American Journal of Applied Chemistry
2013; 1(1) : 1-8
Published online April 2, 2013 (http://www.sciencepublishinggroup.com/j/ajac)
doi: 10.11648/j.ajac.20130101.11
An Ultra-sensitive and selective LC-UV method for the
simultaneous determination of pravastatin, diltiazem,
naproxen sodium and meloxicam in API, pharmaceutical
formulations and human serum
Najma Sultana1, Muhammad Saeed Arayne2, Saeeda Nadir Ali2,*
1
2
Department of Pharmaceutical Chemistry, University of Karachi, Karachi-75270, Pakistan
Department of Chemistry, University of Karachi, Karachi-75270, Pakistan
Email address:
saeeda_khowaja@hotmail.com (S. Nadir Ali)
To cite this article:
Najma Sultana, Muhammad Saeed Arayne, Saeeda Nadir Ali. An Ultra-Sensitive and Selective LC-UV Method for the Simultaneous
Determination of Pravastatin, Diltiazem, Naproxen Sodium and Meloxicam in API, Pharmaceutical Formulations and Human Serum .
American Journal of Applied Chemistry. Vol. 1, No. 1, 2013, pp. 1-8. doi: 10.11648/j.ajac.20130101.11
Abstract: The aim of the present work was to develop a sensitive liquid chromatographic method for the quantitation of
pravastatin and diltiazem along with naproxen and meloxicam using ultraviolet detector. The separation of components was
achieved on Purospher Star, C18 (5 µm, 25 x 0.46 cm) column using methanol-water (80:20 v/v) as mobile phase and pH
adjusted 3.4 with 85% o-phosphoric acid. Related parameters that may influence the enrichment efficiency of speration of
drugs such as the kind and volume of elute, sample flow rate, sample pH, and volume of the drug samples were investigated. Detection was performed at ambient temperature at 220 nm by pumping the mobile phase at the flow rate 1.0 mL min-1.
The experimental results indicated a good linearity (R 2 > 0.9947) over the concentration range of 0.5-20 µg mL-1 for pravastatin, naproxen and meloxicam and 0.75-24 µg mL-1 for diltiazem. The method was compared by programming the detector adjusting the wavelength with time to match the individual analyte's chromophore which enhanced sensitivity with
linear range 0.25-8.0, 0.5-16, 0.4-12 and 0.2-4.0 µg mL-1 for pravastatin, diltiazem, naproxen and meloxicam respectively.
The LOD values shifted down from 33, 70, 50 and 80 ng mL-1 to 15, 42, 20 and 10 ng mL-1 for pravastatin, diltiazem, naproxen and meloxicam respectively. Validation of the method showed good precision and accuracy for the proposed method.
All the results indicated that this procedure could allow the simultaneous determination of these four compounds in API,
pharmaceutical formulations and serum at trace levels. The method can be successfully applied for the determination of these
drugs in human serum, clinical laboratories and in pharmaceutical formulations without diode array detector and without
interference of excipients or endogenous components of serum.
Keywords: Pravastatin, Diltiazem, Naproxen, Meloxicam, RP-HPLC, Time Program
1. Introduction
Pravastatin (PRA) (figure.1: I) is a potent statin which
averts the cholesterol production in the liver by blocking
the enzyme HMG-CoA reductase that produces cholesterol.
It shows significant protection against cisplatin-induced
nephrotoxicity in mice [1] and also found to reduce edema
in spontaneously hypertensive rats when administered
intravenously after middle cerebral artery occlusion [2].
Diltiazem hydrochloride (DTZ) ((figure.1: II) is a non dihydropyridine calcium channel blocker antagonist, frequently used as antihypertensive [3] and possesses antianginal and antiarrythmic properties [4]. Non-steroidal an-
ti-inflammatory drugs (NSAIDs) are widely prescribed for
the treatment of inflammatory conditions such as rheumatoid arthritis, osteoarthritis and ankylosing spondilytis [5].
They are known to relief symptoms of primary dysmenorrhea, fever and also possess mild antiplatelet effect. They
are useful in sepsis-induced acute pneumonia [6], in retarding metastases of mammary carcinoma [7] and in preventing oxidative lesions of lungs caused by phosgene [8].
Naproxen (NAP) (figure.1: III) and meloxicam (MEL)
(figure.1: IV), are potent inhibitors of cyclooxygenase enzyme are novel NSAIDs of aryl acetic acid group and oxicam class respectively.
Literature review reveals that a number of analytical
2
Najma Sultana et al.: An Ultra-sensitive and selective LC-UV method for the simultaneous determination
of pravastatin, diltiazem, naproxen sodium and meloxicam in API, pharmaceutical formulations and human serum
methods have been reported for the determination of these
drugs in pharmaceutical formulations, human serum and
body fluids by using different analytical techniques. PRA
has been determined by capillary electrophoretic method
[9], liquid chromatography [10], gas chromatography employing chemical ionization mass spectrometry (GC/CIMS)
[11], HPTLC [12] and enzyme immunoassay [13]. Reported methods for the determination of DTZ and its metabolites include spectrophotometry [14], amperometry [15],
HPLC [16], HPTLC [17], capillary gas chromatography
[18] and liquid–liquid micro-extraction [19]. Different
techniques have been reported for the determination of
NAP i.e. chip-based immunoaffinity capillary electrophoresis [20], HPLC [21], HPTLC [22], LC–MS/MS [23] and
GC/MS [24]. For the determination of MEL, spectrophotometric [25], Voltammetric [26], fluorimetric [27], electrochemical [28], HPLC [29] and capillary electrophoresis
[30] are reported. A number of methods for individual or
simultaneous determination of these and many other [31-33]
drugs have also been developed by our research group like
liquid chromatographic method for simultaneous determination of ceftriaxone sodium [34] with statin, lisinopril
with statin [35] and NSAIDs [36], NSAID with statin [37,
38], DTZ with non steroidal anti-inflammatory drugs [39],
MEL [40], , NAP [41].
component. It can successfully be applied for the determination of these drugs with good percent recovery in human
serum and pharmaceutical formulation with out any chromatographic interference from tablet excipients or serum.
2. Experimental
2.1. . Standards and Reagents
PRA (Bristol Meyer Squib), DTZ (Abbott Laboratories
Pvt Ltd), NAP (PharmEvo Pvt Ltd) and MEL (AGP Pharma
Pvt Ltd) were used as reference standards. Pravachol® (20
mg), Calcard® (60mg), Anex® (275 mg) and Melfax®
(15mg) were purchased from local pharmacy. HPLC
grade methanol, 85% o-phosphoric acid (Merck Darmstadt,
Germany) and double distilled de-ionized water were used
throughout the analysis. Drug free human serum was voluntarily donated by healthy person and collected at Fatmid
Foundation Karachi.
2.2. Instrumentation
All chromatographic experiments were carried out using
HPLC system consisting of Shimadzu LC-20 AT VP solvent delivery pump, rheodyne manual injector fitted with a
20 µL loop and a SPD-20A Shimadzu UV visible detector.
Chromatographic separation was performed by using a Purospher Star, C18 (5 µm, 25 x 0.46 cm) column. Another
column of same specification Discovery, C18 (5 µm, 25 x
0.46 cm) was used for ruggedness studies. Shimadzu
CBM-102 communication Bus Module was used for data
integration. Data acquisition was performed by Shimadzu
Class-GC 10 software (version 2). Shimadzu 1800
UV-visible spectrophotometer was used for the determination of individual λmax and isosbestic point of drug analytes.
2.3. HPLC Conditions and Method Optimization
Fig. 1. Chemical structures
Present study describes a rapid, ultra-sensitive liquid
chromatographic method for the simultaneous determination of PRA, DTZ, NAP and MEL. The developed method
has many advantages; being low cost without using expensive reagents and chemicals. Diode array detector has since
long been used to enhance the sensitivity of such methods
but the method presented is viable giving ultra sensitivity in
simultaneous separation of representatives of statins (PRA),
calcium channel blockers (DTZ) and NSAIDs (NAP and
MEL) in API, pharmaceutical formulation and human serum, without diode array detector. The method employs
adjusting the wavelength with time to match the individual
analyte's absorption maxima. As a result the method is extremely low cost and can be adopted easily. The method has
been validated following the ICH guidelines [42] at isosbestic point of 220 nm and by programming the detector
adjusting the wavelength with time to match the individual
analyte's chromophore to enhance the sensitivity of each
A number of initial chromatographic parameters including different ratios of mobile phase, pH, and flow rates
were evaluated to achieve the best resolution with shortest
retention time and good separation of components. The
suitable mobile phase consisted of methanol-water (80:20
v/v) adjusted to pH 3.4 with o-phosphoric acid (85%). Prior
to delivering into the system it was filtered through a millipore vacuum filter system equipped with 0.45 µm filter
and degassed using an ultrasonic bath (LC 30H). All the
raw materials were eluted isocratically with a flow rate of
1.0 mL min-1 at room temperature. Initially, the spectrophotometric detector was operated at 220 nm and then
comparative studies were carried out by programming the
detector at 237, 240, 230 and 205 nm for PRA, DTZ, NAP
and MEL respectively.
2.4. Preparation of Solutions and Calibration Curves
Stock solution of 50 µgmL-1 of all reference standards
were prepared in methanol:water 80:20 (diluent). Each so-
American Journal of Applied Chemistry 2013, 1(1) : 1-8
lution was sequentially diluted to working standard solutions of 0.5-20 µgmL-1 for PRA, NAP and MEL and
0.75-24 µgmL-1 for DTZ for isosbestic point and 0.25-8.0,
0.5-16, 0.4-12 and 0.2-4.0 µg mL-1 for PRA, DTZ, NAP
and MEL for time program respectively with diluent for the
preparation of calibration curves. These working standard
solutions were prepared once and stored at -20oC, and
analyzed daily for interday and intraday precision of the
method. 20 µL of these solutions was injected into the LC
system. Before injecting into the system all the solutions
were degassed by a sonicator and then filtered through a
0.45 µm filter.
2.5. Sample preparation
2.5.1. Pharmaceutical Preparations
Twenty tablets of each sample, Pravachol® (20 mg),
Calcard® (60 mg), Anex® (275 mg) and Melfax® (15mg),
were individually triturated to obtain homogenous mixture.
The amount equivalent to 10 mg of sample was transferred
into 100 mL volumetric flasks separately and dissolved in
diluent. The resulting solutions were sonicated to facilitate
proper solubilization. These solutions were then filtered to
separate insoluble excipients and the volume of each flask
was completed with diluent. All the solutions were diluted
to get the final concentration of 50 µg mL-1. Each solution
was serially diluted to 0.5-20 µgmL-1 for PRA, NAP and
MEL and 0.75-24 µg mL-1 for DTZ for isosbestic point
and 0.25-8.0, 0.5-16, 0.4-12 and 0.2-4.0 µg mL-1 for PRA,
DTZ, NAP and MEL for time program respectively.
2.5.2. Drug Serum Solutions
Into 1.0 mL of plasma, 9 mL of acetonitrile was added
and vortexed for one minute and followed by centrifugation
for 10 minutes at 10,000 rpm. The resultant deproteinated
supernatant was filtered by a 0.45 µ membrane filter. The
clear solution obtained was spiked with drug solutions to
produce desired concentrations of PRA, DTZ, NAP and
MEL in human serum. 20 µL of these solutions was injected into the LC system. The prepared serum solutions
were stored at -20 oC.
2.6. Method Validation
Following ICH guidelines [38], the developed analytical
method was validated to be efficient for the analysis of studied drugs in bulk drug and in human serum employing
various parameters including system suitability test, linearity, accuracy, precision, specificity, limits of detection and
quantitation, robustness and ruggedness.
2.6.1. Specificity
Specificity of the proposed method was analyzed
through possible interference due to commonly present
excipient or endogenous components of serum. For this
purpose, placebo (without active) of each tablet was prepared in mobile phase. The excipients include starch, carboxymethylcellulose, magnesium stearate, monohydrated
lactose and neutral talc. Placebo, blank serum, tablet sam-
3
ple and spiked serum sample were injected to the system.
2.6.2. System Suitability
The system suitability of the method was accessed by
equilibrating the initial mobile phase composition, followed
by ten consecutive injections of same standard. The system
suitability was evaluated on each day of method validation.
2.6.3. Linearity
Aliquots of stock standard solutions of PRA, DTZ, NAP
and MEL were transferred in to two different series of 25
mL volumetric flasks to get the concentration of 0.5-20 µg
mL-1 for PRA, NAP and MEL and 0.75-24 µgmL-1 for
DTZ for isosbestic point and 0.25-8.0, 0.5-16, 0.4-12 and
0.2-4.0 µg mL-1 for PRA, DTZ, NAP and MEL for time
program respectively and the volumes were made up to the
mark with 80:20 methanol-water (diluent). Calibration
curves were constructed to evaluate linearity.
2.6.4. Precision
The intra-day precision of the method was evaluated at
six concentration levels ranging from 0.5-20 µg mL-1 for
PRA, NAP and MEL and 0.75-24 µgmL-1 for DTZ at
isosbestic point and 0.25-8.0, 0.5-16, 0.4-12 and 0.2-4.0 µg
mL-1 of PRA, DTZ, NAP and MEL for time program respectively. The inter-day precision was assessed at three
consecutive days. Similarly, intra-day and inter-day precision in serum was evaluated at 2.5, 5.0 and 10 µg mL-1 for
PRA, NAP and MEL and 3.0, 6.0 and 12.0 µg mL-1 for
DTZ respectively.
2.6.5. Accuracy
Aliquots of standard tablet solutions and serum sample
were transferred into 25 mL volumetric flasks and final
volumes were completed with mobile phase. The final
concentrations of each series were 2.5, 5.0 and 10 µg mL-1
for PRA, NAP and MEL and 3.0, 6.0 and 12.0 µg mL-1 for
DTZ respectively and percent recovery was calculated.
2.6.6. Detection and Quantitation Limits
The detection and quantitation limits were calculated
from slope and standard deviation of calibration curve by
employing equation LOD = 3.3 SD / α and LOQ = 10 SD /
α respectively, where SD is the standard deviation and α is
the slope of curve.
2.6.7. Ruggedness
The ruggedness of the method was evaluated on two different columns of same specification Discovery, C18 (5 µm,
25 x 0.46 cm) and Purospher STAR, C18 (5 µm, 25 x 0.46
cm) while taking all other parameters constant including
mobile phase, pH, flow rate and wavelength of detector.
The analysis was carried out in the same lab and on the
same instrument.
2.6.8. Robustness
The optimum parameters were varied like mobile phase
ratio 80:20 methanol water ± 2.0, pH 3.4 ± 0.2, flow rate
1.0 ± 0.1 mL min-1 and detector wavelength 220 ± 2.0 nm
4
Najma Sultana et al.: An Ultra-sensitive and selective LC-UV method for the simultaneous determination
of pravastatin, diltiazem, naproxen sodium and meloxicam in API, pharmaceutical formulations and human serum
at fixed concentration of 2.5 µgmL-1 for PRA, NAP and
MEL and 3.0 µgmL-1 for DTZ to assess the robustness of
the method by six replicate analyses.
3.. Results and Discussion
3.1. Method Development and Optimization
The use of statin and calcium channel blocker with
NSAIDs demands the need of simultaneous determination of
these drugs in human serum and dosage forms as no method
has yet been developed. Consequently, the aim of present
study was to develop a rapid, sensitive, consistent and least
expensive liquid chromatographic method for the simultaneous analysis of PRA, DTZ, NAP and MEL in bulk drug,
human serum and in pharmaceutical formulations. The
present work reports a sensitive high-performance liquid
chromatographic method for the quantitation of the PRA and
DTZ along with NAP and MEL with ultraviolet detection.
The optimum parameters were investigated and developed
method was validated.
The analysis was carried out at isosbestic point as well as
by programming the detector. The chromatographic parameters were initially evaluated by varying the mobile phase
compositions, pH and flow rate. Different ratios of methanol-water (80:20, 70:30, 60:40, and 50:50) with pH range
2.8-3.6 were investigated. Asymmetric peaks were obtained
with greater tailing factor. The methanol-water (80:20 v/v)
with pH 3.4 adjusted with 85% o-phosphoric acid at flow
rate 1.0 mL min-1 resulted in symmetric peaks with good
resolution and tailing factor with in acceptable limit. The
UV scan showed absorbance maxima at 237, 240, 230 and
205 nm for PRA, DTZ, NAP and MEL respectively, therefore the detector wavelength was adjusted to 220 nm for
analysis at isosbestic point where as the detector was programmed at individual wavelength for time program method.
UV spectra and typical chromatogram showing the peaks of
PRA, DTZ, NAP and MEL at isosbestic point as well as at
time program method are represented in figures 2 and 3.
Fig. 3. Representative chromatogram of (1) PRA,(2) DTZ (3) NAP and (4)
MEL
3.2. Method Validation
The developed method is rapid and sensitive and the
analysis is complete within 7.5 minutes. The parameters
employed to certify the validity of developed analytical
method were suitability test, linearity, accuracy, precision,
specificity, limits of detection and quantitation, robustness
and ruggedness.
3.2.1. Specificity
Blank and spiked excipients and serum samples were
analyzed to evaluate the specificity of the method and check
the possible interference of excipients or endogenous components of serum. The chromatogram represented in figure 4
shows satisfactory peaks of components and confirms no
interfering peaks of excipient or serum at the retention times
for PRA, DTZ, NAP and MEL.
Fig. 4. Chromatograms of (a) solvent, (b) blank serum, (c) reference
standard and (d) serum sample spiked with standards for (1) PRA, (2)
DTZ (3) NAP and (4) MEL.
Fig. 2. Absorption spectra of (1) PRA, (2) DTZ (3) NAP and (4) MEL
3.2.2. System Suitability Test
The %RSD of all the drugs was found to be less than 2
indicating the suitability of the system. Moreover, the system suitability parameters including number of theoretical
plates (> 2000) and tailing factor (< 2) for ten consecutive
injections were found to be in the range. The system suitability data is presented in table 1.
American Journal of Applied Chemistry 2013, 1(1) : 1-8
Table 1. System suitability Parameters
Drug
tR
Isosbestic point
PRA
3.22
DTZ
4.22
NAP
5.27
MEL
6.61
Time program
PRA
3.20
DTZ
4.24
NAP
5.30
MEL
6.66
k’
N
T
Res
Α
0.66
1.17
1.71
2.40
4181
5252
4738
5098
1.84
1.69
1.83
1.90
2.10
4.64
3.87
3.97
1.54
1.79
1.46
1.40
0.97
1.59
2.24
3.07
4325
5356
4791
5290
1.62
1.56
1.71
1.62
2.22
4.75
3.92
4.05
1.39
1.64
1.41
1.37
5
tR=retention times, k’=capacity factor, N=theoretical plates, T=tailing
factor, Res=resolution, α=separation factor
3.2.3. Linearity
Calibration curves were constructed with six different
concentrations in the range of 0.5-20 µg mL-1 for PRA,
NAP and MEL and 0.75-24 µg mL-1 for DTZ at isosbestic
point. The correlation coefficients determined between peak
area and concentration of drugs in each case were greater
than 0.998. The regression characteristics including slope,
intercept, standard error and standard error of estimate are
tabulated in table 2.
Table 2. Regression characteristics and sensitivity of the method
Drug
Isosbestic point
PRA
DTZ
NAP
MEL
Time program
PRA
DTZ
NAP
MEL
Serum
PRA
DTZ
NAP
MEL
Linearity
µgmL-1
Intercept
Slope
R2
SEa
SEEb
LOD
ngmL-1
LOQ
ngmL-1
0.5-20
0.75-24
0.5-20
0.5-20
24767
23910
27256
28040
10416
10762
16358
20627
0.9993
0.9980
0.9996
0.9981
0.1647
0.2895
0.1054
0.2293
0.2433
0.4447
0.1641
0.3682
33
70
50
80
100
220
140
230
0.25-8.0
0.5-16
0.40-12
0.2-4.0
65866
65552
133113
58881
53986
33788
52484
188793
0.9986
0.9997
0.9980
0.9981
1.0260
1.2155
0.8406
0.1076
1.4798
1.8185
1.2332
0.1740
15
42
20
10
46
128
70
20
-
14864
17962
30659
35352
13007
11898
16107
18763
1.0000
0.9989
1.0000
0.9979
0.2293
0.3674
0.0349
0.5333
0.3682
0.2496
0.0226
0.3466
60
90
70
150
180
270
210
440
a=standard error estimate, b= standard error
3.2.4. Precision
Precision of the proposed method was analyzed with respect to repeatability (intra-day) and intermediate (inter-day)
precision over a period of three days for bulk drug and human serum, and was expressed as percent relative standard
deviation. The %RSD values for PRA, DTZ, NAP and MEL
were found to be 0.14-1.95, 0.52-1.90, 0.13-1.98 and
0.16-1.80 at isosbestic point respectively assuring that the
proposed method has high precision (table 3).
Table 3. Precision of the proposed method
Conc
PRA
Isosbestic point
0.5
1.25
2.5
5
10
20
Time program
0.25
0.5
1.0
2.0
4.0
8.0
Serum
2.5
5
10
%RSD
Conc
DTZ
%RSD
Conc
NAP
%RSD
Conc
Mel
%RSD
0.46
1.80
0.14
1.19
1.95
0.29
0.75
1.5
3
6
12
24
0.84
1.90
1.23
0.54
0.52
0.62
0.5
1.25
2.5
5
10
20
1.38
0.13
1.42
1.21
1.98
0.43
0.5
1.25
2.5
5
10
20
1.38
0.33
1.11
1.80
1.31
0.16
0.75
0.29
0.41
0.14
0.08
0.21
0.5
1.0
2.0
4.0
8.0
16
1.02
0.95
0.39
0.69
0.20
0.67
0.4
0.8
1.5
3.0
6.0
12
0.67
0.94
0.20
0.27
0.14
0.94
0.15
0.25
0.5
1.0
2.0
4.0
1.00
0.79
0.25
0.16
1.36
0.13
1.60
0.83
0.99
3
6
12
1.28
1.45
1.48
2.5
5
10
1.10
1.03
1.54
2.5
5
10
0.49
0.87
1.77
6
Najma Sultana et al.: An Ultra-sensitive and selective LC-UV method for the simultaneous determination
of pravastatin, diltiazem, naproxen sodium and meloxicam in API, pharmaceutical formulations and human serum
thod. The recovery data ranged from 99.65-100.87,
98.85-100.55, 99.88-101.14 and 98.56-101.40 for PRA,
DTZ, NAP and MEL (table 4) showed that the method is
suitable for the simultaneous determination of these drugs in
pharmaceutical formulation and in human serum.
3.2.5. Accuracy
Recovery tests at three concentration levels (n=5) at
isosbestic point for pharmaceutical formulation and human
serum were carried out in order to evaluate the accuracy by
adding known amount of PRA, DTZ, NAP and MEL to the
unknown sample followed by analysis using proposed me-
Table 4. Recovery in pharmaceutical formulations and serum
Conc
%Rec
Conc
PRA
%Rec
Conc
DTZ
%Rec
Conc
NAP
%Rec
Mel
Pharmaceutical formulation
2.5
100.87
3
100.44
2.5
98.88
2.5
101.40
5
99.83
6
99.85
5
99.30
5
100.22
10
99.65
12
98.85
10
100.21
10
99.72
2.5
100.57
3
100.55
2.5
101.14
2.5
98.56
5
99.79
6
99.77
5
101.48
5
100.81
10
100.50
12
99.99
10
100.84
10
100.02
Serum
3.2.6. Detection and Quantitation Limits
Limit of detection and limit of quantitation [43] are the
concentration of samples that give peak height three times or
ten times to the baseline noise and were found to be 33, 70,
50 and 80 ng mL-1 and 100, 220, 140 and 230 ng mL-1 for
PRA, DTZ, NAP and MEL at isosbestic point. The data is
presented in table 2.
3.2.7. Ruggedness
The ruggedness was assessed on two different columns
Discovery, C18 (5 µm, 25 x 0.46 cm) and Purospher STAR,
C18 (5 µm, 25 x 0.46 cm) on the same instrument by the
same analyst. The analytical results were unaffected by
change in column parameter and proved to be stable and
reliable for everyday analysis (table 5).
Table 5. Robustness and ruggedness
PRA
Parameters
NAP
T
N
5125
1.45
6880
1.41
1.79
5240
1.16
6484
1.38
5802
1.47
5292
2.00
6594
1.80
5892
-
5125
1.73
6656
1.64
-
4374
-
3853
2.00
4618
1.75
4923
1.92
6163
1.62
5330
1.87
6740
1.61
78:22
4825
1.68
6073
1.70
5065
1.29
6681
1.62
80:20
4810
-
6067
1.65
5350
-
6745
1.52
82:18
4671
-
5917
-
5153
1.99
6550
1.20
Purospher STAR
4533
1.20
5988
1.65
4890
1.93
6760
1.62
Discovery
3454
-
4360
1.53
3861
1.81
4798
1.51
Flow rate
(ml min-1)
Mobile phase
(MeOH:H2O)
T
N
T
3.2
4765
1.59
6079
1.73
3.4
4873
1.71
6033
3.6
4896
1.95
0.9
4712
-
1.0
3560
1.1
MEL
N
pH
Column
DTZ
N
N = Theoretical plates, T = Tailing factor
3.2.8. Robustness
Minor variations in analytical parameters were introduced
and any difference between the results was observed. The
method was found to be robust for changes in flow rate from
0.90 to 1.10 mL min-1, mobile phase composition, methanol-water proportions 78:82, 80:20 and 82:12 and mobile
phase pH in the range of 3.2-3.6. Results indicated that the
method is quite suitable and can successfully be applied for
routine analysis (table 5).
3.3. Pharmaceutical Formulations and Serum
The assay of drugs in serum samples and in pharmaceutical formulation was carried out by preparing the samples as
described earlier. The samples of desired concentrations
were prepared for serum and pharmaceutical formulation
and assayed in triplicates, which showed no significant
difference in the results obtained for regression characteristics (table 2), precision (table 3) and recovery (table 4)
American Journal of Applied Chemistry 2013, 1(1) : 1-8
without interference of excipients or serum components,
confirming that the method is reliable for everyday analysis
in human serum and in pharmaceutical formulation.
3.4. Comparison of Isosbestic Method with Time Program
Method
The method was compared by programming the detector
adjusting the wavelength with time to match the individual
analyte's absorption maxima which enhanced sensitivity and
linearity range. The LOD values shifted down drastically.
In this respect, the detector was fixed at 237 nm for 0-3.5
min, 240 nm for 3.6-4.5 min, 230 nm for 4.6-5.5 min and
205 nm for 5.6-7.0 min respectively. The respective chromatogram showing the difference in peak height at isosbestic point as well as after programming the detector is presented in figure 5. A drastic change was observed when the
sample of same concentration was injected at isosbestic
point as well as at time program method. The sensitivity and
linear range was enhanced 0.25-8.0, 0.5-16, 0.4-12 and
0.2-4.0 µg mL-1 for PRA, DTZ, NAP and MEL respectively,
which is considerably lower than the range established at
isosbestic point. The LOD values were also shifted to 15, 42,
20 and 10 ng mL-1 from 33, 70, 50 and 80 ng mL-1 indicating the high sensitivity of method. Moreover, the LOQ
values shifted to 46, 128, 70 and 20 ng mL-1 from 100, 220,
140 and 230 ng mL-1 for PRA, DTZ, NAP and MEL respectively. The inter-day and intra-day %RSD of the method
was found to be 0.08-0.75, 0.20-1.02, 0.14-0.94 and
0.13-1.00 which is in acceptable limits. Overall, the method
was found to be highly sensitive and precise and can conveniently be applied for the analysis of these drugs in human
serum and pharmaceutical formulation.
Fig. 5. Comparison of (1) PRA, (2) DTZ (3) NAP and (4) MEL at
isosbestic point and time program
4.. Conclusion
Liquid chromatographic method for the simultaneous
determination of PRA, DTZ, NAP and MEL has been developed and validated at isosbestic point of 220 nm and by
programming the detector adjusting the wavelength with
time to match the individual analyte's λmax (237, 240, 230
7
and 205 nm); which enhanced the resolution and separation.
The method was compared by programming the detector
adjusting the wavelength with time to match the individual
analyte's chromophore which enhanced sensitivity with
linear range of each molecule.
The determination at time program method is advantageous as it gives lesser quantitation limit and requires small
sample volume and does not require expensive diode array
detector. The method was found to be linear with correlation
coefficient greater than 0.998 in each case. The proposed
method can be successfully applied for the determination of
these drugs with good percent recovery values in human
serum and pharmaceutical formulation with out any chromatographic interference from tablet excipients or endogenous components of serum.
Acknowledgement
Mrs Saeeda Nadir Ali would like to acknowledge Higher
Education Commission, Pakistan for providing indigenous
scholarship for Ph.D research work.
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