Abstract：
BACKGROUND: To date, clinical laboratories face challenges in quantifying retinol from DBS samples. Disputes arise throughout the whole detection process, encompassing the storage condition, the release strategy as well as the selection of internal standards.
METHODS: We incubated DBS with ascorbic acid solution. Then, retinol-d4 in acetonitrile was introduced to incorporate isotopic internal standard and promote protein precipitation. Afterward, sodium carbonate solution was added to ionize cytochromes (such as bilirubin), which amplified the difference of their hydrophobicity to retinol. Subsequently, cold-induced phase separation could be facilitated to separate retinol from the impurities. In the end, the upper layer was injected for LC-MS/MS analysis.
RESULTS: By comparing the detected retinol content in whole blood and DBS samples prepared from the same volume, we confirmed the established pretreatment was capable to extract most of retinol from DBS (recovery >90 %). Thereafter, we verified that within DBS, retinol possessed satisfying stability without antioxidation. Indoor-light exposure and storage duration would not cause obvious degradation (<10 %). Following systematic validation, the established method well met the criteria outlined in the relevant guidelines. After comparing with detected DBS results to the paired plasma samples, 54 out of 60 met the acceptance limit for cross-validation of ±20 %.
CONCLUSIONS: We realized precise quantification of retinol from one 3.2 mm DBS disc. By circumventing conventional antioxidation, liquid-liquid/solid-phase extraction and organic solvent evaporation, the pretreatment could be completed within 15 min consuming only minimal amounts of low-toxicity chemicals (ascorbic acid, acetonitrile, and sodium carbonate). We expect this contribution holds the potential to significantly facilitate the evaluation of patients\' vitamin A status by using DBS samples in the future.
METHODS: We incubated DBS with ascorbic acid solution. Then, retinol-d4 in acetonitrile was introduced to incorporate isotopic internal standard and promote protein precipitation. Afterward, sodium carbonate solution was added to ionize cytochromes (such as bilirubin), which amplified the difference of their hydrophobicity to retinol. Subsequently, cold-induced phase separation could be facilitated to separate retinol from the impurities. In the end, the upper layer was injected for LC-MS/MS analysis.
RESULTS: By comparing the detected retinol content in whole blood and DBS samples prepared from the same volume, we confirmed the established pretreatment was capable to extract most of retinol from DBS (recovery >90 %). Thereafter, we verified that within DBS, retinol possessed satisfying stability without antioxidation. Indoor-light exposure and storage duration would not cause obvious degradation (<10 %). Following systematic validation, the established method well met the criteria outlined in the relevant guidelines. After comparing with detected DBS results to the paired plasma samples, 54 out of 60 met the acceptance limit for cross-validation of ±20 %.
CONCLUSIONS: We realized precise quantification of retinol from one 3.2 mm DBS disc. By circumventing conventional antioxidation, liquid-liquid/solid-phase extraction and organic solvent evaporation, the pretreatment could be completed within 15 min consuming only minimal amounts of low-toxicity chemicals (ascorbic acid, acetonitrile, and sodium carbonate). We expect this contribution holds the potential to significantly facilitate the evaluation of patients\' vitamin A status by using DBS samples in the future.
摘要：
背景：迄今为止，临床实验室在从DBS样品中定量视黄醇方面面临挑战.纠纷贯穿于整个检测过程,包括储存条件，发布策略以及内部标准的选择。
方法:我们用抗坏血酸溶液孵育DBS。然后，在乙腈中引入视黄醇-d4以掺入同位素内标并促进蛋白质沉淀。之后,加入碳酸钠溶液电离细胞色素（如胆红素），放大了它们与视黄醇的疏水性差异。随后，冷诱导的相分离可以促进从杂质中分离视黄醇。最后,注入上层用于LC-MS/MS分析。
结果：通过比较全血和从相同体积制备的DBS样品中检测到的视黄醇含量，我们证实建立的预处理能够从DBS中提取大部分视黄醇(回收率>90%)。此后,我们验证了在DBS中，视黄醇具有令人满意的稳定性而不抗氧化。室内光照和储存时间不会引起明显的降解(<10%)。经过系统验证，所建立的方法完全符合相关指南中概述的标准。在将检测到的DBS结果与配对的血浆样本进行比较后，60人中有54人达到了±20%的交叉验证验收极限。
结论：我们实现了对一张3.2mmDBS圆盘的视黄醇的精确定量。通过规避传统的抗氧化，液-液/固相萃取和有机溶剂蒸发，预处理可以在15分钟内完成，只消耗最少的低毒性化学品(抗坏血酸，乙腈,和碳酸钠)。我们预计这一贡献具有在未来使用DBS样本显著促进患者维生素A状态评估的潜力。
方法:我们用抗坏血酸溶液孵育DBS。然后，在乙腈中引入视黄醇-d4以掺入同位素内标并促进蛋白质沉淀。之后,加入碳酸钠溶液电离细胞色素（如胆红素），放大了它们与视黄醇的疏水性差异。随后，冷诱导的相分离可以促进从杂质中分离视黄醇。最后,注入上层用于LC-MS/MS分析。
结果：通过比较全血和从相同体积制备的DBS样品中检测到的视黄醇含量，我们证实建立的预处理能够从DBS中提取大部分视黄醇(回收率>90%)。此后,我们验证了在DBS中，视黄醇具有令人满意的稳定性而不抗氧化。室内光照和储存时间不会引起明显的降解(<10%)。经过系统验证，所建立的方法完全符合相关指南中概述的标准。在将检测到的DBS结果与配对的血浆样本进行比较后，60人中有54人达到了±20%的交叉验证验收极限。
结论：我们实现了对一张3.2mmDBS圆盘的视黄醇的精确定量。通过规避传统的抗氧化，液-液/固相萃取和有机溶剂蒸发，预处理可以在15分钟内完成，只消耗最少的低毒性化学品(抗坏血酸，乙腈,和碳酸钠)。我们预计这一贡献具有在未来使用DBS样本显著促进患者维生素A状态评估的潜力。
