In this study, a one-step method for liquid-liquid extraction has been compared against a two-step procedure for testosterone assays in terms of accuracy, specificity, recovery, lipid removal and baseline noise, using QCs and unknown samples. The difference in accuracy was less than 5% for adult sera, while it was less than 10% for prepubescent sera. To compare specificity, the ion ratio transition of 289 → 97 to 289 → 109 was monitored for all QCs and unknown samples; no interference in the testosterone peak was observed for any tested sample prepared by either the one-step or two-step procedure. The baseline comparison of LC–MS/MS chromatograms of samples indicated that samples prepared by the one-step procedure were of the same quality as those prepared by the two-step procedure; however, recovery in unaltered serum using the one-step procedure was approximately 15% greater across the low to high concentration range. Furthermore, recovery using the one-step procedure was more consistent between stripped and unstripped serum. Lipids were removed efficiently in the two-step procedure as verified by monitoring the typical phospholipid MRM transition of m/z 496 → 184. For every sample processed by the one-step procedure, a one-minute online column wash with 95% methanol was able to remove 95% of the bound lipids, thereby providing a column life-time approximately equivalent to that for the two-step procedure. The presented data indicate that the one-step procedure could replace the two-step procedure while maintaining accuracy, saving time, increasing recovery, and minimizing the potential for errors with the fewer steps required.

在这项研究中，使用质量控制和未知样品，在准确性、特异性、回收率、脂质去除和基线噪声方面，对液-液提取的一步法与睾酮测定的两步法进行了比较。成人血清的准确度差异小于 5%，而青春期前血清的准确度差异小于 10%。为了比较特异性，对所有 QC 和未知样品的离子比从 289 → 97 到 289 → 109 的转变进行了监测；对于通过一步或两步程序制备的任何测试样品，没有观察到睾酮峰受到干扰。样品 LC-MS/MS 色谱图的基线比较表明，一步法制备的样品与两步法制备的样品具有相同的质量；然而，在低浓度到高浓度范围内，使用一步法在未改变的血清中回收率大约高出 15%。此外，使用一步法的回收率在剥离血清和未剥离血清之间更加一致。通过监测 m/z 496 → 184 的典型磷脂 MRM 转变，验证了两步程序中脂质的有效去除。对于通过一步程序处理的每个样品，使用 95% 甲醇进行一分钟的在线柱清洗能够去除 95% 的结合脂质，从而提供大约相当于两步程序的色谱柱寿命。所提供的数据表明，一步程序可以取代两步程序，同时保持准确性、节省时间、提高恢复率，并通过所需的更少步骤最大限度地减少潜在错误。