A New HPLC Method for 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
Key Words: High-pressure liquid chromatography, Nifedipine
INTRODUCTION
Nifedipine,
1-4-dihydro-2-6-dimethyl-4-2(2-nitrophenyl)-3,5-pyridine-
dicarboxylic acid dimethyl ester, is a calcium channel blocker belonging
to the
dihydropyridine class of compound. It is widely used in the treatment of vascular diseases
such as hypertension, angina pectoris and Raynaud’s phenomenon (1,2). Nifedipine, itself
is a highly non-polar compound and absorbed completely from the gastrointestinal tract,
predominantly from the jejunum but has a very low bioavailability mainly due to largely
presystemic metabolism, following absorption, nifedipine is further metabolized in the
small intestine and in the liver to more polar compounds which are primarily eliminated
by the kidney (3,4 and 5). Nifedipine is highly sensitive to chemical oxidation on
exposure to ultra-violet rays (UV and visible). The photodegradation products and other
analogues are deprived of calcium channel blocking properties. Nifedipine is handled
very carefully under the sodium lamp(6,7).
In earlier published HPLC methods for determination of nifedipine in biological
samples lack full validation process outlined by FDA, Health Canada and other related
European agencies and usually omit accuracy especially for lowest quantifiable
concentration and the photostability study of the nifedipine (3,4,6,7,9,10).
In this study, we report a rapid, sensitive and selective reversed-phase HPLC assay which
follows the guidelines set up for the complete analytical method validation and which is
suitable for processing large numbers of nifedipine plasma samples taken during the
clinical and non-clinical studies.
EXPERIMENTAL
Chemical and Reagents
Nifedipine hydrochloride was kindly presented by Bayer (Wuppertal, Germany).
Internal Standard (Diazepam) was obtained from Sigma Chemicals (St. Louis, MO,
USA). Methanol and Acetonitrile were of HPLC grades (BDH chemicals, Poole, U.K.).
Other chemicals and reagents used were of analytical grade.
Instrumentation
A Waters HPLC system (Millipore Waters Division, Milford, MA, USA) was
used, consisting of Model M-45 solvent delivery Pump , an autoinjection ( Model WISP
712), model 481 UV-VIS Variable wavelength detector set at 240nm and a data module
integrator (Model 746 ). Analytical separation was accomplished using a Supelcosil LC
-18 , 5um, 15cm.x 4.6mm Column (Supelco) with a guard precolumn of the same
packing.
Chromatographic Conditions
The mobile phase consisted of acetonitrile-methanol-Water (35:17:48, v/v)
adjusted to approximately pH 3.8 with phosphoric acid. The mixture was filtered through
a 0.22um membrane filter (Millipore Corp. MA, USA) under vacuum. The flow rate was
1.0ml/min. during analysis. The chromatograms were recorded and integrated at a speed
of 0.25 cm.min-1. Sample collection , preparation and analysis were conducted very
strictly at room temperature under sodium lamp.
Internal Standard
A Stock solution of diazepam was prepared by dissolving 10mg in 100ml of
methanol. The solution was further diluted with methanol to prepare 1ug/ml of working
solution and stored at 4c.
Standard Solution of Nifedipine Hydrochloride
The standard stock solution of nifedipine hydrochloride was prepared by dissolving 10mg
in 100ml of methanol. The solution was further diluted with methanol to prepare 1ug/ml
of working standard solution. The solution was protected from light, wrapped with
aluminum foil and stored at -20c. The solution was stable for at least 3 months.
Sample Preparation
This assay is a modification of a previously reported nifedipine HPLC assay (ElSayed et al). Nifedipine working standard (1ug/ml) was added to a 15ml graduated glass
centrifuge tube in volumes of 0, 2, 3, 4, 5, 6, 7, 8 and 9ul.. Drug free plasma (10 ml) was
added to the tubes, provide calibration standards of 0.0 (no nifedipine added), 10, 25, 50,
75, 100, 125, 150 & 200 ng/ml. Standard Solutions were distributed in disposable
polypropylene microcentrifuge tubes (15 ml, Eppendorf) in volumes of 1.1 ml and stored
at -70c pending analysis. To 1 ml of plasma sample, in a glass tube (150x16 mm), was
added 100ul aliquot of the internal standard (Diazepam , 1 ug/ml), vortex mixed for 15
seconds. The sample was then alkalinized by addition of 100 µl of 1N NaOH and
vortexing for 30 seconds. 5ml of HPLC grade diethyl ether was added to the glass test
tube and the mixture was again vortexed for 60 seconds and subsequently centrifuged for
10 minutes at 4000 rpm.. The supernatant layer (organic layer) was transferred to another
glass centrifuge tube and the contents were evaporated to complete dryness using
laboratory supplied air. The residue was reconstituted in 250ul mobile phase, vortexed for
30 seconds and transferred to 1.5ml disposable Eppendorf tubes and centrifuged in a
microcentrifuge at 13,000 rpm for 3 minutes to precipitate any particulate matter. A 80 ul
aliquot was injected onto the HPLC.
Preparation of Calibration Curves
Fresh and drug-free plasma (1 ml) was used for construction of calibration curves
by spiking with 0, 10, 25, 50, 75, 100, 125, 150 and 200 ng/ml of required volume of
freshly prepared working standard solution of nifedipine to it for each run of the assay.
Plasma samples were prepared as described and the ratio of peak area of nifedipine to be
assayed to peak area of internal standard were calculated. The calibration curves were
constructed by weighted least squares regression analysis. Data were presented as
mean±S.D.
Validation of the Assay Method
Photostability of Nifedipine
Two concentrations (80ng/ml and 175ng/ml of nifedipine) were used for the
photostability study in organic solvent, plasma and whole blood. The nifedipine
preparations were divided into two- one 50 ml volumetric flasks containing nifedipine
dissolved in organic solvent, plasma and whole blood was kept in dark room (under
sodium lamp) and another volumetric flask was exposed to normal laboratory light at
room temperature (25°C) on a normal winter day for the following time periods: 0, 10,
20, 30, 40 and 60 minutes for organic solvent and up to 120 minutes for plasma and
whole blood. Samples were taken from both sources (light and dark), internal standard
was added and the samples were analyzed as per the method described earlier.
Specificity
The different commonly used drugs were used for interference using the same
chromatographic conditions as for nifedipine (Table !).
RESULTS and DISCUSSION
In Fig. 1 typical chromatograms were shown which were obtained after extraction
of drug-free and fresh plasma and blank plasma to which only internal standard has been
added and plasma containing 25ng/ml of nifedipine. It seems that no interfering
substances has been co-extracted. The photo-degradation products could not be appeared
in plasma samples of volunteers who received nifedipine indicating that protection from
normal laboratory light has been adequate. In this reported assay peaks illustrating
nifedipine and internal standard were eluted approximately at 6.60 minutes and 14.40
minutes respectively. Several published nifedipine methods have already described the
required sensitivity and resolution of nifedipine without interference from tested
metabolites and photodegradation products. This method is also not optimized for
detection and quantitation of nifedipine metabolites in human plasma samples, as known
metabolites are inactive (1,6).
Nifedipine concentrations and peak area ratios of nifedipine/diazepam were linear
throughout the range of nifedipine concentrations (10-200ng/ml) studied. The calibration
curves
of
nifedipine
was
typically
described
by:
Y=-0.0119(+0.0165)
=
0.0052(+0.0002)x, (r=0.99+0.005;n=7) where Y corresponds to the peak area ratio of
nifedipine to the internal standard and x to added nifedipine concentration over a
concentration range 10 to 200ng/ml.
There was minor day-to-day variability in slopes and intercepts, and excellent
linearity was observed for all calibration curves (r>0.994). The coefficient of variation for
slopes was 4.23%.None of the commonly used drugs (Tab.1)
interfered with the
nifedipine assay.
The lowest measurable concentration was about 7.5ng/ml using a signal -to-noise
ratio of three.
The results in table1 described accuracy (% error) and precision (coefficient of
variation, CV%) of the method. The within-day precision was evaluated by replicate
analysis (^ samples each) of plasma samples containing nifedipine at three different
known concentration (low, medium and high). The coefficient of variation (CV%) ranged
from 2.22-3.47%. The day-to-day precision in plasma samples was similarly evaluated
for six week period. The coefficient of variation ranged from 2.34- 7.06%.
The results in table 2 show analytical recovery (absolute and relative) of
nifedipine in plasma was evaluated at three different concentrations (low, medium, high)
by assaying the exact amount of nifedipine concentration in organic solvent and in
plasma adding the same amount of internal standard. The peak areas of extracted versus
unextracted nifedipine samples were compared under identical chromatographic
conditions. The Absolute Recoveries of nifedipine in plasma ranged from 88.6 - 93.3%
(extraction of 15, 80 and 175ng/ml concentrations, n= , replicates). We agree to the fact
that extraction recovery was less than 100% but the choice of diethylether as extraction
solvent provided adequate sensitivity to plasma samples over the several other organic
solvents (e.g. hexane, dichloromethane, ethylacetate, etc.) and also minimized the
endogenous interfering peaks and base line noise. Chemical stability factor of nifedipine
was not evaluated by many authors while validating their assays. For this purpose, three
nifedipine concentrations (low, medium, high) were selected and the blank plasma was
spiked with the concentration and all three were kept in a freezer at -70c. The plasma
samples were analyzed over 6 weeks period with a fixed time interval between removing
from freezer to thawing of the samples. The stability of nifedipine in frozen plasma
samples was verified by the results outlined in Table 3.
CONCLUSION
In our assay procedure, the chromatogram run (15minutes) is higher than other
published methods but this is not the only factor which should be considered in selecting
a full validated method. Several other namely photo- and chemical stability have a major
role in determination of exact nifedipine concentration in clinical samples. Based upon
practical aspects of evaluating a method, our method provide a sensitive, selective and
precise HPLC assay to measure nifedipine concentrations in pharmacokinetic,
pharmacotoxic and other related studies
REFERENCES
1. E.M. Sorkin, S.P. Clissold and R.N. Brogden, Drugs 30 ,182 (1985).
2. Hardman J.G., Limbird L.E., Molinoff P.B., Ruddon R.W., Gilman A.G. (1996):
Goodman and Gilman, The Pharmacological Basis of Therapeutics, 9th Edn. New
York: McGraw Hill,635.
3. Dokladalova J, Tykal JA, Coco SJ, Durkee PE, Quercia GT and Korst JJ, J.
Chromatogr. 231, 451 (1982).
4. J.H.M. Schellens, I.M.M. Van Haelst, J.B. Houston and D.D. Breimer, Xenbiotica
21(4),547 (1991).
5. Kleinbloesen C H., van Brummeln P., Faber H., Danhof M., Vermeulen N.P.E.,
Breimer D.D. Biochem. Pharmacol. 33, 3721 (1984).
6. P.A. Soons, J.H.M. Schellens, M.C.M. Roosenmalen and D.D. Breimer, J. Pharm.
Biomed. Anal. 9(6), 475 (1991).
7. W. Snedden, P.G. Fernandez and C. Nath, Can. J. Physiol. Pharmacol. 64, 290
(1986).
8. V.P.Shah, K.K.Midha, S.Dighe, I.J.McGilvery, J.P. Skelly, A. Yacobi, T. Layloff,
C.T. Viswanathan, C.E. Cook, R.D. McDowall, K.A. Pittman and S.Spector, Eur. J.
Drug Metab. Pharmacokinet. 16(4),249 (1991).
9. N.D. Huebert, M. Spedding and K.D. Haegele, J. Chromatogr.353 ,175 (1986).
10. El-Sayed Y.M., Niazi E.M., Khidr S.H. . J.Clin.Pharm. Ther. 18,325 (1993).
Table 1: Within-day and day-to-day accuracy and precision of nifedipine in plasma.
Added
Concentration
(ng/ml)
Within-day*
Day-to-day**
Measured
Conc.
(ng/ml)
15.53±0.35
(n=8)
Accuracy
%
Precision
%
3.5
2.22
80
85.09±3.95
(n=8)
6.36
175
179.13±6.22
(n=8)
2.36
15
Measured
Conc.
(ng/ml)
14.76±1.04
(n=8)
Accuracy
%
Precision
%
-1.58
7.06
4.64
80.04±2.77
(n=8)
0.05
3.46
3.47
166.34±3.90
(n=8)
-4.95
2.34
* Mean value represents different plasma samples for each concentration.
Table 2: Analytical Recovery of Nifedipine from Plasma.
Concentration
(ng/ml)
Mean Peak Area Ratio
Aqueous
Plasma
Absolute Recovery
%
Relative Recovery (%)
Mean
Range
15
93.3±5.0
102.7
96.0-107.3
80
90.1±3.9
98.0
94.0-103.3
175
88.6±2.7
99.3
91.0-102.3
Table 3:
Freeze-thaw stability of nifedipine in plasma.
Added Concentration
(ng/ml)
Measured Concentration*
(ng/ml)
Accuracy
%
Precision
%
15
15.45±0.64
3.00
4.15
80
80.68±2.91
0.84
3.60
175
176.11±6.44
0.64
3.66
Table 4:
Retention times for commonly used drugs using the current method.
Drug
Amidarone
Benzamide
Benzamide
Carbamazepine
Chloramphenicol
Clomipramine
Diltiazem
Fluvoxamine
Furosemide
Glibenclamide
Haloperidol
Hydrazoline
Meclofenamate
Metoclopramide
n-Cetylpyridinium
Nitrazepam
Phenacetin
Procainamide
Propranolol
Propyl Paraben
Quinidine
S-guanidine
Thiopentane
Thymol
Tolbutamide
Retention time (min)
3
5
ND
ND
ND
ND
ND
ND
3
12
ND
4
5
2
ND
4.5
2
ND
4.5
4
4.5
4.5
3
3
4
Table 5:
Dark
80 ng/ml
Aqueous
Plasma
Blood
After 120 min
After 60 min
Light
175 ng/ml
80 ng/ml
175 ng/ml
LIST OF FIGURES
Fig.1 Liquid chromatograms of:
Plasma Samples,
A, Blank plasma; B, Blank plasma spiked with the internal standard; C,
Plasma extract from healthy subject following oral administration of
Nifedipine capsule (Nifedipine Conc. =
µg/ml)
DIZ = ………….. (Internal Standard).
Fig.2 Nifedipine plasma concentrations following oral administration of a capsule
formulation of Nifedipine (10 mg) to three healthy male adults (closed circles)
and one sustained release tablet formulation to one male adult (open circles).