Brazilian Journal of Analytical Chemistry - BRJAC - Brazilian Journal ...

ma samples in three cycles was studied with control
concentrations in quintuplicate. Samples were frozen
at - 20°C and thawed in three cycles of 24, 36 and
48 h. In addition, the long-term stability of FLU in QC
samples was also evaluated by analysis after 134 days
of storage at - 20°C. Autosampler stability was studied
over a 12-24 h storage period in the autosampler rack
with control concentrations.
rEsults
sElECtivity
The analysis of flunarizine and cinarizine using the selective
reaction monitoring (SRM) function was highly
selective, with neither interfering compounds nor significant
ion supression from endogenous substances
observed at the retention times for flunarizine and IS,
as shown in Figure 2.
fi g u rE 2. rEPrEsEntativE srm C h ro m a t o g ra m s o f f l u n a r i z i nE a n d CinarizinE
in B l a n k human P l a s m a.
Figure 3 shows the mass spectrum of FLU in the sequential
mode: m/z 404.60 generating ion fragments
at m/z 202.90 at collision energies of 20 eV. The intensities
of other fragments are negligible.
fi g u rE 3. ms-ms sPECtra o f f l u n a r i z i nE BasE PEak at m/z 404.6 a n d
daugthEr PEak m/z 202.9. Co l l i s i o n E n E rg y 20Ev.
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determination o f f l u n a r i z i n e in plasma by liquid
C h ro m a t o g r a p h y-eleCtrospray t a n d e m m a s s speCtrometry.
Chromatograms obtained from plasma spiked with
flunarizine (0.30 ng/mL) are shown in Figure 4.
fi g u rE 4. Ch ro m a t o g ra m o f f l u n a r i z i nE o B t a i nE d f ro m P l a s m a sPikEd w i t h
0.30 n g/ml.
The matrix effect was evaluated directly by extracting
blank plasma and then spiking it with the analyte at the
LOQ concentration. There was no difference observed
in the signal for the solution and the spiked extract at
the LOQ concentration.
linEarity, PrECision a n d a C C u ra C y
Calibration curves were plotted as the peak area ratio
(flunarizine/cinarizine) versus flunarizine concentration.
The assay was linear in the concentration range of 0.30-
150.00 ng/mL; R.S.D.s were less than 10%. The relative
error of the mean of measured concentrations ranged
from 7.33 to 9.00%. The determination coefficients (r 2 )
were greater than 0.98 for all curves (Table I).
sP i kE d P l a s m a
C o n C E n t r a t i o n
(n g/ml)
taBlE i. CaliBration CurvE f ro m BatCh
o n E d u r i n g m E t h o d validation.
Co n C E n t r a t i o n mEasurEd
(m E a n) (n g/ml)
r.s.d. (%) a
(n=3)
rE l a t i v E
E r ro r b
0.30 0.29 1.72 -3.33
1.50 1.58 6.83 5.33
5.00 5.30 5.20 6.00
15.00 15.37 1.23 2.46
50.00 47.78 2.74 -4.44
75.00 69.48 2.07 -7.36
150.00 148.56 6.83 - 0.96
a standard deviation / mean concentration measured
b [( Mean concentration measured – spiked plasma concentration) /
spiked plasma concentration ] × 100.
Precision and accuracy for this method were controlled
by calculating the intra-batch and inter-batch variation
at three concentrations (0.90, 60.00 and 120.00 ng/mL)
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