Zic hilic guard column

To quantify succinate and malonate a LCMS-8060 mass spectrometer (Shimadzu, UK) with a Nexera X2 UHPLC system (Shimadzu, UK) was used as described before [29 (link)]. Briefly, metabolites were separated using a SeQuant® ZIC®-HILIC column (3.5 μm, 100 Å, 150 x 2.1 mm, 30 °C column temperature; MerckMillipore, UK) with a ZIC®-HILIC guard column (200 Å, 1 x 5 mm) at a flow rate of 200 μl/min with mobile phases of A) 10 mM ammonium bicarbonate and B) 100% acetonitrile. The MS was operated in negative ion mode with multiple reaction monitoring (MRM) and spectra acquired using Labsolutions software (Shimadzu, UK). Succinate and malonate were quantified using standard curves relative to [¹³C₄]-succinate or [¹³C₃]-malonate internal standard.

Samples were analysed using an LCMS-8060 mass spectrometer (Shimadzu, UK) with a Nexera X2 UHPLC system (Shimadzu, UK). Sample separation was achieved using a SeQuant®ZIC®-HILIC column (3.5 μg, 100 Å, 150 × 2.1 mm, 30 °C column temperature; MerckMillipore, UK) with a ZIC®-HILIC guard column (200 Å, 1 × 5 mm). A flow rate of 200 μl/min was used with mobile phases of 10 mM ammonium bicarbonate (pH unchanged) and B) 100% acetonitrile. The mass spectrometer was operated in negative ion mode with multiple reaction monitoring (MRM). Sample spectra were acquired using Labsolutions software (Shimadzu, UK) and the peak area for each compound of interest measured relative to the internal standard. Sample concentrations were then calculated from a standard curve of known compound concentrations produced by LC-MS/MS in a similar manner.

LC–MS/MS analysis of succinate was performed using an LCMS-8060 mass spectrometer (Shimadzu, UK) with a Nexera X2 UHPLC system (Shimadzu). Samples were stored in a refrigerated autosampler (4°C) before injection of 5 μL using a 15 μL flowthrough needle. Separation was achieved using a SeQuant^® ZIC^®-HILIC column (3.5 μm, 100 Å, 150 mm × 2.1 mm, 30°C column temperature; MerckMillipore, UK) with a ZIC^®-HILIC guard column (200 Å, 1 mm × 5 mm). A flow rate of 200 μL/min was used with mobile phases of buffer A: 10 mM ammonium bicarbonate and buffer B: 100% acetonitrile. A gradient of 0–0.1 min, 80% MS buffer B; 0.1–4 min, 80–20% B; 4–10 min, 20% B, 10–11 min, 20–80% B; 11–15 min, 80% B was used. The mass spectrometer was operated in negative ion mode with multiple reaction monitoring and spectra were acquired using LabSolutions software (Shimadzu), with compound quantities calculated from relevant standard curves in MS extraction buffer and comparing against [¹³ (link)C₄]-succinate internal standard.

LC-MS was performed on an Agilent Technologies
(Santa Clara, CA) Infinity 1290 LC system coupled via a dual-source
AJS electrospray interface to an Agilent Technologies 6560B ESI Ion
Mobility Q-TOF. Glycopeptide standards were analyzed with a SeQuant
ZIC-HILIC column (20 × 2.1 mm², 3.5 μm particles;
Merck, Darmstadt, Germany), with 0.1% (v/v) formic acid as eluent
A and ACN as eluent B, using a linear gradient from 90–50%
B over 5 min and maintaining 50% B for 8 min. MS was performed in
positive ion mode for glycans 1 and 3 and
negative ion mode for glycans 2 and 4. O-glycans, released from BSM oxidatively or by β-elimination,
were separated with a ZIC-HILIC column (150 × 2.1 mm², 3 μm particles) with a ZIC-HILIC guard column (20 ×
2.1 mm², 3 μm particles; Merck, Darmstadt, Germany)
using 5 mM ammonium formate (pH 6.5) as eluent A and ACN as eluent
B. Chromatographic separation was achieved using 85% B for 5 min,
followed by a linear gradient to 50% B over 25 min at 0.2 mL/min. O-glycans released by β-elimination were additionally
analyzed with the same gradient using 10 mM ammonium bicarbonate (pH
7.8) as eluent A to preserve sulfated moieties better. MS analysis
was performed in negative ion mode with a capillary voltage of 3500
V, nozzle voltage of 2000 V, nebulizer pressure of 40 psi, drying
gas flow rate of 300 °C at 8 L/min, and sheath gas temperature
of 300 °C at 11 L/min.