Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Uppsala University, BMC Box 574, SE-751 23, Uppsala, Sweden.
Anal Bioanal Chem. 2019 Jul;411(17):3919-3928. doi: 10.1007/s00216-019-01870-7. Epub 2019 May 18.
In this study, a special interest was focused on the stability of diazepam and nordiazepam in aqueous samples at acidic and neutral pH. The aim of the study was to isolate and illustrate one of the many possible sources of error that can be encountered when developing and validating analytical methods. This can be of particular importance when developing multi-analyte methods where there is limited time to scrutinize the behavior of each analyte. A method was developed for the analysis of the benzodiazepines diazepam and nordiazepam in treated wastewater. The samples were extracted by solid phase extraction, using SPEC C18AR cartridges, and analyzed by the use of liquid chromatography, with a C18 stationary phase, coupled to tandem mass spectrometry. Environmental water samples are often acidified during storage to reduce the microbial degradation of the target compounds and to preserve the sample. In some cases, the samples are acidified before extraction. In this study, it was found that a chemical equilibrium between nordiazepam and a transformation product could cause inaccurately high extraction recovery values when the samples were stored at low sample pH. The stability of nordiazepam was shown to be low at pH 3. Within 12 days, 20% of the initial concentration of nordiazepam was transformed. Interestingly, the transformed nordiazepam was shown to be regenerated and reformed to nordiazepam during sample handling. At a sample pH of 7, diazepam and nordiazepam were stable for 12 days. It was concluded that great care must be taken when acidifying water samples containing nordiazepam during storage or extraction. The storage and the extraction should be conducted at neutral pH if no internal standard is used to compensate for degradation and conversion of nordiazepam. The developed method was validated in treated wastewater and applied for the quantification of diazepam and nordiazepam in treated wastewater samples.
在这项研究中，特别关注酸性和中性 pH 条件下水中的地西泮和去甲西泮的稳定性。研究的目的是分离和说明在开发和验证分析方法时可能遇到的许多可能的误差源之一。当开发多分析物方法时，这可能特别重要，因为没有足够的时间仔细研究每个分析物的行为。开发了一种用于分析处理废水中的苯二氮䓬类药物地西泮和去甲西泮的方法。样品通过固相萃取，使用 SPEC C18AR 小柱进行提取，并通过液相色谱法分析，采用 C18 固定相，与串联质谱法耦合。环境水样在储存过程中通常会酸化，以减少目标化合物的微生物降解并保存样品。在某些情况下，样品在提取前酸化。在本研究中，发现当样品在低 pH 值下储存时，去甲西泮与转化产物之间可能存在化学平衡，导致不准确的高提取回收率值。去甲西泮在 pH 3 时稳定性低。在 12 天内，初始浓度的 20%去甲西泮被转化。有趣的是，在样品处理过程中，转化的去甲西泮被证明会再生并重新形成去甲西泮。在 pH 7 时，地西泮和去甲西泮在 12 天内稳定。结论是，在储存或提取含有去甲西泮的水样时，必须非常小心地酸化。如果不使用内标来补偿去甲西泮的降解和转化，则应在中性 pH 下进行储存和提取。开发的方法在处理后的废水中进行了验证，并应用于处理后的废水样品中地西泮和去甲西泮的定量。
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