Franke Adrian A, Li Xingnan, Dabalos Chester, Lai Jennifer F
University of Hawaii Cancer Center, Analytical Biochemistry Shared Resource, Honolulu, Hawaii.
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii.
Drug Test Anal. 2020 Jun;12(6):846-852. doi: 10.1002/dta.2783. Epub 2020 Mar 11.
Native circulating oxytocin (OT) levels in non-pregnant/non-lactating/non-medicated humans are very low (≤ 8 pg/mL). The lower limit of detection (LLOD) of our previous liquid chromatography mass spectrometry (LC-MS) method (10-25 pg/mL) precluded their quantification in serum and urine. Thus, we sought to improve the LC-MS sensitivity of OT measurements in these matrices by hydrophobic tagging and solid phase extraction (SPE). In the former approach, OT was reduced then alkylated with N-alkyl acetamide (C12, C14, C16, and C18) tags or derivatized using sulfonyl chloride-based reagents. In the latter approach, native OT in serum and urine was concentrated by offline SPE using gradient acetonitrile washings after first crashing with acetonitrile. Peak urinary eluate fractions were further concentrated online then analyzed by orbitrap-based LC-MS with electrospray ionization. All hydrophobic OT derivatives had lower sensitivity than native OT. Washing with a water-acetonitrile gradient during SPE improved the LLOD of OT in spiked serum to 2.5 pg/mL, while adding a subsequent online-concentration step improved the LLOD in spiked urine to 1-5 pg/mL and allowed us to detect OT in urine from lactating women. We were unable to improve the sensitivity of OT measurements by hydrophobic tagging or by derivatization using sulfonyl chloride-based reagents. However, we were successful in improving the sensitivity of native OT measurements in serum and urine 2- and 5-fold, respectively, from our previous orbitrap-based LC-MS method. Offline SPE was mandatory for both matrices and a subsequent online-concentration step was required for urine.
非妊娠/非哺乳/未用药的人体内天然循环催产素（OT）水平非常低（≤8 pg/mL）。我们之前的液相色谱质谱法（LC-MS）的检测下限（LLOD）为10 - 25 pg/mL，这使得无法对血清和尿液中的OT进行定量。因此，我们试图通过疏水标记和固相萃取（SPE）来提高这些基质中OT测量的LC-MS灵敏度。在前一种方法中，OT先被还原，然后用N-烷基乙酰胺（C12、C14、C16和C18）标签进行烷基化，或使用基于磺酰氯的试剂进行衍生化。在后一种方法中，血清和尿液中的天然OT先用乙腈沉淀，然后通过离线SPE使用梯度乙腈洗涤进行浓缩。尿液洗脱峰馏分进一步在线浓缩，然后通过基于轨道阱的电喷雾电离LC-MS进行分析。所有疏水OT衍生物的灵敏度均低于天然OT。在SPE过程中用水-乙腈梯度洗涤可将加标血清中OT的LLOD提高到2.5 pg/mL，而增加后续的在线浓缩步骤可将加标尿液中的LLOD提高到1 - 5 pg/mL，并使我们能够检测哺乳期妇女尿液中的OT。我们无法通过疏水标记或使用基于磺酰氯的试剂进行衍生化来提高OT测量的灵敏度。然而，我们成功地将血清和尿液中天然OT测量的灵敏度分别比我们之前基于轨道阱的LC-MS方法提高了2倍和5倍。离线SPE对两种基质都是必需的，而尿液则需要后续的在线浓缩步骤。
