Use of a five‐channel multiplexed electrospray quadrupole time‐of‐flight hybrid mass spectrometer for metabolite identification

Abstract

Metabolism data provided with reduced cycle time has become of increasing importance for the early evaluation of DMPK properties of drugs in discovery. In this regard, quadrupole time-of-flight hybrid mass spectrometers (Q-TOF) can provide very reliable metabolite identification via accurate mass measurement of ions and the consequent access to the elemental composition of the metabolite. However, due to their cost, they are often used for drug metabolism studies on later stage drug candidates or to address challenging metabolism questions. A new prototype, consisting of a five-channel multiplexed electrospray ionization (ESI) source on a Q-TOF with one channel used for lock-mass compound infusion, was evaluated for metabolite identification. The goal was to increase the sample throughput of a single ESI-MS system by a factor of 4, while maintaining efficient metabolite separation in high-performance liquid chromatography (HPLC) as well as adequate sensitivity and mass accuracy, and ultimately improve the speed and quality of metabolism studies supporting drug discovery. The analytical performance of the system was assessed by evaluating the sensitivity and mass accuracy (using real in vitro and in vivo samples), inter-channel differences in retention times, MS/UV response, and cross-talk among channels. The sensitivity using the multiplexed ESI source was on average 2-fold lower than with single ESI, correlating well with previous literature data. The mass accuracy was comparable to that obtained using single ESI in both MS and MS/MS modes, making the metabolite identification process using the multiplexed ESI source as reliable as with single ESI. Compound-dependent differences in ionization efficiencies were observed among channels, and were minimized by analyzing related samples on the same channel. Finally, the level of cross-talk among channels was acceptable (around 0.3%) and comparable to levels previously published for quantitative applications using multiplexed ESI. The paper also focuses on the advantages and disadvantages of this new approach compared to other approaches in the literature in the field of metabolite identification.

Pulsed Nano-Electrospray Ionization: Characterization of Temporal Response and Implementation with a Flared Inlet Capillary

“…Throughput has been increased by a factor of four while mass measurement accuracies of below 5 ppm were maintained for a variety of metabolites by coupling an ESI source to each of four HPLC effluent streams and using a fifth channel to introduce a lock mass for calibration. [14] A dual sprayer setup on the front end of a TOF instrument was used to achieve mass accuracies of 3 ppm for ions below mass-to-charge ( m/z ) 1000 using sub-picomole amounts of sample. [15] Each sprayer was independently and sequentially sampled by alternately switching the high voltage (HV) applied to each sprayer.…”

“…The use of HV switching also allows for shorter transit times between sampling from different sprayers (~1 ms) as opposed to the use of a mechanical switching method (~100 ms); where the former is also advantageous to source stability, sample consumption, and selective sprayer sampling. [4,14,15]…”

Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time‐of‐flight mass spectrometry

“…Liquid chromatography coupled with electrospray ionization orthogonal acceleration time‐of‐flight mass spectrometry (LC/TOFMS) enables accurate mass determination of components of complex mixtures on a routine basis 1–3. Within small molecules, the methodology has recently proven particularly useful in screening combinatorial chemistry libraries4, 5 and identifying metabolites6, 7 related to drug discovery. Other current applications include screening for anabolic steroids in illegal cocktails8 and fungal metabolites in culture extracts,9 elucidating unknown compounds in environmental water,10 and determining organic acids in aerosol particles from coniferous forests 11…”

Method for rapid metabolite profiling of drug candidates in fresh hepatocytes using liquid chromatography coupled with a hybrid quadrupole linear ion trap

“…The three‐dimensional and the linear ion traps offer a high duty cycle for scanning, ideal for structural identification 3–8. The use of accurate mass measurement using quadrupole time‐of‐flight (Q‐TOF),9, 10 Fourier transform mass spectrometry (FTMS), and the most recent hybrid linear ion trap Fourier transform ion cyclotron resonance (LTQ‐FT) and OrbiTrap (LTQ‐OrbiTrap),11, 12 has greatly improved the capabilities to identify the structures of metabolites. Despite the advances in the instrumentation, metabolite identification is usually performed using the end points of microsome or hepatocyte incubations at concentration ranges higher than the K m of most drug candidates.…”