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.     

Guideline on bioanalytical method validation

在创新药物研发过程中,生物基质（如血清、血浆、血液、尿液、唾液）中的药物浓度测定是一个重要的方面。其数据可用于支持新活性物质的应用和仿制药及已授权药品的变更申请。动物的毒代动力学研究和临床试验,包括生物等效性研究的结果为原料药或产品的安全性和有效性提供关键性的数据支持。因此,应用经过充分验证并记录到一个满意标准的生物分析方法以得到可靠的结果,这是非常重要的。2012年2月1日,欧洲药品管理局（EMA）开始实施最新的《生物样品分析方法验证指南》（Guideline on bioanalytical method validation）,本指南适用于动物的毒代动力学研究和所有阶段的临床试验中获得的生物样品中的药物浓度的生物分析方法的验证。本文摘录其方法学验证部分,抛砖引玉,供相关研究人员参考。     

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.     

Multi-element method for determination of trace elements in sunscreens by ICP-AES

An inductively coupled plasma atomic emission spectrometric (ICP-AES) method was developed for multi-element analysis of sunscreen creams and lotions. The objective was the simultaneous determination of Ti (TiO₂ being is the only authorized inorganic UV filter in the European Union) and several minor, trace or toxic elements (Al, Zn, Mg, Fe, Mn, Cu, Cr, Pb and B) in the final products. Two alternative pretreatment procedures were examined: (i) total acid digestion in closed pressurized vessels prior to sample introduction into the plasma and (ii) direct introduction of sample in the form of emulsified slurry. The latter was proved inefficient for several types of creamy samples due to their high viscosity and insolubility. Several acid mixtures were examined for wet digestion because of the complex and fatty matrix of creams and lotions. Plasma parameters like nebulizer argon gas flow rate and radiofrequency incident power were optimized in order to improve the atomization. The recovery of the proposed acid digestion method was evaluated using spiked samples. The calculated recoveries were 95.0% for Ti, 98.2% for Zn and 101.3% for Fe, and the detection limits were 0.2 μg g⁻¹ for Ti, 0.2 μg g⁻¹ for Zn and 0.5 μg g⁻¹ for Fe, respectively. Possible interference from the presence of Ti on the sensitivity of each analyte was examined. Finally the method was applied successfully to several commercial sun protection products and the results were compared with those obtained by atomic absorption spectrometry as reference method.     

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.     

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.     

Development and validation of a fast and sensitive bioanalytical method for the quantitative determination of glucocorticoids—Quantitative measurement of dexamethasone in rabbit ocular matrices by liquid chromatography tandem mass spectrometry

A sensitive, selective, accurate and robust LC–MS/MS method was developed and validated for the quantitative determination of glucocorticoids in rabbit ocular tissues. Samples were processed by a simple liquid–liquid extraction procedure. Chromatographic separation was performed on Phenomenex reversed phase C18 gemini column (50 mm × 4.6 mm i.d.,) with an isocratic mobile phase composed of 30% of acetonitrile in water containing 0.1% of formic acid, at a flow rate 0.2 mL/min. Dexamethasone (DEX), prednisolone (PD) and hydrocortisone (HD) were detected with proton adducts at m/z 393.20 → 355.30, 361.30 → 147.20 and 363.20 → 121.0 in multiple reaction monitoring (MRM) positive mode respectively. Finally, 50 μL of 0.1% novel DEX mixed micellar formulation was topically administered to a rabbit eye and concentrations were measured. The method was validated over a linear concentration range of 2.7–617.6 ng/mL. Lower limit of quantitation (LLOQ) of DEX and PD was measured in the concentration range of 2.7 and 11.0 ng/mL respectively. The resulting method demonstrated intra and inter-day precision within 13.3% and 11.1% and accuracy within 19.3% and 12.5% for DEX and PD, respectively. Both analytes were found to be stable throughout freeze–thaw cycles and during bench top and postoperative stability studies (r² > 0.999). DEX concentrations in various ocular tissue samples i.e., aqueous humor, cornea, iris ciliary body, sclera and retina choroid were found to be 344.0, 1050.07, 529.6, 103.9 and 48.5 ng/mg protein respectively. Absorption of DEX after topical administration from a novel aqueous mixed micellar formulation achieved therapeutic concentration levels in posterior segment of the rabbit eye.     

Development and validation of an accurate HPLC method for the quantitative determination of picroside II in tablets

A simple, sensitive and accurate high performance liquid chromatographic method (HPLC) with UV detection was developed and validated to determine picroside II in a new tablet formulation with paeoniflorin as internal standard. Chromatographic separation was achieved on an Agilent XDB C18 column (250 x 4.6 mm I.D., 5 microm) using a mobile phase consisting of acetonitrile-water-acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 ml/min. The UV detection wavelength was set at 265 nm. Linear calibration curves were obtained in the concentration range of 0.10-100 microg/ml with the limit of quantification (LOQ) of 0.10 microg/ml. The within- and between-run precisions in terms of % relative standard deviation (RSD) were lower than 5.7% and 6.3%, respectively. The accuracy in terms of % relative error (RE) ranged from -2.3% to 5.0%. This validated method was successfully applied to the determination of the content of picroside II in a new tablet formulation.     

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.     

Cost-effective and business-beneficial computer validation for bioanalytical laboratories

Computerized system validation is often viewed as a burden and a waste of time to meet regulatory requirements. This article presents a different approach by looking at validation in a bioanalytical laboratory from the business benefits that computer validation can bring. Ask yourself the question, have you ever bought a computerized system that did not meet your initial expectations? This article will look at understanding the process to be automated, the paper to be eliminated and the records to be signed to meet the requirements of the GLP or GCP and Part 11 regulations. This paper will only consider commercial nonconfigurable and configurable software such as plate readers and LC-MS/MS data systems rather than LIMS or custom applications. Two streamlined life cycle models are presented. The first one consists of a single document for validation of nonconfigurable software. The second is for configurable software and is a five-stage model that avoids the need to write functional and design specifications. Both models are aimed at managing the risk each type of software poses whist reducing the amount of documented evidence required for validation.     

Development and validation of rapid and sensitive HPLC method for the quantitative determination of doxorubicin in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 254?nm has been developed for the determination of doxorubicin in human plasma. Plasma samples were extracted by a selective one-step liquid–liquid extraction using dichloromethane. Doxorubicin and the internal standard epirubicin were separated using a column packed with C₁₈ material, using a mobile phase consisting of water:acetonitrile (75:25v/v). The calibration graph for doxorubicin was linear in the range 0.2–10?μg/mL, with a correlation coefficient, R²?=?0.9986. Lower limit of quantitation was 0.2?μg/mL, using 1?mL plasma samples. The extraction recovery ranged from 98.5–101.1%, and the recovery rate was consistent for drug and internal standard examined at each level. The interday and intraday precision values ranged between 0.5–2%. Validation data showed that the assay for doxorubicin is sensitive, selective, accurate, and reproducible. The assay has been used in population pharmacokinetics study.     

ICP-MS法测定双黄连注射液中微量元素的含量

目的:建立双黄连注射液微量元素的测定方法,并对其所含微量元素进行含量测定。方法:样品经微波消解后,利用电感耦合等离子体质谱(ICP-MS)测定双黄连注射液中Be、B、Al、V、Cr、Mn、Fe、Co、Ni、Cu、Zn、As、Cd、Ba、Hg、Ti、Pb等17种微量元素的含量。结果:有毒金属元素Cu、Cd、Hg、Pb、As含量均符合美国药品和功能性食品的标准,微量元素中B、Al、Fe等元素含量相对较高。结论:所建立的测定方法快捷、准确、灵敏度高,可以用于双黄连注射液的微量元素的含量测定,并为其质量控制研究提供了一定的参考依据。