Automated open-access liquid chromatography high resolution mass spectrometry to support drug discovery projects

Journal of Pharmaceutical and Biomedical Analysis 178 (2020) 112908

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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba

Automated open-access liquid chromatography high resolution mass spectrometry to support drug discovery projects Alberto Fontana a,∗ , Laura Iturrino a , David Corens b , Antonio L. Crego c,∗ a

Analytical Sciences, Janssen Research & Development, A Division of Janssen-Cilag, S.A. C/Jarama 75A, 45007, Toledo, Spain Analytical Sciences, Janssen Research & Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, B-2340, Beerse, Belgium Department of Analytical Chemistry, Physical Chemistry, and Chemical Engineering, Faculty of Sciences, University of Alcalá, Ctra. Madrid-Barcelona, Km. 33.600, 28871, Alcalá de Henares, Madrid, Spain b c

a r t i c l e

i n f o

Article history: Received 7 June 2019 Received in revised form 20 September 2019 Accepted 1 October 2019 Available online 5 October 2019 Keywords: Automation Open-access liquid chromatography Open-access high resolution mass spectrometry Drug Discovery

a b s t r a c t The need of a continuous productivity increases in medicinal chemistry laboratories of the pharmaceutical industry motivated the development, over the years, of new software solutions to enable Open-Access in many analytical techniques such as NMR or LC, among others, to characterize and assess the purity of new molecules. These approaches have been widely spread in LC with low resolution MS systems, but similar automated platforms have been rather less explored with high resolution MS. In this work, an improved Automated Open-Access methodology on an UHPLC with DAD coupled to ESI and quadrupole time-offlight MS system is described. Detailed reports from standard UHPLC-MS runs containing chromatograms and different spectra (MS with different fragmentation) are automatically sent to the chemists. High resolution MS data is typically achieved within ± 1 mDa mass accuracy regardless of sample concentration. Upon training, chemists log-in samples into the system by selecting appropriate methods, being able to interpret the results by themselves in 95% of the cases. The instrument is working unattended, except for a limited number of samples (5%) which require more complex experiments. To the best of our knowledge, this is the first time a completely automated Open-Access LC-HRMS approach has been implemented for medicinal chemists of a pharmaceutical industry. © 2019 Elsevier B.V. All rights reserved.

1. Introduction The use of LC coupled to MS has been widely spread in many laboratories, being an essential tool to make decisions in a big number of scientific disciplines [1,2]. Over the years, the need of the industry for doing rapid molecular mass assessment using MS has been addressed by the development of Open-Access tools that allow access of non-expert users to the LC–MS instrumentation. Since its introduction in the early 1990´s, these Open-Access tools have been typically developed for medicinal chemists enabling them to introduce their samples on the systems by applying generic conditions set by the LC–MS specialists. Once the samples are analyzed, automated reports are typically sent by email to the users allowing the chemists to interpret the data on their own. Obviously, the LC–MS Open-Access environment increased very significantly the throughput in many chemistry laboratories [3–6]. These Open-

∗ Corresponding authors. E-mail addresses: [email protected] (A. Fontana), [email protected] (A.L. Crego). https://doi.org/10.1016/j.jpba.2019.112908 0731-7085/© 2019 Elsevier B.V. All rights reserved.

Access platforms have been commonly used with low resolution MS (LRMS) systems in early drug discovery for reaction monitoring and for purity assessment of samples synthesized by medicinal chemists from the pharmaceutical industry. In recent years, OpenAccess approaches on LRMS systems are still being reported [7,8]. Opposite to this, on high resolution MS (HRMS) systems, this methodology has not been so widely spread. In the past, the reason of the limited Open-Access HRMS applications could have been related to its limitations, as the technique was not fully reliable with old HRMS instruments. For instance, mass accuracy on these systems was affected by laboratory temperature fluctuations. Also, only a narrow dynamic range was allowed: analyte concentrations had to be in the same order compared to the concentration of the reference material used for correcting mass deviation. As result, on old HRMS instruments, the acquisitions frequently caused oversaturation in MS peaks producing low mass accuracy and isotopic pattern disturbances, so the HRMS had to be searched on scans not belonging to the maximum area for the MS peak. Specially, this automation was not feasible on highly concentrated samples from medicinal chemistry laboratories (typically 0.1 mg/mL or 0.3 mM for an average molecular mass of 300 umas) as this concentration

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is needed for the DAD purity determination that is also assessed together with the HRMS. During the 2000 decade, only three Open-Access HRMS works were reported. In 2004 Sirtori [9] developed an automated method for HRMS determination with a quadrupole time-of-flight (QTOF) for compounds from a medicinal chemistry department. Flow injection mode (FIA) was run on samples with concentrations of 130 nM. The workflow generated an excel data sheet through automated processing within accuracies of ±5 ppm only for 70% of the samples analyzed. Thomas [10], also in 2004, reported an Open-Access LC-HRMS set up for medicinal chemists with high concentrated samples (around 0.1 mg/mL) achieving mass accuracies between ± 3 mDa and ±5 ppm for small molecules either by FIA-HRMS or by LC-HRMS analysis. The protocol used allowed the chemist either to generate and interpret data for themselves or otherwise to provide high-quality data which they can review with an expert. However, the workflow followed was not automated; to obtain a reduced number of possible formulas, chemists needed to manually limit the number of elements present in the target molecule to be confirmed by HRMS. Also, the process required manual combination of a number of scans from the acquisition spectra to obtain lists of feasible elemental compositions. Finally, in 2007 Williams [11] reported a protein Open-Access LC-HRMS using a TOF platform. Automated processing protocols were applied, resulting in an email confirmation to the chemist if the sample was auto-approved. For samples submitted for final quality control, mass measurement accuracy within ±60 ppm was typically achieved throughout the 10.000–100.000 Da mass range. During the 2010 decade, HRMS instruments have continuously evolved during the years increasing their performance and capabilities. In new instruments, resolution power has continuously increased around 1 to 5-fold compared to older systems, allowing mass accuracies currently below ±2 ppm and mass-scale drift produced by laboratory temperature fluctuations are no longer affecting the data. Dynamic ranges have also been increased and thus, MS oversaturation affects the accuracy of the measured accurate mass ions to a lesser extent: analyte concentration can vary 4–5 orders of magnitude compared to the concentration of the reference material. Together with the evolution of the HRMS instrumentation, there has been a big transformation in many HRMS software packages which have significantly increased automation on data processing. Currently, many LC-HRMS vendors offer a diverse number of software tools to perform automated complex MS determinations on systems typically developed for biological samples, with compounds in concentrations of ␮g/mL range. In this way, in 2014, Taibon [12] reported a UHPLC-QTOF method for qualitative and quantitative assessment of destruxins, which are bioactive metabolites produced by fungal culture broths. Also, Llorca, in 2016 [13] described an LC–MS with Orbitrap mass spectrometer approach for the screening of a number of pharmaceuticals and their known transformation products followed by data processing with specific software and manual confirmation to correct any possible misinterpretation of the data. All these systems are used by specialized LC–MS teams and have not been reported in an Open-Access environment for synthetic chemists. Thus, on new instruments, highly concentrated samples from medicinal chemistry laboratories could still require a method development to obtain fully automated HRMS in combination with the DAD purity assessment. The additional technological advances on new HRMS instruments have provided the development of powerful analytical methods. In addition to an MS/MS experiment that can be run on a QTOF, new systems allow to perform MS experiments applying different energies at the collision cell prior to the analysis in the TOF. On these MS experiments, first a low collision energy is used on a wide mass range to obtain molecular ions and secondly, a

high collision energy is used to produce fragment ion data, which is often essential for structure elucidation. The former MS acquisition with high fragmentation would be equivalent to an MS/MS so hereafter will be named in this manuscript as “pseudo MS/MS”. Thus, Plumb in 2006 [14] reported on a QTOF system this strategy for biomarker structure elucidation. In 2013, Ramirez-Ambrosi [15] reported a QTOF system to improve the characterization of phenolic compounds in complex plant samples applying two collision energies. Some of the structures identified through the fragmentation obtained with the pseudo MS/MS acquisition had not been reported before on MS-TOF experiments. Likewise, in 2013, Rosano [16] published a toxicology application comparing the detection rate of postmortem drugs with the pseudo MS/MS acquisition on a QTOF with the MS/MS on a tandem triple quadrupole mass detector. In that work, the benefit of the pseudo MS/MS on not targeted screening for unknown drugs was shown, increasing the detection rates from a 72% with MS/MS to a 99% with this strategy. In this paper, the implementation of an automated Open-Access methodology on an UHPLC coupled to HRMS using a QTOF system for samples coming from medicinal chemistry programs at Janssen pharmaceutical laboratories is described. Different MS methods were developed to achieve a universal MS method valid for a huge diversity of compounds, typically prepared on a concentration of 0.1 mg/mL. MS experiments, applying two collision energies, allow chemists users both to confirm HRMS of the compounds obtained from the synthesis and to interpret the fragment ions on them. To the best of our knowledge this the first time that a full automated type of Open-Access platform is being used by end users on a UHPLC-HRMS system in a medicinal chemistry program of a pharmaceutical industry. 2. Experimental 2.1. Chemicals and samples Deionized water (18 Mcm) was produced by a Milli-Q® Integral 5 system (Millipore SAS, Molsheim, France). Acetonitrile and Methanol for LC MS (LiChrosolv® ) were purchased from MercK (Darmstadt, Germany). Leucine Enkephalin and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA). A quality control sample (QC) that was reported in 2011 by our group [17] was used to test the configuration and conditions on the UHPLCQTOF system. All compounds of the QC sample were obtained from Sigma-Aldrich (see Table 1). It was composed of seven standards (0.1 mg /mL each). The solution was stored in an amber 30 mL vial refrigerated at 5  C, for one month without showing significant degradation. Finally, samples from Medicinal Chemistry were dissolved in either methanol or acetonitrile and were filtered through 0.2 ␮m filters. Concentration was around 0.1 mg/mL. 2.2. Instrumentation conditions The UHPLC measurements were carried out using an IClass UPLC® system from Waters® (Milford, MA, USA) equipped with a binary solvent delivery pump, an autosampler with flow through needle injector, a column compartment with 2 column positions, a DAD detector and a sample organizer module for sample introduction into the system. A standard reversed phase gradient was performed on an Acquity UPLC® BEH C18 column (2.1 mm x 50 mm, 1.7 ␮m) from Waters® (Milford, MA, USA) at 50  C using as components of the mobile phase: a solution of 6.5 mM ammonium acetate in water with 5% acetonitrile (A) and acetonitrile (B). The UHPLC method was used for purity assessment of final compounds before sending for biological screening. It consisted on a 5 min run. Gradient com-

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Table 1 Structure and properties of the compounds from the QC mixture (values of log P and pka were obtained with ACD / Percepta software). o

N

Compound / Structure

Formula and Molecular Mass

pKa

Log P

1

C8 H10 N4 O2 194.0804

0.5

0.28

2

C6 H5 NO3 139.0269

7.2

3

C14 H11 ClN2 O4 S 338.0128

9.6 / 10.8

0.41

4

C19 H16 O4 308.1049

4.5

3.11

5

C21 H23 ClFNO2 375.1401

8.0 / 13.9

6

C15 H10 O2 222.0681

–

3.42

7

C15 H16 O2 228.1150

–

2.92

position was: 5% B at initial conditions, to 95% B at 4.6 min, kept for 0.4 min. Re-equilibration of the system was achieved by a 0.5 min pre-run. Other conditions were: a flow rate at 1.0 mL/min, giving 12,500 psi at initial conditions, an injection volume of 1 or 2 ␮L, and the DAD scanned from 200 to 450 nm with a 2.4 nm resolution at a scan rate of 40 points per second. The MS detector coupled to the UHPLC system was the Xevo® G2-S QTOF (Waters® , Milford, MA, USA). It was equipped with an ESI interface and a second, orthogonal, LockSprayTM probe for TOF mass correction. MS Data were collected in centroid mode at positive or negative polarity with the LockSprayTM frequency set at 10 s and averaged over 3 spectra for 1 s. Both polarities were performed by default to all compounds with ionization voltage between 0.25 kV and 2 kV for ESI + and 2 kV for ESI-. Nitrogen was used as cone gas and as desolvation gas at flow rates of 80 L/h or 250 L/h and 600 L/h, respectively. Desolvation temperature of 200  C or 400  C was used. Sampling cone voltage was 25 V for both positive and negative ionization modes. A solution of Leucine Enkephalin (1 ng/␮L) in 1:1 acetonitrile/water was used as MS internal calibrant in both positive and negative modes (ESI+: m/z [M+H]+ = 556.2771 and a fragment of m/z = 278.1141; ESI-: m/z [M−H]− = 554.2615 and a fragment of m/z = 236.1035). An isocratic pump (HP1100, Agilent Technologies, Waldbronn, Germany) was used to pump the calibrant at 0.3 mL/min flow, which was splitted to bring 20–30 ␮L/min into the MS system. The MS resolution obtained was typically 40.000. Two MS experiments, MS and MS/MS were developed on both polarities. The MS one was used routinely for most of the compounds analyzed. Acquisition mass range was m/z 50 to 1200 at 0.1 s per scan. Both experiments employed two collision energies in the collision cell: (a) low collision energy to obtain molecular ions without fragmentation using 6 eV; and (b) high collision energy to obtain the fragment ions data using an energy ramp

1.71

3.48

Ions ESI+: [M+H]+ (low detection)

ESI-: no detection ESI+: no detection ESI-: [M-H]− ESI+: [M + Na]+ ; [M+H]+ and [M-H2 O]+ (low detection)

ESI-: [M-H]ESI+: [M+H]+

ESI-: [M-H]− ESI+: [M+H]+

ESI-: [M-H]− and [M + AcO]− ESI+: [M+H]+

ESI-: no detection ESI+: [M + Na]+ ; [M+H]+ (low detection) ESI-: no detection

from 10 to 50 eV. Argon was used as collision induced gas at 0.5 bars. 2.3. Software Data was acquired under WindowsTM 7 running MassLynxTM / OpenLynxTM 4.1 software (Waters® , Milford, MA, USA). MassFragment 3.0 was also installed to allow interpretation of fragment ions by the LC–MS expert if needed. To let users to log-in samples, OALogin was installed on a standalone pc connected to the acquisition pc. The electronic notebook software (ELN) from Janssen was also installed on the log-in pc, to obtain the exact monoisotopic mass of the expected compound. The mass is introduced on the login process by chemist users either for exact mass confirmation on the MS or for obtaining specific fragment ions from a precursor ion. OpenLynx Browser 4.1 was installed at chemist´s PCs to allow them to visualize the reports that had been automatically sent. 2.4. Open-Access methodology Open-Access methods are developed by LC–MS specialists to allow chemists to use a combination of different LC–MS acquisition conditions. On these methods, generic parameters must be defined to allow an automatic processing of the data. The most important ones are: the number of scans to be combined at the top of each MS peak, the delay for the MS peak with the corresponding DAD one to provide correct identifications of the ions reported at each DAD peak (especially important for doing correct identification of partially co-eluting components present in a sample), the different adducts that are feasible and the thresholds for the integration of the peaks and for the ion abundances (to allow the automatic identification of the expected compounds). Once a run is finished, a report is generated through an automated processing of the data.

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In ESI mode, the presence of adducts and dimers due to the mobile phase compositions need to be considered. The most probable adducts and dimers have been included in the processing file to automatically identify all the related ions for an expected monoiosotopic molecular weight. These are for positive mode, [M+H]+ , [M + Na]+ , [M + NH4 ]+ , and [2M+H]+ , and for negative mode, [M−H]− , [M + AcO]− and [2M−H]− . For small molecules coming from synthetic chemistry, typically only mono-charged ions are detected, so m/z corresponds to the measured accurate mass ions.

Table 2 MS conditions with ESI + for the different parameters (cone voltage was kept 25 V and desolvation gas flow was kept 600 L/h). Changes of conditions in each of them are highlighted in a box.

3. Results and discussion The pharmaceutical compounds are evaluated in drug discovery by LC–MS using the quantification of DAD relative peak areas to estimate a relative purity for the different compounds in a mixture. Purities of 95% by DAD are considered the standard criterion for acceptance of biological screening of the compounds. Medicinal chemists in general use concentrations around 0.1 mg/mL that yield adequate absorptions in UV to determine the purity of a compound, but this concentration give relatively high response in MS which could cause oversaturation in the HRMS detection. Therefore, the implementation of a completely automated Open-Access method that combines DAD and HRMS detection on these highly concentrated samples needs to be developed. 3.1. Implementation of an open-access LC-HRMS method The QC mixture described in Table 1 and a group of about twenty compounds from the Janssen library (their structures are not shown) were used to develop the HRMS conditions. The QC compounds have chemical properties that satisfy known pharmaceutical drug-like properties, according to the Lipinsky´s rules [18]. On one hand they cover a broad range of elution times and showed different response on their ionizations, thus it is possible to daily check both the column performance and to check the MS detection for different polarities. On the other hand, the m/z range studied with these seven compounds considered Lipinski´s rule regarding molecular mass for a compound to have good drug-like properties (a value adequate should be below 500). Initially, 1 mL/min was sent to the ESI interface using an ionization voltage of 2.0 kV, a cone voltage of 25 V, a desolvation temperature of 400  C, a desolvation gas flow of 600 L/h and a cone gas flow of 80 L/h. With this “starting method” the two polarities were tested. Very high mass accuracies at the top of the different peaks were obtained (precisions below ± 1 mDa) for all the compounds of the QC mixture. However, when using these MS conditions with the group of compounds from the Janssen library, oversaturation at the top of the MS peaks was observed in ESI + mode for 25–30 % of the compounds tested which required manual combination of non-oversaturated scans with lower intensities. This problem was not detected in ESI- runs due to lower response in this mode of ionization. Subsequent dilutions of the oversaturated samples in ESI + achieved an accurate mass determination below ± 2 mDa mass accuracy, confirming that the problem was related to high concentrations. Obviously, this approach was not valid for an Open-Access environment. To implement an efficient Open-Access process, reanalysis of samples should be avoided. Thus, new MS methods were tested upon modifications of several MS parameters (see Table 2) in order to reduce the intensity of ionization. First, a split of the mobile phase was configured allowing only 200 ␮L/min flow to get into the MS keeping the same MS starting parameters (named “new method A”). As one fifth of the amount of analyte was entering the MS a comparable intensity reduction was expected. Opposite to this, on the QC mixture, only 25% intensity

reduction was observed for good ionized compounds (like warfarine, haloperidol and flavone) while low ionized ones (caffeine, chlorthalidone and nabumetone) gave increased detection up to fifteen times. These results may be attributed to the better efficiency obtained in the ESI + ionization at low flows which have affected more significantly on the ionization for the former type of compounds. Therefore, the new method A was found to be appropriate only for low ionizable compounds (with low basicity) from the Janssen library that gave poor ESI + detection with the “starting method”. An example of the utility of this new method is shown in Fig. 1. A very low basic compound gave almost no detection on the “starting method” while the ionization was improved about twelve times when the “new method A” was used. Second, a “new method B” was evaluated to obtain a more significant reduction on the ionization, by keeping the split of 1/5 and applying the following MS changes: ionization voltage was decreased from 2.0 kV to 0.5 kV to reduce the charges on the spray, desolvation gas flow temperature was reduced from 400  C to 200  C to reduce the evaporation of the droplets and finally, cone gas flow coming in the opposite direction of the spray, was increased from 80 to 250 L/h to reduce the flow brought into the MS. All these modifications obtained about a 50% intensity reduction for all compounds of the QC mixture regarding the “starting method” and achieved automated good mass accuracies for 85–90 % of the compounds coming from the Janssen library. An example of the utility of this new method is shown in Fig. 2. As it is shown in this figure, the isotopic pattern obtained with the “starting method” seemed to belong to a compound with two chlorine atoms (about 64% in the abundance of the third peak of the isotopic pattern, due to 37 Cl), while the compound had only one chlorine. The “new method B” corrected the saturation of the ions and therefore, both the isotopic pattern and the mass accuracy were correctly reported (only 32% in the abundance of the third peak of the isotopic pattern). Finally, to obtain the desired mass accuracies for the other 10–15 % of the compounds, a “new method C” was developed by further reduction of the ionization voltage to 0.25 kV. Thus, as it is shown in Fig. 3, the “new method B” did not result adequate, being the peak for 13 C in the isotopic pattern (second ion) the most intense one, due to a drift in the mass of one uma, while the “new method C” corrected the saturation of the ions. By reducing the ionization, both an adequate isotopic pattern and a better mass accuracy were achieved. Good mass accuracies were obtained in the 98% of the reports when more than 400 compounds synthesized by chemists were analyzed by one of the three new methods described above. The “new method B” was the method valid for most of the samples. The “new method C” was used if oversaturation was observed on the “new method B”, resulting adequate for highly ionizable com-

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Fig. 1. TICs and spectra for a compound with low basicity and an elemental composition of C9 H6 BrIN2 O2 using different methods in ESI+: isotopic pattern and exact mass deviations (Da) from automatic Open-Access LC-HRMS reports.

Fig. 2. TICs and spectra for a compound with an arylic nitrogen and an elemental composition of C14 H14 ClN3 using different methods in ESI+: isotopic pattern and exact mass deviations (Da) from automatic Open-Access LC-HRMS reports.

Fig. 3. TICs and spectra for a very basic compound with an alkylic nitrogen and an elemental composition of C27 H36 N2 O3 using different methods in ESI+: isotopic pattern and exact mass deviations (Da) from automatic Open-Access LC-HRMS reports.

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Fig. 4. Comparison of S/N ratios on the different ESI + MS methods for nine compounds synthesized by Janssen chemists.

Fig. 5. TICs and spectra for a compound with an elemental composition of C25 H34 N2 O4 S using different methods in ESI+: isotopic pattern and exact mass deviations (Da) from automatic Open-Access LC-HRMS reports.

pounds with very basic alkylic nitrogens. Finally, the “new method A” was only used for low ionizable compounds, with electrowithdrawing groups such as amide or other less basic N (aniline, ␲-deficient heterocycles. . .), that did not give good ionization on “new method B” or when chemists suspected a low ionization for a given compound. The use of these three methods avoided the need of diluting a sample that previously would have failed in the initial standard MS conditions but implied to reanalyze a sample if the method chosen by chemists was not suitable. Therefore, implementation of a more universal method was searched to improve the effectiveness of our Open Access protocol. The “new method C” improved both the mass accuracy and isotopic patterns for the compounds that failed with the “new method B” but resulted in very low signal to noise ratio (S/N) for less ionizable compounds which could be even not integrated, and their spectra not reported. Thus, a new method should increase the S/N compared to the “new method C” while being less sensitivity than “new method B” to avoid signal oversaturation. The method that fulfilled both requirements was named “new method D” and consisted in preserving the same MS parameters of “new method C” except for cone gas flow value, which was decreased to 80 L/h, as it was initially set in the “starting method”, to allow the entrance of more ions into the MS. Fig. 4 shows a comparison of the S/N obtained for nine compounds from chemists taken from different chemical series (structures not shown). For the nine compounds, the “new method D” increased the S/N ratio from one to two orders of magnitude

regarding “new method C”. Also, the “new method D” provided S/N values very similar to the ones on “new method B”, allowing adequate identification of the signals as it is shown in Fig. 5. This final method D is called “universal Open-Access ESI + method”. On the other hand, although as discussed previously the oversaturation problem was not detected when working on ESI-, a more universal ESI- method was put in place in Open-Access mode, keeping desolvation temperature, cone gas flow and split respect to the universal ESI + method with an ionization voltage of 2 kV. Finally, the QC mixture was analyzed daily using both universal Open-Access methods to check reproducibility on the mass accuracy and on the retention time during four months without MS calibration in ESI + or ESI- modes. We would like to highlight the excellent reproducibly on the accuracy of the MS system for the QC mixture (see Table 3). The results show that the exact mass deviation for the compounds of the QC mixture either in ESI + or in ESI- were usually lower than ± 1 m Da in four months. In addition, it is important to note that both universal MS methods using two collision energies. Thus, medicinal chemists can visualize in the reports (see Fig. 6) both the DAD chromatogram with the purity (%) of a compound, the two total ion chromatograms (TICs) and the two MS spectra, which are the spectrum at low collision energy to obtain the accurate mass of the compound and the spectrum at high collision energy (pseudo MS/MS) to get the information on the different fragments that will help to confirm the compound analyzed.

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Table 3 Accurate mass for the most intense ion (Da), mass accuracy in parenthesis (mDa), average retention time (RT) for the compounds of the QC mixture (at 0.1 mg/mL), and theirs RSD (%). Month

Caffeine ESI+

pNitrophenol ESI-

Chlorthalidone Chlorthalidone Warfarine ESI+ ESIESI+

Warfarine ESI-

Haloperidol ESI+

Haloperidol ESI-

Flavone ESI+

Nabumetone ESI+

Jan

195.0878 (+0.1) 195.0879 (+0.2) 195.0880 (+0.3) 195.0878 (+0.1) 0.41 1.43 %

138.0194 (-0.2) 138.0194 (-0.2) 138.0193 (-0.3) 138.0193 (-0.3) 0.70 1.17 %

361.0028 (+0.7) 361.0028 (+0.7) 361.0027 (+0.6) 361.0029 (+0.8) 0.85 0.59 %

307.0971 (-0.5) 307.0969 (-0.7) 307.0971 (-0.5) 307.0977 (+0.1) 0.94 0.53 %

376.1482 (+0.8) 376.1480 (+0.6) 376.1486 (+1.2) 376.1478 (+0.4) 1.70 0.76 %

374.1320 (-0.8) 374.1330 (+0.2) 374.1325 (-0.3) 374.1318 (-1.0) 1.70 0.76 %

223.0758 (+0.4) 223.0758 (+0.4) 223.0758 (+0.4) 223.0758 (+0.4) 1.96 0.73 %

251.1046 (+0.3) 251.1048 (+0.5) 251.1044 (+0.1) 251.1044 (+0.1) 2.11 0.67 %

Feb Mar Apr RT RSDRT

337.0052 (+0.3) 337.0053 (+0.2) 337.0053 (+0.2) 337.0052 (+0.3) 0.85 0.59 %

309.1129 (-0.7) 309.113 (+0.8) 309.1126 (+0.4) 309.1130 (+0.8) 0.94 0.53 %

Fig. 6. Visualization of a complete Open-Access LC-HRMS report for a sample containing a main compound with elemental composition C22 H23 N7 O3 : DAD, TICs and the two MS spectra at low and high collision energies for the expected compound are shown.

3.2. Possibilities of implementation of MS/MS experiments in open-access LC-HRMS As described above, the pseudo MS/MS acquisition can be used by chemists to identify the different fragments for each compound from the spectrum at high collision energy on an MS experiment. Alternatively, conventional MS/MS experiments can also be applied. Both experiments typically provide very similar MS spectrum if the expected structure is present and the compound is well ionized, as shown in Fig. 7. In the spectrum at low collision energy, m/z signals for the [M+H]+ ion (compound with an elemental composition of C22 H28 N6 O3 ) were m/z 425.2300 on the MS experiment and m/z 425.2305 on the MS/MS experiment. The spectrum at high collision energy on both experiments gave a main fragment with elemental composition of C9 H10 NO2 (related to the loss of C13 H18 N5 O) at m/z 164.0711 on the MS experiment and 164.0708 on the MS/MS experiment. However, the use of each experiment has its own benefits or drawbacks depending on the sample that is being analyzed. Thus, the MS experiment is needed to characterize unexpected compounds with different molecular mass to the desired structure. Obviously, for a compound of a different mass to the one being searched, an MS/MS experiment will not provide the correct information because the precursor ion of the MS/MS experiment is

erroneous. This situation is illustrated in Figs. 8 and 9 where the sample analyzed contained two compounds, an expected compound at 0.84 min with an elemental composition of C16 H14 N8 O and an unexpected one at 1.35 min with an elemental composition of C21 H22 N8 O2 . The MS experiment (see Fig. 8) provided in the report the correct [M+H]+ ions for the two compounds in the two collision energy acquisitions, allowing the chemist to identify the unknown compound, which corresponded to the desired structure where a hydrogen was substituted by a C5 H9 O group. In the low collision energy acquisition, the ionization on the expected compound gave a [M+H]+ ion of m/z 335.1371, while the unexpected compound gave a [M+H]+ ion of m/z 419.1943 and a fragment ion [M-C5 H8 O]+ with m/z 335.1372. On the spectrum at high collision energy, the two most intense fragments for the two compounds were: a fragment which corresponded to the elemental composition of [C7 H5 N4 O]+ at m/z 161.0457 or 161.0461 and a fragment which corresponded to the elemental composition of [C9 H11 N4 ]+ at m/z 175.0977 or 175.0981. In addition, for the compound at 1.35 min also the fragment at m/z 335.1377 was detected. However, the MS/MS experiment (see Fig. 9) using as precursor ion the expected m/z 335.1 provided in the report the same ion for the two compounds at the two collision energy acquisitions, because the ionization on the unexpected compound produced the fragment ion [M-C5 H8 O]+ at m/z 335.1370 previously described. Based

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Fig. 7. DAD chromatograms and ESI + spectra from automatic Open-Access LC-HRMS reports for a compound with an elemental composition of C22 H28 N6 O3 at 0.92 m in. (a) MS experiment; and (b) MS/MS experiment using m/z 425.2 as precursor ion.

Fig. 8. DAD chromatograms and ESI + spectra from an automatic Open-Access LC-HRMS report for a sample containing two compounds (at 0.84 min and 1.35 min): MS experiment.

on these results the chemist could have assigned this result to a mixture of regioisomers. Because of this advantage of the MS experiment, it has been selected as the standard method on our Open-Access platform to help chemist users to identify unexpected compounds that could be present in the samples. On the other hand, despite the limitation described above for MS/MS experiment, this experiment can be the best option to elucidate the structures of a mixture of coeluting compounds, avoiding the need of a purification prior to the HRMS analysis. This coelution is not so frequent on the UHPLC separation but if it happens, by running separated MS/MS acquisitions for different precursor ions, the specific fragment ions for each precursor ion can be obtained in each of these experiments. Fig. 10 provides the reports in ESI + of a mixture of two compounds with elemental compositions of C13 H19 N3 O and C10 H20 N2 O. The major signal in the DAD contained two peaks that were coeluting. On the MS experiment (Fig. 10a), the expected compound with elemental composition of C13 H19 N3 O was identified in the low energy MS spectrum ([M+H]+ ion at m/z

234.1611, along with two other minority ions (m/z 185.1651 and m/z 123.0918). The ion at m/z 123.0918 was identified as a fragment ion of the expected compound ([C7 H11 N2 +H]+ ), while the ion at m/z 185.1651 was not a feasible fragment ion for this compound. On the MS/MS experiment for precursor ion m/z 234.2 (Fig. 10b), the ion at m/z 123.0919 was detected in the high energy MS spectrum, but the ion at m/z 185.2 was not detected in any of the two spectra, so it was confirmed that it belonged to another compound. In addition, on the MS/MS experiment for precursor ion m/z 185.2 (Fig. 10c), the main fragment ion detected in the high energy MS spectrum at m/z 112.0757 (related to the loss of a C4 H10 N group) helped to assign this compound to an elemental composition of C10 H20 N2 O ([M+H]+ ion at m/z 185.1652). To make this method more adequate, a single MS/MS experiment with two precursor ions for the analysis of two targets that are coeluting could be applied. Chemists would need to receive additional training on data analysis to be able to interpret those results. The inherent difficulty in the analysis of these experiments by non-expert users discourages the implementation

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Fig. 9. DAD chromatograms and ESI + spectra from an automatic Open-Access LC-HRMS report for a sample containing two compounds (at 0.84 min and 1.35 min): MS/MS experiment using m/z 335.1 as precursor ion.

Fig. 10. DAD chromatograms and ESI + spectra from automatic Open-Access LC-HRMS reports for a sample with two compounds that were partially coeluting (at 0.35 min and 0.40 min): (a) MS experiment; (b) MS/MS experiment using m/z 234.2 as precursor ion; and (c) MS/MS experiment precursor using m/z 185.2 as precursor ion.

of more complex automatic MS/MS experiments in an Open-Access environment. 3.3. Application to Medicinal Chemistry programs using MS experiments in open-access LC-HRMS Chemists in several Janssen chemistry projects have used the Open-Access LC-HRMS methodology described for over three years. Although there is a broad diversity in the chemistry classes, the “Universal Open-Access ESI + method” allowed generating OpenAccess LC-HRMS determinations in 98% of the compounds that ionized on this polarity. For the other 2% of the compounds (typically low ionizable structures in ESI+), the “new method A” achieved the HRMS determination. On the other hand, the “Universal OpenAccess ESI- method” is also being acquired routinely on all the samples to identify any compound or impurity that exclusively ionize in this polarity, which can happen in 5% of the samples. Up to

date, more than 3.000 LC-HRMS analyses have been reported with mass accuracy usually lower than ± 1 mDa and a typical concentration of 0.1 mg/mL. Table 4 shows the lead compounds of four chemical series of compounds synthesized in different Medicinal Chemistry projects from Neuroscience and Infectious Disease therapeutic research groups at Janssen, that have been analyzed by LC-HRMS using the Open-Access platform described in section 3.1. In 2016, Mc Gowan et al. [19] published the discovery of fortyone novel amino-pyrimidine structures (serie 1 in Table 4) to treat Hepatitis B Virus. The “Universal Open-Access ESI + method” was used for all the compounds reported. In 2017, Rombouts et al. [20] reported the discovery of N-(Pyridin-4-yl)-1,5-naphthyridin2-amines (serie 2 in Table 4) as potential Tau Pathology PET Tracers for Alzheimer´s disease. The “Universal Open-Access ESI + method” was applied to the thirty-four compounds described. In 2017, Van Gool et al. [21] reported new 1,3,5-trisubstituted pyrazoles as negative allosteric modulators of the metabotropic glutamate

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A. Fontana, L. Iturrino, D. Corens et al. / Journal of Pharmaceutical and Biomedical Analysis 178 (2020) 112908

Table 4 Lead compounds synthesized on different Medicinal Chemistry projects at Janssen, analyzed by Open-Access LC-HRMS (values of log P and pka were obtained with ACD Labs 2014 / Percepta software). o

Serie N

Lead compound / Molecular Mass

pKa

Log P

7.0

2.17

6.9

3.14

3

4.2

4.51

4

6.0

4.85

1

subtype 2 and 3 (mGlu2/3) receptors (serie 3 in Table 4). The “Universal Open-Access ESI + method” was applied to all the compounds (eighteen) that were analyzed by LC-HRMS. Finally, in 2018, Wright et al. [22] reported a new methodology for the synthesis of new trifluoromethyl-sulfonimidamides (serie 4 in Table 4). In this publication, the “Universal Open-Access ESI- method” was used for eighteen compounds, while the “Universal Open-Access ESI + method” was used for only one compound. The diversity of these chemical series can be concluded based on the molecular mass, pKa and log P values for the different lead compounds that are shown. 4. Conclusions An automated Open-Access LC-HRMS methodology for the analysis of medicinal chemistry samples on a UHPLC-QTOF has been developed. On this implementation, complete automated reports are sent to chemists, containing a DAD to determine the purity of the compound analyzed and two HRMS spectra at two collision energies for its unambiguous identification. The low collision energy typically obtains molecular ions while the high collision energy obtains fragment ions data. Different ESI + conditions were developed to obtain a reduction in the intensity of the ionization which was needed in those cases in which oversaturation of the signal produced shifts in the HRMS measurement and bad isotopic patterns due to the high sample concentration needed to determine the purity of a compound by DAD. After several changes of the initial MS conditions, a “universal MS ESI + method” was found, valid for a huge diversity of compounds. This universal ESI + method achieved good mass accuracies within ± 1 mDa and S/N, providing automatic identifications for 98% of the compounds that ionized on this polarity. Only the 2% of these compounds dont´ give good ionization on the universal method (typically low ionizable structures in ESI+) and needed the use of another MS method, also developed in this work. Additionally, a “universal Open-Access ESI- method” was also developed to analyze routinely all the samples, to identify compounds that exclusively ionize in this polarity, which can happen in 5% of the samples.

On one hand, the use of MS experiments with two collision energies in the Open-Access LC-HRMS methodology developed allows chemists to confirm the compounds obtained from the synthesis thanks to the fragment ions obtained in the spectra. The MS/MS experiments give very similar results on samples in which the expected structure is present and is well ionized, but as the MS experiment helps the elucidation of unexpected compounds in the samples, this experiment has been selected as the standard in the Open-Access LC-HRMS methodology developed. On the other hand, the benefit of using the MS/MS experiment by the specialist from the analytical team in cases of coelution of unknown compounds has also been shown. This method allows the elucidation of these structures by the identification of the specific fragment ions from each molecular ion and may help the chemist to decide if it is interesting or not to purify two compounds that are coeluting. Finally, as result of the implementation described, the instrument is being used by chemist end-users, working unattended in Open-Access mode, enabling them to perform HRMS validations of novel synthetic compounds in 95% of the cases with no assistance from the analytical team. The remaining 5% of the cases are usually analyzed by the LC–MS specialist and consist on the identification of coeluting compounds or on the determination of unknown compounds that contain uncommon elements not suspected to be isolated from the reactions. To the best of our knowledge this the first time that a full automated type of Open-Access platform is being used by end users on a UHPLC-HRMS system in a medicinal chemistry program of a pharmaceutical industry. Declaration of Competing Interest None. Acknowledgement We would like to thank Luis Miguel Font, Maria Victoria Pérez and Jose Manuel Alonso for their contributions. Also, we thank María García for the text corrections and the Neuroscience team working at the Janssen Research & Development, a Division of Janssen-Cilag, S.A. for their good collaboration in the laboratory. This research did not receive any specific grant from funding agencies in the public, commercial, or non-for-profit sectors. References [1] H. Lee, Pharmaceutical applications of liquid chromatography coupled with mass spectrometry, J. Liquid Chrom. Related Technol. 28 (2005) 1161–1202. [2] A. Marín, K. Burton, A. Rivera-Sagredo, A. Espada, C. Byrne, C. White, G. Sharman, L. Goodwin, Optimization and Standardization of Liquid Chromatography-Mass Spectrometry Systems for the Analysis of Drug Discovery Compounds, J. Liquid Chrom. Related Technol. 31 (2008) 2–22. [3] F. Pullen, G. Perkins, K. Burton, R. Ware, M. Teague, J. Kiplinger, Putting mass spectrometry in the hands of the end user, J. Am. Soc. Mass Spectrom. 6 (1995) 394–399. [4] L. Taylor, R. Johnson, R. Raso, Open access atmospheric pressure chemical ionization Mass spectrometry for routine sample analysis, J. Am. Soc. Mass Spectrom. 6 (1995) 387–393. [5] L.M. Mallis, A. Sarkahian, J.M. Kulishoff, W.L. Watts, Open-access liquid chromatography mass spectrometry in a drug discovery environment, J. Mass Spectrom. 37 (2002) 889–896. [6] A. Coddington, J. Van Antwerp, H. Ramjit, Critical considerations for high-reliability open access LC/MS, J. Liquid Chrom. Related Technol. 26 (2003) 2839–2859. [7] 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. [8] J. Gao, S.S. Ceglia, M.D. Jones, J. Simeone, J. Van Antwerp, L.-K. Zhang, C.W. Ross III, R. Helmy, A novel compact mass detection platform for the open access environment in drug discovery and early development, J. Pharm. Biomed. Anal. 122 (2016) 1–8. [9] M. Colombo, F.R. Sirtori, V. Rizzo, A fully automated method for accurate mass determination using high-performance liquid chromatography with a

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