Ultra biphenyl column

An Agilent 1200 Series HPLC system coupled to an AB Sciex API4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer was used to monitor two product ions, one for quantitation and one for identity confirmation, using scheduled multiple reaction monitoring for each steroid and internal standard. Chromatography was conducted on a Restek (Bellefonte, PA, USA) Ultra Biphenyl column (250 mm x 4.6 mm, 5.0 μm particle size) with acetonitrile and methanol both containing 0.1 % formic acid (volume fraction) for reproductive hormones. A flow of 500 μL/min was used for a solvent gradient of 20 % acetonitrile increased to 45 % over 30 min., then increased to 80 % over 1 min and held for 4 min, then washed with 100 % acetonitrile for 5 min and re-equilibrated at 20 % for 10 min. Corticosteroids were separated on an Agilent (Santa Clara, CA, USA) Eclipse Plus C18 column (21 mm X 150 mm, 5.0 μm particle size) with methanol and water both containing 0.1 % acetic acid (volume fraction). Column conditions were as follows: flow rate of 250 μL/min, and isocratic method of 46 % methanol for 20 min, a wash of 100 % methanol for 13 min, and re-equilibrated for 10 min. Additional information on compound and instrument parameters can be found in Boggs et al. (Boggs et al., 2017 (link)).

Residual drug solution from used implants was extracted from the silicone plugs on the devices using a 20 G needle before the device was opened and left in 100% methanol overnight. TAF residual was brought up to 10 ml in 100% methanol using a volumetric flask while FTC was brought up to 25 ml. The concentrations of TAF and FTC were measured by adapting a previously described method for HPLC with UV detection (Hitachi Chromaster) (24 (link)). A Restek (Bellefonte, PA, USA) Ultra-biphenyl column (2.1 × 100 mm, 5 μm) controlled at 30°C was used as the stationary phase. A solution of 1% acetic acid, 3% acetonitrile, and water was used as mobile phase preparation A while acetonitrile was used as mobile phase B to follow the gradient program listed in Supplemental Table 1 with a flow rate of 0.50 ml/min and a detection wavelength of 260 nm. Injection volume was 20 μl, and total run time was 14 min. The retention times for TAF and FTC were 9 minutes and 4.5 minutes, respectively.

Analyte concentrations were assayed with a validated liquid chromatogrphy tandem spectrometry (LC-MS/MS) method using a Waters Acquity™ UPLC, a Restek Ultra Biphenyl column (100mm x 2.1mm, 5μm) and API5500 Qtrap triple quadrupole mass spectrometer (Applied Biosystem/MDS SCIEX, Foster City, CA). Sample solutions were prepared by 200-fold dilution in LC-MS grade water containing 100 μL of the internal standarad (320 ng/mL ertapenem). The working sample solution was injected (5 μL for piperacillin and 10 μL for tazobactam and avibactam quantitation) into the LC-MS/MS and eluted by a gradient of mobile phase A (0.1% vol/vol formic acid in water) and mobile phase B (0.1% vol/vol formic acid in acetonitrile) at a flow rate of 0.35 mL/min. Multiple reaction monitoring (MRM) transitions of m/z 518.1➔143.2 (positive mode), 300.9➔168.2 (positive mode) and 264.1➔95.6 (negative mode) were used for the identification of piperacillin, tazobactam and avibactam respectively. The linear range of quantification was 0.0625–128 μg/mL; intra-day and inter-day variability were < 10%. The resulting concentration-time profiles were characterized by a 1-compartment pharmacokinetic model with zero order input using ADAPT 5 [11 ].