Journal of Pharmaceutical and Biomedical Analysis 122 (2016) 1–8
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
A novel compact mass detection platform for the open access (OA) environment in drug discovery and early development Junling Gao a , Scott S. Ceglia a,∗ , Michael D. Jones b,c,∗∗ , Jennifer Simeone c , John Van Antwerp c , Li-Kang Zhang a , Charles W. Ross III a , Roy Helmy a a
Department of Process and Analytical Chemistry, Merck & Co. Inc., Rahway, New Jersey 07065, USA Analytical and Environmental Sciences, School of Biomedical Sciences, King’s College, London, SE1 9NH, UK c Waters Corporation, Milford, MA 01757 USA b
a r t i c l e
i n f o
Article history: Received 9 September 2015 Received in revised form 6 January 2016 Accepted 7 January 2016 Available online 15 January 2016 Keywords: Compact mass detector Open-access UPLC-MS Process Development Drug discovery
a b s t r a c t A new ‘compact mass detector’ co-developed with an instrument manufacturer (Waters Corporation) as an interface for liquid chromatography (LC), specifically Ultra-high performance LC® (UPLC® or UHPLC) analysis was evaluated as a potential new Open Access (OA) LC–MS platform in the Drug Discovery and Early Development space. This new compact mass detector based platform was envisioned to provide increased reliability and speed while exhibiting significant cost, noise, and footprint reductions. The new detector was evaluated in batch mode (typically 1–3 samples per run) to monitor reactions and check purity, as well as in High Throughput Screening (HTS) mode to run 24, 48, and 96 well plates. The latter workflows focused on screening catalysis conditions, process optimization, and library work. The objective of this investigation was to assess the performance, reliability, and flexibility of the compact mass detector in the OA setting for a variety of applications. The compact mass detector results were compared to those obtained by current OA LC–MS systems, and the capabilities and benefits of the compact mass detector in the open access setting for chemists in the drug discovery and development space are demonstrated. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Mass spectrometry is an essential tool for supporting research across the biopharmaceutical industry [1–3]. Historically, MS experimentation was carried out by highly-trained analytical chemists using sophisticated instrumentation and complex software packages making it less than optimal for efficiently supporting fast-paced synthetic research programs. Until recently most of the mass spectrometers on the market were expensive, possessed a large footprint, and required skilled-operation thereby prohibiting widespread deployment within synthetic chemistry laboratories. In recent years however, much focus has been placed on bringing mini-MS instrumentation or compact mass detectors that mimic the simplicity of a UV detector [4–6] to market. As MS instrumentation has become more robust and easier to use, MS experimentation
∗ Corresponding author at: Department of Process and Analytical Chemistry, Merck & Co. Inc., Rahway, New Jersey 07065, USA. ∗∗ Corresponding author at: Waters Corporation, Milford, MA 01757, USA. E-mail addresses: scott [email protected] (S.S. Ceglia), Michael D [email protected] (M.D. Jones). http://dx.doi.org/10.1016/j.jpba.2016.01.017 0731-7085/© 2016 Elsevier B.V. All rights reserved.
has become more mainstream with primary analysis and interpretation responsibilities falling onto non-experts with consultation and advanced experimentation provided by analytical MS experts as needed. Open access liquid chromatography–mass spectrometry (OA LC–MS) networks are one such area where these compact mass detectors could be extremely impactful, as there are large quantities of synthesized samples and substantial numbers of potential “non-expert” end-users. As described in the paper by Coddington et al. [7] in 2003, a walk-up non-expert instrument must have the following criteria: robustness, ease of use, chromatographic and mass spectral fidelity, long LC column life, and exceptional uptime. Chromatography and mass spectrometry instrumentation quickly improved through the early 2000s with shorter chromatography analysis times, enhanced resolution and detection limit, and faster data acquisition scan speeds to match the peak widths associated with UHPLC chromatography. As a result, “walk-up” or open access (OA) LC–MS environments have seen tremendous growth in the biopharmaceutical and other industries. In fact, dedicated OA software packages are now available from many of the leading instrument manufacturers (e.g., Waters Corporation OpenLynx, MassLynx; Agilent EasyAccess; Bruker Compass OpenAccess; Shimadzu Scientific
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Instruments Open Solutions and QuantAnalytics). Using these OA LC–MS systems, chemists routinely obtain molecular weight information of their samples, monitor reaction progress, and carry out purity assessments of intermediates and products. Automated sample analyses are rapidly conducted by use of short LC columns and fast gradients with typical analysis times of 2 min or less. As the LC–MS instrument performance had exceeded the basic needs of the discovery space, it was determined the next step was not additional features, but rather improvement in the more physical aspects of the instrument along with simplification of the user interface. Whether in the drug discovery space or early development stage, OA LC–MS systems are now critical analytical tools used by bench chemists on a daily basis to drive research [8,9]. Therefore, incorporation of a “robust” compact mass detector into existing OA platforms would deliver significant improvements in efficiency and cost. Implementing novel technology into the everyday workflow of a pharmaceutical research environment must however be carefully planned out. Any disruptions caused by reductions in throughput, lack of system robustness, or difficulties related to the ‘ease of use’ would be readily identified by researchers and extremely detrimental. As such, assessments of the compact mass detector in ‘applied use’ scenarios were essential to properly evaluate the compact mass detector with regards to these potential concerns. A small subset of discovery chemists was enlisted to test the compact mass detector in ‘real world’ pharmaceutical applications by monitoring reactions and evaluating compound purities over an extended period of time. Comparisons of the compact mass detector to that of the traditional mass spectrometers currently employed were performed during this assessment period. Experiments using commercially available reference compounds, quantitation studies, and some HTS applications were conducted to evaluate and compare the baseline performance of the compact mass detector. The results of these studies were used to determine the overall feasibility, robustness, and flexibility of this novel technology within an OA environment as set forth by the co-development collaboration between Merck and Waters Corporation. 2. Materials and methods 2.1. Instrumentation A mass spectrometer consists of a single quadrupole in which electrical potentials of RF and DC are applied to opposite pairs of a linear array of four parallel cylindrical rods, where ions are separated by mass-to-charge (m/z) ratios and filtered depending on their trajectory in the oscillating electrical fields which are applied to the cylindrical rods. This research explores a novel technology typically utilized in higher-end triple quadrupole mass spectrometers but employed with a thought-free user interface design to easily attain electrospray based nominal mass measurements. The concepts of tuning parameters, manual calibrations, and various ionization choices have been eliminated to facilitate a fitfor-purpose practicality of providing masses for a detected analyte analogous in workflow and visual outputs of a simple UV detector and wavelength determination. For this reason, the instrument will be referred to as a mass detector due to the streamlined approach and simplified user-interface when compared to those user-inputs required for a traditional mass spectrometer. The size comparison of the mass detector, to that of a traditional mass spectrometer further lend itself to being defined as a compact mass detector, since the footprint is more comparable to that of an optical detector. The compact mass detector design utilizes dual off-axis ion guides for elimination of neutral noise to provide increased detection limit and robustness. The design incorporates a conjoined stacked ring ion guide and second stage quadrupole ion guide (Fig. 1). The
Fig. 1. Cross-sectional view of the compact mass detector ion guide and analyzer technology (compliments of Waters Corporation).
design reduces contamination, which contributes to improvement in method robustness where complex matrices are being analyzed. 2.2. Open access instrument configurations Experiments were performed using ACQUITY H-Class UPLC and ACQUITY ‘Classic’ UPLC instrumentation (Waters Corporation, Milford, Massachusetts, USA). The ACQUITY H-Class was configured with a sample organizer for increased sample capacity required during high throughput screening assessments. Each UPLC configuration was coupled with a Waters QDa compact mass detector. An ACQUITY SQD (Waters Corporation) mass spectrometer coupled to the UPLC configurations was used as the traditional mass spectrometer for comparative assessments. MassLynx Open Access OpenLynx 4.1 (Waters, Milford) application manager software was used to enable walk-up functionality and data visualization. The UPLC systems were configured with ACQUITY BEH C18 columns (1.0 mm × 50 mm; 1.7 m) on both the ACQUITY H-Class and on the ACQUITY Classic UPLC. Each column compartment was maintained at 50 ◦ C. The ACQUITY H-Class automatically metered the mobile phase for a linear gradient elution composition of 95:5 to 5:95 (0.05% TFA/water: 0.05% TFA/acetonitrile) over 2.0 min. The ACQUITY ‘Classic’ UPLC used a linear gradient method composition of 95:5 to 5:95 (0.05% TFA/water: 0.05%TFA in acetonitrile). The flow rate was maintained at 0.3 mL/min. The injection volume of each sample was 1.0 L. 2.3. Chemicals Acetonitrile and methanol (LC/MS Optima Grade) were obtained from Fisher Scientific (FairLawn, NJ, USA). Sulfadimethoxine, terfenadine, verapamil, leucine-enkephalin, 2-fluro-5-trifluromethoxy phenyl acetic acid and L-1-BOC-nipecotic acid were obtained from Sigma–Aldrich (St. Louis, MO, USA). Human insulin was obtained from FUJIFILM Diosynth Biotechnologies (Morrisville, NC, USA). (R)-
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4-oxo-4-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4][4,3a]pyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenyl) butan-2-aminium (sitagliptin) and (R)-3-amino-1-(3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4][4,3-a]pyrazin-7(8H)-yl-5,5,6,6-d4)-4-(2,4,5trifluorophenyl)butan-1-one (tetra Deutero-labeled sitagliptin) were synthesized at Merck Research Laboratories. (Rahway, NJ, USA) QC 96-well plates were obtained from Chembridge Inc. (San Diego, CA, USA). 2.4. Standard and sample preparation Standard stock solutions of each compound of interest were prepared at a concentration of 10 mM in methanol. A series of standard solutions, 0.001, 0.005, 0.01, 0.05, 0.1, 0.25, and 0.5 g/mL were prepared by dilution of the stock solution. For the quantitation studies, stock solutions of sitagliptin and internal standard (IS, tetra deutero-labeled sitagliptin) were prepared at 10 mM and 2 mM in methanol, respectively. A series of sitagliptin standard solutions, 0.01, 0.05, 0.10, 0.50, 1.00, 2.50, and 5.00 M were prepared by dilution of the stock solution. Each dilution was spiked with 0.05 M tetra-deutero labeled sitagliptin as internal standard. 2.5. MS parameters ESI MS analysis was performed on the compact mass detector in both positive-ion and negative ion full scan modes during the “applied use” robustness assessment and HTS testing. The compact mass detector and traditional mass spectrometer were operated in ESI-SIR positive mode for the linearity and dynamic range assess-
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ments and comparison testing. Electrospray ionization capillary voltages and probe temperatures were 800 V and 600 ◦ C for the compact mass detector, and 3000 V and 450 ◦ C for the traditional mass detector in positive mode. Both systems used the same cone voltage, 20 V. Source temperatures were 120 ◦ C and 140 ◦ C for the compact MS detector and traditional mass spectrometer, respectively. The ESI-SIR parameters were the same for both systems and optimized for the signal-to-noise ratio of target ion signal. Quantitation studies using an internal standard were performed on the compact mass detector in ESI-SIR positive mode. MS analysis of human insulin was performed on the compact mass detector and traditional mass spectrometer (SQD) in full scan ESI positive mode.
3. Results/discussion 3.1. Performance assessment of the compact mass detector 3.1.1. “Applied use” robustness assessment The UPLC coupled to the compact mass detector was run in a “test” setting over the course of approximately 1 month, during which time over 700 small molecule samples were run by multiple discovery scientists, spanning 13 different internal research programs. System uptime was determined to exceed 95%, with no mechanical issues reported. The only downtime during this testing period was intentional (at the onset) to make minor modifications to instrument settings in order to minimize the presence of sodium adduct [M+Na]+ signal intensities. During this testing phase the compact mass detector was evaluated to verify that typical and
Fig. 2. Signal-to-noise evaluation of sulfadimethoxine (A), terfenadine (B), verapamil (C) at 1 ng/mL and 0.05 g/mL for leucine enkephalin (D) in support of LOD assessments for the compact mass detector.
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Fig. 3. Detection of sulfadimethoxine by ESI-SIR for the traditional MS spectrometer. Signal-to-noise assessment at 1 ng/mL for the traditional MS spectrometer.
reliable results were being generated for research compounds containing a multitude of functional groups including halogenated species, where characteristic isotopic patterns are regularly used for identification purposes. In all instances, the experimental data generated using the compact mass detector proved reliable and comparable to data generated side-by-side with current OA UPLCMS systems. 3.1.2. Linearity and dynamic range assessment Four commercially available compounds (sulfadimethoxine, terfenadine, verapamil and leucine-enkephalin) were analyzed at various concentration ranges and calibration curves were generated. Each compound was quantified separately and three replicas for each concentration were evaluated. The use of single ion recording (SIR) mode enhanced the measurement of target masses and reduced the interferences during quantitation. The mean calibration curves gave correlation coefficients of R2 = 0.9946, 0.9828, and 0.9930 for sulfadimethoxine, terfenadine and verapamil, respectively, over the concentration range of 0.001–0.50 g/mL. Leucine-enkephalin showed good linearity (R2 = 0.9990) over the 0.05–5.0 g/mL concentration range. Reproducibility for the compact mass detector across the linearity range over multiple injections (N = 3) resulted in RSDs of typically less than 5%, with slightly larger RSDs appearing at the lower concentration limits where system linearity begins to degrade. Specific limits of detection (LOD) values for terfenadine, verapamil, sulfadimethoxine and leucine-enkephalin were not formally obtained since the lower concentration limit tested for each compound was still above the required LOD S/N = 3 (Fig. 2). 3.1.3. Comparison of the compact mass detector versus the traditional mass spectrometer Sulfadimethoxine was analyzed at various concentration ranges and a calibration curve was generated on a similar walk-up instrument with a traditional mass spectrometer. The traditional mass spectrometer performed slightly better than the compact mass detector yielding a correlation coefficients of 0.9996. Data reproducibility on the compact mass detector appeared to be slightly better than the traditional mass spectrometer for most concentrations tested, except at the lower concentration range where the compact mass detector gave a 9.47% RSD and the traditional mass spectrometer resulted a 4.74% RSD. While this discovery is significant enough to mention, typically research conducted in
the discovery and early development space is not compound limited, and sample preparation rarely occurs in the sub-micro molar concentrations. Comparison of the SIR chromatograms for sulfadimethoxine run on the compact mass detector (Fig. 2) and on the traditional mass spectrometer (Fig. 3) clearly shows that the compact mass detector has a cleaner baseline. Visual comparisons of the linearity data, signal-to-noise, and LOD values are usually fit for purpose and allow scientists to determine if an instrument is a suitable and equivalent substitute. The values of the linearity data from both instruments were further explored from a statistical perspective via Bland–Altman techniques [10]. The regression-based difference plot constructed from the terfenadine linearity data (average mean of n = 3) suggest a high correlation with an R2 = 0.9916 (Fig. 4). Re-plotting the dataset in Fig. 4, where the difference (SQD − QDa) is plotted against the average (QDa + SQD)/2 allows for easier observations of outliers and trends as it relates to the error and bias of the results. The resulting Bland–Altman plot suggests good agreement at a 95% confidence interval (Fig. 5). All points are within the upper and lower limits of agreement with no observed outliers or trends.
Fig. 4. Comparison of QDa and SQD responses for terfenadine measured over the concentration range of 0.001–0.50 g/mL.
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5
10000
5000
0 0
50000
100000
150000
200000
250000
300000
QDa - SQD
-5000
-10000
-15000
-20000
-25000
(SQD + QDa) / 2 Fig. 5. Bland–Altman plot comparing the difference vs. the average of the terfendine linearity results.
3.1.4. Quantitation using an internal standard assessment Quantitation and calibration (mean = 3) of Merck compound sitagliptin, with isotopically labeled (tetra deutero-labeled) internal standard by ESI-SIR for a concentration range of 0.01–5.0 M using the compact mass detector is shown in Table 1. The data generated for the calibration curve was linear, with a correlation coefficient of R2 = 0.9993. The slope and intercept describing the relationship were found to be 24.65 and −0.327 respectively. Additionally, sample data generated for a 0.5 M sample was shown (Fig. 6) to be accurate, demonstrating the ability for the compact mass detector to also be utilized in the early discovery space for quantitative applications. 3.1.5. Biopharmaceutical detection of multiply charged ions One of the initial concerns for the compact mass detector (m/z = 1250 Da max) was the limited mass range as compared to more traditional mass spectrometers currently populating the walk-up environment, which typically cover a wider range (up to m/z = 4000 Da). Injections of Human insulin on the traditional mass spectrometer did provide more comprehensive spectral data (Table 2) as shown by the 3 multiply charged species (Fig. 7A) versus only 2 multiply charged species on the compact mass
Fig. 6. Quantitation of sitagliptin spiked with isotopically labeled internal standard by ESI-SIR using compact mass detector MS detector. SIR chromatograms of sitagliptin and internal standard at 0.5 M.
Table 1 Quantitation results of sitagliptin (MK-0431) with internal standard. Conc. (M)
(ASG /AInt ) (Injection 1)
(ASG /AInt ) (Injection 2)
(ASG /AInt ) (Injection 3)
Mean n=3
Std. dev n=3
% RSD n=3
0.01 0.05 0.10 0.50 1.00 2.50 5.00
0.243 1.282 2.222 12.656 22.382 65.633 123.472
0.283 1.216 2.367 12.475 21.664 60.408 121.598
0.284 1.216 2.215 11.516 22.073 62.784 122.504
0.270 1.238 2.268 12.216 22.040 62.942 122.525
0.023 0.038 0.086 0.613 0.360 2.616 0.937
8.66 3.08 3.78 5.02 1.63 4.16 0.76
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Table 2 Observed charge states of insulin. Human insulin MW 5803 [Da] System
Mass scan range
[M+3H]3+ Da
[M+4H]4+ Da
[M+5H]5+ Da
[M+6H]6+ Da
Traditional MS Compact mass det.
50–1900 Da 50–1250 Da
n/a n/a
1452
1162 1162
968 968
Fig. 7. Multiple charged states of human insulin by (A) traditional MS and (B) compact mass detector.
detector (Fig. 7B). Multiply charged species were however still easily identifiable with the compact mass detector. 3.2. High throughput screening (HTS) assessments Much of the work performed in the early discovery and development space requires rapid processing and data analysis turnaround times in order to meet stringent timelines. High throughput screening is vital in this arena and as such we placed the compact mass detector into our OA workflow to see how it performed. In HTS, the OA system is equipped with an expanded capacity sample management module to enable multiple sample plates to be placed in the queue and run as part of the automated process initiated by the analyst. Software modifications enable processing parameters to be tailored to meet desired “pass/fail” criteria so that rapid assessments via color-coded “hits” based on the mass data can be made instantaneously without having to sift through the multitude of individual sample results. The compact mass detector performance in process optimization, identification, and library QC projects was also evaluated. 3.2.1. Catalysis screening and process optimization As a process optimization tool, the compact mass detector based OA platform was employed for a difficult cross-coupling reaction (Fig. 8), where initial experimental attempts running standard literature procedures by chemists at the bench resulted in no desired
product. A catalyst screening plate (4 × 6) was set up and run through the HTS work flow. Identification of “hits” shown in green (A1; A2, D2) was immediately possible via this simplified output, which enabled chemists to rapidly focus attention on those catalysis conditions that afforded positive results. These hits were then further elaborated into a scalable solution that could be successfully run in the synthetic labs to support discovery research projects. 3.3. HTS library QC comparison A 96-well compound library was acquired from ChemBridge Inc., in order to evaluate both mass measurement systems via the typical OA-login procedure in a discovery laboratory. The objective was to evaluate the ‘hit’ ratio for the target compounds for plates from each instrument and investigate differences in the resulting reports. Results for the plate screening for each mass measurement instrument are shown (Fig. 9). The discrepancies between red visual indicators and green indicators for each of the instruments results were investigated. Differences were observed regarding retention time, peak width, background noise, and ion fragmentation patterns. The retention times observed in the conventional mass spectrometer were later than the retention times observed for the compact mass detector (Fig. 10). This difference can be explained by the lower post extra-column volume provided by the compact mass detector probe. The lower post extra-column volume on the compact mass detector also explains the lower observed
Fig. 8. Catalysis reaction screening with plate results.
J. Gao et al. / Journal of Pharmaceutical and Biomedical Analysis 122 (2016) 1–8
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Fig. 9. Traditional mass spectrometer vs. compact mass detector results for a QC HTS plate screening. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
bandspread of the analyte peak (Fig. 11). The traditional mass spectrometer yielded a peak width of 0.036 min (2.16 s measured with 12 scans per peak) while the compact mass detector yielded a peak width of 0.021 min (1.26 s with 14 scans per peak). The stock solution of each of the samples was diluted in 100% DMSO, and the plate was generated using dilutions of the stock solution in a 1:10 ratio with methanol, therefore it was expected that a high concentration of DMSO would be visible in the total ion chromatographic trace (TIC). Interestingly, the lower background noise for the compact mass detector resulted in a lower interference from the DMSO diluent than that observed by the traditional mass spectrometer (Fig. 10). It is hypothesized that the unique design of the compact mass detector and location of the point of ionization contribute to this significant decrease in observed background noise. Investigating the spectral patterns of the compounds from each set of results also revealed some unique differences. The traditional mass spectrometer yielded greater fragmentation for numerous target analytes. The increased fragmentation of some of the target analytes yielded ‘not found’ results for the targeted mass, whereas the compact mass detector yielded a ‘hit’ result for the same analytes in many cases. The two instrument designs apply the ‘cone’ voltage differently as described by the vendor and therefore it
was expected that more fragmentation would occur within the compact mass detector if the settings were matched to the same settings as the traditional mass spectrometer. The compact mass detector applies the cone voltage downstream in the ion path as compared to that of the conventional mass spectrometer, which is applied near the orifice, hence the need for different method parameter settings between the instruments. Instrument method settings for the compact mass detector were set to the defaults, while cone voltage settings for the traditional mass spectrometer were varied at decreasing voltages of 20, 15, and 10 V to inhibit in-source collision induced dissociations from occurring, and therefore try to best match those results generated by the compact mass detector. Unfortunately, the in-source collision induced dissociation occurred in the traditional mass spectrometer even at the lower voltages and thus resulted in an overall lower hit rate due to lower intensity mass spectral measurements for some of the target compounds. It should be noted that three control samples were inserted into the plate to yield ‘not found’ results. These are represented by the three red visual indicators at the end of the bottom three rows of each plate. These three controls were empty wells and indicated in the software to find a mass that did not exist in the well. This concept was initiated to validate the comparisons
Fig. 10. Total ion chromatogram comparison of the compact mass detector (top) and the traditional mass spectrometer (bottom) for a given analyte in the ChemBridge QC HTS plate. Retention time shift due to volume differences and lower DMSO and background noise are observed.
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Fig. 11. Extracted ion chromatogram for the targeted analyte measured at m/z—329 Da for each instrument used to determine the differing effects of extra-column volume of the probe configurations on peak width.
between the data collection accuracy and data processing between the two instrument HTS experiments.
4. Conclusion The comprehensive assessment and evaluation of this novel compact mass detector succeeded in demonstrating that the instrument meets the needs and addresses the challenges facing a pharmaceutical drug discovery team. The compact mass detector provides a higher level of simplicity to the OA environment versus traditional single quadrupole mass spectrometers on the market. Initial set-up, calibrations, and MS setting adjustments are all but eliminated for the compact mass detector. Mass data generated with the compact mass detector were comparable to conventional mass spectrometer data for a multitude of samples run in the OA environment over the course of this evaluation. In some cases, the performance of the compact mass detector surpassed expectations, as in the case of the library QC plates where minimal in-source fragmentation occurred for the compact mass detector and resulted in a more accurate overall plate assessment as compared to more traditional mass spectrometers. In the biopharmaceutical assessment of insulin, where the limited MS scan range of the compact mass detector was an initial concern, adequate data for multiply charged species was produced. Coupling the compact MS detector with standard deconvolution software could therefore expand its capabilities to include a wide variety of larger molecular weight applications not explored herein. Use of the compact mass detector for quantitation type applications, as was the case for the sitagliptin studies, was also shown to be accurate and beneficial. Overall, the compact mass detector has proven to be a valuable and reliable mass detection and compound confirmation tool for the open access environment in the pharmaceutical discovery and development space where speed and uptime is crucial, unit mass resolution is a necessity, and sample availability is rarely a concern. The benefits of the compact mass detector for use in a wide variety of applications including standard small molecule analysis, HTS and QC type analyses, as well as biological samples and quantitation studies have been demonstrated.
Acknowledgements The authors would to acknowledge Alexey Makarov, Yong Liu, Ray McClain and Chris Welch at Merck for their support and contributions to our discussions. We would also like to acknowledge Eva Gallea, Ed Aig, Howard Read, and the many folks at Waters Corporation who helped in various capacities to advance the development of this new technology. References [1] S. Caron, N. Murray Thomson, Pharmaceutical process chemistry: evolution of a contemporary data-rich laboratory environment, J. Org. Chem. 80 (2015) 2943–2958. [2] S. Khater, C. West, Development and validation of a supercritical fluid chromatography method for the direct determination of enantiomeric purity of provitamin B5 in cosmetic formulations with mass spectrometric detection, J. Pharm. Biomed. Anal. 102 (2015) 321–325. [3] D. Spaggiari, F. Mehl, V. Desfontaine, A. Grand-Guillaume Perrenoud, S. Fekete, S. Rudaz, D. Guillarme, Comparison of liquid chromatography and supercritical fluid chromatography coupled to compact single quadrupole mass spectrometer for targeted in vitro metabolism assay, J. Chromatogr. A 1371 (2014) 244–256. [4] G.F. Verbeck, V.M. Bierbaum, Focus on harsh environment and field-portable mass spectrometry: editorial, J. Am. Soc. Mass Spectrom. 26 (2014) 199–200. [5] X. Bu, J. Yang, X. Gong, C.J. Welch, Evaluation of a compact mass spectrometer for routine support of pharmaceutical chemistry, J. Pharm. Biomed. Anal. 94 (2014) 139–144. [6] S.E. Hamilton, F. Mattrey, X. Bu, D. Murray, B. McCullough, C.J. Welch, Use of a miniature mass spectrometer to support pharmaceutical process chemistry, Org. Process Res. Dev. 18 (2014) 103–108. [7] A. Coddington, J. Van Antwerp, H. Ramjit, Critical considerations for high-reliability open access LC/MS, J. Liq. Chromatogr. Related Technol. 26 (2003) 2839–2859. [8] L.O. Hargiss, M.J. Hayward, Use of an electrospray time-of-flight mass spectrometer for a broad range of open access applications including accurate mass analysis for elemental compositions, in: Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, United States, August 26–30 ANYL-149, 2001. [9] A. Malcolm, S. Wright, R.R.A. Syms, R.W. Moseley, S. O’Prey, N. Dash, A. Pegus, E. Crichton, G. Hong, S.A. Holmes, A. Finla, P. Edwards, S.E. Hamilton, C.J. Welch, A miniature mass spectrometer for liquid chromatography applications, Rapid Commun. Mass Spectrom. 25 (2001) 3281–3288. [10] D.G. Altman, J.M. Bland, Measurement in medicine: the analysis of method comparison studies, J. R. Stat. Soc. Ser. D 32 (1983) 307–317.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
A novel compact mass detection platform for the open access (OA) environment in drug discovery and early development Junling Gao a , Scott S. Ceglia a,∗ , Michael D. Jones b,c,∗∗ , Jennifer Simeone c , John Van Antwerp c , Li-Kang Zhang a , Charles W. Ross III a , Roy Helmy a a
Department of Process and Analytical Chemistry, Merck & Co. Inc., Rahway, New Jersey 07065, USA Analytical and Environmental Sciences, School of Biomedical Sciences, King’s College, London, SE1 9NH, UK c Waters Corporation, Milford, MA 01757 USA b
a r t i c l e
i n f o
Article history: Received 9 September 2015 Received in revised form 6 January 2016 Accepted 7 January 2016 Available online 15 January 2016 Keywords: Compact mass detector Open-access UPLC-MS Process Development Drug discovery
a b s t r a c t A new ‘compact mass detector’ co-developed with an instrument manufacturer (Waters Corporation) as an interface for liquid chromatography (LC), specifically Ultra-high performance LC® (UPLC® or UHPLC) analysis was evaluated as a potential new Open Access (OA) LC–MS platform in the Drug Discovery and Early Development space. This new compact mass detector based platform was envisioned to provide increased reliability and speed while exhibiting significant cost, noise, and footprint reductions. The new detector was evaluated in batch mode (typically 1–3 samples per run) to monitor reactions and check purity, as well as in High Throughput Screening (HTS) mode to run 24, 48, and 96 well plates. The latter workflows focused on screening catalysis conditions, process optimization, and library work. The objective of this investigation was to assess the performance, reliability, and flexibility of the compact mass detector in the OA setting for a variety of applications. The compact mass detector results were compared to those obtained by current OA LC–MS systems, and the capabilities and benefits of the compact mass detector in the open access setting for chemists in the drug discovery and development space are demonstrated. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Mass spectrometry is an essential tool for supporting research across the biopharmaceutical industry [1–3]. Historically, MS experimentation was carried out by highly-trained analytical chemists using sophisticated instrumentation and complex software packages making it less than optimal for efficiently supporting fast-paced synthetic research programs. Until recently most of the mass spectrometers on the market were expensive, possessed a large footprint, and required skilled-operation thereby prohibiting widespread deployment within synthetic chemistry laboratories. In recent years however, much focus has been placed on bringing mini-MS instrumentation or compact mass detectors that mimic the simplicity of a UV detector [4–6] to market. As MS instrumentation has become more robust and easier to use, MS experimentation
∗ Corresponding author at: Department of Process and Analytical Chemistry, Merck & Co. Inc., Rahway, New Jersey 07065, USA. ∗∗ Corresponding author at: Waters Corporation, Milford, MA 01757, USA. E-mail addresses: scott [email protected] (S.S. Ceglia), Michael D [email protected] (M.D. Jones). http://dx.doi.org/10.1016/j.jpba.2016.01.017 0731-7085/© 2016 Elsevier B.V. All rights reserved.
has become more mainstream with primary analysis and interpretation responsibilities falling onto non-experts with consultation and advanced experimentation provided by analytical MS experts as needed. Open access liquid chromatography–mass spectrometry (OA LC–MS) networks are one such area where these compact mass detectors could be extremely impactful, as there are large quantities of synthesized samples and substantial numbers of potential “non-expert” end-users. As described in the paper by Coddington et al. [7] in 2003, a walk-up non-expert instrument must have the following criteria: robustness, ease of use, chromatographic and mass spectral fidelity, long LC column life, and exceptional uptime. Chromatography and mass spectrometry instrumentation quickly improved through the early 2000s with shorter chromatography analysis times, enhanced resolution and detection limit, and faster data acquisition scan speeds to match the peak widths associated with UHPLC chromatography. As a result, “walk-up” or open access (OA) LC–MS environments have seen tremendous growth in the biopharmaceutical and other industries. In fact, dedicated OA software packages are now available from many of the leading instrument manufacturers (e.g., Waters Corporation OpenLynx, MassLynx; Agilent EasyAccess; Bruker Compass OpenAccess; Shimadzu Scientific
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Instruments Open Solutions and QuantAnalytics). Using these OA LC–MS systems, chemists routinely obtain molecular weight information of their samples, monitor reaction progress, and carry out purity assessments of intermediates and products. Automated sample analyses are rapidly conducted by use of short LC columns and fast gradients with typical analysis times of 2 min or less. As the LC–MS instrument performance had exceeded the basic needs of the discovery space, it was determined the next step was not additional features, but rather improvement in the more physical aspects of the instrument along with simplification of the user interface. Whether in the drug discovery space or early development stage, OA LC–MS systems are now critical analytical tools used by bench chemists on a daily basis to drive research [8,9]. Therefore, incorporation of a “robust” compact mass detector into existing OA platforms would deliver significant improvements in efficiency and cost. Implementing novel technology into the everyday workflow of a pharmaceutical research environment must however be carefully planned out. Any disruptions caused by reductions in throughput, lack of system robustness, or difficulties related to the ‘ease of use’ would be readily identified by researchers and extremely detrimental. As such, assessments of the compact mass detector in ‘applied use’ scenarios were essential to properly evaluate the compact mass detector with regards to these potential concerns. A small subset of discovery chemists was enlisted to test the compact mass detector in ‘real world’ pharmaceutical applications by monitoring reactions and evaluating compound purities over an extended period of time. Comparisons of the compact mass detector to that of the traditional mass spectrometers currently employed were performed during this assessment period. Experiments using commercially available reference compounds, quantitation studies, and some HTS applications were conducted to evaluate and compare the baseline performance of the compact mass detector. The results of these studies were used to determine the overall feasibility, robustness, and flexibility of this novel technology within an OA environment as set forth by the co-development collaboration between Merck and Waters Corporation. 2. Materials and methods 2.1. Instrumentation A mass spectrometer consists of a single quadrupole in which electrical potentials of RF and DC are applied to opposite pairs of a linear array of four parallel cylindrical rods, where ions are separated by mass-to-charge (m/z) ratios and filtered depending on their trajectory in the oscillating electrical fields which are applied to the cylindrical rods. This research explores a novel technology typically utilized in higher-end triple quadrupole mass spectrometers but employed with a thought-free user interface design to easily attain electrospray based nominal mass measurements. The concepts of tuning parameters, manual calibrations, and various ionization choices have been eliminated to facilitate a fitfor-purpose practicality of providing masses for a detected analyte analogous in workflow and visual outputs of a simple UV detector and wavelength determination. For this reason, the instrument will be referred to as a mass detector due to the streamlined approach and simplified user-interface when compared to those user-inputs required for a traditional mass spectrometer. The size comparison of the mass detector, to that of a traditional mass spectrometer further lend itself to being defined as a compact mass detector, since the footprint is more comparable to that of an optical detector. The compact mass detector design utilizes dual off-axis ion guides for elimination of neutral noise to provide increased detection limit and robustness. The design incorporates a conjoined stacked ring ion guide and second stage quadrupole ion guide (Fig. 1). The
Fig. 1. Cross-sectional view of the compact mass detector ion guide and analyzer technology (compliments of Waters Corporation).
design reduces contamination, which contributes to improvement in method robustness where complex matrices are being analyzed. 2.2. Open access instrument configurations Experiments were performed using ACQUITY H-Class UPLC and ACQUITY ‘Classic’ UPLC instrumentation (Waters Corporation, Milford, Massachusetts, USA). The ACQUITY H-Class was configured with a sample organizer for increased sample capacity required during high throughput screening assessments. Each UPLC configuration was coupled with a Waters QDa compact mass detector. An ACQUITY SQD (Waters Corporation) mass spectrometer coupled to the UPLC configurations was used as the traditional mass spectrometer for comparative assessments. MassLynx Open Access OpenLynx 4.1 (Waters, Milford) application manager software was used to enable walk-up functionality and data visualization. The UPLC systems were configured with ACQUITY BEH C18 columns (1.0 mm × 50 mm; 1.7 m) on both the ACQUITY H-Class and on the ACQUITY Classic UPLC. Each column compartment was maintained at 50 ◦ C. The ACQUITY H-Class automatically metered the mobile phase for a linear gradient elution composition of 95:5 to 5:95 (0.05% TFA/water: 0.05% TFA/acetonitrile) over 2.0 min. The ACQUITY ‘Classic’ UPLC used a linear gradient method composition of 95:5 to 5:95 (0.05% TFA/water: 0.05%TFA in acetonitrile). The flow rate was maintained at 0.3 mL/min. The injection volume of each sample was 1.0 L. 2.3. Chemicals Acetonitrile and methanol (LC/MS Optima Grade) were obtained from Fisher Scientific (FairLawn, NJ, USA). Sulfadimethoxine, terfenadine, verapamil, leucine-enkephalin, 2-fluro-5-trifluromethoxy phenyl acetic acid and L-1-BOC-nipecotic acid were obtained from Sigma–Aldrich (St. Louis, MO, USA). Human insulin was obtained from FUJIFILM Diosynth Biotechnologies (Morrisville, NC, USA). (R)-
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4-oxo-4-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4][4,3a]pyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenyl) butan-2-aminium (sitagliptin) and (R)-3-amino-1-(3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4][4,3-a]pyrazin-7(8H)-yl-5,5,6,6-d4)-4-(2,4,5trifluorophenyl)butan-1-one (tetra Deutero-labeled sitagliptin) were synthesized at Merck Research Laboratories. (Rahway, NJ, USA) QC 96-well plates were obtained from Chembridge Inc. (San Diego, CA, USA). 2.4. Standard and sample preparation Standard stock solutions of each compound of interest were prepared at a concentration of 10 mM in methanol. A series of standard solutions, 0.001, 0.005, 0.01, 0.05, 0.1, 0.25, and 0.5 g/mL were prepared by dilution of the stock solution. For the quantitation studies, stock solutions of sitagliptin and internal standard (IS, tetra deutero-labeled sitagliptin) were prepared at 10 mM and 2 mM in methanol, respectively. A series of sitagliptin standard solutions, 0.01, 0.05, 0.10, 0.50, 1.00, 2.50, and 5.00 M were prepared by dilution of the stock solution. Each dilution was spiked with 0.05 M tetra-deutero labeled sitagliptin as internal standard. 2.5. MS parameters ESI MS analysis was performed on the compact mass detector in both positive-ion and negative ion full scan modes during the “applied use” robustness assessment and HTS testing. The compact mass detector and traditional mass spectrometer were operated in ESI-SIR positive mode for the linearity and dynamic range assess-
3
ments and comparison testing. Electrospray ionization capillary voltages and probe temperatures were 800 V and 600 ◦ C for the compact mass detector, and 3000 V and 450 ◦ C for the traditional mass detector in positive mode. Both systems used the same cone voltage, 20 V. Source temperatures were 120 ◦ C and 140 ◦ C for the compact MS detector and traditional mass spectrometer, respectively. The ESI-SIR parameters were the same for both systems and optimized for the signal-to-noise ratio of target ion signal. Quantitation studies using an internal standard were performed on the compact mass detector in ESI-SIR positive mode. MS analysis of human insulin was performed on the compact mass detector and traditional mass spectrometer (SQD) in full scan ESI positive mode.
3. Results/discussion 3.1. Performance assessment of the compact mass detector 3.1.1. “Applied use” robustness assessment The UPLC coupled to the compact mass detector was run in a “test” setting over the course of approximately 1 month, during which time over 700 small molecule samples were run by multiple discovery scientists, spanning 13 different internal research programs. System uptime was determined to exceed 95%, with no mechanical issues reported. The only downtime during this testing period was intentional (at the onset) to make minor modifications to instrument settings in order to minimize the presence of sodium adduct [M+Na]+ signal intensities. During this testing phase the compact mass detector was evaluated to verify that typical and
Fig. 2. Signal-to-noise evaluation of sulfadimethoxine (A), terfenadine (B), verapamil (C) at 1 ng/mL and 0.05 g/mL for leucine enkephalin (D) in support of LOD assessments for the compact mass detector.
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Fig. 3. Detection of sulfadimethoxine by ESI-SIR for the traditional MS spectrometer. Signal-to-noise assessment at 1 ng/mL for the traditional MS spectrometer.
reliable results were being generated for research compounds containing a multitude of functional groups including halogenated species, where characteristic isotopic patterns are regularly used for identification purposes. In all instances, the experimental data generated using the compact mass detector proved reliable and comparable to data generated side-by-side with current OA UPLCMS systems. 3.1.2. Linearity and dynamic range assessment Four commercially available compounds (sulfadimethoxine, terfenadine, verapamil and leucine-enkephalin) were analyzed at various concentration ranges and calibration curves were generated. Each compound was quantified separately and three replicas for each concentration were evaluated. The use of single ion recording (SIR) mode enhanced the measurement of target masses and reduced the interferences during quantitation. The mean calibration curves gave correlation coefficients of R2 = 0.9946, 0.9828, and 0.9930 for sulfadimethoxine, terfenadine and verapamil, respectively, over the concentration range of 0.001–0.50 g/mL. Leucine-enkephalin showed good linearity (R2 = 0.9990) over the 0.05–5.0 g/mL concentration range. Reproducibility for the compact mass detector across the linearity range over multiple injections (N = 3) resulted in RSDs of typically less than 5%, with slightly larger RSDs appearing at the lower concentration limits where system linearity begins to degrade. Specific limits of detection (LOD) values for terfenadine, verapamil, sulfadimethoxine and leucine-enkephalin were not formally obtained since the lower concentration limit tested for each compound was still above the required LOD S/N = 3 (Fig. 2). 3.1.3. Comparison of the compact mass detector versus the traditional mass spectrometer Sulfadimethoxine was analyzed at various concentration ranges and a calibration curve was generated on a similar walk-up instrument with a traditional mass spectrometer. The traditional mass spectrometer performed slightly better than the compact mass detector yielding a correlation coefficients of 0.9996. Data reproducibility on the compact mass detector appeared to be slightly better than the traditional mass spectrometer for most concentrations tested, except at the lower concentration range where the compact mass detector gave a 9.47% RSD and the traditional mass spectrometer resulted a 4.74% RSD. While this discovery is significant enough to mention, typically research conducted in
the discovery and early development space is not compound limited, and sample preparation rarely occurs in the sub-micro molar concentrations. Comparison of the SIR chromatograms for sulfadimethoxine run on the compact mass detector (Fig. 2) and on the traditional mass spectrometer (Fig. 3) clearly shows that the compact mass detector has a cleaner baseline. Visual comparisons of the linearity data, signal-to-noise, and LOD values are usually fit for purpose and allow scientists to determine if an instrument is a suitable and equivalent substitute. The values of the linearity data from both instruments were further explored from a statistical perspective via Bland–Altman techniques [10]. The regression-based difference plot constructed from the terfenadine linearity data (average mean of n = 3) suggest a high correlation with an R2 = 0.9916 (Fig. 4). Re-plotting the dataset in Fig. 4, where the difference (SQD − QDa) is plotted against the average (QDa + SQD)/2 allows for easier observations of outliers and trends as it relates to the error and bias of the results. The resulting Bland–Altman plot suggests good agreement at a 95% confidence interval (Fig. 5). All points are within the upper and lower limits of agreement with no observed outliers or trends.
Fig. 4. Comparison of QDa and SQD responses for terfenadine measured over the concentration range of 0.001–0.50 g/mL.
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5
10000
5000
0 0
50000
100000
150000
200000
250000
300000
QDa - SQD
-5000
-10000
-15000
-20000
-25000
(SQD + QDa) / 2 Fig. 5. Bland–Altman plot comparing the difference vs. the average of the terfendine linearity results.
3.1.4. Quantitation using an internal standard assessment Quantitation and calibration (mean = 3) of Merck compound sitagliptin, with isotopically labeled (tetra deutero-labeled) internal standard by ESI-SIR for a concentration range of 0.01–5.0 M using the compact mass detector is shown in Table 1. The data generated for the calibration curve was linear, with a correlation coefficient of R2 = 0.9993. The slope and intercept describing the relationship were found to be 24.65 and −0.327 respectively. Additionally, sample data generated for a 0.5 M sample was shown (Fig. 6) to be accurate, demonstrating the ability for the compact mass detector to also be utilized in the early discovery space for quantitative applications. 3.1.5. Biopharmaceutical detection of multiply charged ions One of the initial concerns for the compact mass detector (m/z = 1250 Da max) was the limited mass range as compared to more traditional mass spectrometers currently populating the walk-up environment, which typically cover a wider range (up to m/z = 4000 Da). Injections of Human insulin on the traditional mass spectrometer did provide more comprehensive spectral data (Table 2) as shown by the 3 multiply charged species (Fig. 7A) versus only 2 multiply charged species on the compact mass
Fig. 6. Quantitation of sitagliptin spiked with isotopically labeled internal standard by ESI-SIR using compact mass detector MS detector. SIR chromatograms of sitagliptin and internal standard at 0.5 M.
Table 1 Quantitation results of sitagliptin (MK-0431) with internal standard. Conc. (M)
(ASG /AInt ) (Injection 1)
(ASG /AInt ) (Injection 2)
(ASG /AInt ) (Injection 3)
Mean n=3
Std. dev n=3
% RSD n=3
0.01 0.05 0.10 0.50 1.00 2.50 5.00
0.243 1.282 2.222 12.656 22.382 65.633 123.472
0.283 1.216 2.367 12.475 21.664 60.408 121.598
0.284 1.216 2.215 11.516 22.073 62.784 122.504
0.270 1.238 2.268 12.216 22.040 62.942 122.525
0.023 0.038 0.086 0.613 0.360 2.616 0.937
8.66 3.08 3.78 5.02 1.63 4.16 0.76
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Table 2 Observed charge states of insulin. Human insulin MW 5803 [Da] System
Mass scan range
[M+3H]3+ Da
[M+4H]4+ Da
[M+5H]5+ Da
[M+6H]6+ Da
Traditional MS Compact mass det.
50–1900 Da 50–1250 Da
n/a n/a
1452
1162 1162
968 968
Fig. 7. Multiple charged states of human insulin by (A) traditional MS and (B) compact mass detector.
detector (Fig. 7B). Multiply charged species were however still easily identifiable with the compact mass detector. 3.2. High throughput screening (HTS) assessments Much of the work performed in the early discovery and development space requires rapid processing and data analysis turnaround times in order to meet stringent timelines. High throughput screening is vital in this arena and as such we placed the compact mass detector into our OA workflow to see how it performed. In HTS, the OA system is equipped with an expanded capacity sample management module to enable multiple sample plates to be placed in the queue and run as part of the automated process initiated by the analyst. Software modifications enable processing parameters to be tailored to meet desired “pass/fail” criteria so that rapid assessments via color-coded “hits” based on the mass data can be made instantaneously without having to sift through the multitude of individual sample results. The compact mass detector performance in process optimization, identification, and library QC projects was also evaluated. 3.2.1. Catalysis screening and process optimization As a process optimization tool, the compact mass detector based OA platform was employed for a difficult cross-coupling reaction (Fig. 8), where initial experimental attempts running standard literature procedures by chemists at the bench resulted in no desired
product. A catalyst screening plate (4 × 6) was set up and run through the HTS work flow. Identification of “hits” shown in green (A1; A2, D2) was immediately possible via this simplified output, which enabled chemists to rapidly focus attention on those catalysis conditions that afforded positive results. These hits were then further elaborated into a scalable solution that could be successfully run in the synthetic labs to support discovery research projects. 3.3. HTS library QC comparison A 96-well compound library was acquired from ChemBridge Inc., in order to evaluate both mass measurement systems via the typical OA-login procedure in a discovery laboratory. The objective was to evaluate the ‘hit’ ratio for the target compounds for plates from each instrument and investigate differences in the resulting reports. Results for the plate screening for each mass measurement instrument are shown (Fig. 9). The discrepancies between red visual indicators and green indicators for each of the instruments results were investigated. Differences were observed regarding retention time, peak width, background noise, and ion fragmentation patterns. The retention times observed in the conventional mass spectrometer were later than the retention times observed for the compact mass detector (Fig. 10). This difference can be explained by the lower post extra-column volume provided by the compact mass detector probe. The lower post extra-column volume on the compact mass detector also explains the lower observed
Fig. 8. Catalysis reaction screening with plate results.
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Fig. 9. Traditional mass spectrometer vs. compact mass detector results for a QC HTS plate screening. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
bandspread of the analyte peak (Fig. 11). The traditional mass spectrometer yielded a peak width of 0.036 min (2.16 s measured with 12 scans per peak) while the compact mass detector yielded a peak width of 0.021 min (1.26 s with 14 scans per peak). The stock solution of each of the samples was diluted in 100% DMSO, and the plate was generated using dilutions of the stock solution in a 1:10 ratio with methanol, therefore it was expected that a high concentration of DMSO would be visible in the total ion chromatographic trace (TIC). Interestingly, the lower background noise for the compact mass detector resulted in a lower interference from the DMSO diluent than that observed by the traditional mass spectrometer (Fig. 10). It is hypothesized that the unique design of the compact mass detector and location of the point of ionization contribute to this significant decrease in observed background noise. Investigating the spectral patterns of the compounds from each set of results also revealed some unique differences. The traditional mass spectrometer yielded greater fragmentation for numerous target analytes. The increased fragmentation of some of the target analytes yielded ‘not found’ results for the targeted mass, whereas the compact mass detector yielded a ‘hit’ result for the same analytes in many cases. The two instrument designs apply the ‘cone’ voltage differently as described by the vendor and therefore it
was expected that more fragmentation would occur within the compact mass detector if the settings were matched to the same settings as the traditional mass spectrometer. The compact mass detector applies the cone voltage downstream in the ion path as compared to that of the conventional mass spectrometer, which is applied near the orifice, hence the need for different method parameter settings between the instruments. Instrument method settings for the compact mass detector were set to the defaults, while cone voltage settings for the traditional mass spectrometer were varied at decreasing voltages of 20, 15, and 10 V to inhibit in-source collision induced dissociations from occurring, and therefore try to best match those results generated by the compact mass detector. Unfortunately, the in-source collision induced dissociation occurred in the traditional mass spectrometer even at the lower voltages and thus resulted in an overall lower hit rate due to lower intensity mass spectral measurements for some of the target compounds. It should be noted that three control samples were inserted into the plate to yield ‘not found’ results. These are represented by the three red visual indicators at the end of the bottom three rows of each plate. These three controls were empty wells and indicated in the software to find a mass that did not exist in the well. This concept was initiated to validate the comparisons
Fig. 10. Total ion chromatogram comparison of the compact mass detector (top) and the traditional mass spectrometer (bottom) for a given analyte in the ChemBridge QC HTS plate. Retention time shift due to volume differences and lower DMSO and background noise are observed.
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Fig. 11. Extracted ion chromatogram for the targeted analyte measured at m/z—329 Da for each instrument used to determine the differing effects of extra-column volume of the probe configurations on peak width.
between the data collection accuracy and data processing between the two instrument HTS experiments.
4. Conclusion The comprehensive assessment and evaluation of this novel compact mass detector succeeded in demonstrating that the instrument meets the needs and addresses the challenges facing a pharmaceutical drug discovery team. The compact mass detector provides a higher level of simplicity to the OA environment versus traditional single quadrupole mass spectrometers on the market. Initial set-up, calibrations, and MS setting adjustments are all but eliminated for the compact mass detector. Mass data generated with the compact mass detector were comparable to conventional mass spectrometer data for a multitude of samples run in the OA environment over the course of this evaluation. In some cases, the performance of the compact mass detector surpassed expectations, as in the case of the library QC plates where minimal in-source fragmentation occurred for the compact mass detector and resulted in a more accurate overall plate assessment as compared to more traditional mass spectrometers. In the biopharmaceutical assessment of insulin, where the limited MS scan range of the compact mass detector was an initial concern, adequate data for multiply charged species was produced. Coupling the compact MS detector with standard deconvolution software could therefore expand its capabilities to include a wide variety of larger molecular weight applications not explored herein. Use of the compact mass detector for quantitation type applications, as was the case for the sitagliptin studies, was also shown to be accurate and beneficial. Overall, the compact mass detector has proven to be a valuable and reliable mass detection and compound confirmation tool for the open access environment in the pharmaceutical discovery and development space where speed and uptime is crucial, unit mass resolution is a necessity, and sample availability is rarely a concern. The benefits of the compact mass detector for use in a wide variety of applications including standard small molecule analysis, HTS and QC type analyses, as well as biological samples and quantitation studies have been demonstrated.
Acknowledgements The authors would to acknowledge Alexey Makarov, Yong Liu, Ray McClain and Chris Welch at Merck for their support and contributions to our discussions. We would also like to acknowledge Eva Gallea, Ed Aig, Howard Read, and the many folks at Waters Corporation who helped in various capacities to advance the development of this new technology. References [1] S. Caron, N. Murray Thomson, Pharmaceutical process chemistry: evolution of a contemporary data-rich laboratory environment, J. Org. Chem. 80 (2015) 2943–2958. [2] S. Khater, C. West, Development and validation of a supercritical fluid chromatography method for the direct determination of enantiomeric purity of provitamin B5 in cosmetic formulations with mass spectrometric detection, J. Pharm. Biomed. Anal. 102 (2015) 321–325. [3] D. Spaggiari, F. Mehl, V. Desfontaine, A. Grand-Guillaume Perrenoud, S. Fekete, S. Rudaz, D. Guillarme, Comparison of liquid chromatography and supercritical fluid chromatography coupled to compact single quadrupole mass spectrometer for targeted in vitro metabolism assay, J. Chromatogr. A 1371 (2014) 244–256. [4] G.F. Verbeck, V.M. Bierbaum, Focus on harsh environment and field-portable mass spectrometry: editorial, J. Am. Soc. Mass Spectrom. 26 (2014) 199–200. [5] X. Bu, J. Yang, X. Gong, C.J. Welch, Evaluation of a compact mass spectrometer for routine support of pharmaceutical chemistry, J. Pharm. Biomed. Anal. 94 (2014) 139–144. [6] S.E. Hamilton, F. Mattrey, X. Bu, D. Murray, B. McCullough, C.J. Welch, Use of a miniature mass spectrometer to support pharmaceutical process chemistry, Org. Process Res. Dev. 18 (2014) 103–108. [7] A. Coddington, J. Van Antwerp, H. Ramjit, Critical considerations for high-reliability open access LC/MS, J. Liq. Chromatogr. Related Technol. 26 (2003) 2839–2859. [8] L.O. Hargiss, M.J. Hayward, Use of an electrospray time-of-flight mass spectrometer for a broad range of open access applications including accurate mass analysis for elemental compositions, in: Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, United States, August 26–30 ANYL-149, 2001. [9] A. Malcolm, S. Wright, R.R.A. Syms, R.W. Moseley, S. O’Prey, N. Dash, A. Pegus, E. Crichton, G. Hong, S.A. Holmes, A. Finla, P. Edwards, S.E. Hamilton, C.J. Welch, A miniature mass spectrometer for liquid chromatography applications, Rapid Commun. Mass Spectrom. 25 (2001) 3281–3288. [10] D.G. Altman, J.M. Bland, Measurement in medicine: the analysis of method comparison studies, J. R. Stat. Soc. Ser. D 32 (1983) 307–317.
