干血点采样技术和干血浆采样技术在麦考酚酸药动学分析中的应用

目的探讨干血点采样技术（dried blood spots,DBS）和干血浆采样技术（dried plasma spots,DPS）在麦考酚酸药动学分析中的应用。方法色谱柱为Ecosil C18（150mm×4.6mm,5μm）,流动相为0.01moL/L甲酸水（0.1%甲酸）-乙腈-甲醇（25：45：30）,体积流量为0.4mL/min,柱温为30℃,进样量10μL;质谱检测采用ESI离子源,正离子MRM方式检测;考察可能的影响因素并进行临床研究。结果 DBS法和DPS法方法学和临床实验数据均良好。结论 DBS方法和DPS法较一般处理方法简便,用血量少,储存运输方便,在一些药物的药动学分析中能代替一般的处理方法。     

EPO transgene detection in dried blood spots for antidoping application

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.     

干血点采样技术和干血浆采样技术在麦考酚酸药动学分析中的应用

目的 探讨干血点采样技术（dried blood spots,DBS）和干血浆采样技术（dried plasma spots,DPS）在麦考酚酸药代动力学分析中的应用。方法 色谱柱为Ecosil C₁₈（150 mm×4.6 mm,5 μm）,流动相为0.01 moL/L甲酸水（0.1%甲酸）- 乙腈-甲醇（25∶45∶30）,体积流量为0.4 mL/min,柱温为30 ℃,进样量10 μL;质谱检测采用ESI离子源,正离子MRM方式检测;考察可能的影响因素并进行临床研究。结果 DBS法和DPS法方法学和临床实验数据均良好。结论 DBS方法和DPS法较一般处理方法简便,用血量少,储存运输方便,在一些药物的药动学分析中能代替一般的处理方法。     

Analysis of 3,4-methylenedioxymetamphetamine: whole blood versus dried blood spots

Analysis of dried blood spots is an increasingly accepted method in therapeutic drug monitoring, whereas its application by analogy to forensic samples has not been studied in detail. Therefore, we investigated whether determination of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolite 3,4-methylendioxyamphetamine (MDA) from dried blood spots (DBS) is as reliable as that from whole blood specimens. Analysis was performed by liquid chromatography-tandem mass spectrometry following liquid-liquid extraction of blood and corresponding DBS samples from 20 volunteers participating in a controlled driving experiment under the influence of MDMA. The assay was checked for carryover, ion suppression/enhancement, linearity of response, lower limits of detection (LLOD) and quantitation, extraction efficiency and the within-run and between-run assay imprecision for both whole blood and DBS. The LLODs were 2.0 and 1.6 ng/mL for MDMA in whole blood and DBS, respectively, using a volume of 100 μL. LLODs of MDA were determined to be 0.25 ng/mL in whole blood specimens and 0.12 ng/mL in DBS. Extraction efficiency and imprecision did not differ significantly between the two methods for both MDMA and MDA. The mean concentration ratio of corresponding whole blood and DBS samples, t-test, and the Bland-Altman difference plot were used to test hypothesis of equality. Statistical analyses revealed that methods did not significantly differ for MDMA or MDA. Thus, DBS analysis has potential as a precise and inexpensive alternative to whole blood analysis of MDMA.     

The effects of iron injection on blood doping biomarkers in dried blood spots

Iron supplementation is not considered as a doping method; however, it can affect the levels of several biomarkers of the hematologic module of the athlete biological passport (ABP), such as the reticulocyte percentage (%RET) and hemoglobin (HGB) level. Thus, iron injection could be a confounding factor in antidoping analyses. Previous studies have suggested that the HGB level and the expression levels of reticulocyte-related-mRNAs, such as 5′-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1), could be promising biomarkers for the ABP and detectable in dried blood spots (DBSs). Therefore, in this study, we examined the impact of iron injection on the levels of these potential biomarkers in DBSs. Reticulocyte-related-mRNAs analyses were performed by RT-qPCR. Ferritin level in DBS was measured with enzyme-linked immunosorbent assay (ELISA) method. Notably, there were no significant effects of iron supplementation on the levels of ALAS2 and CA1 mRNAs but by contrast, the %RET and immature reticulocyte fraction (IRF) measured in whole blood increased significantly following iron injection. As expected, iron supplementation increased the ferritin level significantly in both serum and DBS samples. In conclusion, these findings reinforce the specificity of reticulocyte-related mRNAs in DBSs as biomarkers of blood doping to target in antidoping analyses.     

A fast screening method for the detection of CERA in dried blood spots

Continuous erythropoietin receptor activator (CERA) is a third-generation erythropoiesis-stimulating agent that was developed for the treatment of anemia. However, misuse of CERA for doping in endurance sports has been reported. Previous studies have shown blood as the matrix of choice for the detection of CERA, due to its high molecular weight. The use of dried blood spots (DBSs) for anti-doping purposes constitutes a complementary approach to the standard urine and venous blood matrices and could facilitate sample collection and increase the number of blood samples available for analysis due to reduced costs of sample collection and transport. Here, we investigated whether CERA could be indirectly detected in extracts of single DBSs using an erythropoietin-specific immunoassay that is capable of providing results within approximately 2 h. Reconstituted DBS samples were prepared from mixtures of red blood cell pellets and serum samples. The samples were collected in a previous clinical study in which six healthy volunteers were injected with a single, 200 μg dose of CERA. Using a commercially available ELISA kit, CERA was detected in the DBSs with a detection window of up to 20 days post-injection. Furthermore, in order to demonstrate the fitness-for-purpose, three authentic doping control serum samples, which were identified as containing CERA, were analyzed by the presented methodological approach on DBS. The testing procedure described here could be used as a fast and cost-effective method for the detection of CERA abuse in sport.     

Perforated dried blood spots: a novel format for accurate microsampling

Dried blood spots (DBS) in their current format encounter challenges in bioanalysis using fixed areas, including but not limited to, waste of DBS samples (only a fraction is used for analysis), the need for sample punching leading to concerns of sample carryover, uncertainty for accurate recovery assessments and hematocrit (HCT) effects. Here we describe a novel concept, namely perforated dried blood spots (PDBS), for accurate microsampling that addresses previous challenges. PDBS discs were prepared from regular filter paper, with a diameter of 6.35 mm and a thickness of 0.83 mm. An accurate amount of blood sample (5-10 μl), was deposited, dried and stored on the PDBS discs. Upon sample analysis, PDBS samples are simply pushed by single-use pipette tips into 96-well plates. The proof-of-concept study was carried out on a PDBS LC-MS/MS assay development and validation under GLP criteria for the quantitation of lansoprazole in human whole-blood (K(3)EDTA). Particularly, the effect of HCT on the accuracy of quantitation was found to be related to recovery from PDBS samples. In all, PDBS was proved to be a viable alternative to conventional DBS, offering additional advantages of complete sample utilization, no requirement for punching, ease of recovery assessments, and elimination of sampling influence due to HCT levels.     

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。     

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.     

Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application

The World Anti-Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of the class of prohibited substances called erythropoietin (EPO) receptor agonists (ERAs) from DBSs are not yet described. The aim of this study was to find optimal conditions (sample volume and storage) to sensitively detect endogenous erythropoietin (hEPO) and prohibited ERAs from DBSs and compare detection limits to WADA-stipulated minimum required performance levels (MRPLs) for ERAs in serum/plasma samples. Venous whole blood was spotted onto Whatman 903 DBS cards with primarily 60 μl of blood, but various volumes from 20 to75 μl were tested. All samples were immunopurified with MAIIA EPO Purification Gel kit (EPGK) and analysed with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western blot. Sixty-microliter DBSs allowed the detection of the four main ERAs (BRP, NESP, CERA and EPO-Fc) at concentrations close to WADA's MRPLs described for 500 μl of serum/plasma. Different storage temperatures, from −20°C to 37°C, were evaluated and did not affect ERA detection. A comparison of the detection of endogenous EPO from the different anti-doping matrices (urine, serum and DBSs produced from upper arm capillary blood) from five participants for 6 weeks was performed. Endogenous EPO extracted from DBSs showed intra-individual variations in male and female subjects, but less than in urine. Doping controls would benefit from the stability of ERAs on DBSs: It can be a complementary matrix for ERA analysis, particularly in the absence of EPO signals in urine.     

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.