Zorbax extend c18 analytical column

The samples were reduced using 5 mM TCEP for 20 min, and alkylated by 50 mM chloroacetamide for 30 min in the dark at room temperature. The proteins were first digested with endo-proteinase LysC (Roche, 11047825001, 1:100 w/w) at 35 °C for an hour. The mixture was then diluted to 2 M urea with tryptic digestion buffer (100 mM Tris-HCl, 1 mM CaCl₂, pH 8.5) and the proteins were further digested by trypsin (Thermo, PRV5113, 1:20 w/w) for 20 hours at 35 °C. The resulting peptides from total crude lysate and soluble fractions were purified by STop-And-Go Extraction (STAGE) high-capacity tips using C18 resin (Phenomenex)^{50 (link)}.
Roughly 100 μg of peptides of each sample was fractionated by offline high pH reversed-phase chromatography using a Zorbax Extend-C18 analytical column, 5 μm, 4.6 × 50 mm (Agilent), on a 64 minute gradient (followed by a 21 minute equilibration with buffer A) with a 50 μL/min flow rate, where Buffer A contained 5 mM NH₄HCO₂, pH 10 and 2% acetonitrile and Buffer B contained 5 mM NH₄HCO₂, pH 10 and 90% acetonitrile. Ninety-six fractions were collected at 40 seconds/fraction. The resulting fractions were pooled in a non-contiguous manner, as previously described^{51 (link)}. The fractionated samples were pooled into 9 fractions.

Cephapirin was analyzed using Agilent 1290 UPLC coupled with Agilent 6490 Triple Quad tandem mass spectrometry (Agilent, Santa Clara, CA, USA). Electrospray negative ionization in multiple-reaction monitoring mode was used. Zorbax Extend C₁₈ analytical column (4.6×50 mm, 5 µm particle size, Agilent, Santa Clara, CA, USA) coupled with Zorbax Extend C₁₈ guard column (4.6×12 mm, 5 µm particle size, Agilent, Santa Clara, CA, USA) was used for chromatographic separation. Sampler and column compartments were kept at 8 and 40°C, respectively. The injection volume was 10 µL. A gradient elution program consisting of two mobile phases (mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in MeOH; Table 2) was used at a flow rate of 0.5 mL min⁻¹. Mass spectrometry parameters are listed in Table 3. Cephapirin in positive and spiked samples were qualified by comparing LC-MS/MS spectra of samples with those of cephapirin standards. Accepted variation in mass to charge ratio was 10%, and acceptable variation was set as 20% for the ratio of qualifier and quantifier ions. Cephapirin concentration in tested samples was quantified using the calibration curve of seven matrix-matched cephapirin standards (0.7, 1, 2, 4, 5, 10, and 20 µg L⁻¹ matrix solution). Matrix-match standards were prepared using the SPE cleaned-up extracts of blank feces or urine samples.