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.     

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.     

Advances in validation, risk and uncertainty assessment of bioanalytical methods

Bioanalytical method validation is a mandatory step to evaluate the ability of developed methods to provide accurate results for their routine application in order to trust the critical decisions that will be made with them. Even if several guidelines exist to help perform bioanalytical method validations, there is still the need to clarify the meaning and interpretation of bioanalytical method validation criteria and methodology. Yet, different interpretations can be made of the validation guidelines as well as for the definitions of the validation criteria. This will lead to diverse experimental designs implemented to try fulfilling these criteria. Finally, different decision methodologies can also be interpreted from these guidelines. Therefore, the risk that a validated bioanalytical method may be unfit for its future purpose will depend on analysts personal interpretation of these guidelines. The objective of this review is thus to discuss and highlight several essential aspects of methods validation, not only restricted to chromatographic ones but also to ligand binding assays owing to their increasing role in biopharmaceutical industries. The points that will be reviewed are the common validation criteria, which are selectivity, standard curve, trueness, precision, accuracy, limits of quantification and range, dilutional integrity and analyte stability. Definitions, methodology, experimental design and decision criteria are reviewed. Two other points closely connected to method validation are also examined: incurred sample reproducibility testing and measurement uncertainty as they are highly linked to bioanalytical results reliability. Their additional implementation is foreseen to strongly reduce the risk of having validated a bioanalytical method unfit for its purpose.     

Optimization and validation of a SPE-HPLC-PDA-fluorescence method for the simultaneous determination of drugs used in combined cardiovascular therapy in human plasma

This paper reports the chemometrical optimization and the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-Fluo) method for the simultaneous analysis, in human plasma, of drugs usually combined in cardiovascular therapy. Separation of chlorthalidone (CLTD), valsartan (VAL), valsartan-M1 (VAL-M1), fluvastatin (FLUV) and the internal standard (IS) candesartan cilexetil was performed on a dC18 Atlantis column (100 mm × 3.9 mm, 3 μm) using a gradient with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% of formic acid and 10 mM of ammonium formate at pH 4.1. UV and fluorimetric (valsartan, its metabolite and fluvastatin) detectors were used. The sample preparation consisted of protein precipitation using acetonitrile suited to a solid-phase extraction (SPE) on a Strata-X cartridge for sample clean-up. Method validation was developed following the recommendations for bioanalytical method validation of International Conference on Harmonisation (ICH) and Food and Drug Administration (FDA) organizations. The method showed good linearity (31–3000 μg/l for chlorthalidone, 20–1000 μg/l for valsartan-M1, 10–5000 μg/l for valsartan and 14–1000 μg/l for fluvastatin), precision and accuracy. Recoveries were in the range of 78–91%. This method allowed the determination of these drugs in human plasma samples obtained from patients under cardiovascular treatment.     

基于液质联用技术的高通量靶标磷脂组学的分析研究

目的建立高通量靶标磷脂组学分析方法,研究尿液中的磷脂轮廓并筛选出尿液样本中潜在的磷脂标志物。方法用改良的甲基叔丁基醚法提取尿液中磷脂,用高效液相-电喷雾-四级杆-线性离子阱串联质谱技术对尿液中目标磷脂进行半定量分析,用7种磷脂标准品和15种随机选取的内源性磷脂化合物进行方法学考察,包括专属性、灵敏度、精密度、基质效应、携带效应、稳定性和回收率。对穿插在检测过程中的质控(QC)样品进行主成分分析,考察数据的可靠性。结果磷脂酰胆碱、溶血磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰甘油、磷脂酰肌醇和鞘磷脂的定量下限分别为0.25,0.25,2.00,2.00,1.00,1.00ng·mL^-1和625.00 pg·mL^-1。连续进样过程中的随机峰的保留时间精密度RSD为0.72%~3.44%,相对峰面积RSD为0.71%~10.53%,7种磷脂化合物的回收率均在54.05%~105.73%内,尿样经室温放置12 h、制备后放置24 h、冻融2次以及-80℃下存放1个月后样本均保持稳定。QC样品在第一主成分图显示检测数据稳定可靠。结论该检测分析方法在磷脂组学研究中具有简单、稳定和敏感的特点,可应用于后续大样本量的磷脂组学研究及潜在尿磷脂标志物的定量研究。     

Biomarker method validation in anticancer drug development

Over recent years the role of biomarkers in anticancer drug development has expanded across a spectrum of applications ranging from research tool during early discovery to surrogate endpoint in the clinic. However, in Europe when biomarker measurements are performed on samples collected from subjects entered into clinical trials of new investigational agents, laboratories conducting these analyses become subject to the Clinical Trials Regulations. While these regulations are not specific in their requirements of research laboratories, quality assurance and in particular assay validation are essential. This review, therefore, focuses on a discussion of current thinking in biomarker assay validation. Five categories define the majority of biomarker assays from 'absolute quantitation' to 'categorical'. Validation must therefore take account of both the position of the biomarker in the spectrum towards clinical end point and the level of quantitation inherent in the methodology. Biomarker assay validation should be performed ideally in stages on 'a fit for purpose' basis avoiding unnecessarily dogmatic adherence to rigid guidelines but with careful monitoring of progress at the end of each stage. These principles are illustrated with two specific examples: (a) absolute quantitation of protein biomarkers by mass spectrometry and (b) the M30 and M65 ELISA assays as surrogate end points of cell death.     

Forensic application and evaluation of a commercially available pregabalin immunoassay test in serum on an Olympus AU480

Misuse of pregabalin and its forensic relevance is steadily increasing. The aim of this study was to evaluate the usability of the commercially available ARKTM Pregabalin II Assay (ARK Diagnostics) for serum analysis of forensic samples. Overall, 156 samples were tested by both the immunoassay and a validated liquid chromatographic–tandem mass spectrometric (LC-MS/MS) method. Sensitivity was 100%, and specificity was 98.7% (n = 79 positive cases confirmed by LC-MS/MS in a range of 380–37,000 ng/mL). A good correlation (R² = 0.73) could also be shown between quantitative immunoassay and LC-MS/MS results. In conclusion, the assay shows excellent reliability for screening of forensic serum samples.     

Bioanalytical Method Validation for Macromolecules in Support of Pharmacokinetic Studies

The development and validation of ligand binding assays used in the support of pharmacokinetic studies has been the focus of various workshops and publications in recent years, all in an effort to establish a guidance document for standardization of these bioanalytical methods. This summary report of the workshop from 2003 focuses on the issues discussed in presentations and notes points of discussion and areas of consensus among the participants.     

Regulatory recommendations for clopidogrel: Focus on validation of a reliable bioanalytical method and its application to a bioequivalence study

A rapid, simple, and sensitive liquid chromatography tandem mass spectrometric (LC–MS/MS) method for the determination of clopidogrel in human plasma was developed and validated using clopidogrel-d4 as the internal standard (IS). The analyte and the IS were extracted from 500 µl aliquots of human plasma via solid phase extraction. The precursor to product ion transitions monitored for clopidogrel and IS were m/z 322.2 → 212.0 and 326.2 → 216.0, respectively. The method was fully validated for sensitivity, accuracy and precision, linearity, recovery, matrix effect, dilution integrity, and stability. The Linearity range was 20.4–10,772.6 pg/mL with mean correlation coefficient (r) ≥ 0.9977. The back conversion was also evaluated during method validation as per EMA recommendation. Results proved that clopidogrel was accurately and reliably estimated by the method without any evidence of back conversion of clopidogrel acid. The method was successfully applied to a bioequivalence study of 75?mg clopidogrel bisulfate tablet formulation in 32 healthy male volunteers under fasting conditions. The ratios of least-squares means (with 90% confidence intervals) for the pharmacokinetic parameters C_max, AUC_0–t and AUC_0–α were 107.98% (94.52–123.35%), 100.25% (91.28–110.09%), and 100.49% (91.37–110.51%), respectively.     

Toolbox for the Extraction and Quantification of Ochratoxin A and Ochratoxin Alpha Applicable for Different Pig and Poultry Matrices

Ochratoxin A (OTA) is one of the major mycotoxins causing severe effects on the health of humans and animals. Ochratoxin alpha (OTα) is a metabolite of OTA, which is produced through microbial or enzymatic hydrolysis, and one of the preferred routes of OTA detoxification. The methods described here are applicable for the extraction and quantification of OTA and OTα in several pig and poultry matrices such as feed, feces/excreta, urine, plasma, dried blood spots, and tissue samples such as liver, kidney, muscle, skin, and fat. The samples are homogenized and extracted. Extraction is either based on a stepwise extraction using ethyl acetate/sodium hydrogencarbonate/ethyl acetate or an acetonitrile/water mixture. Quantitative analysis is based on reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Method validation was successfully performed and the linearity, limit of quantification, accuracy, precision as well as the stability of the samples, were evaluated. The analyte recovery of the spiked samples was between 80 and 120% (80–150% for spiked concentrations ≤ 1 ng/g or ng/mL) and the relative standard deviation was ≤ 15%. Therefore, we provide a toolbox for the extraction and quantification of OTA and OTα in all relevant pig and poultry matrices.     

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.     

Precision,Accuracy, and Data Acceptance Criteria in Biopharmaceutical Analysis

Accuracy and precision are the most important criteria in the assessment of an analytical method, and monitoring quality control during sample analysis is essential to ensure the validity of reported results. Various approaches to testing accuracy, precision, and quality control were applied to 10 analytes from seven chromatographic bioanalytical methods. These methods include fixed interval bias and significance testing for accuracy; fixed interval percentage relative standard deviation (%RSD) and analysis of variance (ANOVA) approaches for precision; ±20% fixed range, 99% confidence interval, multiple rules, and range chart for individuals approaches for quality control acceptance criteria. Quality control approaches were also applied to the entire run and to a bracketed approach whereby results are considered valid only if bracketed by acceptable quality control. Accuracy and precision were assessed for six runs of each analyte at three concentrations established to represent the calibration range of the analytical method. Quality control acceptance criteria were evaluated using all data sets from each of the analytical methods collected during the course of running various numbers of real samples. The data suggest that the fixed interval bias criteria for accuracy was a more liberal method of accuracy assessment because three of the seven methods would have been rejected according to the significance testing criteria whereas all were acceptable by the fixed internal bias criteria. Precision can be effectively assessed for between- and within-run data by criteria set on unconfounded %RSD values or by separation of the sources of variation using an ANOVA approach applied to confounded data. The percentage of samples rejected for the 99% confidence interval applied to brackets, the multiple rules approach applied to the entire run, and the individual range chart approach applied to brackets were comparable and were found to be 7.0, 6.2 and 8.3 percent respectively. The ±20% fixed range criteria applied to the two-thirds of the run resulted in just 2.9% sample rejection and was not considered comparable to the other methods.     

Planar chromatography: Current status and future perspectives in pharmaceutical analysis — I. Applicability, quantitation and validation

The most important features of capillary action planar chromatographic methods are summarized. Certain properties such as case in operation, sensitivity to experimental conditions, stationary and mobile phase selection, phase system optimization, separation efficiency and detection possibilities are discussed only briefly. Other aspects such as the applicability of planar chromatographic methods in pharmaceutical analysis, quantitation and validation are considered in more detail. The advantages and limitations of different evaluation techniques (visual comparison, spot elution techniques and in situ densitometry) are also discussed. Validation steps specific to planar chromatographic methods are emphasized.     

Chiral purity assay for Flindokalner using tandem mass spectrometry: method development, validation, and benchmarking

The present work demonstrates the application and validation of a mass spectrometry method for quantitative chiral purity determination. The particular compound analyzed is Flindokalner, a Bristol-Myers Squibb drug candidate for post-stroke neuroprotection. Chiral quantification of Flindokalner was achieved using tandem mass spectrometry (MS/MS) and the kinetic method, a gas phase method used for thermochemical and chiral determinations. The MS/MS method was validated and benchmarked against two separate chromatographic techniques, chiral high performance liquid chromatography with ultra-violet detection (LC/UV) and achiral high performance liquid chromatography with circular dichroism detection (LC/CD). The chiral purity determination of Flindokalner using MS/MS proved to be rapid (3 min run time for each sample) and to have accuracy and precision comparable to the chiral LC/UV and achiral LC/CD methods. This method represents an alternative to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful in rapid screening experiments.     

Procedural elements involved in maintaining bioanalytical data integrity for good laboratory practices studies and regulated clinical studies

This article describes procedural elements involved in ensuring the integrity of bioanalytical data. These elements can be divided into 3 areas. First, there are those ensuring the integrity of the analyte until analysis, through correct sample collection, handling, shipment, and storage procedures. Incorrect procedures can lead to loss of analyte via instability, addition of analyte through contamination or instability of related metabolites, or changes in the matrix composition that may adversely affect the performance of the analytical method. Second, the integrity of the sample identity needs to be maintained to ensure that the final result reported relates to the individual sample that was taken. Possible sources of error include sample mixup or mislabeling, or errors in data handling. Finally, there is the overall integrity of the documentation that supports the analysis, and any prestudy validation of the method. This includes a wide range of information, from paper and electronic raw data, through standard operating procedures and analytical procedures and facility records, to study plans and final reports. These are critical to allow an auditor or regulatory body to reconstruct the study.     

Does the perceived accuracy of urine drug testing impact clinical decision-making?

Abstract

Background: Urine drug testing techniques have different rates of false-positive and false-negative test results. However, clinicians may have highly varying perceptions of test accuracy and may compensate for perceived inaccuracy by incorporating other factors into their interpretation of observed test results. Thus, there is the potential for adverse consequences from decisions based on inaccurate test results or interpretation. Methods: We surveyed 466 members of the American Society of Addiction Medicine to examine clinicians’ perceptions of the accuracy of 2 types of urine drug tests, immunoassay (IA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS), and the extent to which behavioral and demographic factors influence the interpretation of test results. Participants read 4 brief vignettes describing positive and negative test results in hypothetical patients who differed along several dimensions (gender, age, race/ethnicity, comorbid mental disorder, court-ordered versus voluntary status, treatment compliance). Outcome variables include likelihood of renewed drug use, likelihood of test error, whether to request additional testing, and whether to report the violation to a probation officer. Results: The strongest predictor of study outcomes was treatment compliance (consistent versus inconsistent attendance), as this was the only independent variable to generate effect sizes of medium strength. Significant effect sizes were also found for type of test used (IA versus LC-MS/MS), legal status (court-mandated versus voluntary), presence of a comorbid mental disorder, treatment history, and race, although effect sizes for these variables were small and less consistently observed. Conclusions: These results highlight the potential for error in clinician judgments about urine drug testing. Not only were participants likely to underestimate the accuracy of “confirmatory” LC-MS/MS testing, but vignettes suggested that a number of historical and demographic factors may influence interpretation of test results.     

Establishment and intra‐/inter‐laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation

A reactive oxygen species (ROS) assay was previously developed for photosafety evaluation of pharmaceuticals, and the present multi‐center study aimed to establish and validate a standard protocol for ROS assay. In three participating laboratories, two standards and 42 coded chemicals, including 23 phototoxins and 19 nonphototoxic drugs/chemicals, were assessed by the ROS assay according to the standardized protocol. Most phototoxins tended to generate singlet oxygen and/or superoxide under UV–vis exposure, but nonphototoxic chemicals were less photoreactive. In the ROS assay on quinine (200 µm ), a typical phototoxic drug, the intra‐ and inter‐day precisions (coefficient of variation; CV) were found to be 1.5–7.4% and 1.7–9.3%, respectively. The inter‐laboratory CV for quinine averaged 15.4% for singlet oxygen and 17.0% for superoxide. The ROS assay on 42 coded chemicals (200 µm ) provided no false negative predictions upon previously defined criteria as compared with the in vitro/in vivo phototoxicity, although several false positives appeared. Outcomes from the validation study were indicative of satisfactory transferability, intra‐ and inter‐laboratory variability, and predictive capacity of the ROS assay. Copyright © 2012 John Wiley & Sons, Ltd.