Luna nh2 hplc column

After overnight fasting (about 12 h), BMAT was isolated from the femurs based on the protocol of Tencerova and the co-authors with modifications²⁴ . Briefly, the femurs were longitudinally bisected using a Dremel rotary tool with a 409 cutoff wheel, and the BMAT was then removed using a stainless-steel spatula. The BMAT was snap-frozen for further metabolomic analysis.
The extraction of metabolites was performed using the prechilled (−20 °C) extraction buffer containing MeOH-ACN-H₂O (43:43:16, v/v/v) and stable isotope standards as described before^{25 ,26 (link)}. The metabolomic analysis was performed on an Agilent 1290 HPLC coupled with a SCIEX QTRAP 5500 triple quadrupole mass spectrometer (LC-MS/MS)^{26 (link)}. The metabolites were separated on a Luna NH₂ HPLC column (250 mm × 2.0 mm, 5 μm, Phenomenex, USA). The MS was performed by multiple reaction monitoring (MRM) in both negative and positive mode with rapid polarity switching (50 ms). A total of 420 metabolites covering all the major metabolic pathways were targeted^{21 (link)}. Data were log 2 transformed before statistical analysis. Multivariate analysis (Partial least squares discriminant analysis, PLS-DA), pathway analysis, and ANOVA simultaneous component analysis were performed using Metaboanalyst 4.0 (www.metaboanalyst.ca). The details were described in the Supplementary method.

Levels of ACs and AAs were simultaneously semiquantified, together with specific FA species and key metabolic intermediates, in 3 µL of plasma or PBMC suspension (~14,000 cells) by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) based on a previously used method [91 (link),92 (link),93 (link)]. The analysis was performed on UltiMate 3000 Rapid Separation system (RSLC) (Thermo Fisher Scientific, Waltham, MA, USA) coupled to the 5500 QTRAP mass spectrometer (AB Sciex LLC, Framingham, MA, USA). Briefly, samples were mixed with internal standards in 100 µL of acetonitrile with 0.1% acetic acid, vortexed, precipitated proteins removed by centrifugation at 12,000 g, 4 °C, 10 min, and immediately analyzed. The internal deuterated standards for ACs and AAs (MassChrom Newborn Screening Kit, Chromsystems, Gräfelfing, Germany) were used. Samples (2 L) were separated using Luna NH2 HPLC column (150 × 2.1 mm Phenomenex), gradient elution: A, acetonitrile; B, 20 mM ammonium acetate, pH 9.45, flow 0.3 mL/min; 0 min–5%B, 3.8 min–70%B, 4.3 min–95%B, 8 min–95%B, 9 min–5%B, 12 min–5%B. Analytes were detected in multiple reaction monitoring (MRM) positive/negative ion-switching mode as before [92 (link)]. Data analysis was performed with Analyst software (AB Sciex LLC, Framingham, MA, USA). Peak area raw data were normalized by the plasma volume or PBMC number.

Dry extracts were re-suspended in water (100 μL) and centrifuged (12000 × g, 3 min). The supernatants were used for subsequent analyses. Separation was achieved with a Luna-NH₂ HPLC column (150 mm × 2 mm, 3 μm particle size, 100 Å pore size, Phenomenex) using a binary gradient method (Solvent A: acetonitrile/Solvent B: 10 mM ammonium acetate, pH 9.9). Gradient parameters were as follows: 0–24 min: 20–100% B; 25–34 min: 100% B; 35 min: 20% B. Detection was performed with a tandem mass spectrometry detector with an ESI ion source (Shimadzu LCMS-8040) in multiple reaction monitoring (MRM) mode (Lorenz et al., 2011 (link)). Peak areas were normalized to fully ¹³C-labeled internal standards and absolute quantification of metabolites was achieved with linear calibration curves of the standards. Finally, concentrations were normalized to the amount of biomass. For MRM fragments see Supplementary Table S2.