Tof-HRMS: Rescuing Low-Exposure Bioanalysis Applications from Chemical Noise
The drug development process requires pharmacokinetic (PK) analysis to be performed as part of safety and efficacy studies in both preclinical species and human subjects. For high-sensitivity bioanalysis, liquid chromatography (LC) coupled to a triple quadrupole mass spectrometer (LC-MS/MS) is considered the gold standard for quantification. However, for some low-exposure applications, traditional MS/MS does not provide the resolution necessary to overcome the increased chemical noise due to matrix effects.
As a means to overcome this challenge, Dr. Chris Evans and his colleagues at GlaxoSmithKline (GSK) are using time-of-flight high resolution mass spectrometry (Tof-HRMS) to support the development of a dermal application for acne vulgaris. They report on their validation of a sensitive and robust assay that utilizes Tof-HRMS via Waters SYNAPT G2-Si High Definition mass spectrometer to support a first time in human (FTIH) study in a recent publication in the journal Bioanalysis.
How did they do it?
Flexible and resourceful sample preparation in combination with the innate attributes of LC‑Tof-HRMS enabled these investigators to achieve their required lower limit of quantitation (LLOQ) of 1 pg/mL.
Increase efficiency through sample preparation
Switching from ethyl acetate to methyl-tertbutyl ether increased extraction efficiency by approximately 15%.
Maintain selectivity while injecting more sample
Increasing injection volume from 5 to 25 µL allowed the analysts to meet their LLOQ target with Tof‑HRMS because of its increased selectivity. It follows that using the optimized extraction procedure and then injecting a larger volume would provide more signal for the ion of interest but a corresponding increase in noise. Applying high-resolution detection enables the use of narrower mass windows (as compared to the tandem quadrupoles) thus reducing any noise outside the window which results in an increased S:N ratio. The investigators used an optimal 0.05 Da extraction window providing a good balance between increased selectivity and concomitant loss in intensity.
Maximized assay sensitivity and specificity via acquisition mode
The acquisition mode utilized for data collection on the SYNAPT G2-Si was Tof-MRM (sensitivity mode). This mode is ideal for quantification through the use of target enhancement.
The ability to move analytical methods seamlessly between the unit resolution (triple, a.k.a. tandem, quadrupoles) and mono-isotopic Tof-HRMS augmented method development and provided timely study support and direction. This combination mitigated any potential delays resulting from developing a new, more selective and, by extension, more sensitive validated method to measure the anticipated exposure levels in the clinical studies at lower therapeutic doses.
High resolution mass spectrometry and, in particular, Tof-HRMS, offers several complementary attributes to triple quadrupoles:
- The ability to garner both qualitative and quantitative results from a single HRMS analysis reduces the time for method development.
- The mono-isotopic resolution associated with Tof-HRMS platforms compared to a triple quadrupole’s unit resolution provides increased selectivity, leading to an increased S:N ratio and a subsequent improvement in the LLOQ.
- Tof-MRM mode provides similar quantitative results; in this instance it surpasses the quadrupole. This enables easy transfer of methodology for bioanalysis labs, with maximum sensitivity for ions of interest.
The results in this journal article are consistent with the ability of Tof-HRMS to provide narrower extraction windows that resolve the noise, thus providing increased selectivity. The ability to resolve the analyte from the chemical background noise improves the S:N ratio and consequently the sensitivity of the assay. In this instance, even after the dose was reduced and lower exposure levels were needed mid-study, selectivity enabled the attainment of the required LLOQ and the subsequent development of a robust, validated assay for monitoring a dermal, first-time-in human clinical study.
It is clear from the successes reported in Bioanalysis that Tof-HRMS will continue to have a pivotal place in the bioanalytical tool box for both discovery and development for some time to come.
Take a closer read:
- “Modify on the fly: triple quad to high resolution in support of a dermal clinical study requiring an ultra low LLOQ,” Bioanalysis, Feb. 2016
- “Robust, fit-for-purpose method transfer: why we should apply equivalence testing,” Bioanalysis, April 2015
- Listen to this recent webinar from Dr. Evans on the merits and utility of Tof-HRMS for regulated quantitative bioanalysis: “The ever-expanding role of HRMS in regulated bioanalysis: application to both small molecule and biopharmaceutical study support,” BioanalysisZone, Oct. 2015
- Check out my recent article in LCGC, “Meeting the Challenges of Bioanalysis with Time-of-Flight High Resolution Mass Spectrometry (TOF-HRMS),” where I further discuss the acceptance of Tof-HRMS as a quantitative analysis resource
- Visit our DMPK website to see more applications of HRMS for quantification