Assessment of the drug-like properties of compounds, including absorption, distribution, metabolism and excretion (ADME) via in vitro screening assays, has become a frontline routine practice in drug discovery owing to its effectiveness in guiding lead optimization, and preventing selections of candidates with unfavorable properties for the later, more expensive drug development studies.[1-4] These plate-based profiling assays, commonly operated in a highly automated and parallel fashion, generate large numbers of samples for large numbers of discovery candidates, presenting challenges to the downstream bioanalysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS), which is operated in a sequential manner for every sample injection. Therefore, there have been continuous efforts to improve bioanalytical speed in order to address the ever-increasing throughput and capacity requirements in ADME profiling. One such effort has been the application of multiplexed LC systems in place of the conventional single-stream LC prior to MS analysis.[5, 6] The multiplexed configuration allows for staggered sample injections and elution from multiple columns, followed by alternated MS detection of each of the multiplexed LC channels. The approach can theoretically reduce the injection-to-injection cycle time by n-fold (where n is the number of LCs multiplexed) by significantly reducing the mass spectrometer idle time, while at the same time maintaining the same chromatography integrity as on a single-column system. In addition to multiplexing, further speed-enhancing approaches include the reduction of auto-sampler overhead, utilization of ultra-high pressure liquid chromatography (UPLC), optimization of gradient, and the elimination of MS method downloading time for each injection by acquiring a multiple-injection-chromatogram (MIC) into the same data file. Taken together, these approaches have successfully reduced the bioanalytical cycle time from several minutes per injection to less than 1 min per injection for in vitro ADME support. Several custom-built systems with these features have been reported to date.[7, 8] However, these systems were typically built by integrating components from multiple vendors, and therefore the overall system control has to be performed by custom software that interacts with multiple vendor software. One additional complicating factor for ADME bioanalytical platforms is the integration with hardware/software solution for automated SRM method optimization, as that is the pre-requisite for SRM quantitation of a large number of compounds being profiled. The recently introduced Apricot Designs Dual Arm (ADDA) is the first commercially available system with all these features by integrating with DiscoveryQuant (DQ), a software tool by AB Sciex, to enable streamlined compound optimization, acquisition method creation and sample submission.
The Aria system by Thermo Scientific has been extensively used for multiplexed bioanalysis with LC systems and mass spectrometers from all major vendors, with a single-point software control of multiplexing, running either multiplexed online solid-phase extraction via turbulent flow chromatography,[10, 11] or multiplexed LC without the online extraction. When coupled with a Thermo mass spectrometer, Aria fully integrates with QuickQuan, a Thermo Scientific software tool for automated compound optimization, via infusion or flow injection analysis. We have previously reported the development and application of a multiplexed system employing Aria 1.6.2 on a Quantum Ultra mass spectrometer in support of a high-throughput protein-binding assay. The version of Aria software used then only supported one data file per injection; however, the recently launched Aria 1.6.3 now supports MIC data acquisition. In this study, we report an optimized multiplexed bioanalytical platform by incorporating this new feature of MIC data acquisition to our system and developing a corresponding workflow to achieve fully automated and high-speed bioanalysis, all with single-point software control for the entire integrated system. The application of this system to high-throughput in vitro screening assays is discussed.
RESULTS AND DISCUSSION
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- RESULTS AND DISCUSSION
We previously reported an Aria 1.6.2 controlled, multiplexed LX-2 bioanalytical system consisting a twin-arm auto-sampler for injection, dual-column for staggered parallel chromatography, and LC/MS/MS acquisition via one data file per injection on a Thermo Quantum Ultra mass spectrometer. In the development and implementation of the system, two areas were identified to be the rate-limiting steps to the injection-to-injection cycle time: the first was the auto-sampler pre-injection time (29 s) which included the time for opening and closing of the enclosed cool stack sample drawer for sample aspiration, and the second was the mass spectrometer reset delay (25 s) on the Quantum Ultra mass spectrometer for downloading an acquisition method and opening an acquisition file before each injection. To address the first problem, we replaced the drawer-style sample holders on the CTC auto-sampler with an open-deck manifold and eliminated the auto-sampler overhead time spent on opening and closing the drawers. The strategy effectively reduced the pre-injection time from 29 s to 14 s, leaving only the necessary traveling time of the auto-sampler arm to the designated wells for sample aspiration and to the injector for sample injection.
Collecting data of all the injections from a compound into a single file, or MIC data acquisition, could largely eliminate the 25 s mass spectrometer reset delay for each injection on the previous system using a Thermo Quantum Ultra mass spectrometer. Under this acquisition mode, a data file was created upon the first injection of each compound and remained open for all subsequent injections from this compound, thus eliminating the need to load the acquisition method or open a new data file for each injection. The detector reset delay was only applied to the first injection of each compound, cutting 25 s overhead for each subsequent injection. For many of the ADME assays when up to several dozens of samples are generated for the same compound, application of MIC data acquisition enabled significant runtime reduction. In terms of instrument operation, elimination of method downloading for each injection also reduced chances for communication errors between the peripheral modules and mass spectrometer, which in our experience could be problematic for analysis involving a large number of sample injections.
In addition to the aforementioned reduction of auto-sampler overhead and MS acquisition time, the chromatography gradient time was also trimmed down from 91 s on the previous system to 40 s on the current, with a corresponding reduction in column dimension from 2.1 × 30 to 2.1 × 20 mm to speed up re-equilibration. With all these implemented, the injection-to-injection cycle time achieved was 23 s, which approaches the theoretical maximum efficiency of the LX-2 system running this gradient (40 s/2 = 20 s).
The chromatographic review of MIC data thus acquired was not trivial, since the number of injections and the corresponding injection time had to be taken into consideration by the reviewing software to perform proper peak detection and integration. This was accomplished by using GMSU/QuickCalc software, which parsed the Aria sequence file (.cbt) to obtain this information and subsequently applied it to raw data files during peak integration. The unique data format allowed display of all the chromatographic peaks for a compound and corresponding internal standard altogether in a single view, greatly facilitating the data review process. QuickCalc provided flexibility in peak integration, allowing users the option of modifying chromatographic integration for an individual peak manually, or applying globally a universal set of integration parameters for all the injections of a compound, just as in the case of single file per injection. The utilization of GMSU/QuickCalc has been reported for reviewing MIC data generated on both a multiplexed LC/MS/MS system and an online SPE/MS/MS system.[8, 17]
To evaluate the performance of developed analytical methods and system in terms of reproducibility and carryover, repetitive injections of a reserpine solution in 50:50 water/ methanol at 100 nM were made using the multiplexed LC/MS/MS analysis and MIC data acquisition. The %CV for twelve consecutive injections of reserpine was 4.7% and 7%, respectively, on the two multiplexed channels; a blank solvent injection subsequent to the reserpine injections represented a carryover of 2.9% and 1.8%, respectively; both were comparable to the multiplexed platform we reported previously before the upgrade. The bioanalytical method accuracy, precision and dynamic range were assessed using propranolol prepared at five different concentrations in extracted microsomal matrix spiked with d7-propranolol as internal standard. Four sets of calibration curves of such were separately prepared, each sample of a given set was injected onto both of the multiplexed channels, and measurements from both channels were combined for the intra-assay calibration curve construction. As listed in Table 1, the method demonstrated adequate linear dynamic range (5–2000 nM), intra-assay accuracy, and inter-assay accuracy (96–114%) as well as inter-assay %CV (<6%). Figure 2 plots the internal standard areas acquired in a 13 h overnight run on both of the multiplexed channels. The robustness of the system and method was manifested by a %CV of 8% on each of the multiplexed channels for the measured signal of the internal standard during the entire course of the sample run.
Table 1. Performance of propranolol standard curve
| || ||Propranolol nominal concentration (nM)|
|1||Intra-assay mean (n=4)||4.9||56||492||929||2010|
|2||Intra-assay mean (n=4)||4.9||58||458||985||1903|
|3||Intra-assay mean (n=4)||5.0||58||537||968||1838|
|4||Intra-assay mean (n=4)||5.0||55||492||948||1933|
|All||Inter-assay mean (n=4)||5.0||56||494||957||1921|
The applicability of the platform for metabolic stability assay was evaluated with 288 randomly selected proprietary compounds incubated in six separate assays, each containing 48 compounds. The assay incubation was conducted in 96-well plates and protein precipitation was employed post-incubation for sample extraction, the resulted supernatant was transferred to 384-well plates for LC/MS/MS analysis. The assay generated 12 samples to be injected for each compound, which were grouped into a single file using MIC data acquisition. The %Remaining results were derived from the ratio of analyte to internal standard signal at T10 and T0 of incubation. For all the compounds screened, an MS acquisition window of 15 s was able to capture all the test compounds eluted by the generic chromatographic method with a gradient time of 40 s. Figure 3 represents the typical MIC chromatograms for a test compound. For analysis of each compound, the sample injection sequence was purposely arranged to have T0 and T10 samples of the same replicate injected to the same channel of the multiplexed system, to avoid any differential responses between the two channels for the analyte and the non-isotopically labeled internal standard. In addition, all the T10 samples for all three species were injected prior to the T0 samples to minimize the impact of carryover. The assay samples generated for all the test compounds were subjected to analysis with both the previous LX-2 multiplexed platform, and the current system acquiring data in MIC fashion. The %Remaining values obtained from both systems were compared and the results are presented in Fig. 4, which shows an excellent correlation between the two measurements. Bioanalytical runtime for the metabolic stability assay using the current platform was reduced significantly, from 14.6 to 4.6 min per compound, as illustrated in Fig. 5. This shortened runtime is a combined effect of reduced pre-injection time and MS reset delay, and it translates directly to an appreciable improvement in assay data turnaround, assay throughput and capacity. As illustrated in Table 2, the required runtime of a metabolic stability assay on the previous system allowed for only one assay analysis per overnight run on each instrument, and currently the overnight run can complete three assays.
Table 2. Comparison of platforms
| || ||Current platform||Previous platform|
|Auto-sampler||Format||Open-deck plate holder||Enclosed cool stack|
|Pre-injection time||14 s||29 s|
|LC method||Method time||40 s||91 s|
|MS detection||Format||MIC||One file per injection|
|Reset delay||0 s*||25 s|
|Acquisition window||15 s||42 s|
|Metabolic stability assay||Injection-to-injection cycle time||23 s||73 s|
|Assay analytical time||4.1 h||11.7 h|
Besides the metabolic stability assay, the applicability of the system was also evaluated for in vitro Caco-2 and CYP inhibition (CYP3A4, midazolam substrate) assays using respectively 48 and 224 randomly selected proprietary compounds. All the assay samples generated (in 384-well plates for CYP3A4, and 96-well plates for Caco-2) were injected onto both the current system and a comparing system routinely used for the bioanalytical support of the assays in our lab. Figure 6 illustrates correlations of apparent permeability for the PAMPA assay determined using the current system for analysis with those using the conventional non-multiplexed, single file per injection acquisition. Figure 7 presents correlation of %Inhibition for the CYP inhibition assay between the current multiplexed platform and a RapidFire (Agilent, Santa Clara, CA, USA) direct online SPE/MS/MS system. For both assays the results displayed acceptable linear correlation, demonstrating the feasibility of the platform to additional in vitro ADME assays.
The developed system and methods using multiplexed LC coupled with MIC data acquisition significantly reduced the injection-to-injection cycle time (23 s) than our previously reported platform. This cycle time approaches those reported for the new ADDA system and RapidFire system,[17, 18] with both running direct online SPE in MIC mode. In comparison, advantages of this platform reported herein include its relatively lower cost, its compatibility with various LC and MS components from major vendors, and its single point software control by Aria. In addition, the current system offers flexibility in performing better chromatographic separation without any system modifications. Figure 8 illustrates partial separation of an analyte from its hydroxyl metabolite using the generic chromatographic conditions used for the metabolic stability assay. This chromatography resolution can be further optimized if needed, which would not be possible on a dedicated online SPE system.