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Evaluation of LCMS/MS scrambling ratios for deuterium-labeled Vitamin D metabolites, steroids and other compounds of clinical significance

Authors: Joshua Cooper PhD, Huahua Jian PhD, Derrell Johnson, Isil Dilek PhD, and Uma Sreenivasan PhD
Presented at the American Association for Clinical Chemistry (AACC) Annual Meeting and Clinical Lab Expo, Atlanta, Ga, July 2011

Introduction and objective: A significant clinical challenge with LC-MS/MS is the potential for matrix effects that cause interferences or impact ionization efficiency. Stable isotope-labeled internal standards are frequently used to compensate for matrix effects and to increase the accuracy of quantitation. The use of a labeled internal standard that co-elutes with the drug being monitored can potentially offset patient specific matrix effects (co-eluting concomitant medication, etc.) that may occur at the retention time of the analyte of interest. Complications in the use of deuterium-labeled internal standards can arise from hydrogen-deuterium scrambling in the collision cell at the selected transitions or in the ion source. In this study, we examined deuterium-labeled 25-Hydroxyvitamin D, testosterone, and other compounds of clinical significance by LC-MS/MS at multiple transitions. We investigated reproducibility of the scrambling ratio and influences on scrambling of different LC-MS systems (tandem quadrupole vs. quadrupole time-of-flight), matrix selection, concentration, and deuterium placement in the internal standard.

Methods: LC-MS Systems used were a Waters Alliance UPLC-Xevo G2 Q-Tof system and an Agilent 1290 UHPLC-6460 triple quad system. Various reverse phase columns and mobile phases were used. Samples were analyzed by direct infusion, injection of neat compounds on column and injection of extracted serum samples on column. Serum extraction was conducted using 200µL of serum, adding 200µl of methanol, followed by 1mL of heptane. Samples were then centrifuged, dried down and reconstituted in 100µL of ethanol.

Results: Scrambling was observed on both tandem quad and Q-Tof at select transitions for the deuterium-labeled internal standards studied in both infusion and on column experiments. One example of this scrambling was with D3-25-Hydroxyvitamin D2, for a specific transition 398->380. The scrambling was consistent and was not influenced by matrix, concentration, column or presence of mobile phase. However, scrambling was able to be mitigated or eliminated by selection of another transition, which was also true for the other compounds investigated. The occurrence and extent of scrambling was consistent for each analyte and was dependent on the transition monitored and the ionization system.

Conclusions: Evaluation of scrambling is important in clinical method development to ensure accurate quantitation and reproducible results for critical decision-making in patient care. Awareness of potential scrambling is important for proper internal standard design and selection. Scrambling can be mitigated or eliminated by altering instrument conditions and transition selection or potentially by selecting a transition with consistent scrambling. Deuterium-labeled internal standards are a viable option for LC-MS/MS analysis with selection of the appropriate transition. They also offer a more cost-effective alternative to carbon-13 or nitrogen-15-labeled analogs with benefits such as ready availability and lower cost per test.

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