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.     

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.     

Qualitative threshold method validation and uncertainty evaluation: A theoretical framework and application to a 40 analytes liquid chromatography–tandem mass spectrometry method

Qualitative methods hold an important place in drug testing, filling central needs in screening and analyses, among others, linked to per se legislation. Nevertheless, the bioanalytical method validation guidelines do not discuss this type of method or describe method validation procedures ill‐adapted to qualitative methods. The output of qualitative methods are typically categorical, binary results, such as presence/absence or above cut‐off/below cut‐off. As the goal of any method validation is to demonstrate fitness for use under production conditions, qualitative validation guidelines should evaluate performance based on discrete, binary results instead of the continuous measurements obtained from the instrument (e.g. area). A tentative validation guideline for threshold qualitative methods was developed by in silico modelling of measurements and derived binary results. This preliminary guideline was applied to a liquid chromatography–tandem mass spectrometry method for 40 analytes, each with a defined threshold concentration. Validation parameters calculated from the analysis of 30 samples spiked above and below the threshold concentration (false negative rate, false positive rate, selectivity rate, sensitivity rate and reliability rate) showed a surprisingly high failure rate. Overall, 13 out of the 40 analytes were not considered validated. A subsequent examination found that this was attributable to an appreciable shift in the standard deviation of the area ratio on a day‐to‐day basis, a previously undescribed and unaccounted‐for behaviour in the qualitative threshold method validation literature. Consequently, the developed guideline was modified and used to validate a qualitative threshold method, based on the binary results for performance evaluation and incorporating measurement uncertainty.     

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.     

单克隆抗体药物药代动力学特征、分析方法以及体内分析方法学研究进展

近年来,单克隆抗体药物发展迅速,在肿瘤、免疫、血液等系统疾病领域应用日益广泛。截至2019年其全球处方药物市场占比已达15.3%(1400亿美元)。单克隆抗体药物作为一种大分子蛋白,因其特殊的结构和生理性质,在体内的吸收、分布、代谢及排泄均与小分子药物存在较大差异,具有靶点介导的药物处置、非线性药动学代谢、时间依赖性、较长的半衰期等独特的药代动力学特征,充分了解这些特征有益于该类药物分析方法的开发。单克隆抗体药物在生物体内的处置具有特殊性和复杂性,极大地增加了生物检测的难度,因此必须建立专属、灵敏、准确、可重复的测定分析方法。本文旨在论述单克隆抗体药物的药代动力学特征、常用分析方法及其优缺点、体内分析方法学验证要求等,并逐点与小分子药物进行对比讨论,以期为单克隆抗体药物的分析技术开发提供部分参考。     

GC法测定复方醋酸地塞米松乳膏中薄荷脑含量的不确定度评定

目的 探讨气相色谱法测定复方醋酸地塞米松乳膏中薄荷脑含量的不确定度评定方法.方法 分析测量不确定度的来源,建立数学模型,并计算各分量引入的不确定度,合成标准不确定度和扩展不确定度.结果 扩展不确定度为3.0％(κ=2);测定结果为99.4％±3.3％.结论 所用方法可用于GC法测定药物含量的不确定度评定,使测定结果更加准确.     

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.     

Validation, transfer and measurement uncertainty estimation of an HPLC-UV method for the quantification of artemisinin in hydro alcoholic extracts of Artemisia annua L

Malaria is the world's most important parasitic infection with 500 millions cases annually and almost 2 millions death per year. This disease is more present in Sub-Saharan Africa where 90% of the infections are found. Artemisinin and its semi synthetic derivatives (artemether, artesunate) have actually the most powerful activity on malaria, even in its complicated forms and resistance cases.Various methods have been proposed for detection and quantification of artemisinin in Artemisia annua L. by HPLC-UV, but the plant extracts used for this quantification were extracts obtained with organic solvents (toluene, petroleum ether, hexane). To be able to use crude A. annua extracts prepared at low cost to formulate antipaludic drugs, we chose the use of a mixture of water and ethanol as solvent of extraction, but no adequate analytical method for this kind of extracts is published.The main objectives of this work were first to develop an analytical method for artemisinin quantification in hydro alcoholic extracts of A. annua. Second, this method had to be thoroughly validated by the research and development laboratory and, third, the transfer of this method to the routine laboratory had to be demonstrated. The final aim was to compare the estimation of measurement uncertainty obtained during the method validation with validation standards to measurement uncertainty estimates obtained during the method transfer study with real samples.The method was validated following the accuracy profile methodology and was found to be accurate in the concentration range of 10.0-54.0 μg/ml with CV < 8%. Limit of detection and of quantification were 2.73 and 10.0 μg/ml, respectively. The method was then successfully transferred to a laboratory in Benin by showing that the quality of the results that it will generate during routine application of the method is sufficient. Finally, the measurement uncertainty of the method was estimated from the validation experiments as well as from the transfer study with authentic unspiked samples of A. annua. The comparison of these measurement uncertainty estimations showed that they were coherent. It confirmed thus that the estimation of measurement uncertainty from validation experiments predicts well the measurement uncertainty of real routine samples. This analytical method was thus shown to be convenient for routine analysis of hydro alcoholic extracts of A. annua in Benin.     

高效液相色谱法常规监测中国胃肠间质瘤患者体内的伊马替尼血药浓度（英文）

伊马替尼目前是胃肠间质瘤的一线治疗用药。由于伊马替尼在不同患者体内血药浓度差异较大,目前指南推荐常规监测伊马替尼的血药浓度以提高伊马替尼的疗效,降低不良反应的发生率。本研究建立了一种简单灵敏快速的高效液相色谱法,采用简单的沉淀蛋白法处理血浆样品,处理后的样品通过色谱柱Inert Sustain C18柱(250mm×4.6 mm, 5μM)进行分离,流动相为25 mM NH₄H₂PO₄ (pH=8)–乙腈(61:39, v/v),柱温为40℃,流速为1mL/min,紫外检测波长为265nm。该方法标准曲线在50–10 000 ng/m L浓度范围内线性良好,日间与日内准确度和精密度在–5.81%–6.33%之间。绝对回收率在92.38%–97.86%之间。稳定性测试和样品再分析结果均符合指南要求。该方法成功用于150名中国胃肠间质瘤患者的常规伊马替尼血药浓度监测。     

A synergistic framework for development of green HPLC methods for determination of amlodipine besylate and candesartan cilexetil in presence of their hydrolytic degradation products in two different matrices

The world is changing so fast towards rationalization of resources consumption and employing safer practices for the surrounding environment and humans. Therefore, there is a renewed interest in integrating green analytical chemistry principles and experimental design methodologies for screening and optimizing complex analytical procedures such as HPLC. Thus, this work introduces three sustainable versatile multi-analyte HPLC methods with two types of detection techniques. Firstly, amlodipine besylate and candesartan cilexetil with three of their hydrolytic products were chromatographed on CSH-C₁₈ (150 mm) stationary phase within short run time (about 8.9 min) through gradient elution mode, flow rate 1 mL min^?1 and UV-detection at 210 nm and 254 nm. Face-centered composite design and Derringer's desirability function were implemented to optimize the chromatographic conditions to fit the goals of pharmaceutical and biological analysis via fluorometric detection. A good separation was obtained utilizing HSS-Cayno (250 mm) column and mobile phase composed of ethanol and phosphate buffer, pH 3.5, (55.6: 44.4, v/v) delivered at 1.03 mL min^?1 at 245 and 446 nm as excitation and emission wavelengths, respectively, with quantification range of 1.00–400.00 ng mL^?1 for amlodipine and 1.00–600.00 ng mL^?1 for candesartan cilexetil. The conditions were modified for plasma analysis to a new ratio (45:55, v/v) pH 3.7 and flow rate 0.7 mL min^?1. The methods' greenness profiles have been evaluated and compared with the reported ones via several comprehensive tools.     

药品不良反应信号发现及验证研究方法概述

通过对目前药品不良反应监测方法的概述,了解其研究现状,发现各方法的不足。建议在本领域研究中应将信号检测方法与信号验证性研究两者相结合,取长补短,从而得到确切的不良反应与药品之间的因果关系。     

A scoping review of evaluations of and recommendations for default uncertainty factors in human health risk assessment

Uncertainty factors (UFs) are used to account for uncertainties and variability when setting exposure limits or guidance values. Starting from a proposal of a single UF of 100 to extrapolate from an animal NOAEL to a human acceptable exposure, the aspects of uncertainty and number of UFs have diversified and today there are several risk assessment guidelines that contain schemes of default UFs of varying complexity. In the present work, we scoped the scientific literature on default UFs to map developments regarding recommendations and evaluations of these. We identified 91 publications making recommendations for one or several UFs and 55 publications evaluating UFs without making explicit recommendations about numerical values; these were published between 1954 and 2021. The 2000s was the decade with the largest number of publications, interspecies differences and intraspecies variability being the most frequent topics. The academic sector has been the most active (76 out of 146 publications). Authors from the private sector more often presented UF recommendations, but differences between sectors regarding size of recommendations were not statistically significant. The empirical underpinning of the reviewed recommendations ranges from four to 462 chemicals, that is, relatively low numbers compared with the range of chemicals these default UFs are expected to cover. The recommended UFs have remained remarkably constant, with merely a slight decrease over time. Although chemical specific UFs are preferable, the widespread use of default UFs warrants further attention regarding their empirical and normative basis.     

On the evaluation of reliability,repeatability, and reproducibility of instrumental evaluation methods and measurement systems

ABSTRACT

Validation of instrumental evaluation methods or measurement systems plays an important role in both pharmaceutical and cosmetic research and development. In practice, it is suggested that validation should be performed according to performance characteristics as described in the United States Pharmacopedia and National Formulary (USP/NF, 2000) for analytical methods validation. A validated method or measurement system is expected to achieve a certain degree of accuracy and reliability. However, it is a concern whether the test results obtained are repeatable (with similar test samples) and/or reproducible (under similar but slightly different experimental conditions). In this article, reliability and repeatability/reproducibility of a measurement system estimated within a mixed-effects nested design are monitored by relevant variability acceptance limits. A method based on the concept of empirical power (reproducibility) is used to determine these acceptance limits and thus ensure that there is a high probability of repeatability/reproducibility of the tests results. Formulas or procedures for sample size requirements for comparing the variabilities between products are derived. An example is presented to illustrate the use of the proposed method.     

综合应用Delphi法、风险矩阵法与Borda序值法评估医院病区药品管理风险

目的:探讨病区药品管理风险评估方法,为科学制定病区药品风险应对策略提供依据。方法:以中日友好医院病区药品管理风险为研究对象,采用Delphi法对病区药品管理风险发生的可能性及后果的严重性进行风险分析;应用风险矩阵法评价病区药品管理风险的风险水平;采用Borda序值法对5类风险进行了排序。结果:2轮调研的专家积极系数均为100%,权威系数为(0.86±0.06),第2轮专家咨询结果显示,受访专家对5项风险的风险发生可能性和后果严重性评价的Kendall协调系数Wc分别为0.428(χ2=25.669,P<0.005),0.426(χ2=25.553,P<0.005),Wc值统计学意义显著。确定了5类病区药品管理风险发生的可能性和后果严重性,并确定了5类风险事件的风险水平及其排序,其中"病区药品管理人员专业素质"的风险排序位列第一。结论:综合应用Delphi法、风险矩阵法与Borda序值法是评估医院药品风险的有效方法,具有推广应用价值。     

Variability of blood alcohol content (BAC) determinations: The role of measurement uncertainty,significant figures,and decision rules for compliance assessment in the frame of a multiple BAC threshold law

The measurement of blood‐alcohol content (BAC) is a crucial analytical determination required to assess if an offence (e.g. driving under the influence of alcohol) has been committed. For various reasons, results of forensic alcohol analysis are often challenged by the defence. As a consequence, measurement uncertainty becomes a critical topic when assessing compliance with specification limits for forensic purposes. The aims of this study were: (1) to investigate major sources of variability for BAC determinations; (2) to estimate measurement uncertainty for routine BAC determinations; (3) to discuss the role of measurement uncertainty in compliance assessment; (4) to set decision rules for a multiple BAC threshold law, as provided in the Italian Highway Code; (5) to address the topic of the zero‐alcohol limit from the forensic toxicology point of view; and (6) to discuss the role of significant figures and rounding errors on measurement uncertainty and compliance assessment. Measurement variability was investigated by the analysis of data collected from real cases and internal quality control. The contribution of both pre‐analytical and analytical processes to measurement variability was considered. The resulting expanded measurement uncertainty was 8.0%. Decision rules for the multiple BAC threshold Italian law were set by adopting a guard‐banding approach. 0.1 g/L was chosen as cut‐off level to assess compliance with the zero‐alcohol limit. The role of significant figures and rounding errors in compliance assessment was discussed by providing examples which stressed the importance of these topics for forensic purposes. Copyright © 2014 John Wiley & Sons, Ltd.     

Bioanalytical method development and validation of natamycin in rabbit tears and its application to ocular pharmacokinetic studies

A new selective and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of natamycin in rabbit tears using amphotericin B as internal standard (IS). Chromatographic separation was achieved on a Luna Cyano column (100 mm × 2 mm, 3 μm) using ammonium acetate buffer (pH 4; 3.5mM): methanol (10:90, v/v) as the mobile phase. The run time was 5 min. Detection was performed by negative ion electrospray ionization in multiple reaction monitoring (MRM) mode. The calibration curve was linear over the concentration range from 25 to 800 ng/ml, and lower limit of detection of 12.5 ng/ml. The accuracy and precision of the method were within the acceptable limit of ± 20% at the lower limit of quantitation and ± 15% at other concentrations. Natamycin was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at -70 ± 10 °C. The method was successfully applied to the ocular pharmacokinetic studies of natamycin eye drops in New Zealand rabbit tears.     

Development and validation of a LC-ESI-MS/MS method for simultaneous quantification of olmesartan and hydrochlothiazide in human K3 EDTA plasma and its application to pharmacokinetic biostudy

This is the first publication on a complete validated bioanalytical method for estimation of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human K3 EDTA plasma that chromatographically resolves its olmesartan glucuronide. An API 4000 mass spectrometer was employed in this method where olmesartan d4 (OLMD4) and hydrochlorothiazide ?¹³C, d2 (HCTZD2) served as the internal standard. Sample was prepared by solid phase extraction (SPE) technique using a polymer based, MCX cartridges and chromatographic resolution achieved on Synergi MAX RP-18A, (4.6?×?150?mm, 4?μm) column using a mobile phase of 0.2% formic acid solution/acetonitrile (30:70, v/v). Negative mass transitions (m/z) of OLM, HCTZ, OLMD4, and HCTZD2 were detected in multiple reactions monitoring (MRM) mode at 445.5?→?149.3, 296.0?→?269.0, 449.2?→?149.3, and 299.1→270.0, respectively. The linearity was checked over a concentration range of 4.051–2500.912?ng/mL for OLM and 0.506–304.109?ng/mL for HCTZ. Intra- and inter-run precision of OLM and HCTZ assay at four concentration levels were below 3.7% and 4.3%, and accuracy was within ±4.4% and 3.0%, respectively. Mean recoveries for OLM, HCTZ, and internal standards OLMD4 and HCTZD2 were 75.68, 77.60%, and 80.2, 89.1%, respectively. This method has been successfully applied to pharmacokinetic biostudy.