V.Sreedevi et al.
/ International Journal of Pharma Sciences and Research (IJPSR)
Vol.2(2), 2011, 65-73
LC-MS Method Development and
validation for the estimation of Felodipine
in human plasma and Stability studies of
freeze thaw analyte.
V.Sreedevi* 1, Putta Rajesh Kumar 2, Rajesh Thatavarti 2.
1
Department of Pharmaceutical Analysis, Bhaskara Institute of Pharmacy, Bobbili.
2
Department of Pharmaceutics, V.L.College of Pharmacy, Raichur, Karnataka.
Abstract:
Purpose: A simple reverse phase liquid chromatographic and mass spectroscopic analytical method has been
developed and validated for estimation of felodipine in plasma. Methods: The separation was carried out on
Princeton SPHER C18 (150 x 4.6 mm i.d. of 5) as Stationary phase, Mobile Phase: Acetonitrile : 2mM
ammonium acetate Elution mode : Isocratic A: B= 80:20% v/v Flow rate: 0.8 ml/min using SPD M-10AVP
photo diode array detector at 38.10 nm. Results: The described LC MS method was linear over a concentration
range of 0.8-13.0ng/ml. Pantaprazole was used as internal standard. The felodipine and pantaprazole showed
retention factor of 2.97 respectively. The limit of detection (LOD) and the limit of quantification (LOQ) for
felodipine was 0.10 ng/ml, 0.50 ng/ml and for pantaprazole 0.06, 0.21 ng/ml respectively. The stability of the
drug spiked human plasma samples during three freeze thaw cycles were stable in plasma for about one month
when stored at frozen state. Conclusions: The results of the study showed that the proposed LC MS method is
simple, rapid, precise and accurate, which is useful for the estimation of felodipine in bulk fluids and biological
plasma sample analyte with accuracy and reproducibility.
Keywords: Felodipine, LC MS method, Pantaprazole and Freeze thaw cycles.
Introduction:
Felodipine is slightly yellowish, crystalline powder with melting point 1450C, Photosensitive and
Insoluble in water and is freely soluble in Dichloromethane and ethanol acts as Calcium antagonist-calcium
channel blocker. It is O3-ethyl O5-methyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate with Molecular formula C18H19Cl2NO4, Molecular weight 384.2540 daltons, Log P of 4.36 with
2.5 to 10 mg daily dose. Felodipine Prevents calcium from being released within muscle cells of the small
arteries and thereby causes the muscle to relax and the arteries to dilate or expand [1-4].
Pharmacokinetics: Oral bioavailability is 15%, 1% Urinary excretion with 99% Bound in plasma,
Clearance 0.8 l/min, Volume of distribution of 10 l/Kg and 11h Half-life. Mean peak concentrations following
oral administration of felodipine are reached in 2.5 h. Both peak plasma concentration time curve (AUC)
increases linearly with dose up to 20 mg. Felodipine is taken of 5-10 mg once-a-day, maximum 10mg two times
a day [5-6].
Literature survey revealed that felodipine is estimated by Felodipine by High-performance Liquid
Chromatography-tandem Mass Spectrometry (HPLC-MS/MS), high-performance liquid chromatography
coupled to tandem mass spectrometry, Spectrophotometric, Spectrofluorometric, High-performance liquid
chromatography with amperometric detection, HPLC and Chemometrically-Assisted Spectrophotometric
Estimation, liquid chromatography-tandem mass spectrometry, liquid chromatography/UV diode array
detection/atmospheric pressure chemical ionization mass spectrometry, Several methods have been reported for
quantification of felodipine in plasma as mentioned above. The present investigation reports a simple, rapid,
sensitive, and reproducible LC MS method for analysis of felodipine in plasma, using pantaprazole as internal
standard (IS) [7-12].
The Plan of the present study is as follows: Optimization of chromatographic conditions were
proposed to be developed and optimized like selection of Ionization, selection of initial separation conditions,
nature of the stationary phase, nature of the mobile phase (pH, peak modifier, solvent strength, ratio and flow
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Vol.2(2), 2011, 65-73
rate) and Selection of internal standard. The developed method were also proposed to be validated using the
various validation parameters such as, Accuracy, Precision, Linearity and Range, Limit of detection (LOD) /
Limit of quantitation (LOQ), Selectivity / specificity, Robustness / ruggedness, Stability and System suitability
as per ICH guidelines [13]. The Felodipine present in the biological fluid was proposed to be estimated.
Materials and methods:
Chemicals, reagents and Instrumental Conditions: Working Standard of Felodipine was obtained
from M/s Saimirra Innopharm Chennai, India and Pantaprazole internal standard was gifted by Dr Reddy’s
Laboratories, Hyderabad, India. Tablets were procured from the local market. Acetonitrile of HPLC grade by
Merck, Ammonium Acetate AR grade obtained from Qualigens fine chemicals and Water HPLC grade from
Milli-Q RO system were used. All other reagents used were of HPLC grade. Shimadzu LC2010A HT LCMS
system with following configuration was used i.e. LC-10 AD-vp solvent delivery system (pump), SIL 10 AD-vp
Auto injector, SPD M-10AVP photo diode array detector, CTO 10 vp column oven, GU 14AM degasser, LC –
MS solution data station, Analytical columns of Symmetry (Waters) C18 (150 x 4.6 mm i.d., 5 ), Shimadzu
160A UV-VIS spectrophotometer. Sartorius single pan digital balance (R200D & 1702) Systronics - pH meter,
pH system 361 and Ultra Sonicator were used for investigation.
Ethical approval: The detail of the study was approved by the Institutional Ethical Committee of J.S.S.
College of Pharmacy. The volunteers were also instructed to refrain from consuming alcohol, smoking or other
stimulant drinks during investigation period.
Chromatographic Conditions:
LC Conditions Stationary phase : Princeton SPHER C18 (150x4.6 mm i.d.,5) Mobile Phase:
Acetonitrile:2mM ammonium acetate Elution mode : Isocratic A: B= 80:20% v/v Flow rate : 0.8 ml/min
Injection volume : 10µl using Auto injector. MS Conditions Interface: ESI Operation mode : SIM Polarity :
Negative Probe temperature : Ambient CDL Temperature : 250º C Block Temperature : 200º C Detector voltage
: 1.3kv Nebulizer Gas flow: 1.5 l/min Drying gas : 10 L/min Detection : Felodipine – 382.05 Data station : LC-
MS solution data station Internal Standard: Pantoprazole - 382.10. The mobile phase was filtered through a 0.22
µ membrane and degassed using ultrasonicator. The experiments were carried out at room temperature of about
200C.
Validation of the method Validation is a process which involves confirmation or establishment by
laboratory studies that a method / procedure / system / analyst can give the required accuracy, precision,
sensitivity, ruggedness, etc. In the most basic form, validation of an analytical procedure demonstrates that the
procedure developed is suitable for its intended purpose. Validation of the method was carried out after the
development of the HPLC method. This section describes the procedure followed for the validation of the
methods developed.
Accuracy The accuracy of the drug was calculated by comparing the concentration obtained from the
relative recovery of drug supplemented plasma to the actually added concentration. To drug supplemented
plasma, standard Felodipine solution (Three levels) and internal standard solution were added. The resulting
sample solution was analysed and the response factor was calculated. The absolute recovery of Felodipine was
determined by comparing the response factor of the drug obtained from the plasma with response factor obtained
by the direct injection of Felodipine in mobile phase at three different levels. Recovery studies were carried out
for three levels at six times and the % recovery, mean, standard deviation and % CV was calculated.
Precision The precision of the method was determined by intraday precision and interday precision.
The intraday precision was evaluated by analysis of plasma samples containing Felodipine at three different
concentrations containing internal standard using nine replicate determinations for three occasions. The interday
precision was similarly evaluated over two week period. Precision studies were carried out for three levels at
nine times and three occasions. The mean concentration, standard deviation and % CV were calculated.
Selectivity Method I: The six blank plasma samples obtained from six different volunteers were
analysed and the spectrums were recorded. These spectrums were compared with the spectrums obtained from
standard solutions. Each spectrum was tested for interference. The combination of the sample preparation
procedure and spectrums provided an assay which must be free from significant interfering endogenous plasma
components at the retention times of Felodipine and the internal standard. Method II: This method involves the
peak purity test method using MS spectrum.
Linearity and Range The different concentrations of standard solutions were prepared to contain 0.8 -
13 ng /ml of Felodipine containing 10.00 μg/ml of internal standard. These solutions were analysed and the peak
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Vol.2(2), 2011, 65-73
areas and response factors were calculated. The calibration curve was plotted using response factor Vs
concentration of the standard solutions. The calibration curve was constructed on six different days over a two
weeks period to determine the variability of the slopes and intercepts.
Stability Studies The stability studies of plasma samples spiked with Felodipine were subjected to
three Freeze thaw cycles, Short term stability at room temperature for 3 hrs and Long term stability at 700 C
over four weeks. In addition, stability of standard solutions was performed at room temperature for 6 hr and
freeze condition for four weeks. The stability of triplicate spiked human plasma samples following three freeze
thaw cycles was analysed. The mean concentrations of the stability samples were compared to the theoretical
concentrations. The stability of triplicate short term samples spiked with Felodipine was kept at room
temperature for 1.00 to 3.00 h before extraction. The plasma samples of the long term stability were stored in the
freezer at 700 C until the time of analysis. The mean concentrations of the stability samples were compared to the
theoretical concentrations. The stability of the Felodipine standard solution at room temperature for 6 h and
freezed condition for two weeks were demonstrated by comparing a freshly prepared standard solution. The
stability of the internal standard stock solution was also performed by comparing a freshly prepared standard
solution containing internal standard.
System Suitability Studies The parameters namely column efficiency, resolution, peak asymmetry
factor and capacity factor for the standard solutions was calculated.
Limit of detection Testing method: By HPLC In-house method Based on Signal- to- Noise (3:1)
approach Determination of the signal-to-noise ratio is performed by comparing measured signals from samples
with known low concentrations of Felodipine with those of blank samples (i.e. mobile phase) and establishing
the minimum concentration at which the Felodipine can be reliably detected.
Limit of quantitation Testing method: By HPLC In-house method Based on Signal to Noise (10: 1)
approach Determination of the signal- to- noise ratio is performed by comparing measured signals from samples
with known low concentrations of Felodipine with those of blank samples (i.e. mobile phase) and establishing
the minimum concentration at which the Felodipine can be reliably quantified.
Ruggedness/robustness The ruggedness of the method was studied by changing the experimental
conditions such as, Different operators in the same laboratory, Changing the source of reagents and solvents
(different manufacturers like S.D. Fine Chemicals, Ranbaxy, Qualigens Fine Chemicals) and Changing to
another column of similar type and estimating the drugs using the assay procedure. The separation factor,
resolution time and peak asymmetry factors were then calculated. For demonstrating the robustness of the
method, slight variations in the optimised conditions were made and the standard solution was injected. The
variation made were, 1 % in the ratio of acetonitrile in the mobile phase, 0.05 ml of the flow rate. Then the
separation factor, retention times and peak asymmetry were calculated.
Estimation of felodipine in plasma A Shimadzu LC-MS system was used for the analysis with the
following chromatographic conditions. LC Conditions Stationary phase: Princeton SPHER C18 (150 x 4.6 mm
i.d., 5) Mobile Phase: Acetonitrile: 2mM ammonium acetate Elution mode: Isocratic A: B= 80:20% v/v Flow
rate: 0.8 ml/min Injection volume : 10 µl using Auto injector. MS Conditions Interface : ESI Operation mode :
SIM Polarity : Negative Probe temperature : Ambient CDL Temperature : 250º C Block Temperature : 200º C
Detector voltage : 1.3kv Nebulizer Gas flow : 1.5 l/min Drying gas : 10 l/min Detection : Felodipine – 382.05
Data station : LC-MS solution data station Internal Standard : Pantoprazole – 382.10 The mobile phase was
filtered through a 0.22 µ membrane and degassed using ultrasonicator. The experiments were carried out at room
temperature of about 200C.
Preparation of Felodipine standard stock solution Accurately transferred 100 mg of Felodipine
working standard into a 100 ml volumetric flask and dissolved in acetonitrile and made the final volume with
water and acetonitrile (1:1) to give 1.0 mg/ml solution of Felodipine. Labeled and stored the solution in a
refrigerator below 8°C. Preparation of Felodipine standard solution Standard solution for Calibration curve
Prepared, 10 ml each of 16, 20, 40, 80, 120, 240, 260 ng /ml of Felodipine standard solutions using the
Felodipine standard stock solution and mobile phase and labeled and stored at –2 ± 20 C until analysis. Standard
solution for QC Prepared, 10 ml each of 16, 120 and 240 ng/ml of Felodipine standard solutions using the
Felodipine standard stock solution and mobile phase and stored at –2 ± 20C until analyzed.
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Vol.2(2), 2011, 65-73
Preparation of stock and Calibration curve samples (CC) Using 0.5ml of Felodipine standard stock
solution, 10.0 ml each of 0.8, 1, 2, 4, 6, 12, 13 ng /ml of Felodipine calibration curve samples was prepared and
made up the volume with blank plasma and stored at –7 ± 20 C until processing.
Preparation of Quality control (QC) Samples Using 0.5ml of Felodipine standard stock solution,
10.0 ml each of 0.8, 6 and 12 ng/ml of Felodipine calibration curve samples was prepared and made up the
volume with blank plasma, transferred in to different 2ml centrifuge tubes and stored at –7 ± 20 C until
processing.
Preparation of plasma samples At the time of analysis, the samples were removed from the deep
freezer and kept in the room temperature and allowed to thaw. A volume of 0.5 ml of sample was pipetted into
2.0 ml centrifuge tube with this 500 μl of internal standard solution (10.0 μg/ml) and 0.5 ml of precipitating
agent (10% Perchloric acid) was added. The resulting solution was vortexed for 5 minutes and centrifuged at
4000 r/min for 10 min. Supernatants from the above solutions were separated and used for the analysis.
Results of the investigation of felodipine in plasma by LC MS estimation method:
Figure.1 Typical chromatogram of blank plasma
Figure.2 Mass spectrum of felodipine
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Vol.2(2), 2011, 65-73
Figure.3 Mass spectrum of internal standard
Figure.4 Typical standard chromatogram of felodipine and internal standard
Figure.5 Typical sample chromatogram of felodipine and internal standard
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Vol.2(2), 2011, 65-73
Figure.6 Calibration curve of felodipine
The results observed by LC MS method for recovery studies of felodipine are showed in table no. 1. The
Precision studies were indicated in table no.2. Followed by linearity and range results in table no.3. The table
no.4 indicates Stability of Felodipine in plasma during storage and sample handling followed by LC-MS System
suitability studies for Felodipine in table no.5.
Table 1: Felodipine Accuracy and recovery studies
Amount of drug Amount of
Concentration Relative
recovered Drug recovered
Level of drug added Recovery (%) Recovery
(ng/ml) in (%) in Mobile
ng/ml (%)
plasma sample phase
Mean: 97.57 Mean: 99.05
Level I 0.8 0.76±2.51 CV: 2.92 CV: 1.04 99.36
N: 6 N: 6
Mean: 98.05 Mean: 98.96
Level II 6 5.86±1.96 CV: 1.52 CV: 1.73 98.94
N: 6 N: 6
Mean: 98.72 Mean: 99.27
Level III 12 11.84±1.08 CV: 0.91 CV: 1.01 98.95
N: 6 N: 6
Table 2: Felodipine Precision Studies (ng/ml)
Nominal Concentration (ng/ml)
Sno LQC 0.8 MQC 6 HQC 12
1 0.7956 5.9865 11.99652
2 0.7523 5.8427 11.7532
3 0.7135 5.7312 11.5984
4 0.7026 5.4023 11.8537
5 0.6938 5.5643 11.3764
Mean 0.73 5.710 11.71
± SD 0.40 0.23 0.23
% CV 5.77 4.02 1.96
% Nominal 91.45 95.09 97.58
n 5 5 5
Nominal Concentration (ng/ml)
Sno LQC 0.8 MQC 6 HQC 12
1 0.7853 5.9952 11.8956
2 0.7742 5.6327 11.9534
3 0.7612 5.8348 11.7635
4 0.7398 5.7219 11.7952
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Vol.2(2), 2011, 65-73
5 0.7023 5.5023 11.8234
Mean 0.75 5.74 11.85
± SD 0.03 0.19 0.08
% CV 4.36 3.29 0.65
% Nominal 94.07 95.62 98.72
n 5 5 5
Nominal Concentration (ng/ml)
Sno LQC 0.8 MQC 6 HQC 12
1 0.7562 5.9862 11.8695
2 0.7243 5.8324 11.9532
3 0.7395 5.4975 11.7634
4 0.7481 5.7364 11.5132
5 0.7235 5.4231 11.7627
Mean 0.74 5.70 11.77
± SD 0.01 0.23 0.17
% CV 1.95 4.10 1.41
% Nominal 92.29 94.92 98.10
n 5 5 5
Table.3. Linearity and Range of Felodipine
Drug Concentration (ng/ml) Internal Standard Concentration Response Factor (RSD)
(μg/ml)
0.8 10 0.00011
1 10 0.00014
2 10 0.00023
4 10 0.00055
6 10 0.00082
12 10 0.00164
13 10 0.00178
Table.4. Stability of Felodipine in plasma during storage and sample handling
Nominal Concentration (ng/ml)
Freeze and Thaw LQC 0.8 MQC 6 HQC 12
Cycle 1 0.7523 5.8956 11.9568
Cycle 2 0.7358 5.6327 11.6348
Cycle 3 0.7042 5.4827 11.2672
Mean 0.73 5.67 11.62
S.D (+/-) 0.02 0.21 0.35
C.V. (%) 3.34 3.69 2.97
% Nominal 91.35 94.51 96.83
n 3 3 3
Nominal Concentration (ng/ml)
Short Term Plasma at RT LQC 0.8 MQC 6 HQC 12
After 1 h 0.7953 5.8956 11.8965
After 2 h 0.7423 5.5768 11.5324
After 3 h 0.7369 5.6972 11.2746
Mean 0.76 5.72 11.57
S.D (+/-) 0.03 0.16 0.31
C.V. (%) 4.26 2.81 2.70
% Nominal 94.77 95.39 96.40
n 3 3 3
Nominal Concentration (ng/ml)
Long Term Plasma at 700C LQC 0.8 MQC 6 HQC 12
After 1 week 0.7952 5.8321 11.5632
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After 2weeks 0.6835 5.4937 11.1237
After 4 weeks 0.7035 5.1095 11.0743
Mean 0.73 5.48 11.25
S.D (+/-) 0.06 0.36 0.27
C.V. (%) 8.19 6.60 2.39
% Nominal 90.93 91.31 93.78
n 3 3 3
Nominal Concentration (ng/ml)
Standard stock Solutions LQC 0.8 MQC 6 HQC 12
After 3 h 0.7952 5.9357 11.8634
After 6 h 0.7632 5.8752 11.6716
After 4 Weeks 0.7452 5.5068 11.6381
Mean 0.77 5.77 11.72
S.D (+/-) 0.03 0.23 0.12
C.V. (%) 3.30 4.02 1.04
% Nominal 95.98 96.21 97.70
n 3 3 3
Table.5. LC-MS System suitability studies for Felodipine
S.No Parameters Int Std Drug
1 Theoretical Plate 176532 22158
2 Resolution factor 2.97 2.97
3 Asymmetric factor 1.25 1.01
4 LOD(ng/ml) 0.06 0.10
5 LOQ(ng/ml) 0.21 0.50
Discussion:
Optimisation of chromatographic conditions are intended to take into account the various goals of the
method development and to weigh each goal (resolutions, run time, sensitivity, peak symmetry, etc) accurately,
according to the requirements of HPLC can be used for the estimation of Felodipine in plasma samples. The
optimised conditions for estimation provided a well defined separation between the drug, internal standard and
endogenous components. The blank plasma samples showed no interference at retention time of the drugs and
their internal standards. (Fig1,2,3,4 and 5). In the Validation of the developed method the accuracy was
determined by relative and absolute recovery experiments. The percentage recovery values for Felodipine were
ranged from 97.57 to 98.72 % respectively. Their relative recovery values ranged from 98.94 to 99.36 %. The
coefficient of variation (%) of these values was less than 5 %. It is therefore, derived that the developed methods
are accurate and reliable.
The optimized methods for the estimation of the drugs were precise as it showed < 10 % coefficient of
variation at all concentrations. The six blank plasma samples obtained from six different volunteers were
analysed and the chromatograms were recorded. Endogenous interferences were not detected at the retention
time of selected drugs and internal standard. The peak purity test method using PDA detector was employed for
selectivity studies. Some additional peaks were also observed in the sample chromatograms. These peaks,
however, did not interfere with the drugs and internal standards peaks. These observations show that the
developed assay method is specific and selective. The linearity range for Felodipine was found to be, 0.8, 1.0,
2.0, 4.0, 6.0, 12.0 and 13.0 ng/ml. The results indicated that no significant inter and intra day variability of slopes
and intercepts over the optimised concentration range.
The limit of detection (LOD) value was found to be 0.1 ng/ml for Felodipine and their limit of
quantification (LOQ) value was 0.5 ng/ml. This observation showed that the developed methods have adequate
sensitivity. These values, however, may be affected by the separation conditions (e.g., column, reagents, and
instrumentation and data systems), instrumental changes (e.g., pumping systems and detectors) and use of non
HPLC grade solvents and may result in changes in signal to noise ratios. The ruggedness and robustness of the
methods were studied by changing the experimental conditions. No significant changes in the chromatographic
parameters were observed when changing the experimental conditions (operators, instruments, source of
reagents and column of similar type) and optimised conditions (pH, mobile phase ratio and flow rate). System
suitability parameters such as column efficiency (theoretical plates), resolution factor and peak asymmetry factor
of the optimised methods were found satisfactory.
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Vol.2(2), 2011, 65-73
The stability of the drug spiked human plasma samples at three levels were studied for three freeze thaw
cycles. The mean concentrations of the stability samples were compared to the theoretical concentrations.
Similarly, short term (3 h), long term (4 weeks) and standard solution stability were evaluated. The stability of
the internal standards was also performed. The results showed that the selected drugs were stable in plasma for
about one month when stored at frozen state.
Conclusion:
The developed method for the estimation of Felodipine in plasma is accurate, precise, selective and
linear and is therefore, can be employed for estimation of the drug from the spiked samples of plasma with ease
of sensitivity and reproducibility in the analysis. Hence the current investigation concluded by representing the
significant analytical work which could be used for the felodipine estimation by LC MS method in human
plasma for bio availability and pharmacokinetic studies.
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