During the routine validation of a benzodiazepine method (performed on a Liquid Chromatography - Tandem Mass Spectrometer), it was noted that lorazepam, triazolam, and α-hydroxytriazolam showed a quadratic shift/bias in the calibration curve, particularly at high concentrations. The ultimate cause of this bias was determined to be due to the natural presence of chlorine (Cl) isotopes (35Cl and 37Cl) in these benzodiazepines. The presence of the heavy (37Cl) isoforms of Cl resulted in the analyte's mass being the same as the internal standard which, in turn, caused the internal standard to appear "falsely increased", thus skewing the calibration curve. One solution to this potential issue was to take advantage of this natural phenomenon and use the Cl heavy isoforms of the respectively labeled internal standards.

中文翻译：

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在苯二氮卓测定方法开发过程中氯所带来的挑战。

在苯并二氮杂卓方法的常规验证（在液相色谱-串联质谱仪上进行）中，注意到劳拉西m，三唑仑和α-羟基三唑仑在校正曲线上表现出二次偏移/偏差，特别是在高浓度下。确定这种偏见的最终原因是由于这些苯二氮卓类化合物中自然存在氯（Cl）同位素（35Cl和37Cl）。Cl的重（37Cl）同工型的存在导致分析物的质量与内标物相同，从而使内标物看起来“错误地增加”，从而使校准曲线倾斜。解决该潜在问题的一种方法是利用这种自然现象，并使用分别标记为内标的Cl重同工型。