolution is essential for improving confidence in accurate mass results in the analysis of complex biological samples. This review discusses recent applications of HRMS in analytical toxicology.     

Different approaches to legal requirements on validation of test methods for official control of foodstuffs and of veterinary drug residues and element contaminants in the EC

 In order to ensure food consumer protection as well as to avoid barriers to trade and unnecessary duplications of laboratory tests and to gain mutual recognition of results of analyses, the quality of laboratories and test results has to be guaranteed. For this purpose, the EC Council and the Commission have introducedprovisions – on measures for quality assurance for official laboratories concerning the analyses of foodstuffs on the one hand and animals and fresh meat on the other, – on the validation of test methods to obtain results of sufficient accuracy. This article deals with legal requirements in the European Union on basic principles of laboratory quality assurance for official notification to the EC Commission and on method validation concerning official laboratories. Widespread discussions and activities on measurement uncertainty are in progress, and the European validation standards for official purposes may serve as a basis for world-wide efforts on quality harmonization of analytical results. Although much time has already been spent, definitions and requirements have to be revised and further additions have to be made.     

A need for clearer terminology and guidance in the role of reference materials in method development and validation

 The fact that various definitions and terminology applied to measurements in analytical chemistry are not always consistent and straightforward, by not only answering the question ”what”, but also ”how”, leads to their various interpretations. This results in non-uniform implementation of very basic and essential metrological principles in chemistry. Such a diverse situation is not conducive to the endorsement of harmonised measurements all across the world, to serve as a tool for improving the quality of life in its broadest sense for all its citizens. The discussion in this paper is focused on problems associated with terminology and definitions of ’reference material’ and ’validation’. The role of reference materials in measurement processes for purposes other than calibration and validation principles in analytical chemistry are also discussed in this paper. Where possible, potential solutions are proposed, but more often, questions of essential importance are raised in order to initiate international discussion which will hopefully lead to equally understandable answers. Received: 2 November 2002 Accepted: 3 February 2003 Acknowledgements   The author is grateful to Aleš Fajgelj for his comprehensive comments on the topic described in this paper. Sincere thanks also to Philip Taylor, Ewa Bulska, Emilia Vassileva, Miloslav Suchanek and Margreet Lauwaars for their contribution during fruitful discussions on validation. Presented at the CERMM-3, Central European Reference Materials and Measurements Conference: The function of reference materials in the measurement process, May 30–June 1, 2002, Rogaška Slatina, Slovenia Correspondence to N. Majcen     

QbD‐oriented development and validation of a bioanalytical method for nevirapine with enhanced liquid–liquid extraction and chromatographic separation

The present studies describe the systematic quality by design (QbD)‐oriented development and validation of a simple, rapid, sensitive and cost‐effective reversed‐phase HPLC bioanalytical method for nevirapine in rat plasma. Chromatographic separation was carried out on a C₁₈ column using isocratic 68:9:23% v/v elution of methanol, acetonitrile and water (pH 3, adjusted by orthophosphoric acid) at a flow rate of 1.0 mL/min using UV detection at 230 nm. A Box–Behnken design was applied for chromatographic method optimization taking mobile phase ratio, pH and flow rate as the critical method parameters (CMPs) from screening studies. Peak area, retention time, theoretical plates and peak tailing were measured as the critical analytical attributes (CAAs). Further, the bioanalytical liquid–liquid extraction process was optimized using an optimal design by selecting extraction time, centrifugation speed and temperature as the CMPs for percentage recovery of nevirapine as the CAA. The search for an optimum chromatographic solution was conducted through numerical desirability function. Validation studies performed as per the US Food and Drug Administration requirements revealed results within the acceptance limit. In a nutshell, the studies successfully demonstrate the utility of analytical QbD approach for the rational development of a bioanalytical method with enhanced chromatographic separation and recovery of nevirapine in rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of ethyl sulfate--a marker for recent ethanol consumption--in human urine by CE with indirect UV detection

A CE method for the determination of the ethanol consumption marker ethyl sulfate (EtS) in human urine was developed. Analysis was performed in negative polarity mode with a background electrolyte composed of 15 mM maleic acid, 1 mM phthalic acid, and 0.05 mM cetyltrimethylammonium bromide (CTAB) at pH 2.5 and indirect UV detection at 220 nm (300 nm reference wavelength). This buffer system provided selective separation conditions for EtS and vinylsulfonic acid, employed as internal standard, from urine matrix components. Sample pretreatment of urine was minimized to a 1:5 dilution with water. The optimized CE method was validated in the range of 5-700 mg/L using seven lots of urine. Intra- and inter-day precision and accuracy values, determined at 5, 60, and 700 mg/L with each lot of urine, fulfilled the requirements according to common guidelines for bioanalytical method validation. The application to forensic urine samples collected at autopsies as well as a successful cross-validation with a LC-MS/MS-based method confirmed the overall validity and real-world suitability of the developed expeditious CE assay (sample throughput 130 per day).     

Establishment of an ISO 17025:2005 accredited forensic genetics laboratory in Italy

Forensic genetics is extremely useful for the resolution of criminal cases, identification of missing persons and in paternity/kinship testing. Each and every laboratory that works in the forensic genetics area has developed its own working method independently, however, generally in accordance with international guidelines. More than thirty institutional, public and private forensic laboratories that deal with the identification/paternity testing through DNA in Italy have been surveyed, but to this day, only five public laboratories (four of the police and one of a university hospital) and two private ones are accredited. There are, however, many other laboratories that perform occasional forensic genetics activities that have not been surveyed. The need to achieve the ISO 17025:2005 accreditation may represent for these laboratories an excellent opportunity to improve their activities. Although the DNA analysis for forensic investigation is used in Italy since the beginning of the technique, the quality of the results has been called into question more than once, as it appears by many court cases in which the results of genetic tests have been subject to strong criticisms. Obviously, the ISO 17025:2005 is not sufficient to guarantee the quality of the results. It is essential to show the laboratory working method to the scientific community in order to obtain reliable and robust analytical results that can be used in court to accuse/exonerate individuals accused of a crime or to assign a true biological father to a child. Here, we show a part of the workflow validation process of the internal method used in the Forensic Genetic Service (FGS) of the Diagnostic Genetics Unit (DG) of the Careggi University Hospital. This article outlines some relevant aspects of the methods adopted to ensure robustness, reliability and reproducibility of genetic profiles used for forensic identification.     

Validation and quality assurance of planar chromatographic procedures in pharmaceutical analysis

Within the process of the International Conference on Harmonization (ICH), 2 guidelines were released containing a standardized terminology, a verified model of requirements for the validation of analytical procedures, and some guidance in the practical aspects of conducting validation studies in pharmaceutical analysis. For planar chromatographic procedures, which may be used at different levels either in qualitative identity testing, assays, semiquantitative limit tests, or quantitative determination of impurities, this paper tries to transfer these formal requirements into practical approaches for validation. Basic acceptance criteria for evaluation of validation experiments based on practical experience are proposed. In addition, selected parameters for robustness testing of given procedures and quality assurance of quantitative planar chromatographic testing by control charts is described.     

Method validation procedures for environmental radiochemical measurements at AECS

The present paper describes the experience of the Atomic Energy Commission of Syria in relation to the application of Eurachem Guide on method validation for environmental radiochemical measurements. Methods validated include gamma and alpha spectrometry for natural and artificial radionuclides determination, and fluorometry determination for total uranium. Documents and records were first set to meet trackability and traceability requirements, where internal quality control mechanisms have been adopted. Methods stability was checked by means of Z-score control charts. Internal method validation parameters including method detection limits, repeatability limits, reproducibility limits, recovery coefficient and relative error were estimated. External method validation has been achieved by participating in international intercomparison exercises and proficiency tests organized by EML, IAEA and WU; some results of these activities are presented. Moreover, the application of both internal and external method validation gave the analysts in our laboratories more confidence in their skills, and it was of great assistance for our customers including regulatory authorities to evaluate the fitness of the method for their applications according to internationally agreed procedure. The steps followed here can be used for method validation in other laboratories with similar applications.     

Quality criteria for residue analysis and reference materials. Relationship between legal procedures and materials

Summary For qualitative results objective reliability checks are often not present at all or applicable. Interlaboratory ring testing of methods, as sometimes required, showed often not to be applicable simply because enough adequate laboratories are not available. For instance this situation applied to the large number of methods of unclear reliability status, to be used for residue monitoring of hormonal growth promotors (anabolic agents), which are completely banned within the European Communities since January 1988. This impasse was circumvented in 1987 with the formulation by an international group of analytical experts of a set of quality criteria for common analytical techniques like TLC, GC and HPLC (separation), UV, MS and IR spectrometry (detection) and immunoassays (separation and detection). These criteria, now published, are overviewed, as well as the availability of the control and reference materials belonging to them for actual analytical quality control and for validation of laboratories. Although developed for anabolic agents this new approach is applicable in practice for nearly all organic analytes and since very recently also for heavy metals. This approach has clear consequences for the mandatory quality of legislative residue analyses of food stuffs. As based on, amongst others, the combined experience of regulatory residue chemists within the EC, a collection of experimental selectivity indices is presented to rank the required specificity of regulatory residue methods (ranging from within laboratory orientation to international forensic purposes) in an objective way. Finally an estimate is summarized of the financial consequences of the applicable analytical techniques.     

Determination of parathion in biological fluids by means of direct solid-phase microextraction

A new and simple procedure for the determination of parathion in human whole blood and urine using direct immersion (DI) solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS) is presented. This technique was developed using only 100 μL of sample, and ethion was used as internal standard (IS). A 65-μm Carbowax/divinylbenzene (CW/DVB) SPME fibre was selected for sampling, and the main parameters affecting the SPME process such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH effect were optimized to enhance the sensitivity of the method. This optimization was also performed to allow the qualitative determination of parathion’s main metabolite, paraoxon, in blood. The limits of detection and quantitation for parathion were 3 and 10 ng/mL for urine and 25 and 50 ng/mL for blood, respectively. For paraoxon, the limit of detection was 50 ng/mL in blood. The method showed linearity between the LOQ and 50 μg/mL for both matrices, with correlation coefficients ranging from 0.9954 to 0.9999. Precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries were 35.1% for urine and 6.7% for blood. Other parameters such as dilution of sample and stability were also validated. Its simplicity and the fact that only 100 μL of sample is required to accomplish the analysis make this method useful in forensic toxicology laboratories to determine this compound in intoxications, and it can be considered an alternative to other methods normally used for the determination of this compound in biological media.     

Technical Factors of Quality Management in Gamma-Ray Spectrometry of Environmental Samples

The establishment of a quality management system is the best way to comply with international requirements concerning the achievement of confident and traceable analytical results. Some important points dealing with the technical factors of quality management in gamma-ray spectrometry of environmental samples are discussed. The experience obtained from analytical procedure validation is presented. Results of the application of standardized procedures to the analysis of ALMERA intercomparison samples, as well as the outcome of the utilization of certified reference materials for the quality control of measurement are presented. In fact, the implantation of simple technical principles reports reliable results and allows to elevate the quality of the measurements at a cost relatively low according to the real possibilities of the small laboratories, even in developing countries.     

A harmonized European framework for method validation to support research on emerging pollutants

Any investigation of environmental processes related to chemical substances or their effects depends on reliable, comparable analytical data. This also holds true for the impact of climate change on occurrence, distribution and effects of emerging pollutants, with respect to which there is particular concern regarding the reliability of analytical data, due to lack of harmonization in method validation and requirements for quality assurance and quality control (QA/QC).We present a recent European approach to developing a harmonized framework for method validation, QA/QC and provision of environmental data on emerging pollutants. The validation approach has been tested and improved by three case studies. We outline the main concept of the validation approach as well as the results of the case studies. This European validation framework turned out to be a feasible tool to check the fitness for purpose of analytical methods and to improve the reliability of environmental analytical data, particularly for emerging pollutants.