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。     

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。     

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.     

Single‐dose administration of clenbuterol is detectable in dried blood spots

Clenbuterol is a β₂‐agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time‐consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 μg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post‐ingestion, with additional urine collections on days 7 and 10. Using LC–MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post‐ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7–10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 μg.     

Stability study of the antiviral agent camphecene in dried blood spots at different temperatures

In this study, an optimized procedure of sample preparation for quantitative determination of the antiviral agent camphecene in dried rat blood spots was developed. It has been shown that when using methanol containing 0.1% HCOOH as an extractant, the recovery of the substance increases in comparison with the previously developed method. In addition to this, there is no need to dilute the obtained solutions with water for the analysis of the sample by high-performance liquid chromatography (HPLC) on a column with a reversed-phase sorbent. By using the developed method, the stability of samples of dried rat blood spots containing camphecene in different concentrations at different temperatures was studied. It was found that while the samples were stored at room temperature, apparently, desorption of the substance occurs leading to a loss of more than 15% of its initial amount after 5–10 days. Lowering the temperature increases the stability of samples and their storage at −70°C is possible for 4 weeks.     

Automated high‐throughput analysis of tramadol and O‐desmethyltramadol in dried blood spots

The World Anti‐Doping Agency (WADA) and the International Testing Agency (ITA) recently announced the development and implementation of dried blood spot (DBS) testing for routine analysis in time for the 2022 Winter Olympic and Paralympic Games in Beijing. Following the introduction of a ban on the use of tramadol in competition in March 2019, the Union Cycliste International (UCI) started a pilot study for the manual analysis of tramadol in DBS for antidoping purposes. In this context, we present a fully automated LC–MS/MS‐based method with automated sample preparation using a CAMAG DBS‐MS 500 for the analysis of tramadol and its metabolite O‐desmethyltramadol in DBS. The presented approach reduces manual handling in the laboratory to an absolute minimum, only requiring the preparation of calibration and quality control DBS cards. The method was developed, optimized, and validated before performing cross‐validation with a liquid blood‐based analysis method using authentic samples from forensic cases. During the validation process, the method showed an extraction efficiency of 62%, linearity r² > 0.99, accuracy and precision (within ± 15% and ± 20% at the LLOQ) for the determination of tramadol and O‐desmethyltramadol. Method comparison in liquid blood with 26 samples showed good agreement (90 ± 19% for tramadol and 94 ± 14% for O‐desmethyltramadol). In conclusion, automated analysis of tramadol and O‐desmethyltramadol in DBS provides a fast and accurate solution for antidoping screening. It is suited for high‐throughput analysis, having a run time of about 4 min per sample. Furthermore, with the automated approach, manual sample extraction becomes obsolete.     

Evaluation of dried blood spots as an alternative sample matrix for equine antidoping analysis

Controlling the abuse of prohibited substances such as anabolic steroids, selective androgen receptor modulators, β-adrenoceptor agonists, and blood doping agents is of great interest to racing authorities. The use of dried blood spots (DBS) as an alternative sampling approach may be a feasible approach for controlling the use of these agents. To assess the feasibility of using DBS in equine blood, an 11-min liquid chromatography–mass spectrometry method was developed on a triple quadrupole mass spectrometer following extraction from Whatman 903 DBS cards. A total of 50 compounds across multiple compound classes were detectable with reproducible results. The stability was assessed with good results after almost 3 months of storage at ambient temperatures. These results suggest that the use of DBS may be a feasible alternative sampling approach in equine drug testing.