Ctc pal chilled autosampler

Tumor samples were analyzed by LC/MS/MS using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 HPLC and a CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent). After separation on a C18 reverse phase HPLC column (Agilent, Waters, or equivalent) using an acetonitrile-water gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in MRM mode. The signal was optimized for each compound by ESI positive or negative ionization mode. An MS2 scan or a SIM scan was used to optimize the fragmenter voltage and a product ion analysis was used to identify the best fragment for analysis, and the collision energy was optimized using a product ion or MRM scan. An ionization ranking was assigned indicating the compound’s ease of ionization.

The samples of donor and receiver side transport buffers from the permeability experiments were analyzed using LC/MS/MS. All analyses were performed using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 HPLC and a CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent) at Cyprotex (Waltham, MA). Analytes were separated on a C18 reverse phase HPLC column (Zorbax Stable Bond 3.5 μM; 2.1 × 3 mm HPLC column, (Agilent, Santa Clara, CA) using an acetonitrile-water gradient system. The drugs were loaded onto the separation column (Zorbax Stable Bond 3.5 μM; 2.1 × 3 mm HPLC column) with 98% solvent A (0.1% formic acid), and 2% solvent B (2% acetonitrile and 0.1% formic acid) at flow rate of 1 μL/min. The instrument settings were as follows: drying gas temperature, 350°C; drying gas flow, 11 L/min; nebulizer pressure, 50 psi; capillary voltage, 4000 V. Using this instrument, a precursor ion is selected using the first quadrupole and is sent to the collision cell for fragmentation. The fragments are scanned through the third quadrupole resulting in a product-ion scan MS/MS. An MS dwell time of 20 msec was used for each multiple reaction monitoring (MRM) transition. Peaks were analyzed by mass spectrometry (MS) using ESI ionization in MRM mode (Table VI).

On days 21–22 of the experiment, blood samples (300 μL) were collected from the tip of the tail after exposure to nicotine for 12 h. The trunk blood samples were collected from the site where animal was decapitated at the 12 h nicotine withdrawal time point (i.e., 12 h after the last nicotine exposure). Nicotine and cotinine levels in the blood were measured (Apredica, Watertown, MA) in both nicotine- and saline-exposed rats to ensure blinding and verification of assay sensitivity. Briefly, plasma samples were analyzed with liquid chromatography/mass spectrometry technique using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 HPLC and a CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent). After separation on a Polar silica HILIC (Sepax) HPLC column using an acetonitrile-water gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in multiple reaction monitoring (MRM) mode. The lower limit of quantitation for nicotine and cotinine was 27 nM in plasma.