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%.