A BORDAJE CUANTITATIVO – A NÁLISIS DE DATOS SECUNDARIOS

Male rats (N = 40, 5 rats per age group, standard housing and feeding) in age groups of 1 day, 14 days, 30 days, 60 days, 90 days, 365 days, 547 days and 730 days (Sprague-Dawley strain, Harlan Sprague Dawley, Inc., Indianapolis, IN, USA) were anesthetized with aerosolized isoflurane and fresh brain samples were obtained and partitioned using a dissecting microscope. Tissue samples (0.1-0.2 g) of striatum, ventral midbrain, frontal cerebral cortex, cerebellar cortex, and hippocampus were weighed and then homogenized in 100 µl of ice-cold dissolution buffer (0.1 M

perchloric acid, 0.1 mM sodium bisulfite, and 0.1 mM EDTA) per 10 mg wet weight. All homogenates were centrifuged at 20,000 g for 25 min at 4C. Supernatants were filtered through 0.22 µm pore size polyvinylidene fluoride (PVDF) syringe-driven membrane filters (Millipore Corp., Bedford, MA, USA) and immediately frozen and stored at -80C until the time of analysis. As above, SPE was performed on all samples prior to LC-MS/MS analysis.

4.2.5 HPLC-EC Analysis

4.2.5.1 Chromatography Standard Preparation

Primary stock standard solutions were prepared by dissolving 10 mg of (R/S)SAL, N-methyl-(R/S)SAL, NorSAL, dopamine, DOPAC, and HVA in 25 ml of dissolution buffer. These concentrates were then divided into 1 ml aliquots, frozen, stored at -80C, and thawed prior to use at 4C. Working standards in the nM range were freshly prepared prior to each assay. Standard curves employed for analyte

quantification were generated with a DHBA internal standard. The relationship between concentration and relative response was linear over two orders of magnitude (sample correlation coefficient > 0.98), with an overall electrochemical sensitivity of 0.001 µA. With HPLC-EC, the limit of quantification for all analytes was 0.7 ng/ml.

4.2.5.2 Mobile Phase Preparation

A stock buffer solution containing 75 mM monobasic sodium dihydrogen phosphate, 2 mM SOS, 25 µM EDTA, 20 mM β-cyclodextrin, and 100 µl of TEA was prepared in 1800 ml of HPLC grade water. To prepare the mobile phase, this solution was then mixed with 200 ml of HPLC grade acetonitrile and buffered to pH 3.0 using concentrated ortho-phosphoric acid. The mobile phase was stored at room temperature for no more than 2 days, filtered through a 0.20 µm pore size white nylon filter

membrane (Millipore Corp.), and degassed under vacuum for 30 min prior to use. The chiral mobile phase additive β-cyclodextrin is a cyclic oligosaccharide which

preferentially binds to one face of an enantiomer. This binding changes the hydrogen- bonding patterns of the enantiomers and allows for their separation using a standard reverse-phase column.

4.2.5.3 Liquid Chromatography

HPLC-EC analysis was performed with an ESA Model 5600A CoulArray® system (ESA Inc., Chemlsford, MA, USA), equipped with Shimadzu Model DGU-14A on-line degassing unit (Shimadzu Scientific Instruments, Columbia, MD, USA), an ESA Model 582 pump, and an ESA Model 542 refrigerated autosampler. The detection system consisted of three coulometric array modules, each containing four electrochemical detector cells. Electrode potentials were selected over the range of 0 to +700 mV, with a 50 mV increment against palladium electrodes. Chromatographic separation was

achieved by auto-injecting 30 µl sample aliquots at 5C onto a MetaChem Intersil (MetaChem Technologies, Torrance, CA, USA) reversed-phase C18 column (5 µm particle size, 250X4.6 mm I.D) with an ESA Hypersil C18 pre-column (5 µm particle size, 7.5X4.6 mm I.D.) [Fig. 3A]. A mobile phase flow rate of 1.25 ml/min and analysis time of 45 min were used for all experiments. System control and data acquisition/processing were performed using ESA CoulArray software (version 1.02). All samples were processed in technical triplicate with median values used for analysis.

4.2.6 LC-MS/MS Analysis

4.2.6.1 Chromatography Standard Preparation

Primary stock standard solutions were prepared by dissolving 10 mg of TIQ, 1- methyl-TIQ, N-methyl-TIQ, and 1-benzyl-TIQ in 25 ml of a mixture of 50% water and 50% acetonitrile. These concentrates were then divided into 1 ml aliquots and stored at -80C. Working standards in the nM range were freshly prepared prior to each assay.

4.2.6.2 Liquid Chromatography

LC-MS/MS analysis was performed using an Agilent model 1100 series HPLC system equipped with a Bruker Esquire-LC ion trap tandem mass selective detector (Bruker Daltronics Inc., Billerica, MA, USA), a Model 1327A temperature controlled autosampler, a Model 1316A temperature controlled column compartment, and a Model G1312A binary pump with Model G1322A solvent degassing module, and a Model G1314A variable wavelength UV–vis detector (Agilent Technologies, Santa Clara, CA). Ultraviolet absorbance monitoring was performed at 220 nm. Separation was achieved by auto-injecting 10 µl sample aliquots at 5 C onto a Thermo Hypersil-Keystone

reversed-phase C18 column (5 μm; 50 x 2.1 mm I.D.; Thermo Fisher Scientific, Waltham, MA, USA) in combination with a C18 Hypersil guard column (10 x 2.1 mm I.D.). A binary mobile phase flow rate of 0.7 ml/min and analysis time of 25 min was utilized. Mobile phase A consisted of water with 0.1% formic acid and mobile phase B consisted of acetonitrile with 0.1% formic acid. The following mobile phase gradient was used: 0 min A/B = 95:5, 5 min A/B = 70:30, 10 min A/B = 50:50, 15 min A/B = 70:30, 20 min A/B = 95:5. The column was given a 5 min post-run period to allow for re-equilibration. System control and data acquisition/processing were performed using ChemStation software revision A.08.03 for LC-MS systems (Agilent Technologies).

4.2.6.3 Ion Trap Tandem Mass Spectrometry

Full-scan mode was used to acquire mass spectra, precursor ions, and product ions. EsquireControl software version 6.16 (Bruker Daltronics Inc.) was employed for control of all mass spectrometric conditions. The mass spectrometer was equipped with a heated capillary interface and an electrospray ionization (ESI) source, operating in positive ion mode. The ESI source and MS/MS parameters were automatically optimized. Spray needle voltage was 4.0 kV and capillary temperature was set to 220 C. Sheath and auxiliary nitrogen gas flow were set to 40 and 0 psi, respectively. Automatic gain control was utilized with a maximum isolation time of 300 ms. A relative collision energy of 33% was used for all analyses. The product ions producing the highest intensity were used for quantification to increase analytical sensitivity and selectivity in LC-MS/MS mode. Quantification was based on detector response, defined as the ratio of the base peak ion of interest to the base peak ion of the standard

compound. The relationship between concentration and relative response was linear over two orders of magnitude (sample correlation coefficient > 0.998) for the MS/MS procedure. All samples were processed in technical triplicate with median values used for statistical analyses. With LC-MS/MS, 0.3 ng/ml was the limit of quantification for all analytes.

4.2.7 Parkinson Disease Lifetime Food Intake Questionnaire