Masshunter qualitative analysis 10

HPLC-MS analyses were performed by an Agilent 1200 Series HPLC system coupled to an Agilent 6520A qTOF-MS equipped with an electrospray ionization (ESI) probe (Agilent Technologies, Santa Clara, CA, USA). For the separation, a Zorbax Eclipse XDB-C18 Solvent Saver Plus reversed-phase column (75 × 3.0 mm i.d.; 3.5 µm; Agilent Technologies, Santa Clara, CA, USA) was employed. The mobile phase consisted of water with 0.1% formic acid and 5% acetonitrile (A) and acetonitrile with 0.1% formic acid and 5% water (B). The elution was carried out at 25 °C with a flow rate of 0.5 mL/min using the following linear gradient: 0–100% B (0–10 min), 100% B (10–12 min), and 0% B (12–15 min). The injection volume was 1 µL. Analytes were detected using drying gas (nitrogen) at 350 °C and 12 L/min and nebulizer gas at 40 psi. For collision-induced dissociation (CID), the parameters were as follows: collision gas: high-purity nitrogen; collision energy: 10–40 eV; fragmentor voltage: 110 V; capillary voltage: 3500 V. MS and MS/MS spectra were acquired in the m/z range of 25–700 and 45–600, respectively. Reference masses of m/z 121.050873 and 922.009798 for positive and m/z 112.985587 and 1033.988109 for negative ionization were applied in the internal calibration. The collected data were evaluated with MassHunter Qualitative Analysis 10.0 software (Agilent Technologies, Santa Clara, CA, USA).

Analysis was performed using liquid chromatography tandem quadrupole time of flight mass spectrometry (LC/Q-TOF) as described previously (33 (link)). Samples were analyzed on an Agilent Technologies 1,290 Infinity UHPLC with AJS-ESI 6,545 QTOF with a run time of 48 s, using data dependent acquisition (DDA) for data collection.
Agilent MassHunter Qualitative Analysis 10.0 software was used for data analysis. Retention time (RT, minutes), database scores (DBS), and library search scores (LSS) were used to determine positivity for all drugs in the scope based on validated criteria established during method development (33 (link)). A sample was considered positive if it met or exceeded the established criteria for RT, DBS, and LSS. Any results that met or exceeded the LSS, but did not meet RT or DBS criteria were manually reviewed for positivity. Results which did not meet these criteria were reported as none detected.

MassHunter Qualitative
Analysis 10.0 and IM-MS Browser 10.0 (Agilent Technologies) were used
for evaluation of the QTOFMS and IM-QTOFMS data, respectively. The
measured accurate masses are tabulated in Tables S3 and S4 (Supporting Information).

A glass microvial containing 3 mg of sample was placed inside the TSP attached to an Agilent 7890B gas chromatograph equipped with a Restek Rxi-5 ms column (30 m × 320 μm × 0.25 μm). The mass spectrometer was an Agilent 7250 GC/Q‑TOF equipped with a low-energy-capable EI source (70 eV). The TSP was set at 300 °C and the oven temperature set at 40 °C for one minute, increasing by 20 °C/minute until 320 °C where it was held for five minutes. Helium was used as a carrier gas, at 1.43L/min flow rate and 8.7psi pressure. The equilibration time was 0.5 min and the sample injection was splitless. The mass range was 50 to 650 m/z. All samples were run in triplicate with blanks added between samples. Data analysis was performed using Agilent Mass Hunter Qualitative Analysis 10.0 with compound assignments using NIST Library 17. Only compounds with a match factor (MF) and reverse match factor (RMF) > 700 and where the molecular ion is present with an accuracy mass error below 50 ppm were shortlisted.

Mass Hunter Qualitative Analysis 10.0 (Agilent, Santa Clara, CA, USA) analysis was used to complete and process the chromatographic data. MS Excel 2016 with the Data Analysis add-on was used for statistical analysis and for regression calculation (Microsoft, Redmond, WA, USA). Linear regression parameters for the standard curve were determined using ANOVA. The IC₅₀ and standard deviation values were calculated using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).