HPLC Determination of Nifedipine In Human Plasma
Hisham S. Abou-Auda*, Naushad M. Ghilzai,
Tawfeeg A. Najjar and BadrAddin M. Al-Hadiya
Department of Clinical Pharmacy,
College of Pharmacy,
King Saud University,
P.O. Box 2457,
Riyadh 11451,
Saudi Arabia
Tel: 966-1-467-7470
Fax: 966-1-467-6789
E-mail: hisham@ksu.edu.sa
* To whom correspondence should be addressed.
ABSTRACT
High-pressure liquid chromatography, Nifedipine Key Words:
Introduction
Nifedipine,
1,4-dihydro-2,6-dimethyl
-4-(2-nitrophenyl)
-3,5-pyridine
dicarboxylic acid dimethyl ester, is one of the most potent calcium-channel blockers
belonging to the group of 1,4-dihydropyridines (1). It is widely used in the treatment
of vascular diseases such as hypertension, angina pectoris and Raynaud’s
phenomenon (2-4). Nifedipine, a highly non-polar compound, is absorbed completely
from the gastrointestinal tract, predominantly from the jejunum, but has a very low
bioavailability mainly due to presystemic metabolism (1,5). Following absorption,
nifedipine is further metabolized in the small intestine and liver to more polar
compounds which are primarily eliminated by the kidney (6-8). Nifedipine is a
photolabile compound, undergoing oxidative biotransformation in human body into
pharmacologically inactive metabolites (6,7,9-11).
Most previously reported HPLC methods for determination of nifedipine in
biological samples lack full validation procedures (6,7,12-15) outlined by FDA,
Health Canada and other related European agencies (16).
The present study documents an accurate, sensitive, rapid, selective and
reproducible reversed-phase HPLC assay which meets an accepted full analytical
method validation. The developed method is suitable for the determination of
nifedipine in plasma samples taken during clinical and non-clinical studies.
Materials and Methods
Apparatus
A Waters HPLC system (Milford, MA, USA) was used, consisting of M-45
solvent delivery pump, an autosampler (model WISP-712), model 481 UV/VIS
variable wavelength detector set at 240 nm, in combination with a data module
integrator (model 746). Chromatographic separations were accomplished using a
supelcosil LC-18, 5 um, 15 cm  4.6 mm stainless steel column ( Supelco) with a
guard precolumn of same packing material.
Reagents
Nifedipine hydrochloride was kindly presented by Bayer (Wuppertal,
Germany). Diazepam (as internal standard) was obtained from Sigma Chemicals (St.
Louis, MO, USA). Methanol and acetonitrile were of HPLC grades (BDH Chemicals,
Poole, U.K.). All other chemicals and reagents were of analytical grade.
Standard solutions
Nifedipine hydrochloride (10 mg) was dissolved in 100 ml of methanol in dark
under sodium lamp. This stock solution was diluted with methanol 100-fold to give a
working standard solution of 1ug/ml concentration. The solution was kept protected
from with an aluminum foil wrapping and stored at –70 C. The solution was stable
for at least three months. The internal standard, diazepam (10 mg) was dissolved in
100 ml of methanol to prepare 1 ug/ml of working standard solution and stored at – 70
C (stable for at least one month).
Chromatographic conditions
The mobile phase consisted of acetonitrile–methanol–water (35 : 17 : 48
v/v)adjusted to pH 4.0 with phosphoric acid. The mixture was filtered through a 0.2
um membrane ( Millipore, MA, USA) under vacuum, further degassed by passing
nitrogen.
The mobile phase was pumped isocratically at a flow rate of 1.2 ml/min during
analysis, at ambient temperature. The chromatograms were recorded and integrated at
a speed of 0.25 cm/min. The effluent was monitored at excitation and emission
wavelengths of 330 and 440 nm, respectively, at attenuation 4 and gain X10.
Sample preparation
Sample preparation and analysis were conducted at room temperature under
sodium lamp. Nifedipine working standard ( 1 ug/ml) was added to 15-ml graduated
centrifuge tubes in volumes of 0, 2, 3, 4, 5, 6, 7, 8,and 9 ul. Drug-free human plasma
was added to complete volume to 10 ul , vortex-mixed for 30 sec , to yield final
calibration standard concentrations of 0.0 (no nifedipine added ) ,10 , 25 ,50 ,75 , 100
,125 , 150 and 200 ng /ml . Each of these standard solutions were distributed in
disposable polypropylene microcentrifuge tubes ( 1.5 ml , Eppendorf ) in volumes of
1.2 ml and stored protected from light at –70 C pending analysis . For preparation of
samples for injection onto HPLC system ,a 100–ml aliquot of the internal standard
(diazepam , 1 ng /ml ) was added to 1 ml of plasma sample in a 10–ml glass–
stoppered tubes , and vortex–mixed for 15 seconds. The sample was then alkalinized
by addition of 100 ul of 1N NaOH, vortex-mixed for 30 sec., and 5 ml of diethyl ether
were added. This mixture was vortex-mixed for one minute and centrifuged at 4000
rpm (2400 g) for 10 minutes. The supernatant organic layer was quantitatively
transferred to another 10-ml glass centrifuge tube and the contents were evaporated to
dryness at room temperature under a stream of pure nitrogen. The residue was
reconstituted in 250 ul of mobile phase, vortex-mixed for 30 seconds, transferred to
1.5 ml eppendorf tubes and centrifuged at 13000 rpm for 5 minutes to precipitate any
particulate matter. Aliquots of 80 ul were injected onto the HPLC column.
Assay Validation Results
The validation procedures and results are presented below . All
samples used for the validation tests were prepared by spiking interference–free pools
of heparinized plasma with prepared standards to give the specified final
concentrations .
Figure 1 depicts representative chromatograms for blank plasma, plasma
spiked with internal standard , and plasma containing internal standard and 25 ng / ml
of nifedipine .
Linearity
Plasma standards , prepared as described above were at eight non –zero
concentrations over the range of 10 to 200 ng / ml . Standards were analyzed in
replicates of seven . The average peak area ratios of nifedipine to the internal standard
were plotted against the concentration . Table 1 shows the results from the linearity
study . The slope ( b ) , intercept ( a ) , and pearson correlation coefficient ( r 2 )were
determined by the method of weighted least squares ( linear mode ) and the data are
also presented in table 1 .The weighted linear regression of nifedipine assay in plasma
was characterized as having a slope of 0.0052 and an intercept of
-0.0119 ( r =
0.9970 ,and r 2 = 0.9941 ) . These results indicate the linearity throughout the range of
nifedipine concentrations ( 10 –200 ng / ml ) studied . There was minor day–to–day
variability in slopes and intercepts , and excellent linearity observed for all calibration
curves , ( r > 0.994 ) . The coefficient of variation for slopes was 4.23 % .
Precision and Accuracy
Replicate samples (n = 8 ) spiked at three control concentrations ( 15 , 80 and
175 ng / ml ) were used to assess intraday (within–day ) precisions , as well as
accuracy , of nifedipine assay in plasma . Selection of concentrations for analysis was
made to allow for definition of precision at low , medium and high concentrations of
the linear range . Precision is expressed as the percent coefficient of variation ( % CV
) for the drug / internal standard ratios . Accuracy is expressed as a percent (observed
conc._ /theoretical conc.) X 100 ) . The intra–day ( within–day ) precisions ranged
from 2.22 to 4.64 % CV , and accuracy ranged from 102.4 to106.4 % (table 2 ) . The
day–to–day ( or inter –day ) precisions in plasma samples was similarly evaluated
over a period of six weeks .Precision ranged from 2.34 – 7.06 % and accuracy from
95.1 to 100.1 % ( table 2 ) .
Lower limit of quantitation
Heparinized plasma spiked at 0.00 ( Blank ) , 5.0 , 7.5 , 10 and 20 ng / ml were
used to assess the sensitivity of the method ,based upon both signal intensity and
variability . Peak heights of both drug and noise ( at the drug retention time in the
blank ) were measured normally from the chromatograms . The average signal–to–
noise ratio ( S /N ) was then calculated at each of the concentrations . The lowest
measurable concentration ( LOQ) was found to be 7.5 ng /ml with a % CV of 13 % .
Selectivity ( Specificity)
The selectivity of the method was examined by preparing and analyzing ( in
duplicate ) eight independent pools of heparinized blank plasma , in addition to
different commonly used drugs . No significant chromatographic interferences were
observed ( Table 3 ).
Recovery
The recoveries of nifedipine (relative and absolute ) from plasma and that of
the internal standard (absolute ) were quantitated using the standards (15 ,80 and 175
ng /ml ) for the drug and 1ng /ml for the internal standard , diazepam . The relative
analytical recovery was measured by
adding the drug and internal standard to drug–free plasma ( eight replicates for each
standard ) to achieve the concentrations shown in Table 4 . The spiked plasma was
then analyzed by the developed method . The relative recovery was calculated by
comparing the concentrations obtained from the drug –supplemented plasma with
actual added amounts . As shown in Table 4 , the relative recovery of nifedipine from
plasma ranged from 91.0 to 107.3 % . The absolute recoveries were calculated by
comparing the observed concentrations obtained from the processed standard samples
to direct injections of stock solutions prepared at concentrations which represented
100 % recovery . As shown in Table 4 , the absolute recovery of nifedipine from
plasma ranged from 88.6 to 93.3 % . As observed , the extraction recovery using
diethyl ether was less than 100 % . In spite of this fact , the choice of diethyl ether as
the extracting solvent provided adequate sensitivity to plasma samples over several
other organic solvents , such as hexane , dichloromethane and ethyl acetate . It also
minimized the endogenous interfering peaks and noisy baseline . The internal
standard absolute recovery was found to be consistent from all eight pools tested ,
averaging 92.0 % with a range of 90 .0 – 94.3 % ( Table 5 ).
Stability
The stability of the drug was determined
( 1) In processed and reconstituted sample .
(2)
Through a period of six–week freeze–thaw cycles .
( 1) Processed samples : Two concentrations ( 80 and 175 ng /ml of nifedipine )
reconstituted in mobile phase , plasma and whole blood , were used for photostability
study . The nifedipine preparations were divided into two 50–ml volumetric flasks .
One had been kept in dark and the other kept under fluorescent lighting at room temp
(20 C). Samples analyzed at time periods of 0 , 10 , 20 , 30 , 40 and 60 minutes for
nifedipine in whole blood and up to 120 minutes in aqueous and plasma samples.
From Table (6) whole blood samples were shown to be stable ( on light exposure ) for
30 minutes , which is an optimum (or convenient) period of time for withdrawing
blood for human subject , separating the serum (or plasma ) , then protected from light
with an aluminum foil wrapping ,and eventually kept in freezer pending analysis . On
the other hand , plasma samples were stable for at least 120 minutes exposure to light
. Those in aqueous solution were stable up to 60 min light exposure . Stability in
heparinized plasma through eight freeze–thaw cycles (six–week period at – 20 +_ 5 C
to room temperature, has been confirmed (Table 7) . Samples were removed from
freezer , allowed to stand on the bench top , under room lighting , for 30 minutes to
thaw , and then assayed for the nifedipine content.
Discussion
The method was sensitive enough to detect a concentration as minimum as 3
ng /ml . The minimum quantifiable concentration of nifedipine was determined to be
7.5 ng /ml and the standard curve was linear to at least 200 ng /ml . The developed
assay method was used to analyze samples collected from one healthy volunteer
demonstrating that it can be applied to clinical and pharmacokinetic studies . Based
on problems encountered by several past assay publications and validation , the
present study has served to develop a satisfactory sensitive , specific , accurate and
fully validated assay method of nifedipine in human plasma . The results in Tables 1
and 2 describe precision ( CV ) and accuracy ( % ) of the method . Both accuracy and
precision values throughout the concentration range ( 10 –200 ng /ml ) were
acceptable . The relative recovery results in Table 4 indicated the extraction yield
ability of diethyl ether for nifedipine from plasma ( ~91 +_ X% ) , compared with
other solvents ( e. g. ethyl acetate, dichloromethane etc.)
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