Directly testing the linearity assumption for assay validation

The ICH Q2(R1) (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) guideline for testing linearity in validation of analytical procedures suggests that “linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content.” The EP6‐A guideline recommends more quantitative methods that compare straight‐line and higher‐order polynomial curve fits. In this paper, a new equivalence test is proposed to compare the quality of a straight‐line fit to that of a higher‐order polynomial. By using orthogonal polynomials and generalized pivotal quantity analysis, one may estimate the probability of equivalence between a straight line and a polynomial curve fit either in the assay signal space (the Y values) or in the concentration space (the X values). In the special case of the linear‐to‐quadratic polynomial comparison, an equivalence test may be constructed via a two one‐sided T test. Copyright © 2013 John Wiley & Sons, Ltd.     

Statistical evaluation of non‐profile analyses for the in vitro bioequivalence

For locally acting drug products such as nasal aerosols and nasal sprays, the 2003 US Food and Drug Administration (FDA) draft guidance suggests that bioequivalence between generic and brand‐name products be established through in vitro tests. In addition, for non‐profile analyses based on spray content uniformity, droplet size distribution, spray pattern, priming, and re‐priming, the draft US FDA guidance recommends that the population bioequivalence (PBE) between generic and innovator's products be demonstrated. However, the linearized criterion recommended in the draft FDA guidance does not take into consideration the variations due to batches, samples, and life stages. Hence, under a two‐stage nested random effects model, we apply the methods of modified large‐sample (MLS) and generalized pivotal quantities (GPQs) to construct the upper 95% confidence limit for in vitro PBE criterion with consideration of variance components as the statistical testing procedures for establishing the in vitro BE. A simulation study was conducted to compare empirical size and empirical power among the three methods. A numerical example illustrates the proposed methods. Copyright © 2010 John Wiley & Sons, Ltd.     

反应堆冷却水中微量氢的顶空气相色谱测定

本文建立了用顶空气相色谱法测定反应堆冷却水中微量氢的方法，在气液体积比为１：２，平衡温度为６０℃，平衡时间为４０ｍｉｎ的气液相平衡条件下Ｈ２的检测下限为０．０２ｍＬ／ＬＨ２Ｏ２。对于Ｈ２浓度高于０．１ｍＬ／ＬＨ２Ｏ的冷却水水样，相对标准偏差小于５．０％，适用于反应堆水样的分析。     

Perspectives on updates,clarifications and controversies in chromatographic assay guidance for bioanalytical method validation from major regulatory agencies and organizations

Bioanalysis, a key supporting function for generating data for pre‐clinical and clinical studies in drug development, is under the regulation of local agencies as well as global organizations to ensure the data integrity and quality in submission. As major regulatory agencies and organizations, the US Food and Drug Administration, the European Medicines Agency and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use have been updating their industry guidance for bioanalytical method validation, to keep up with the development new modalities, technologies and regulations. This article summarizes the recent updates and any clarifications and controversies triggered by those updates. Perspectives and recommendations are given based on our own experience as well as commonly accepted practice in the bioanalytical community.     

Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins

The need for reference materials for quality control of analysis of foodstuffs has been stressed frequently. This has been particularly true in the phycotoxins field, where there is a great shortage of both pure calibration standards and reference materials. Worldwide there are very few independent bodies that produce certified reference materials for phycotoxins, the main producers currently being the National Research Council Canada and the Japanese Food Research Laboratory. Limited availability of contaminated shellfish and algae, as well as the time and knowledge necessary for the production of adequate reference materials, continuously lead to limited editions of certified reference materials and even more limited production of in-house reference materials. The restricted availability of in-house quality control materials promotes the rapid use of the limited certified reference materials, which in turn hampers the production of the suite of materials required globally for complete protection of public health. This paper outlines the various options that analysts can pursue in the use of reference materials for internal and external quality control, with a view to optimising the efforts of both reference materials users and reference materials producers. For this purpose, the logical sequence is reviewed from the discovery of a new bioactive compound in shellfish, through initial method development up to regulation for food safety purposes including accepted reference methods. Subsequently, the requirements for and efforts typically spent in the production and characterisation of laboratory reference materials, certified reference materials and other test materials used in inter-laboratory studies or proficiency testing, in the area of marine biotoxins are evaluated. Particular emphasis is put on practical advice for the preparation of in-house reference materials. The intricate link between reference material characterisation and method performance is outlined to give guidance on the appropriate in-house method validation in the rapidly developing field of phycotoxins.      

A validated assay by liquid chromatography–tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti‐HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20‐mM ammonium acetate/MeOH 50:50. After reverse‐phase chromatography, quantification of RPV and EVG, using matrix‐matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic‐labeled compounds RPV‐¹³C6 and EVG‐D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long‐term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter‐day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, ?20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in‐source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti‐HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a LC‐MS assay for the quantification of ikh12 a novel anti‐tumor candidate in rat plasma and tissues and its application in a pharmacokinetic study

IKH12 is a novel histone deacetylase 6 selective inhibitor. A rapid and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of IKH12 in rat plasma and tissue with kendine 91 as internal standard (IS). The samples were prepared by liquid–liquid extraction with tert‐butyl methyl ether. The chromatographic separation was accomplished by using a Zorbax Extend C₁₈ 4.6 × 150 mm, 5 µm column, with a mobile phase consisting of methanol and 0.1% formic acid (75:25 v/v). Multiple reaction monitoring, using electrospray ionization in positive ion mode, was employed to quantitatively detect IKH12 and IS. The monitored transitions were set at m/z 418 → 252 and 444 → 169 for IKH12 and kendine 91, respectively. The calibration curve was linear over the concentration range 2–1000 ng mL^?1. The intra‐ and inter‐assay precision and accuracy of the quality controls and the limit of quantification were satisfactory in all cases (according to European Medicines Agency guidelines). Stability studies showed that plasma samples were stable in the chromatography rack for 24 h and at ?80 °C for 2 months and also after three freeze–thaw cycles. This method was successfully applied to a pharmacokinetic study of IKH12 in rat. Copyright © 2015 John Wiley & Sons, Ltd.     

Tutorial on evaluation of type I and type II errors in chemical analyses: From the analytical detection to authentication of products and process control

Uncertainty is inherent in all experimental determinations. Nevertheless, these measurements are used to make decisions including the performance of the own measurement systems. The link between the decision and the true implicit system that generates the data (measurement system, production process, category of samples, etc.) is a representation of this uncertainty as a probability distribution. This representation leads to the probabilistic formalization of the possibility of making errors. In the context of regulations established by official agencies, it is important to use these statistical decision methods in some cases because the own norm makes them mandatory and, in general, because this is the way of reasonably evaluating whether a working hypothesis is rejected on the basis of the experimental data.The aim of the present tutorial is to introduce some ideas and basic methods for the critical analysis of experimental data. With this goal, the basic elements of the Neyman-Pearson theory of hypothesis testing are formally introduced in connection with the common problems in chemical analysis and, if this is the case, their relation to the norms of regulatory agencies. The notion of decision with ‘enough quality’ is modelled when explicitly considering: (1) the null, H₀, and alternative, H₁, hypotheses. (2) The significance level of the test, which is the probability, α, of rejecting H₀ when it is true, and the power of the test, 1 − β, β being the probability of accepting H₀ when it is false. (3) The difference between H₀ and H₁ that has to be detected with experimental data. (4) The needed sample size. These four concepts should be explicitly defined for each problem and, under the usual assumption of normal distribution of the data, the mathematical relations among these concepts are shown, which allow the analyst to design a decision rule with pre-set values of α and β.To illustrate the unifying character of this inferential methodology, several situations are exposed along the tutorial: the design of a hypothesis test to decide on the performance characteristics of analytical methods, the capability of detection of both quantitative and qualitative analytical methods (including its generalization to the case of multivariate and/or multiway signals), the analytical sensitivity with multivariate signals, the class-modelling and the process control.     

用平行双样测定结果相对偏差限值评估方法验证实验重复性限合理性的研究

分析方法标准验证实验得到的分析方法基本性能参数重复性限和再现性限是日常检测工作质控规范重要依据。以环境监测领域土壤、沉积物及固废样品中无机元素分析为例,考察了已颁布执行的标准文本和在生态环境部官网公开征求意见的分析方法标准中重复性限。将重复性限转化为相对偏差后,与日常检测工作中质控限值进行了比较。根据目前现行有效的平行双样测定结果相对偏差限值,方法验证数据有多大比例符合质控要求？根据方法验证结果,平行双样测定结果相对偏差限值有无改进可能？从上述两个角度进行了研究。研究结果表明：土壤、沉积物、固体废弃物中无机元素的测定,不同文献来源相同分析方法标准和不同分析方法标准,其重复性限转化得到的平行测定相对偏差合格率存在明显区别;用平行测定相对偏差限值可以快速判断标准文本中的重复性限是否合理,审核方法验证数据质量是否满足要求。基于已有标准文本方法验证数据,探讨了修改平行测定结果相对偏差限值可行性。     

A study for quality evaluation of Taxilli Herba from different hosts based on fingerprint-activity relationship modeling and multivariate statistical analysis

In this study, a fingerprint-activity relationship modeling between chemical fingerprints and antirheumatic activity was established, and multivariate statistical analysis was used to evaluate the quality of Taxilli Herba (TH) from different hosts. Characteristic fingerprints of 20 batches of TH samples were generated by high-performance liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (HPLC-Triple TOF-MS/MS), and the similarity analysis was calculated based on thirteen common characteristic peaks by hierarchical clustering analysis (HCA). Subsequently, nine efficacy markers were discovered by combining fingerprints and antirheumatic activity through grey correlation analysis (GCA) and bivariate correlation analysis (BCA). Meanwhile, the content of 5 constituents in 9 markers was determined by high-performance liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry (HPLC-QTRAP-MS/MS). The comprehensive quality of TH was assessed using multivariate statistical analysis, including principal components analysis (PCA) and technique for order preference by similarity to ideal solution (TOPSIS). The results showed that a high dose of TH extract could markedly ameliorate arthritis damage compared to other doses, with flavonoids playing an important role in the antirheumatic activity. The comprehensive quality of samples from Morus alba L. (SS) was superior to those from Liquidambar formosana Hance (FXS). The present study will demonstrate the markers associated with efficacy, and provide an applicable strategy for more comprehensive quality control and evaluation of TH.     

Analysis and evaluation of nucleosides,nucleobases, and amino acids in safflower from different regions based on ultra high performance liquid chromatography coupled with triple‐quadrupole linear ion‐trap tandem mass spectrometry

Safflower has both medicinal and edible values but research on its nutrient composition is still lacking. This study was established for the quantitative determination of 28 nucleosides, nucleobases, and amino acids based on the ultra‐performance liquid chromatography coupled with triple‐quadrupole linear ion‐trap tandem mass spectrometry. Analysis of 30 batches of safflower from different producing areas indicated that the contents of l ‐proline, l ‐asparagine, l (+)‐arginine, l ‐serine, l ‐histidine, uracil, guanosine, and uridine was high in safflower. Principle component analysis and cluster analysis found that samples from different regions could be distinguished well, and samples from the same area could be clustered into one class, different geographical environments may cause the differences of nucleosides, nucleobases, and amino acids in safflower. The analysis of principal component analysis, cluster analysis, and counter propagation artificial neural network show similar results. Then the content of nucleosides, nucleobases, and essential amino acids were compared, and found that the content in safflower from Gansu was higher than those from other regions, and there was a little difference between the samples from Xinjiang, Sichuan, and Yunnan. This research revealed the composition of nucleosides, nucleobases, and amino acids in safflower, and provided a theoretical basis for utilization of safflower.