Agilent 6410 triple quad mass spectrometer

Due to concerns about rapid iodine isotope decay (t_1/2 ~ 13 h for ¹²³I and 8 days for ¹³¹I), nonradioactive mIBG was used and quantified by LC-MS/MS as previously described [25 (link)]. Briefly, an Agilent 6410 Triple Quad mass spectrometer coupled with an Agilent 1290 series HPLC system (Agilent Technologies, Santa Clara, CA, USA), operated in positive electrospray ionization mode (ESI+), was used for the mIBG quantification. Ten microliters of the diluted cell lysate samples at 4 °C with internal standards were injected through a Zorbax Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm) (Agilent Technologies, Santa Clara, CA, USA) maintained at room temperature. For the mobile phase, 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was used. A 5 min ballistic gradient detailed as follows was used for the compound elution: 3% B from 0 to 0.1 min, 3% B to 90% B from 0.1 to 2 min, 90% B from 2 to 3 min, 90% B to 3% B from 3 to 3.1 min, and 3% B from 3.1 to 5 min. The mass-to-charge (m/z) transitions used were 276.1 → 217.0 for mIBG and 494.1 → 369.0 for glyburide.

Solvents were distilled from appropriate drying agents shortly before use. TLC was carried out on DC-plastikfolien Kieselgel 60 F254 and preparative thick-layer (2 mm) chromatography was performed on Merck 60 F254 plates (Merck KGaA, Darmstadt, Germany). (Merck, Merck KGaA, Darmstadt, Germany). NMR spectra were recorded on AV600 and AV300 MHz spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at 150.92 or 75.47 MHz for ¹³C and 600.13 or 300.13 MHz for ¹H nuclei using DMSO-d₆ as the internal standard (labels in the spectra: Trp = tryptophan ring; Pyr = pyrene ring). Mass spectrometry was performed on the Agilent 6410 Triple Quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and high-resolution mass spectra (HRMS) were obtained using a Q-Tof2 hybrid quadrupole time-of-flight mass spectrometer (Micromass, Cary, NC, USA). WHW was purchased from GenScript, Treubstraat 1, 1st floor. 2288EG, Rijswijk, the Netherlands, as a 95% pure white solid (TFA salt). Experimental procedures for preparing known compounds 1, 2 and 8 are given in the Supplementary Information.

An Agilent 1100 liquid chromatograph (Agilent, Waldbronn, Germany), equipped with a diode-array detector and an Agilent 6410 Triple Quad mass spectrometer (Agilent, Waldbronn, Germany), was employed for analysis. Based on the high polarity of betaine, a hydrophilic interaction chromatography column (HILIC) was used for its analysis. A HILIC Kinetex column (100 mm × 4.6 mm, 2.6 μm) (Phenomenex, Torrance, CA, USA) and a mobile phase (flow rate 0.6 mL/min) composed of acetonitrile (A, 75% v/v) and 10 mM ammonium formate buffer (B, 25% v/v), acidified to pH 3 with formic acid, in isocratic elution mode were used. Injection volume was 5 μL. Data were collected with the use of Agilent Mass Hunter, software version B.04.01.
Electrospray ionization (ESI) was applied in the positive ion mode. Nitrogen was used in the ion source and the collision cell. Full-scan mass spectra were recorded within the mass range of m/z 50–500 Da. Additionally, multiple reaction monitoring (MRM) mode was applied for the quantitative analysis. Three main operation parameters of MS/MS detector, namely collision energy (CoE), fragmentor voltage (FV) and temperature of the source (Temp), were optimized by means of a Central Composite Design (see ‘Optimization of MS parameters through experimental design’ section).

Quantification of gemcitabine and its metabolites was performed using an Agilent 1200 series HPLC-system (Agilent Technologies, Waldbronn, Germany) for chromatographic separation and an Agilent 6410 triple-quad mass spectrometer for mass detection. Concentrations of gemcitabine and dFdU in culture media samples were measured according to our previously published method [24 (link)], with optimised lower limits of quantitation (LLOQ) of 0.1 µM for both analytes. Gemcitabine triphosphate (dFdCTP) was quantified in cell lysates with a slightly modified version of our previously published method [25 (link)]. Modification consisted of reduced analysis time to approximately 30 min and with the mass spectrometer operating in positive ionisation mode, since we only quantified dFdCTP and not the endogenous nucleosides that eluted later. dCTP was used as internal standard due to its similar structure and retention time with dFdCTP. Concentrations above the LLOQ of 0.05 µM were normalised to the cell count in each sample and expressed as pmol per 10⁶ cells (abbreviated to pmol/10⁶ throughout the manuscript).