Kinetex biphenyl column

Stability studies were performed in a saline solution containing 10.5% (v/v) mouse urine, as this mimics the in vivo conditions used in the PET imaging studies after the intravesical administration of the radiotracer. In the time between emptying of the mouse bladder and the start of the PET imaging, we observed 10 μL of urine excretion (corresponds to 10.5% of bladder mouse capacity, or ∼105 μL). Urine was collected from mice with UMUC3 orthotopic BCa. To 170 μL of PBS were added 740 kBq (20 μCi; 10 μL) of compound 4. A 20-μL volume of urine was added to the solution and shaken. Stability was tested by reversed-phase HPLC with in-line radiation (Posi-RAM model 4; LabLogic) detection using a Kinetex Biphenyl column (150 × 4.6 mm; 5-μm particle size; Phenomenex) and a mobile phase gradient of 5%–20% acetonitrile (0.1% trifluoroacetic acid) in water (0.1% trifluoroacetic acid) over 20 min. Intact ¹⁸F-compound 4 elutes at 12 min and is 75% intact (derived from radiotracer chromatogram) or 50% intact (derived from HPLC chromatogram) after 30 min of incubation; several metabolites eluted at later time-points after 1 h (full characterization is provided in the supplemental materials, available at http://jnm.snmjournals.org). The percentages of intact radiotracer and metabolites were obtained from calculating the area under the curve.

HPLC analysis was preformed using an Agilent Technologies 1200 series HPLC instrument equipped with a quaternary high-pressure pump, a vacuum degasser, an auto sampler and a diode-array detector (Wilmington, DE, USA). A Phenomenex Kinetex Biphenyl column (50 mm × 4.6 mm i.d., 5 μm particle size) was used to perform chromatographic separation. The mobile phase consisted of (A) 50% ACN-water solution with 0.05% FA and (B) 0.05% FA in water. The gradient elution program was as follows: 0 min 1% A, 8 min 90% A, 8.5 min 1% A, 10 min 1% A. The flow rate was 0.75 mL min⁻¹ and injection volume was 100 μL. UV monitoring was performed at 205 nm.

Celecoxib was measured by LC–MS/MS with a QTRAP 5500 triple-quadruple mass spectrometer (SCIEX, Framingham, MA) in positive electrospray ionization mode by multiple reaction monitoring data acquisition with an Agilent 1200 HPLC (Agilent Technologies, Santa Clara, CA). Chromatography was performed by automated injection on a Kinetex Biphenyl column, 50 × 2.1 mm, 2.6 μm particle size (Phenomenex, Torrance, CA). HPLC flow was maintained at 400 μL/min with mobile phases of A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile. The initial conditions were 50% A, the gradient was ramped to 5% A at 2.5 min, and then returned to 50% A immediately. The total run time was 7 min. Data acquisition and quantification were performed in Analyst 1.6.2 software (Sciex).

LC–MS/MS (Agilent) was performed as previously described (8 (link)) using an Agilent 1200 series LC and 6470 triple quadrupole tandem mass spectrometer. In brief, DMP was quantified using m/z 97.4→56.2 and qualified using m/z 97.4→70.2, and 3,4-HDMP used m/z 99.2→57.2. The same Phenomenex Kinetex biphenyl column (50 × 3 mm, 2.6 µm) was used, and a second Phenomenex Kinetex C18 column (50 × 3 mm, 2.6 µm) was also used to identify whether the stationary phase played a specific role in the chromatography. Perfluorooctanoic acid (0.01%) in water (phase A) and methanol (phase B) was used for quantification and the flow rate was optimized to 0.4 mL/min, with an injection volume of 1 µL. The gradient started with 20% phase B for 0.5 min and then increased to 80% by 4 min, held until 7.5 min and then returned to 20%.

Plasma concentrations of IQ and epi-IQ were quantified using an API 4000 LC/MS/MS system (AB SCIEX, Framingham, MA, USA) equipped with an electrospray ionization interface. The compounds were separated on a Kinetex biphenyl column (100 × 2.1 mm internal diameter, 100-Å pore size, 2.6-μm particle size, Phenomenex, Torrance, CA, USA) in a mobile phase of water:acetonitrile mixture at a 1:3 (v/v) ratio and also including 0.1% formic acid. The column was heated at 30 °C and the mobile phase was delivered at a flow rate of 0.2 mL/min using an HP 1100 series pump (Agilent, Wilmington, DE, USA).
The turbo ion spray interface was operated at 4500 V at 450 °C. Both the precursor and product ions of IQ, epi-IQ, and the IS appeared predominantly as deprotonated ions [M − H]⁻, at an m/z of 357.0 for both epimers and 296.1 for the IS. After collision with nitrogen in Q2, the product ions were scanned in Q3 at an m/z of 151.0 (declustering potential, −50 eV, collision energy, −54 eV, dwell time, 150 ms) for the IQ epimers and 251.7 (declustering potential, −65 eV, collision energy, −18 eV, dwell time, 150 ms) for the IS. The deprotonated precursor ions and related product ions were quantified by selective reaction monitoring using the peak area ratios of each substance. The analytical data were processed using Analyst software (ver. 1.5.2, Applied Biosystems, Foster City, CA, USA).

The purity of isolates was determined through HPLC-DAD analysis (190-400 nm for the HEP fraction or 200–400 nm for the MeOH fraction) on an Elite LaChrom system consisting of an autosampler L-2200, a pump L-2130, a column oven L-2350, a diode array detector L-2455 (all Hitachi, Tokyo, Japan) and a Kinetex^® biphenyl column (100 Å, 5 µm, 4.6 × 250 mm, Phenomenex, Aschaffenburg, Germany) or a Nucleodur^TM C18 Isis column (RP18, 5 µm, 4.6 × 250 mm, Macherey-Nagel, Düren, Germany). For analyses, gradients described in 0 were conducted at a flow rate of 1 mL/min and an injection volume of 5 or 10 µL (acetonitrile, 1 mg/mL). Thus, the purity was calculated as the proportion of the integral of the main peak in the chromatogram using the maxplot adjusted by a blank (EZChrom Elite 3.1.7, Hitachi).

The purity of isolates was determined by HPLC-DAD (190–400 nm) analysis using an Elite LaChrom system consisting of an autosampler L-2200, a pump L-2130, an column oven L-2350, a diode array detector L-2455 (all Hitachi, Tokyo, Japan) and a Kinetex^® biphenyl column (100 Å, 5 µm, 4.6 × 250 mm, Phenomenex, Aschaffenburg, Germany). The gradients described in Section 3.3.6 were used to analyze 5 µL (acetonitrile, 1 mg/mL) with a flow of 1 mL/min and the chromatograms processed with EZChrom Elite 3.1.7 (Hitachi). Thus, the purity was calculated as the proportion of the integral of the main peak in the chromatogram using the maxplot (adjusted by a blank).