Supelco discovery c18

The peptide bradykinin with a sequence of RPPGFSPFR (MW=1060 g/mol) was spiked at different concentrations (1, 10, 100, 1000 nM) in serum. One set of spiked serum was left undepleted, while the other set was depleted by three sequential extractions using 400 μL of 2 mg/mL solution of the positively charged polymeric nanoassemblies. The serum samples were then digested with trypsin. Targeted detection of bradykinin by selected reaction monitoring (SRM) on a Waters TQD triple quadrupole mass spectrometer was employed on the digested serum samples. LC separation of the digest on a Supelco® Discovery C18 (150mm × 2.1mm, 5μm (Sigma-Aldrich)) reversed phase column was done with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in ACN as mobile phase B under the following gradient: 0–5 min (5%B), 5–15 min (5–50%B), 15–20 min (50–95% B), 20–25 min (95% B), 25–25.1min (95–5% B), 25.1–30 min (5% B). The optimized SRM transition for the bradykinin peptide was determined to be the +2 charge for the precursor ion (m/z 530.8) and the y8 product ion (m/z 904.5). SRM for this transition was acquired from 8 to 15 minutes of the LC run with an optimized collision energy of 22 and cone voltage of 44 V. The resulting SRM chromatogram was analyzed and processed using the MassLynx software.

Quantification of vancomycin and PMLA-Vanco was performed by HPLC (Agilent 1100 Series) using an injection volume of 50 μL and a C18 column (Supelco Discovery C18; Sigma-Aldrich, St. Louis, MO) with a 1 mL/min mobile phase ramped from 100% PBS to 100% methanol over 20 min. This protocol was found to uniquely distinguish vancomycin and PMLA-vanco from other film components, namely tannic acid. Vancomycin concentrations were quantified by comparison of fluorescence (λ_ex = 280 nm; λ_em = 355 nm) to standard curves and the degree of conjugation was calculated by comparison of the measured vancomycin in solution to the total mass concentration of PMLA-Vanco in solution.¹H NMR spectra were obtained in dimethyl sulfoxide-d₆ (Sigma-Aldrich) using a Bruker Avance 400 MHz NMR spectrometer. Vancomycin HCl was converted to its free base to more closely recapitulate the compound for comparison with PMLA-Vanco conjugates by previously described methods¹⁶ where vancomycin HCl dissolved in water was precipitated at pH 8, collected by filtration and washed with ethanol and methanol prior to dissolution in water and lyophilization.