A comprehensive method validation strategy for bioanalytical applications in the pharmaceutical industry--2. Statistical analyses.

The first paper in this two-part series described [Lang and Bolton, J. Pharm. Biomed. Anal. 9, 357-361 (1991)] an overall validation strategy for bioanalytical methods. This second paper focuses on the statistical analyses performed on the validation data that will allow the analyst to evaluate the reliability and reproducibility of a bioanalytical method. Based on the validation results, acceptance criteria for the quality control concentrations are established and used during the study proper to determine if the analytical run is valid. After analysing the clinical study samples and accepting the analytical runs, the quality control results are incorporated into databases to update their acceptance limits. This continuous validation process enables the analyst to monitor the method's performance over time and be confident that accurate sample concentrations are being reported. It is important to emphasize that the statistical analyses of the data provide information that should be considered from a practical point of view by the analyst. The analyst should use sound judgement in evaluating the reliability of the method.     

Introduction to micro-analytical systems: bioanalytical and pharmaceutical applications.

This review presents a brief overview of recent developments in miniaturization of analytical instruments utilizing microfabrication technology. The concept 'Micro-Total Analysis Systems micro-TAS)', also termed 'Lab-on-a-chip', and the latest progresses in the development of microfabricated separation devices and on-chip detection techniques are discussed. Applications of micro-analytical methods to bioanalytical and pharmaceutical studies are also described, including chemical reactions, assays, and analytical separations of biomolecules in micro-scale.     

Key elements of bioanalytical method validation for macromolecules

The Third American Association of Pharmaceutical Scientists/US Food and Drug Administration (FDA) Bioanalytical Workshop, which was held May 1 and 2, 2006, in Arlington, VA, addressed bioanalytical assays that are being used for the quantification of therapeutic candidates in support of pharmacokinetic evaluations. One of the main goals of this workshop was to discuss best practices used in bioanalysis regardless of the size of the therapeutic candidates. Since the last bioanalytical workshop, technological advancements in the field and in the statistical understanding of the validation issues have generated a variety of interpretations to clarify and understand the practicality of using the current FDA guidance for assaying macromolecular therapeutics. This article addresses some of the key elements that are essential to the validation of macromolecular therapeutics using ligand binding assays. Because of the nature of ligand binding assays, attempts have been made within the scientific community to use statistical approaches to interpret the acceptance criteria that are aligned with the prestudy validation and in-study validation (sample analysis) processes. We discuss, among other topics, using the total error criterion or confidence interval approaches for acceptance of assays and using anchor calibrators to fit the nonlinear regression models.     

Key elements of bioanalytical method validation for small molecules

Method validation is a process that demonstrates that a method will successfully meet or exceed the minimum standards recommended in the Food and Drug Administration (FDA) guidance for accuracy, precision, selectivity, sensitivity, reproducibility, and stability. This article discusses the validation of bioanalytical methods for small molecules with emphasis on chromatographic techniques. We present current thinking on validation requirements as described in the current FDA Guidance and subsequent 2006 Bioanalytical Methods Validation Workshop white paper.     

Aspects of bioanalytical method validation for the quantitative determination of trace elements

Bioanalytical methods are used to quantitatively determine the concentration of drugs, biotransformation products or other specified substances in biological matrices and are often used to provide critical data to pharmacokinetic or bioequivalence studies in support of regulatory submissions. In order to ensure that bioanalytical methods are capable of generating reliable, reproducible data that meet or exceed current regulatory guidance, they are subjected to a rigorous method validation process. At present, regulatory guidance does not necessarily account for nuances specific to trace element determinations. This paper is intended to provide the reader with guidance related to trace element bioanalytical method validation from the authors' perspective for two prevalent and powerful instrumental techniques: inductively coupled plasma-optical emission spectrometry and inductively coupled plasma-MS.     

Method validation in the bioanalytical laboratory

Bioanalytical methods, based on a variety of physico-chemical and biological techniques such as chromatography, immunoassay and mass spectrometry, must be validated prior to and during use to engender confidence in the results generated. The fundamental criteria for assessing the reliability and overall performance of a bioanalytical method are: the evaluation of drug and analyte stability, selectivity, limits of quantification and detection, accuracy, precision, linearity and recovery. The extent to which a method is validated is dependent on its prospective use, the number of samples to be assayed and the use to which the data are put.

Specific analytical techniques may require additional validation such as antibody-binding characteristics, peak purity determination, evaluation of matrix effects or structural confirmation of the analyte. Ideally each assay should be cross-validated with a method utilizing a highly specific detector such as a mass spectrometer. Once in use, the performance of the method should be monitored using quality control standards. If a method is set up in another laboratory, the performance of the assay should be monitored with quality control standards sent from the originating laboratory.     

A practical approach to method validation in pharmaceutical analysis

Guidelines issued by Regulatory Authorities make it clear that validation of analytical methodology is now widely required in support of registration dossiers. Although some attempts are made at defining terms and some vague indications are sometimes provided within these guidelines, no clear is provided on how validations should be conducted and what results should be expected. In this paper it is attempted to suggest some practical approaches to conducting validation and in particular to the determination of accuracy, linearity and limit of detection/quantitation.     

A simple method for the preparation of liposomes for pharmaceutical applications: characterization of the liposomes.

A new method for the preparation of liposomes is described that avoids the use of pharmaceutically unacceptable solvents and energy-expensive procedures such as sonication. The method is based on the initial formation of a proliposome mixture containing lipid, ethanol and water, which is converted to lipsomes by a simple dilution step. Measurements using 6-carboxyfluorescein as a marker indicate that water-soluble drugs can be trapped with extremely high efficiency (65-80% depending on lipid composition). The structural organization of the proliposome mixture and the final liposomes were characterized using electron microscopy and 31P-NMR.     

Development and validation of bioanalytical method for the determination of asulacrine in plasma by liquid chromatography

Asulacrine (9-[(2-methoxy-4-methylsulphonylamino)phenylamino]-N,5-dimethyl-4-acridinecarboxamide), an analogue of the antileukaemia drug amsacrine, has high antitumour activity in mice and has also shown clinical activity. A simple method is described for the quantitation of asulacrine in plasma by liquid chromatography. Chromatographic separation was achieved on a reversed phase C 18 column (250 mm x 4.6mm, particle size 5 microm, Gemini) using isocratic elution (acetonitrile and 0.01 M sodium acetate buffer pH 4.0, 45/55, v/v) at a flow rate of 1 ml/min. Asulacrine and internal standard (the ethylsulphonanilide analogue) were measured using UV detection at 254 nm. The total chromatographic run-time was 8 min with asulacrine and internal standard eluting at approximately 4.7 and approximately 6.5 min, respectively. Limit of quantification was 0.1microg/ml. The linearity range of the method was 0.1-10 microg/ml (r2=0.9995). Mean recoveries from plasma were 100-105%. Intra-batch and inter-batch precision was 7.1 and 7.8%, respectively, and intra-batch and inter-batch accuracy (relative error) was 4.9 and 8.4%, respectively (n=8 in all cases). The bench top, freeze thaw, short-term storage and stock solution stability evaluation indicated no evidence of degradation of asulacrine. The validated method is simple, selective and rapid and can be used for pharmacokinetic studies in mice.     

Conceptual and statistical issues in the validation of analytic dilution assays for pharmaceutical applications

The discovery research, and development of a pharmaceutical product relies on the availability of validated assays for assessing product characteristics and drug effects in vivo and in vitro. Development of a validated assay is a multifaceted activity that provides many interesting challenges for bioanalytical chemists and statisticians. In paper this the similarity condition for fundamental validity of an analytic dilution assay is reviewed as a basic concept underlying the validation of assays for pharmaceutical applications. The distinction between the validity and the acceptability of an assay is considered in terms of the characteristics evaluated during four stages of validation. Recent guidelines on the validation of analytical procedures published by the U.S. Food and Drug Administration (1,2) are appraised from a statistical perspective and statistical issues in the validation process are discussed.     

Application issues in bioanalytical method validation,sample analysis and data reporting

Although some degree of consensus has been reached concerning the requirements for acceptable method validation, the procedures used to establish them vary significantly between laboratories. Also, issues arising from application of these requirements during validation and subsequent sample analysis need to be addressed. The purpose of this paper is to discuss application issues concerning prerequisites to method validation, and all validation criteria for evaluation of method reliability and overall performance. Other poorly addressed issues such as re-validation, cross-validation, partial sample volume, multicomponent analysis and reporting will also be discussed. Although many issues discussed are of a general nature, the scope of this presentation is primarily to address issues arising from the validation and routine application of chromatographic methods.     

Development and validation of a high-performance liquid chromatographic method for bioanalytical application with rimonabant

A simple and feasible high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of rimonabant in human plasma. The chromatographic separation was carried out in a Hypersil BDS, C₁₈ column (250 mm × 4.6 mm; 5 μm). The mobile phase was a mixture of 10 mM phosphate buffer and acetonitrile (30:70, v/v) at a flow rate of 1.0 ml/min. The UV detection was set at 220 nm. The extraction recovery of rimonabant in plasma at three quality control (QC) samples was ranged from 84.17% to 92.64%. The calibration curve was linear for the concentration range of 20–400 ng/ml with the correlation coefficient (r²) above 0.9921. The method was specific and sensitive with the limit of quantification of 20 ng/ml. The accuracy and precision values obtained from six different sets of QC samples analyzed in separate occasions ranged from 88.13% to 106.48% and 0.13% to 3.61%, respectively. In stability tests, rimonabant in human plasma was stable during storage and assay procedure. The method is very simple, sensitive and economical and the assay was applied to human plasma samples in a clinical (pharmacokinetic) study of rimonabant.     

EBF recommendation on the validation of bioanalytical methods for dried blood spots

Over the last few years bioanalysts, pharmacokineticists and clinical investigators have rediscovered the technique of dried blood spots. The revival has provided pharmaceutical R&D a wealth of opportunities to optimize the drug-discovery and development process with respect to animal and patient ethics, new scientific insights and costs savings. On the bioanalytical front, multiple experiments have been performed and a lot of experience has been gained. Nevertheless, the technique still has a number of bioanalytical challenges. The European Bioanalysis Forum discussed the advantages and hurdles of the technique and summarized their current thinking in a recommendation on the validation of bioanalytical methods for dried blood spots, which can be used as a cornerstone for further discussions and experiments.     

Development and validation of a bioanalytical LC-UV method with solid-phase extraction for determination of valproic acid in saliva

A bioanalytical HPLC method with UV detection for the determination of the antiepileptic drug valproic acid in human saliva has been developed and validated. Saliva represents an alternative matrix for therapeutic monitoring of antiepileptic drugs due to the increasing interest in free drug concentration. The proposed method involved solid-phase extraction for sample preparation and yielded very good mean recoveries of 99.4 % and 97.9 % for valproic acid and IS, respectively. The calibration function for valproic acid was linear over the concentration range of 1.0-50.0 μg mL(-1) (R(2) = 0.9989). Within-run and between-run precision and accuracy were studied at four concentrations and RSDs were less than 7.3 and 2.2 %, while accuracy values were higher than 96.8 and 97.5 %, respectively. The described method provides sensitivity, linearity, precision, accuracy and is suitable for analyses of valproic acid in saliva samples.     

Comprehensive validation scheme for in situ fiber optics dissolution method for pharmaceutical drug product testing

There has been a growing interest during the past decade in the use of fiber optics dissolution testing. Use of this novel technology is mainly confined to research and development laboratories. It has not yet emerged as a tool for end product release testing despite its ability to generate in situ results and efficiency improvement. One potential reason may be the lack of clear validation guidelines that can be applied for the assessment of suitability of fiber optics. This article describes a comprehensive validation scheme and development of a reliable, robust, reproducible and cost-effective dissolution test using fiber optics technology. The test was successfully applied for characterizing the dissolution behavior of a 40-mg immediate-release tablet dosage form that is under development at Novartis Pharmaceuticals, East Hanover, New Jersey. The method was validated for the following parameters: linearity, precision, accuracy, specificity, and robustness. In particular, robustness was evaluated in terms of probe sampling depth and probe orientation. The in situ fiber optic method was found to be comparable to the existing manual sampling dissolution method. Finally, the fiber optic dissolution test was successfully performed by different operators on different days, to further enhance the validity of the method. The results demonstrate that the fiber optics technology can be successfully validated for end product dissolution/release testing.