Agilent 6495 triple quadrupole mass spectrometer

Participants selected to continue with the trial were asked to input an address through the digital platform, to be sent a DBS sample collection kit. The DBS sample collection kit used in the Delta Trial was a Conformité Européene-marked device under Article 22 of the Medical Device Regulation 2017/745 and contained relevant materials and instructions to allow participants to complete and submit DBS samples.
A standardized MS-based targeted proteomic biomarker screening method, based on previously published work [48 (link),91 (link)], was developed for the Delta Trial. For this method, 203 candidate peptides representing 120 proteins were selected for inclusion, in many cases based on their previous association with psychiatric disorders, including depression, BD, and schizophrenia [91 (link)]. These proteins were first extracted and digested from the DBS samples [48 (link)], and then, unique surrogate peptides representing candidate proteins were monitored through multiple reaction monitoring using an Agilent 1290 liquid chromatography system coupled with an Agilent 6495 Triple Quadrupole Mass Spectrometer.

IR780-oleyl was quantified by LC–MS/MS using an Agilent 1290 HPLC system coupled to an Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). An Ascentis Express C8 column (75 × 2.1 mm, 2.7 μm particles size, Sigma) with a 5 × 2.1 mm guard column of the same material was used for chromatographic separation. Mobile phase A was 10 mM ammonium acetate/0.1% formic acid in water and mobile phase B was 10 mM ammonium acetate/0.1% formic acid in a 5:2 (v/v) mix of acetonitrile and 2-propanol. The flow rate was 0.4 mL/min, and a chromatographic gradient was applied as follows: linear increase from 40 to 95% B over 7 min, 95% B for 2 min, linear increase to 100% over 2 min, 100% B for 3 min, before 1 min column re-equilibration (total run time: 15 min). Injection volume was 2.5 μL. The MS was operated in positive electrospray ionization (ESI) mode (Agilent Jetstream), using the m/z 858.4 → 521.1 transition for IR780-oleyl quantification.
The IR780-oleyl standard was the same as used for nanoparticle synthesis (see above). Standard solutions were prepared by dilution in acetone-added 1 mg/mL Miglyol 812 N (Cremer Oleo GmbH & Co. KG, Hamburg, Germany) to limit IR780-oleyl adhesion to vial walls and pipet tips. Standard curves ranging from 0.1 to 1000 ng/mL were fitted to a quadratic regression model using weighting factor 1/x.

An Agilent 1290 Infinity UHPLC system (Agilent Technologies, Tokyo, Japan) was used for LC, and an Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies) was used for MS. A 100 mm × 3.0 mm InertSustain AQ-C18 column with a particle size of 1.9 μm (GL Science, Tokyo, Japan) was used, and the temperature was set to 60 °C. Distilled water (DW) containing 0.1% formic acid was used for mobile phase A, and acetonitrile containing 0.1% formic acid was used for mobile phase B. The analytes were separated at a flow rate of 0.8 mL/min. The gradient was started at 10% B, increased linearly to 95% B from 0.5 min to 4 min, maintained at 95% B for 1 min, and then returned to 10% B and equilibrated for 0.5 min before the next injection. The injection volume was 20 μL, and electrospray ionization (ESI) was performed in positive mode. The retention time (RT), multiple reaction monitoring (MRM) transition, and collision energy (CE) for each analyte are listed in Table 2.

Metabolites were measured as described previously (44 (link)). Briefly, an Agilent 1290 Infinity II ultrahigh performance liquid chromatographic (UHPLC) system (Agilent Technologies) was used for liquid chromatography (LC). The column was an Acquity BEH (ethylene bridged hybrid) amide column (30 by 2.1 mm with 1.7-µm particle size) (Waters GmbH). The temperature of the column oven was 30°C, and the injection volume was 3 µl. LC solvent A was water with 10 mM ammonium formate and 0.1% formic acid (vol/vol), and LC solvent B was acetonitrile with 0.1% formic acid (vol/vol). The gradient was 90% LC solvent B at 0 min, 40% LC solvent B at 1.3 min, 40% LC solvent B at 1.5 min, 90% LC solvent B at 1.7 min, and 90% LC solvent B at 2 min. The flow rate was 0.4 ml min⁻¹. An Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies) was used for mass spectrometry. The source gas temperature was set at 200°C, with 14 liters min⁻¹ drying gas and a nebulizer pressure of 24 lb/in². The sheath gas temperature was set at 300°C, and flow was set at 11 liters min⁻¹. Electrospray nozzle and capillary voltages were set at 500 and 2,500 V, respectively. Isotope ratio mass spectrometry with ¹³C internal standard was used to obtain absolute quantitative data. Ornithine was calibrated with external standards due to low concentrations in the ¹³C internal standard.

Levels of GlcNAc-6P and UDP-GlcNAc in the heart were measured using targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Briefly, fresh heart samples were vortexed for 30 s, homogenized in extraction solution (acetonitrile: methanol: water = 2:2:1) for 4 min, and sonicated for 5 min on ice, which were then centrifuged at 4 °C for 15 min to obtain the supernatants. Next, the supernatants or standard samples were separated by a Waters Premier Amide (2.1 mm × 100 mm, 1.7 μm) on an Agilent 1290 Infinity II series UHPLC System (Agilent Technologies, Palo Alto, CA, USA) using 0.1% ammonia and 10 mmol/L ammonium formate (phase A), and acetonitrile (phase B) as the mobile phases (300 μL/min). The metabolites were detected using an Agilent 6495 Triple Quadrupole Mass Spectrometer (Agilent Technologies), equipped with an AJS electrospray ionization interface, and then normalized to tissue weight. Typical ion source parameters were: capillary voltage = +3000 V, nozzle voltage = +1500 V, gas (N₂) temperature = 250 °C, gas (N₂) flow = 11 L/min, sheath gas (N₂) temperature = 400 °C, sheath gas flow = 12 L/min, nebulizer = 35 psi.