2.3. In vivo quantification of BK-(1-9) fragments by multiple reaction monitoring (MRM) mass spectrometry analysis
Adult male Wistar rats were anesthetized with i.p. administration of urethane (1300 mg kg-1). A polyethylene catheter was inserted into the inferior vena cava through the femoral vein fori.v. administration (0.1 mL) to add the standards, BK-(1-9) (7µg kg-1 or ~2nmol per animal) and heavy-isotope labelled [Pro3(13C5;15N)]-BK-(1-9) (7µg kg-1 or ~2nmol per animal) diluted in sterile saline (0.9% NaCl). Blood (~ 3 mL) was collected directly from the left ventricle following 15 seconds or 5 minutes of the BK-(1-9) or [Pro3(13C5;15N)]-BK-(1-9) i.v. administration. Samples from control animals (no peptide administration) were used to provide as zero-time (time = 0 s) values. This experimental group was important to define the circulating concentration of any endogenous BK peptide fragments evaluated in this experiment (i.e., BK-(1-7) and BK-(1-5)). The blood was collected in a tube containing the protease inhibitors: 1 mmol L-1 p-hydroxymercuribenzoate, 30 mmol L-1 1,10-phenanthroline, 1 mmol L-1phenylmethylsulfonyl fluoride (PMSF), 1 mmol L-1pepstatin A, and 7.5% EDTA – 140 μL mL-1 of blood. Plasma samples were collected by centrifugation (2000 x g at 4°C for 20 minutes) and stored at −80°C until further use. The peptides were extracted from plasma using the HF Bond Elut C18 (Variant, Palo Alto, CA, USA). The C18 resin was activated by 2 sequential washes with 10 mL of 99% acetonitrile (ACN) / 0.1% trifluoroacetic acid (TFA), followed by 10 mL of 0.1% TFA. Then, the resin was washed with 3 mL of 0.1% TFA / 0.1% BSA, 10 mL of 10% acetonitrile / 0.1% TFA, and 3 mL of 0.1% TFA. The sample (1.2 mL) was loaded and washed with 20 mL of 0.1% TFA and then 3 mL of 20% acetonitrile / 0.1% TFA. The peptides were eluted with 3 mL of 99% ACN / 0.1% TFA into low-binding polypropylene tubes (Eppendorf, Hamburg, Germany) and dried down using a SpeedVac (Eppendorf, Hamburg, Germany). Each sample was resuspended in 60 µL 0.1% formic acid (concentration factor of the sample = 20-fold) and then loaded (5 µL) onto the ACQUITY I-Class UPLC system (Waters, Milford, MA, USA) coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-ESI-MS/MS Xevo TQ-S, Waters, Milford, MA, USA). The chromatographic separation was done in a C18 column (ACQUITY UPLC BEH C18 Column, 130Å, 1.7 μm, 2.1 mm X 100 mm, Waters, Milford, MA, USA) and each chromatography lasted for 5.5 min. Solvent A was made of 0.1% formic acid in H2O and solvent B of 0.1% formic acid in ACN. The chromatographic gradient was as follows (expressed as % of solvent B): i) 3-40% in 3.5 min, ii) 40-99% in 0.01 min, iii) 99% for 0.99 min, iv) 99-3% in 0.01 min, and v) 3% for 0.99 min. Regarding the MS analysis, the main parameters were as follows: i) capillary = 3.5 kV; ii) cone = 20V; iii) temperature of the desolvation gas (hydrogen) = 550oC. The collision energy (CE) (argon gas) was tuned for each target peptide spanning from 10 to 20 CE. The following transitions were monitored by the MS in the MRM mode. BK-(1-9) [RPPGFSPFR]: 354.4 419.3 and 354.4 408.2. BK-(1-7) [RPPGFSP]: 379.42 555.3 and 379.42 527.4. BK-(1-5) [RPPGF]: 278.1 408.4 and 278.1 166.1. For the heavy-isotope labelled peptides, we used the following transitions. [Pro3(13C5;15N)]-BK-(1-9) [RPP(13C5;15N)GFSPFR]: 356.2 419.3. [Pro3(13C5;15N)]-BK-(1-7) [RPP(13C5;15N)GFSP]: 382.2 561.3. [Pro3(13C5;15N)]-BK-(1-5) [RPP(13C5;15N)GF]: 290.2 414.2. The calibration curve was obtained using a stock solution containing a mixture of synthetic BK-(1-9), BK-(1-7) and BK-(1-5). The applied calibration curve model (y = ax + b) proved accurate over the concentration range 10 to 1000 pg mL-1(r2 = 0.968). The limit of quantitation (LOQ) was below 10 pg per sample. The inter- and intra-variability of this method was < 20%.