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Archive of SID
Iranian Journal of Pharmaceutical Sciences
Summer 2007: 3(3): 161-170
www.ijps.ir
R
Original Article
Development and Application of a Validated Liquid
Chromatography-Mass Spectrometry Method for the
Determination of Dexchlorpheniramine
Maleate in Human Plasma
Aravindaraj Joghee Rajua,*, Gopinath Rama, Rajan Sekara, Mahesh Kumar Siddaiahb,
Nanjan Moola Jogheeb, Suresh Bhojrajc
aCADRAT, JSS College of Pharmacy,bTIFAC CORE, JSS College of Pharmacy, cPrincipal, JSS College of
Pharmacy, Rock lands, Ootacamund, India
Abstract
A convenient liquid chromatographic-single Quadrupole mass spectrometric
(LC-MS) method was developed and validated for dexchlorpheniramine maleate (INN
name: chlorphenamine) determination in human plasma. The need for just a single
liquid-liquid extraction with ethyl acetate and being highly sensitive were the
advantages of this method. The linearity was also excellent over the range of 1 to
150 ng.ml-1 of dexchlorpheniramine maleate concentration. The method was
statistically validated for its selectivity, linearity, precision and robustness. This method
was successfully applied to the analysis of chlorpheniramine maleate in clinical
studies.
Keywords: Bioequivalence; Dexchlorpheniramine maleate; LC-MS.
Received: February 5, 2007; Accepted: April 11, 2007.
1. Introduction
Chlorpheniramine maleate (RS-CPM) (3-
(4-chlorophenyl)-N,N-dimethyl-3-(2-pyridyl)
propylamine monomaleate) (Figure 1) is a
highly potent and widely used antihistaminic
drug. It has been widely used for symptomatic
relief of common colds and allergic diseases.
Its activity is predominantly attributed to the
dextrorotary S-enantiomer [1]. The European
Pharmacopoeia III describes an HPLC method
for the determination of the enantiomeric
purity of dexchlorpheniramine maleate (S-
CPM), allowing the presence of 2% (m/m) of
R-enantiomer in the tested sample. Pharmaco-
kinetic studies have revealed that plasma
chlorpheniramine concentrations in humans
are low, for example, the maximum levels of
6.2 and 7.0-8.2 ng/ml after a single oral
administration of 2 and 4 mg [2]; and 5.8-11.3
and 3.9 ng/ml after a 4 and 2.67 mg
administration, respectively. Some devised
methods have been reported to determine
human plasma RS-chlorpheniramine by
using gas chromatography (GC), gas
chromatography-mass spectrometry (GC-MS)
and high-performance liquid chromatography
(HPLC) [3-9].
This paper describes development and
*Corresponding author: Aravindaraj joghee Raju, Senior Research
Associate, CADRAT, JSS College of Pharmacy, Rocklands,
Ootacamund - 643 001, India.
Tel (+91)423-2447135; Fax (+91)423-2447135
Email: dr_aravindraju@yahoo.co.in
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AJ Raju et al./ IJPS Summer 2007; 3(3): 161-170
162
validation of a simple, specific, rapid and
sensitive liquid chromatography-mass
spectrometry (LC-MS) method for the
determination of S-CPM in human plasma
with a limit of quantification (LOQ) of 1.0
ng/ml for S-CPM during a 5.0 min. run time,
using simvastatin (STA) (Figure 1) as an
internal standard. In addition, this method
was applied to S-CPM quantification of a
single dose administration of tablets
containing 6 mg of S-CPM in a crossover
bioequivalency study of S-CPM in healthy
male human subjects.
2. Materials and methods
2.1. Chemicals and reagents
The reference standards of S-CPM (purity:
99.67%) and STA (purity: 98.44%) were
obtained from M/s, Orchid pharmaceuticals
(Chennai, India) and Cadila Pharma
(Ahmedabad, India). Highly purified water
was prepared in-house using a Milli-Q water
purification system obtained from Millipore
(India) Pvt. Ltd. (Bangalore, India). Gradient
grade methanol and acetonitrile were
purchased from E. Merck Ltd. (Mumbai,
India). Ammonium acetate and formic acid
were purchased from Qualigens Fine
Chemicals (Mumbai). Drug free (blank)
heparinized human plasma was obtained from
the local Nursing hospital (Ootacamund,
India) and was stored at (-) 20 °C prior to use.
2.2. Calibration curves
The stock solutions of S-CPM and internal
standard were prepared in acetonitrile at free
base concentration of 1000 μg.ml-1.
Secondary and working standard solutions
were prepared from stock solutions by dilution
by water:acetonitrile (50:50, v/v). These
diluted working standard solutions were used
to prepare the calibration curve and quality
control samples. Blank human plasma was
screened prior to spiking to ensure it was free
from endogenous interference at retention
times of S-CPM and internal standard of S-
CPM (Figure 2). An eight point standard
curve of S-CPM was prepared by spiking the
blank plasma with appropriate amount of S-
CPM. The calibration curve ranged from 1.0
to 150.0 ng.ml-1. Quality control samples,
prepared at three concentration levels of 5.0,
25.0, 75.0 and 125.0 ng.ml-1 for S-CPM were
made with blank plasma. The samples were
vortexed and stored at (-) 70±2 °C for further
processing.
2.3. Sample preparation
A 0.5 ml aliquot of human plasma sample
was mixed with 0.1 ml of the internal standard
working solution (2500.0 ng.ml-1 of STA)
and 1.0 ml of borate buffer of pH 9.00 were
added and mixed. The resulting solution was
vortexed and extracted by ethyl acetate (3×2
ml). The upper organic layer was separated,
evaporated and the drug was reconstituted
using 0.5 ml of the mobile phase and
analyzed.
Figure 1. Structure of dexchlorpheniramine and simvastatin.
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Determination of dexchlorpheniramine
2.4. Instrumentation
Chromatographic separation was carried
out on a Shimadzu HPLC (Shimadzu
Corporation, Japan) with phenomenex (Luna)
- ODS (100x4.6 mm i.d., 5 μm). The mobile
phase consisting of a mixture of methanol
(10 mM) and ammonium acetate (90:10 v/v)
was delivered with a flow rate of 0.5 ml/min.
under ambient temperature. The total running
time for each sample analysis was 6.0 min.
Mass spectra were obtained using a Single
Quadrupole mass spectrometer (Shimadzu,
Japan) equipped with Atmospheric Pressure
Chemical Ionization (APCI) source and
Electrospray ionization (ESI). The mass
spectrometer was operating in the selected
ion-monitoring (SIM) mode. Sample
introduction and ionization was done in the
positive ion mode. The spray voltage and
capillary temperature were 1.3 KV and 400
°C, respectively. The mass transition ion-pair
was selected as m/z 274.9 for S-CPM (Figure
3) and m/z 302.9 for STA. The data acquisition
was ascertained by LC-MS solution data
station. For quantification, the peak area ratios
of the target ions of the drugs to those of the
internal standard were compared with
weighted (1/c) least squares calibration curves
in which the peak area ratios of the calibration
standards were plotted versus their
concentrations.
2.5.Validation
The method was validated according to
FDA guidelines [10, 11] and was validated for
selectivity, sensitivity, linearity, precision,
accuracy, and stability. The selectivity of the
method was evaluated by comparing the
chromatograms obtained from the samples
containing S-CPM and the internal standard
STA with those obtained from blank samples.
Sensitivity was determined in terms of LLOQ
“lower limit of quantification” where the
response of LLOQ should be at least five
times greater than the response of interference
in blank matrix at the retention time or mass
transitions of the analyte. The linearity of
different concentrations of standard solutions
was prepared to contain 1 to 150 ng.ml-1 of S-
CPM containing 2500.0 ng.ml-1 of STA.
These solutions were analysed and the peak
areas and response factors were calculated.
The calibration curve was plotted using
response factor against concentration of the
standard solutions. The standard curve fitting
was determined by applying the simplest
model that adequately describes the
concentration-response relationship using
appropriate weighing and statistical tests for
goodness of fitting. The precision of the
method was determined by intraday precision
and interday precision. The intraday precision
was evaluated by analysis of blank plasma
sample containing S-CPM at three different
concentrations namely low, medium and high
quality control concentrations using nine
replicate determinations for three occasions.
The interday precision was similarly evaluated
over a two-week period.
The accuracy of the developed method
was determined by relative and absolute
recovery experiments. The relative recovery
of the drug was calculated by comparing the
concentration obtained from the drug
supplemented plasma to the actually added
concentration. Recovery studies were carried
163
Table 1. Precision studies of dexchlorpheniramine maleate samples (ng.ml-1).
Intra-assay Inter -assay
Quality control Nominal Mean concentration Mean concentration
sample concentration (ng.mL-1) SD % CV N (ng.mL-1) SD % CV N
LLOQ 5.00 4.519 0.330 7.28 5 4.573 0.385 8.42 5
LQC 25.00 24.472 0.383 1.57 5 24.661 0.402 1.63 5
MQC 75.00 73.686 1.370 1.86 5 73.902 1.530 2.07 5
HQC 125.00 123.221 1.433 1.16 5 123.225 1.434 1.16 5
S.D: Standard deviation; CV: Coefficient of variance; N: Total number of observations for each concentration; LLOQ: Lower limit of
quantification; LQC: Lower quality control; MQC: Middle quality control; HQC: High quality control.
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AJ Raju et al./ IJPS Summer 2007; 3(3): 161-170
164
out six times for three levels and the
percentage recovery, mean, standard deviation
and coefficients of variation were calculated.
As a part of the method validation, stability
and partial volume analysis were evaluated.
The room temperature stock solution stability,
refrigerated stock solution stability, freeze
thaw stability, short term stability and long
term stability were determined. The room
temperature stock solution stability was
carried out at 0, 3 and 8 h by injecting four
replicates of prepared stock dilutions of S-
CPM equivalent to middle quality control
sample concentration and the stock dilution
of the internal standard equivalent to the
working concentration. Comparison of the
mean area response of S-CPM and internal
standard at 3 and 8 h was carried out against
the 0 h value. Refrigerated stock solution
stability was determined at 7, 14 and 27 days
by injecting four replicates of prepared stock
dilutions of the analyte equivalent to the
middle quality control sample concentration
and the stock dilution of internal standard
equivalent to the working concentration. The
stability studies of plasma samples spiked
with S-CPM were subjected to three freeze-
thaw cycles, short term stability at the room
temperature for 3 h and long term stability at
(-)70 °C over 4 weeks. In addition, stability
of standard solutions was performed at room
temperature for 6 h 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 S-CPM was kept at the 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
(-) 70 °C until the time of analysis.
3. Results and discussion
3.1. Method development
The goal of this work was to develop and
validate a simple, rapid and sensitive assay
method for the quantification of S-CPM,
suitable to determine the pharmacokinetics of
this compound in clinical studies. To achieve
this goal, during method development
different options were evaluated to optimize
sample extraction, detection parameters and
chromatography. The standard solutions of S-
CPM were analysed by LC-MS system using
direct injection probed with ESI and APCI
interfaces. From the mass spectrum recorded,
Table 2. Stability of dexchlorpheniramine maleate in human plasma samples.
Sample concentration Concentration found % CV
(ng.ml-1) (n = 6) (mean±S.D.) (ng.ml-1)
Short-term stability (1, 2, 3 h)
25 24.3937±0.46660 1.91
75 73.3997±0.60909 0.83
125 124.4027±1.95214 1.58
Long-term stability (4 weeks)
25 24.7066±0.51219 2.07
75 73.7361±1.18283 1.60
125 124.7353±0.63385 0.51
Stock solution stability (7, 14, 21 Days)
25 25.0503±0.10986 0.44
75 74.9663±0.11445 0.15
125 124.0693±0.92361 0.74
Freeze thaw stability (3 Cycle)
25 24.7416±0.3771 1.52
75 73.1361±1.06265 1.49
125 124.7513±0.58869 0.47
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Determination of dexchlorpheniramine
the detection molecular ion selected was 274.9
for S-CPM (Figure 4).
3.2. Optimization of the chromatographic
conditions
The chromatographic conditions,
especially the composition of mobile phase,
were optimized through several trials to
achieve good resolution and symmetrical
peak shapes for the analyte and the IS, as
well as a short run time. Modifiers, such as
ammonium acetate and acetic acid alone or in
combination with different concentrations
were added. It was found that a mixture of
acetonitrile-water (containing 10 mm
ammonium acetate and 0.5% acetic acid)
(90:10, v/v) could achieve this purpose and
was finally adopted as the mobile phase. The
percentage of acetic acid was optimized to
maintain this peak shape while being
consistent with good ionization and
fragmentation in the mass spectrometer. After
careful comparison of several columns, a
Phenomenex (Luna)-ODS column (100×4.6
mm, i.d., 5μm) was finally used with a flow
rate of 0.5 ml/min. to produce good peak
shapes and permit a run time of 2.0 min. In
order to produce a spectroscopically clean
sample and avoid the introduction of non-
volatile materials onto the column and MS
system, LLE was used for the sample
preparation in this work. Clean samples are
essential for minimizing ion suppression and
matrix effect in LC-MS analyses.
Different reversed phase stationary phases
(C4, C8, and C18) were used and the
chromatograms were recorded. Based on the
retention and peak shape, Phenomenex Luna
ODS column was selected for S-CPM.
Liquid-liquid extraction (LLE) was used
for the sample preparation in this work. LLE
can be helpful in producing a spectroscopical-
ly clean sample and avoiding the introduction
of non-volatile materials onto the column
and MS system. Clean samples are essential
for minimizing ion suppression and matrix
effect in LC-MS analyses. Six organic
solvents, diethyl ether, ethyl acetate, hexane,
dichloromethane, chloroform and butyl tert-
methyl ether, and their mixtures in different
combinations and ratios were evaluated.
Finally, an ethyl acetate was found to be
optimal, which can produce a clean
chromatogram for a blank plasma sample
and yield the highest recovery for the analyte
from the plasma.
3.3. Validation
Estimation of the S-CPM in plasma
samples from the volunteers was carried out
using optimized chromatographic conditions.
The validation parameters such as accuracy,
precision (repeatability and reproducibility),
linearity and range, sensitivity (limit of
detection and limit of quantitation),
robustness/ruggedness, stability, selectivity/
specificity and system suitability studies were
evaluated. The validation results are given
165
Figure 2. LC-MS chromatogram of blank plasma sample. Figure 3. LC-MS chromatogram of dexchlorpheniramine
maleate and internal standard sample.
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166
in Table 1.
The reference standard solution with
internal standard, matrix blank without the
internal standard, zero sample [Matrix blank
with internal standard], spiked calibration
standards, quality control samples were
analysed and chromatograms were recorded.
The mobile phase used for the assay provided
a well defined separation between the drug,
the internal standard and endogenous
components. The blank plasma samples
showed no interference at retention time of the
drugs and their internal standards (Figure 2).
3.4. Accuracy
The accuracy of the optimised methods
was determined by relative and absolute
recovery experiments (Table 1). The
percentage recovery values for S-CPM
ranged from 89.06 to 91.32% and their
relative recovery values ranged from 88.07 to
91.33 %. The coefficient of variation (%) of
these values was less than 10.00%. It is
concluded that the developed methods are
accurate and reliable.
3.5. Precision
The optimized method for the estimation
of S-CPM was found to be precise (Table 2).
This was evident from the coefficieny of
variation values, which were less than 10.00%
in all concentrations.
3.6. Specificity
Specificity of the method was analysed
by six blank plasma samples and the recorded
chromatograms. These chromatograms were
compared with the chromatograms obtained
from standard solutions. Each chromatogram
was tested for interference. The combination
of the sample preparation procedure and
chromatography provided an assay which is
free from significant interfering endogenous
plasma components at the retention times of
the S-CPM and the internal standard. These
observations show that the developed assay
method is specific and selective.
3.7. Linearity
It was observed that the optimised methods
were linear within a specific range of S-CPM
concentration. The calibration curves were
plotted between response factor and
concentration of the standard solutions. The
linearity range were found to be 1 to 150
ng.ml-1. The calibration curves were
Table 3. Mean pharmacokinetic properties of dexchlorpheniramine maleate obtained from studied subjects (N=24) after
administration of a single 6-mg dose of reference and test dexchlorpheniramine maleate formulations.
Pharmacokinetic parameters* Test Reference
(N=24) (N=24)
Cmax, ng.ml-1 25.3421 (2.0605) 22.1150 (4.4148)
tmax, h 3.2708 (1.1514) 6.7500 (0.9891)
AUC0-t (ng.h.ml-1) 234.1008 (22.8974) 241.5723 (33.8520)
keli 0.1000 (0.0135) 0.1177 (0.0225)
AUC0-Q(ng.h.ml-1) 262.8429 (23.6587) 270.3695 (35.5413)
t1/2 (h) 7.0192 (0.9464) 6.0855 (1.0840)
Tmax: Time of maximum concentration; Cmax: Maximum concentration; AUC: Area under the concentration time curve; T1/2: Half-life; keli:
Elimination rate constant; AUC0-t: Area under the plasma concentration-time curve, last available measurement; AUC0-α: Area under the plasma
concentration-time curve from time 0 to infinity.
*Values in the parenthesis indicate standard deviation.
Figure 4. Mass spectrum of dexchlorpheniramine maleate in
positive mode Scan.
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Determination of dexchlorpheniramine
constructed on 11 different days over a period
of four weeks to determine the variability of
the slopes and intercepts. The results indicated
no significant interday variability of slopes
and intercepts over the optimised
concentration range.
3.8. Limit of detection
The limit of detection (LOD) values was
found to be 0.25 ng.ml-1 for S-CPM and their
limit of quantification (LOQ) values were
1.00 ng.ml-1. These observations indicate that
the developed methods have adequate
sensitivity. These values, however, may be
affected by the separation conditions (eg.
column, reagents, and instrumentation and
data systems), instrumental changes (eg.
pumping systems and detectors) and use of
non HPLC grade solvents may results in
changes in signal-to-noise ratios.
3.9. Ruggedness and robustness
The ruggedness and robustness of the
methods were studied by changing the
experimental conditions. No significant
changes in the chromatographic parameters
were observed when the experimental
(operators, instruments, source of reagents
and column of similar type) and optimised
conditions (pH, mobile phase ratio and flow
rate) were changed.
3.10. Stability studies
The stability of plasma samples which had
been spiked with selected drugs were studied
by subjecting the samples to three freeze-
thaw cycles; short and long term stabilities at
room temperature were measured within 3 h
and within 4 weeks at (-)70 °C, respectively
(Table 2). In addition, stability of standard
solutions was measured at the room
temperature and freeze condition within 6 h
and within 4 weeks, respectively. The mean
concentrations of samples were compared to
the theoretical concentrations. The results
indicated that selected drugs in plasma
samples can be stored in freezing condition for
1 month without degradation. The results of
stability studies of short term storage of
plasma and also sample solution at room
temperature and freeze thaw cycles show that
no S-CPM degradation happened and
therefore, plasma samples could be handled
without special precautions.
3.11. System suitability studies
System suitability parameters such as
column efficiency (theoretical plates),
resolution factor and peak asymmetry factor
of the optimised methods were found to be
satisfactory. Theoretical plates of the columns
ranged from 18432 to 22987 and their
resolution factor was 2.46. Similarly, the peak
asymmetry factors ranged from 1.01 to 1.09.
All these observations supported the system
suitability for the evaluation of selected drugs.
In conclusion, this is an accurate, precise,
selective and linear method for S-CPM
estimation in plasma and hence can be used
for bioavailability and bioequivalency studies.
3.12. Application of the developed method
The proposed method was applied to the
determination of S-CPM in plasma samples
from an on going project bioequivalence
studies of sustained release formulation. Open
label, balanced, randomized, two-treatment,
two sequence, two-period, single dose,
crossover bioequivalence study of marketed
repetabs containing 6 mg of S-CPM (reference
sample) against SR tablets containing 6 mg of
S-CPM (test sample) manufactured by Sipali
167
Table 4. Results of statistical analysis of the bioequivalency study of test and reference dexchlorpheniramine maleate
formulations.
AUC0-t (ng.h/ml-1) AUC0- α(ng.h/ml-1)C
max, (ng ml-1)
Ratio (%) 105.8 104.7 111.00
Geometric CI (%) 92.36-107.06 95.51-107.06 93.69-107.74
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168
Chemicals, India in healthy, adult, male,
human subjects under fasting conditions was
conducted in accordance with the current
good clinical practice (GCP) and FDA
guidelines. The study was performed on
healthy, willing, 24 male volunteers 18-45
years of age, after they had been informed of
the purpose, protocol and risk involved in
the study. All subjects gave written informed
consent and the protocol was approved by
local ethics committee. The venous blood
samples 6 ml including (1 ml discarded
heparinised blood) were withdrawn via an
indwelling cannula at pre-dose and at 0.5, 1.0,
1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, 18.0 and 24
h following drug administration in each period
of the study. The samples were collected in
pre-labeled vacutainers containing sodium
citrate as the anti coagulant and centrifuged
at 3000 rpm for 15 min. at 15 °C and plasma
was collected in pre-labeled sample collection
tube. A wash out period of 7 days was
observed between the two phases of the study.
The samples were stored in the deep freezer
at (-)70±5 °C until analyzed by a validated
LC-MS method.
The pharmacokinetic parameters namely
maximum plasma concentration (Cmax), time
point of maximum plasma concentration
(Tmax), area under the plasma concentration-
time curve from 0 h to the last measurable
concentration (AUC0-t), area under the plasma
concentration time curve from 0 h to infinity
(AUC0-Q), elimination rate constant (λZ) and
half-life of drug elimination during the
terminal phase (t1/2) were calculated using
PK solution software statistical analysis of
pharmacokinetic parameters was carried out
using SPSS 12.0.1 for un-transformed and
ln-transformed pharmacokinetic parameters
Cmax, AUC0-t and AUC0-Q (Tables 3 and 4).
Based on the statistical results of 90.0%
confidenct intervals for the ratios of the means
of ln-transformed pharmacokinetic parameters
Cmax, AUC0-t and AUC0-Qconclusion was
drawn as to whether the test product was
bioequivalent to the reference product.
Bioequivalence was to be concluded if the
90.0% confidence interval fell within the
bioequivalency range 80.0-125.0 % for
Cmax, AUC0 - t and AUC0- Q.
The mean (±S.D.) plasma maximum
concentration obtained for S-CPM in
reference and test formulation is 22.1150
(4.4148) ng.ml-1 and 25.3421 (2.0605)
ng.ml-1 (Table 3), respectively.
4. Conclusions
A simple, specific, rapid and sensitive
analytical method for the determination of
S-CPM in human plasma has been developed.
The developed LC-MS method was
successfully applied for the bioequivalency
studies. The method provided excellent
specificity and linearity with a of
quantification limit of 1.00 ng.ml-1 for S-
CPM.
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