Ultra performance lc system

The UHPLC-HRMS analysis of PFCAs,
PFSAs, FOSAs, diPAPs, and F-53B was performed as described by Göckener
et al.^{31 (link)} Briefly, sample extracts were
analyzed with a C18 reverse phase column on an Ultra Performance LC
system by Waters Corporation (Milford, MA, U.S.A.) coupled to a Q
Exactive Plus HRMS system (Thermo Fisher, Waltham, U.S.A.) operated
in electrospray negative mode. The composition of the technical F-53B
product was determined to be 89.6% 6:2 Cl PFESA and 8.2% 8:2 Cl-PFESA.
The analysis and quantification of monoPAPs was conducted as described
by Göckener et al. but the mobile phases were modified with
2 mM ammonium acetate and 0.01% ammonia to increase the ionization
efficiency.^{31 (link)} A list of all substances
analyzed by UHPLC-HRMS including their acronyms, exact masses, and
the corresponding internal standard used for quantification is shown
in Table S2. All limits of quantification
(LOQ) are listed in Table S3.

MCFA levels in the plasma, liver, adipose tissue, muscle, cecum, and NC and HFD samples were determined following a previously described protocol with modifications (12 (link)). The samples containing an internal control (C19:0) were homogenized in methanol and mixed with chloroform and water to extract lipids. The samples were centrifuged at 2,000g and 17°C for 10 minutes. The supernatant containing MCFAs was collected and dried. The samples were resuspended with chloroform/methanol (1:3, v/v) and subjected to LC-MS/MS analysis using an ultra-performance LC system (UPLC, Waters) equipped with an Acquity UPLC system coupled to a Waters Xevo TQD mass spectrometer. The samples were separated on an ACQUITY UPLC BEH C18 column (2.1 × 150 mm, 1.7 μm; Waters) using a methanol gradient in 10 mM ammonium formate aqueous solution.

Apo(a) enrichment with D₃-leucine was measured as described by Zhou et al. (27 (link)). In brief, 200 μl of the LDL fraction or equal volumes of LDL (100 μl) and HDL (100 μl) fractions isolated from plasma by ultracentrifugation were desalted. Isolated lipoprotein fractions were then treated with dithiothreitol to open disulfide bonds, alkylated with iodoacetamide, and digested using trypsin. A multiple reaction monitoring method was used to monitor the following precursor-product ion transitions of a peptide specific to apo(a): (LFLEPTQADIALLK): 786.7 > 1069.7 (M0) and 788.2 > 1069.7 (M3). Two microliteres of the digested samples were analyzed using a nanoAcquity ultra-performance LC system coupled with an ionKey source integrated to a Xevo TQ-S triple quadrupole tandem mass spectrometer (Waters, Milford, Massachusetts). The separation was achieved using an iKey Peptide BEH C18 separation device (130 Ǻ, 1.7 μm, 150 μm × 100 mm) maintained at 60°C. The gradient was 90% A (0.1% formic acid in water)/10% B (0.1% formic acid in acetonitrile) ramped linearly to 10% A at 6 min, held for 3 min, and then reequilibrated to initial conditions (total run time: 12 min; flow rate: 3 μl/min). The multiple reaction transitions were monitored with a collision energy of 24 eV.