Matrix effect and recovery terminology issues in regulated drug bioanalysis

Understanding the meaning of the terms used in the bioanalytical method validation guidance is essential for practitioners to implement best practice. However, terms that have several meanings or that have different interpretations exist within bioanalysis, and this may give rise to differing practices. In this perspective we discuss an important but often confusing term - 'matrix effect (ME)' - in regulated drug bioanalysis. The ME can be interpreted as either the ionization change or the measurement bias of the method caused by the nonanalyte matrix. The ME definition dilemma makes its evaluation challenging. The matrix factor is currently used as a standard method for evaluation of ionization changes caused by the matrix in MS-based methods. Standard additions to pre-extraction samples have been suggested to evaluate the overall effects of a matrix from different sources on the analytical system, because it covers ionization variation and extraction recovery variation. We also provide our personal views on the term 'recovery'.     

2种新型基因工程长效人胰岛素类似物

甘精胰岛素和地特胰岛素是2种新型的人胰岛素类似物。虽然他们都通过基因工程的方法生产,药效能持续24h,无明显的波峰和波谷,是真正意义上的基础胰岛素,但是这2种长效胰岛素的分子结构、长效机制、生产工艺各不相同,在临床使用上也有各自的利弊。     

Derivatization methods for quantitative bioanalysis by LC-MS/MS

LC with atmospheric pressure ionization MS is essential to a large number of quantitative bioanalyses for a variety of compounds, especially nonvolatile or highly polar compounds. However, in many instances, weak ionization, poor LC retention and instability of certain analytes hinder the development of the LC-MS/MS method. Chemical derivatization has been used for different classes of analytes to improve their ionization efficiency, chromatographic separation and chemical stability. This work presents an overview of chemical derivatization methods that have been applied to the quantitative LC-MS/MS analyses of nine classes of molecules, including aldehydes, amino acids, bisphosphonate drugs, carbohydrates, carboxylic acids, nucleosides and their associated analogs, steroids, thiol-containing compounds and vitamin D metabolites, in biological matrices.     

Receptor signaling and endocytosis are differentially regulated by somatostatin analogs

Upon hormone stimulation, the sst2 somatostatin receptor couples to adenylyl cyclase through G(i/o) proteins and undergoes rapid endocytosis via clathrin-coated pits. In this study, we determined the relationship between the ability of ligands to induce sst2 receptor internalization and inhibit adenylyl cyclase. Immunocytochemical studies demonstrated that peptide agonists [such as somatostatin-14, cortistatin-17, octreotide, vapreotide, KE108 (Tyr0-cyclo[d-diaminobutyric acid-Arg-Phe-Phe-d-Trp-Lys-Thr-Phe]), and SOM230 (cyclo[diaminoethylcarbamoyl-hydroxyproline-phenylglycine-d-Trp-Lys-(4-O-benzyl)-l-Tyr-Phe])] and nonpeptide agonists (such as L-779,976), stimulated the rapid endocytosis of sst2 receptors in human embryonic kidney 293 and CHO-K1 cells. In contrast, two antagonists did not induce receptor endocytosis by themselves and completely blocked agonist stimulation. Using a quantitative enzyme-linked immunosorbent assay to measure sst2 receptor sequestration, we found that peptide agonists varied by more than 100-fold in their potencies but exhibited the same efficacy as somatostatin14. In contrast, L-779,976 did not induce maximal receptor internalization. It is interesting that although betaarrestin-2 was recruited to cell surface sst2 receptors after stimulation with either somatostatin14 or L-779,976, the betaarrestin-receptor complex dissociated earlier in the endocytic pathway with the nonpeptide ligand. Although all agonists, including L-779,976, produced the same maximal inhibition of cyclic AMP, the potency ratio for inhibition of cyclic AMP and stimulation of receptor endocytosis varied by 15-fold. In general, native peptides showed similar potencies for cyclic AMP inhibition and receptor endocytosis, whereas short therapeutic analogs were substantially more potent at inhibiting cyclic AMP synthesis. These results demonstrate that the activity of somatostatin analogs to regulate receptor endocytosis and signaling are not tightly linked and provide compelling evidence for the induction of agonist specific states of the sst2 receptor.     

A sensitive chemiluminescent enzyme immunoassay for the bioanalysis of carboxyl-terminal B-chain analogues of human insulin.

Quantification of analogues of human insulin in biological matrices is complicated by differences in their immunoreactivity and the presence of both the analogue and endogenous concentrations of insulin in test samples. To facilitate pharmacokinetic comparisons of carboxyl-terminal B-chain analogues of human insulin, we undertook development of a sensitive ELISA. The ELISA detection method was optimized systematically to permit routine analysis of 10-microl serum samples. Accordingly, a noncompetitive 'sandwich' chemiluminescent ELISA was validated for the quantification of carboxyl-terminal B-chain insulin analogues in human serum over a concentration range from 5 to 3125 pM. The mean bias (RE%) within the validated range varied from -10.3 to 4.3%, with an intermediate precision (inter-assay CV%) from 4.2 to 11.5%. The two-sided 90% expectation tolerance interval for total measurement error was within +/-25% of the nominal concentration for all levels of validation samples. Insulin lispro, human insulin, proinsulin, despentapeptide insulin (DPI) and porcine insulin displayed comparable crossreactivity in the ELISA. Potential utility of the new assay for insulin bioanalysis in nonhuman species was investigated by assessing the pharmacokinetic profile of DPI in rats following administration of a single subcutaneous dose. The sensitive chemiluminescent detection method is simple to perform and should be readily adaptable for ELISAs of other therapeutic proteins.     

2010 white paper on recent issues in regulated bioanalysis & global harmonization of bioanalytical guidance

The 4th Calibration and Validation Group Workshop on Recent Issues in Regulated Bioanalysis, a 2-day full immersion workshop, was organized by the Calibration and Validation Group. Contract research organizations, pharmaceutical companies and regulatory agencies came together to discuss several 'hot' topics concerning bioanalytical issues and regulatory challenges and to reach a consensus among panelists and attendees on many points regarding method validation of small and large molecules.     

人胰岛素注射液中锌含量测定的两种方法比较

目的：建立用酸解法测定人胰岛素注射液中锌含量的方法。方法：将供试品与盐酸混合蒸干后，使与蛋白络合的锌转化成氯化锌，再与锌试剂反应、比色。结果：平均加样回收率为97．47％，RSD为0．39％。本法与法定的测定结果基本一致。结论：本法简便、重现性好，可用于控制人胰岛素注射液中锌的质量。     

肽核酸及其类似物的合成方法概述

肽核酸（PNA）作为一种反义核酸类化合物，在分子水平的疾病诊治上，有其独特的优越性，现在日益受到关注。综述了PNA类化合物在基本合成思路和几种主要合成方法，另外还介绍了几种PNA类似物比较特殊的合成方法。     

Evaluation of non-isothermal methods in stability studies of human insulin pharmaceutical preparations

The purpose of this research was to study the thermal stability of a human insulin pharmaceutical preparation using non-isothermal conditions and comparison with classical isothermal experiments. The isothermal studies were performed in the temperature range 20–60 °C, whereas non-isothermal stability studies were performed using a linear increasing temperature program, heating rate 0.25 °C per hour and temperature interval 30–70 °C.     

2009 White Paper on recent issues in regulated bioanalysis from the 3rd Calibration and Validation Group Workshop

The 3rd Calibration and Validation Group Workshop on Recent Issues in Regulated Bioanalysis was organized by the Calibration and Validation Group as a 1.5-day full immersion workshop for contract research organizations, pharmaceutical companies and regulatory agencies to discuss several 'hot' topics concerning bioanalytical issues and regulatory challenges. A consensus was reached among panelists and attendees on many points regarding method validation of small molecules.     

Simplified quantification of insulin,its synthetic analogs and C‐peptide in human plasma by means of LC‐HRMS

The quantification of peptide hormones by means of liquid chromatography (LC) coupled to mass spectrometry (MS) or other techniques (e.g. immunoassays) has been a challenging task in modern analytical chemistry. Especially for insulin, its synthetic analogs, and C‐peptide, reliable determinations are urgently needed due to their diagnostic value in the management of diabetes and insulin resistance and because of the illicit use of insulin as a performance‐enhancing agent in professional sports or as an effective toxin in forensic toxicology. The concomitant measurement of C‐peptide and insulin offers an established tool for the diagnostic workup of hypoglycemia (endogenous vs. exogenous hyperinsulinemia), characterizing hepatic insulin clearance, and the assessment of beta‐cell function (insulin secretion). Thus, the present approach offers the possibility to determine human insulin and its synthetic analogs (lispro, glulisine, aspart, glargine metabolite, degludec, detemir, porcine, and bovine) and C‐peptide simultaneously after sample preparation utilizing protein precipitation and a mixed‐mode cation‐exchange solid‐phase extraction, and subsequent detection by LC‐high resolution MS. The method was fully validated regarding the following parameters: specificity, limit of detection (0.2 ng/mL), limit of quantification (0.6 ng/mL), recovery (40–90%), accuracy (78–128%), linearity, precision (< 21%), carry over, robustness, and matrix effects. The proof‐of‐concept was shown by analyzing authentic plasma samples from adults with class II obesity and prediabetes collected in the course of an oral glucose tolerance test. All sample preparation steps were controlled by two stable isotope‐labeled internal standards, namely [[²H₁₀] Leu _{B6, B11, B15, B17}]‐insulin, and [[¹³C₆] Leu _{26, 30}] C‐peptide.     

Regulated drug bioanalysis for human pharmacokinetic studies and therapeutic drug management

Regulated drug bioanalysis (i.e., determination of drug concentrations in biological matrices for regulated studies) usually refers to animal toxicokinetics, bioavailability/bioequivalence and clinical pharmacokinetic studies. However, there is another important regulated drug bioanalysis - therapeutic drug management (TDM). In the USA, TDM is regulated by Clinical Laboratory Improvement Amendments. In this article, we review and compare human pharmacokinetic sample analysis and TDM sample analysis. The US FDA/Bioanalytical Method Validation Guidance and the American Association for Clinical Chemistry/TDM Roundtable Recommended Generic Assay Validation Guidance are also compared. Some regulated drug bioanalysis issues, such as terminology, validation concepts and acceptance criteria, are discussed. Fostering interaction between bioanalysts from pharmaceutical science and clinical chemistry and reducing the regulatory gaps between different agencies for drug bioanalysis is our objective.     

猪胰岛素与重组人胰岛素的高效液相色谱分析

目的鉴别猪胰岛素与重组人胰岛素。方法采用C8、C1 8及常规孔径 (6～ 1 0nm)与大孔径 (30nm)色谱柱用梯度洗脱和等度洗脱对猪胰岛素与重组人胰岛素的分离方法进行研究。结果用梯度洗脱代替英国药典中的洗脱体系能达到相同的分离效果。结论用常规C1 8柱通过梯度洗脱可分离猪胰岛素与重组人胰岛素     

胰岛素及其类似物治疗糖尿病进展

目的了解胰岛素及其类似物在治疗糖尿病中的应用进展。方法汇总文献资料,对胰岛素及其类似物在药代动力学、药理作用、疗效、不良反应等方面的研究进行总结。结果速效胰岛素、长效胰岛素和吸入胰岛素有独特的药代动力学;艾塞那肽和普兰林肽可以降低餐后高血糖,还可以延缓胃排空,减少能量摄入,使体质量降低。结论仍需要努力开发出更好的胰岛素制剂以便于糖尿病患者使用。     

Relaxation by adenosine and its analogs of potassium-contracted human coronary arteries

Summary The present study was an attempt to characterize the adenosine receptor in human coronary arteries, and to establish the dependence of the relaxations mediated by this receptor on a functional endothelium. Human coronary arteries were obtained from organ donors. Adenosine and its analogs (5-N-ethyl-carboxamido-adenosine, NECA; N⁶-l-phenylisopropyladenosine, LPIA; 2-chloroadenosine, CAD), all inhibited the contraction induced by 25 mmol/l KCl in a concentration-dependent manner and the order of potency was found to be NECA > CAD > L-PIA > adenosine. These relaxations were antagonized by 8-phenyltheophylline (8PT). At higher concentrations of KCl, the relaxations were attenuated. In rings which relaxed in response to endothelium-dependent relaxing agents (bradykinin and A23187), NECA and CAD produced relaxations similar to those produced in rings which did not show endothelium-dependent responses. The results suggest that the coronary adenosine receptor (probably A₂) mediates relaxations which may not be dependent on the relaxing function of the endothelium. Send offprint requests to S. J. Mustafa at the above address