Author: Hsuan-Chieh (Joyce) Liao, PhD, DABCC, Assistant Professor, Department of Laboratory Medicine and Pathology, University of Washington, Seattle
The widespread use of opioid drugs has contributed to significantly escalating rates of addiction, overdoses, and drug-related deaths. Therefore, targeted urine drug testing plays an important role in supporting the care of patients with chronic pain or addiction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides excellent sensitivity and specificity and remains the definitive choice for confirmatory urine drug testing. Over the past 10 years, LC-MS/MS opioid test requests in our laboratory increased from 631 in 2011 to 12,125 in 2019, indicating the need for clinical laboratories to develop a more scalable approach to opioid confirmatory testing. Yet, the complexities associated with operating LC-MS/MS pose major challenges to clinical laboratories, especially when compared with commercialized immunoassays. The high initial cost of instruments, the individual injection cycle time, and the requirement for chromatographic review further limit the capacity of the technique. The expertise of medical laboratory scientists (MLS) and clinical chemists is crucial for running LC–MS/MS, especially for method development, assay validation, and troubleshooting. While the Clinical and Laboratory Standards Institute (CLSI) provides guidance for the development and verification of LC-MS/MS methods, external quality assurance parameters and individual assay acceptability still have to be defined by laboratory directors, which practically limits most mass spectrometry techniques as high complexity laboratory-developed testing.
In our recently published study1, we leveraged upgraded instrumentation to develop and validate a simplified “dilute-and-shoot" LC-MS/MS opioid assay, which includes 17 quantitative analytes and 7 qualitative glucuronides in the testing panels. By modifying the chromatographic gradient, we were able to resolve and eliminate the isobaric interfering peaks in the codeine channel caused by oxycodol, an oxycodone metabolite. With the utilization of alternative mass transitions and greater dynamic ranges on the new instruments, the analytical measurement ranges for quantitative analytes span four orders of magnitude, with the low limit of quantification for fentanyl at 0.25 ng/mL. The precision, accuracy, linearity, correlation, and carryover were well comparable with the previous assay and peer groups. Additionally, we established quality assurance parameters, which include minimum internal standard peak areas, signal-to-noise ratios, relative retention time, and ion ratios. To eliminate manual review, improve turnaround time, and meet increasing clinical demand, we also implemented SMACK software for automated quality control and data analysis2. The upgraded systems provided better specificity and greater dynamic ranges, which eliminated the need for extra injections and chromatogram reviews. As a result, we are able to achieve 2 to 3 days turnaround time and double testing capacity with the new method and automated data analysis. This improvement leads to better result interpretations and patient care, and our experiences can serve as a roadmap for other clinical laboratories aiming to expand their testing menu or transition to new instrumentation.
探讨新一代阿片类药物质谱检测方法如何改进实验室工作流程和提高测试能力
阿片类药物的广泛使用导致成瘾率、药物滥用、和毒品相关的死亡率显着上升，因此相关的尿液药物检测在支持慢性疼痛或成瘾患者的护理方面有着至关重要的角色。液相层析串联质谱仪（LC-MS/MS）具有优异的灵敏度和特异性，仍然是确认药物测试的最佳方法。在过去的十年中，我们实验室鸦片类药物测试量从2011年的631项增加到2019年的12,125项，这表明临床实验室急需开发更具可扩展性的临床质谱检测方法。然而，与商业化免疫分析相比，层析质谱仪操作的复杂性对临床实验室构成了重大挑战。质谱仪器的高初始成本、液相色谱的分离时间、以及对峰形检查的要求进一步限制了该技术在常规临床检验的发展。人力方面，医学实验室科技人员（MLS）和临床化学家的专业知识是运行质谱仪的先决条件，特别是在方法开发、测试验证和故障排除方面。尽管临床和实验室标准协会（CLSI）为层析质谱仪方法的开发和验证提供了指南，但是大部分的质谱检测仍需要实验室主管定义相关的质量保证参数和各个检测结果的可靠性，这实际上也限制了质谱分析技术为高复杂度且难以商业化的实验室开发测试。
在我们最近发表的研究中(1)，我们利用升级的仪器开发和验证了一种简化的“稀释-进样”阿片类药物检测方法。该测试面板中包括17种阿片类分析物定量分析和定性7种葡萄糖醛酸酯的定性分析。通过优化色谱梯度条件，我们成功分离了与可待因同分子量的干扰物- oxycodol(一种羟考酮oxycodone的代谢物)，并利用新仪器更广的侦测范围，有效的将所有的定量分析范围扩展到四个数量级，芬太尼的定量分析下限更可达到 0.25 ng/mL，同时并维持了与之前检测方法相似的精密度、准确度、线性、相关性和残留比率。此外，我们还建立了不同的质量保证参数，包括最小内标峰面积、信噪比、相对保留时间和离子比。为了消除繁琐的审查、提高周转时间并满足日益增长的临床需求，我们还采用了监控自动质量控制和数据分析的SMACK软件(2)。升级后的方法及系统提供了更好的特异性和更广的侦测范围，消除了额外的进样和色谱图审核的需求。因此，透过优化检测方法和采用自动数据分析软件，我們将周转时间缩短到2-3天并极大提高了实验室测试能力。这一方法的改进使结果分析更加有效率并提供更好的患者护理，而我们实验室的经验也为其他有类似苦恼的临床实验室提供了很好的范例。
References:
1. Liao HC, Laha TJ, Baird GS, Chan KK, Hoofnagle AN. A Second-generation opioid LC-MS/MS assay improves laboratory workflow and capacity. J Chromatogr B Analyt Technol Biomed Life Sci. Nov 1 2022;1210:123438. doi:10.1016/j.jchromb.2022.123438
2. Pablo A, Hoofnagle AN, Mathias PC. Listening to your mass spectrometer: An open-source toolkit to visualize mass spectrometer data. J Mass Spectrom Adv Clin Lab. Jan 2022;23:44-49. doi:10.1016/j.jmsacl.2021.12.003
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