Chapter – III Levofloxacin Introduction
169
LEVOFLOXACIN- AN ANTIBACTERIAL AGENT
3.01 Drug Profile
Levofloxacin chemically known as (-)-(S)-9fluoro-2,3-dihydro-3-methyl-10-
(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]1,4benzoxazine-6-carboxylic acid
hemihydrate. It is the L-isomer of the racemate, ofloxacin, a quinolone antimicrobial
agent. Levofloxacin is a broad-spectrum antibiotic that is active against both Gram-
positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase [221-
222], a type II topoisomerase, and topoisomerase IV, which is an enzyme necessary to
separate replicated DNA, thereby inhibiting cell division.
Physical Properties:
The Chemical formula : C18H20FN3O4 • ½ H2O
Molecular weight : 370.38grams/mole
Apperance : Light yellowish-white to yellowwhite
crystal
Solubility : Water and methanol.
Structure:
N
O
F
O
OH
O
H
N
N
Chapter – III Levofloxacin Introduction
170
Formulations: Levofloxacin is available by prescription in tablet form, injection
solution (Oral 250mg/10ml) as well as used in prescription eye and ear drops.
1) Levoflox -Protec (Cipla) :250 mg, 500mg Tablets
2) Fynal –Mankind (Discover): 250mg, 500mg, 750mg Tables; 100ml Injection
3) Leon -Dr.Reddy’s : 250mg, 500mg, 750mg Tablets
Literature Survey:
Some Spectrophotometric methods have been reported in the literature for the
analysis of Levofloxacin such as ion-ion pair complex formation reactions with some
acid-dyes [223-225], oxidative coupling reactions [226-227] and complexation
formation reactions [228-230] derivative spectrophotometry of their Cu(II)
complexes[231], charge-transfer complexation with n-acceptors [232-237] and ternary
complex formation with eosin and palladium have been strongly reported. Other
methods include titrimetry [237-239] atomic absorption spectrometry [240] florimetry
[241-243] luminescence [244-245] conductometry [246] voltametry and polarography
[247-248] flow injection analysis [249] HPTLC [250-252] and capillary
electrophoresis [253-254]. Several workers [255-263] developed chromatographic
methods for simultaneous quantification of levofloxacin in combination with other
drugs present in human plasma and in chicken. Baietto et al [255] developed a HPLC
method for the determination of levofloxacin in combination with linezolid,
rifampicin, and moxifloxacin .A UPLC method is developed by Park et al [256] for
the determination of levofloxacin in blood plasma in humans. Zhou et al [257] have
developed method for the determination of levofloxacin in human plasma and its
applications to bioequivalence studies.
Chapter – III Levofloxacin Introduction
171
Different authors have developed HPLC methods to determine levofloxacin in
combination with rifampicin [258], with moxifloxac [259] in plasma and amniotic
fluid. Siewart.S [260] developed a HPLC method for the assay of LEF in plasma and
dialysate for pharmacokinetics. Ji et al [261] developed a hydrophilic interaction LC-
MS for the determination of LEF in human plasma, [262] combination with
mexifloxacin, with cefepime, garenoxacinand moxifloxacin [263], Djabarouti, S [264]
developed a HPLC method to determine levofloxacin present in plasma, bone tissues
and bronchoalveolar lavage. Liu, P.Y et al [265] developed a HPLC method to
determine levofloxacin present in chicken.
Hurtado et al [266] developed a HPLC and UV Spectrophotometric method
for the determination of levofloxacin present in formulations. Patel and co-workers
[267] developed column high performance liquid chromatographic and derivative
spectrophotometric methods for the determination of levofloxacin in combination
with ornidazole. A rapid liquid chromatography-tandem mass spectrometry method
for the determination of broad mixtures of pharmaceuticals in surface water is
developed by Conley and co-workers [268]. Kothekar et al [269] also developed
HPLC method to determine levofloxacin combination with ambroxol HCl. Santoro,
M.I.R.M et al [270] reported a HPLC method for the assay of levofloxacin in
combination with gatifloxacin, lomefloxacin and pefloxacin.
Chapter-III Levofloxacin Part-A: HPLC-Method
172
DETERMINATION OF LEVOFLOXACIN BY REVERSE PHASE-
HPLC
3.02 Introduction
A simple method of separating an individual component from a mixture and to
determine its purity is chromatography. The same technique is also used in assay of a
component. Several workers worked on estimation of levofloxacin in combination with
other drugs, in biological fluids and formulations [258-270]. The reported methods are
too cost, time consuming and not applicable in low concentrations, hence the author has
attempted in developing a new HPLC method for quantification of Levofloxacin in
pharmaceutical formulations. The main advantage of the method is less expensive and
time required for the quantification is reduced.
3.03 Experimental
Shimadzu LC- 20AT Prominance liquid chromatographic system is used for
the analysis. Shimadzu LC- 20AT system equipped with binary gradient pump, UV-
VIS SPD 20A detector, Column Oven and controlled by spinchrom software.
Chromatographic conditions:
Column : C18 (250mm, 4.6mm-ID; 5µm particle
size)
Detector : UV-VIS SPD 20A Prominance
Wavelength (λmax) : 295nm
Flow Rate : 1.0ml/min.
Injection Volume : 20 μl
Chapter-III Levofloxacin Part-A: HPLC-Method
173
Temperature : 30oC
Run Time : 20 min.
Retention Time : 3.918min.
3.04Method Development
(i)Materials and Reagents
All reagents and chemicals used are of HPLC grade. Acetonitril, methanol,
and phosphoric acid 85% are purchased from Merck, India and HPLC grade water is
used to prepare the mobile phase. Stock solutions of levofloxacin and sample
solutions are prepared in the mobile phase. All solutions are filtered through 0.45 µm
membrane filter and degassed using a sonicator. Gift samples of levofloxacin and
working reference standard from Chandra Lab Hyderabad are used. The formulations
are purchased locally from market.
(ii) Preparation of Solutions
Buffer solution (0.1% o-phosphoric acid, Merck; v/v): About 1.0ml of o-phosphoric
acid is transferred into 1000ml volumetric flask made up to the mark with HPLC
grade distilled water and degassed by sonication.
Mobile phase(Buffer, Methanol and Acetonitrile;70:12:18 v/v): Mobile phase is
prepared by mixing accurately measured volumes of 350ml of buffer,60ml of
methanol and 90ml of acetonitrile in a 1000ml beaker, stirred well, filtered through
0.45μ or finer porosity membrane filter, sonicated and used for the analysis.
Chapter-III Levofloxacin Part-A: HPLC-Method
174
Drug Standard Solutions
Stock solution: About 12.5mg of Levofloxacin is accurately weighed, transferred into
a cleaned and dried 100ml volumetric flask, dissolved in atetonitrile and made up to
the mark with mobile phase. The solution is sonicated, filtered through 0.45μ or finer
porosity membrane and used for the analysis.
Working standard solution (12.5μg/ml): Working standard solution is prepared daily
by accurately measuring 5ml of the stock solution into 50ml volumetric flask and
made up to the mark with mobile phase.
Preparation of test solutions: Tablets of 250mg/500mg are powdered and mixed
thoroughly; an amount of the powder equivalent to 12.5mg of the drug is accurately
weighed and transferred into a 100ml volumetric flask dissolved in methanol, shaken
well and filtered. The filtrate is evaporated to dryness carefully and the residue is
dissolved in 10ml of acetonitrile and sonicated and made up to 100ml with the mobile
phase
(iii) Procedure:
Chromatographic conditions are fixed for the Shimadzu HPLC system. The
mobile phase is allowed to pump into the column C18 (250mm, 4.6mm-ID; 5µm
particle size) at a flow rate of 1.0 ml/min., keeping the column in thermostat at a
constant temperature of 30oC about 30min. The response of the system is recorded
against time at maximum wavelength 295nm. After the elution of the mobile phase
through the column, it is found that the base line of the chromatogram is almost
parallel to x-axis. Working standard solution of levofloxacin (20μl) is injected into the
system, the mobile phase is allowed to pass through the column for 20minutes and the
Chapter-III Levofloxacin Part-A: HPLC-Method
175
chromatogram is recorded. Typical chromatograms for the standard and
pharmaceutical formulation are represented in Fig.3.01 and Fig.3.02; P: 181.
(iv)Optimization of the proposed method:
The developed method is optimized and optimum conditions are established
by varying the parameters such as concentration of the standard, type of column,
temperature of the column, flow rate, injection volume, composition of the mobile
phase (polarity), pH of the buffer one at a time, keeping the others fixed and
observing the effect on the retention time, tailing factor and other system suitability
parameters.
3.05. Method Validation:
Validation of analytical methods is important in the analysis of pharmaceutical
formulations. The ability to control the quality is dependent upon the ability of the
analytical methods, as applied under well-defined conditions and at an established
level of sensitivity, to give a reliable demonstration of all deviation from target
criteria. Analytical method validation is now required by regulatory authorities for
marketing authorizations and guidelines have been published. It is important to isolate
analytical method validation from the selection and development of the method. The
most widely applied validation characteristics are given below
3.05(i) System Suitability and System Precision:
Working standard solution (15.5μg/ml) of levofloxacin is injected six times
into the HPLC system and the chromatograms are recorded. The system suitability
parameters and system precision are evaluated based on the area of the peaks and
Chapter-III Levofloxacin Part-A: HPLC-Method
176
found to be within the limits. The results are incorporated in Table-3.01(a), P: 183.
The % RSD for peak areas of six replicate injections is determined and found to be
1.089. The results are summarized in Table-3.01(b), P: 183.
3.05(ii) Linearity of detector response:
Linearity between the response of the detector and the different concentrations
of the standard drug solutions is established by plotting a graph to concentration
versus average area of two peaks. The regression parameters are calculated. A series
of six working standard solutions are prepared in the concentration range of about
25.0μg/ml to 150μg/ml corresponding to 25% to 150% of target concentration. Each
solution is injected into the system and recorded the chromatogram under the test
conditions. A graph is plotted to concentration in µg/ml on X-axis versus response on
Y-axis Fig.3.03 (a), P: 182. The detector response is found to be linear with a squared
correlation coefficient of 0.9998. The results are summarized in Table-3.02, P: 183.
3.05(iii) Precision of test method:
Commercial formulations of levofloxacin tablets of 250mg and 500mg are
successfully analyzed. The precision of the test method is evaluated by taking six
replicate measurements. The average percent of assay of levofloxacin in tablets is found
to be 100.53% and 99.99% and %RSD is found to be 0.818 and 0.564 respectively. The
results are summarized in Table-3.03, P: 184.
3.05(iv) Accuracy of the method:
A study of levofloxacin from spiked placebo is conducted. Different
concentration solutions of levofloxacin formulations are prepared equivalent to about
Chapter-III Levofloxacin Part-A: HPLC-Method
177
75%, 100%, and 125% of the target concentration. Sample solutions are prepared in
triplicate for each spike level and assayed as per test method. The % recovery is found
to be 100.52(±0.828), 100.38(±0.891) and 100.08(±1.047). The results are
summarized in Table-3.04, P: 184.
3.05(v) Linearity of the test method:
A series of six different concentration solutions of the drug are prepared as per the
test method and for each concentration the chromatogram is recorded. The amount of
drug recovered is estimated. A linear plot is drawn to average amount of levofloxacin
added (mg) versus average of levofloxacin recovered (mg) in accuracy (Fig.3.03 (b), P:
182). The results of the recovery experiments by the developed method are summarized
in Table-3.05, P: 185.
3.05 (vi)Limit of Detection and Limit of Quantitation
Limit of detection and Limit of quantitation (LOD and LOQ) are calculated based
on the standard deviation of the response and the slope of the calibration curve. The
results of LOD and LOQ are summarized in Table-3.02, P: 183.
3.05(v) Ruggedness
Ruggedness is the degree of reproducibility of results obtained by the analysis of
the same sample under a variety of normal test conditions different, laboratories,
instruments, reagents, assay temperatures, small variations in mobile phase, different
days etc
(a) Intra Day & Inter Day variability: This study is conducted on day-1 and day-2
using same columns on the same HPLC system by assaying six different sample
Chapter-III Levofloxacin Part-A: HPLC-Method
178
preparations of Levofloxacin formulations under similar conditions. The system
suitability parameters are evaluated as per the standard method and found to be within
limits. The average % assay was found to be 99.98 and 100.21 with a relative standard
deviation of 0.695% and 0.576% respectively. The results are summarized in Table-
3.06(a), P: 185. Comparison of the results obtained on two days shows that the assay
method is rugged for day to day variability.
(b) System to System variability: System to system variability study is conducted on
two HPLC systems by using the same column by assaying six separately prepared
sample solutions of levofloxacin formulations under similar conditions. The system
suitability parameters are evaluated as per the test method on both the systems and
found to be within limits. The average % assay for the two systems is found to be
100.22and 100.27 with a relative standard deviation of 0.454% and 0.587%
respectively. The results are summarized in Table-3.06(b), P: 186. Comparison of the
results obtained on two systems shows that the assay method is rugged for system to
system variability.
(c) Column to Column variability: This study is conducted using two columns on the
same HPLC system by assaying six separately prepared sample solutions of
levofloxacin formulations under similar conditions. The system suitability parameters
are evaluated as per the test method on both the systems and found to be within limits.
The average % assay for the two systems was found to be 100.24and 100.25 with a
relative standard deviation of 0.562% and 0.547% respectively. The results are
summarized in Table-3.06(c), P: 186. Comparison of the results obtained on two
columns shows that the assay method is rugged for column to column variability.
Chapter-III Levofloxacin Part-A: HPLC-Method
179
3.05(vi) Robustness
Robustness is a measure of the capacity of an analytical method to remain
unaffected by small but deliberate variations in method parameters such as per cent
organic content in the mobile phase, pH of the mobile phase, buffer concentration,
temperature, injection volume and column temperature. These parameters may be
evaluated one factor at a time or simultaneously as part of a factorial experiment. It
also provides some indication of the reliability of an analytical method during normal
usage
Six separately prepared sample solutions are injected into the HPLC system
with flow rates of 0.9ml/min. and 1.1ml/min, at two different wavelengths 290nm and
300nm, keeping the column at 28oC and 32
oC, using mobile phase of different
composition and maintaining buffer solution at different pH. The instrument response
is recorded against time in each case. The system suitability parameters are evaluated
as per the test method and found to be within limits and are shown in Table-3.07, P:
187.
3.06 Results and Discussion:
Reverse phase high performance liquid chromatography method is chosen for
the analysis of levofloxacin in pure and pharmaceutical formulations. The optimized
chromatographic conditions are adjusted before running the system. The
chromatograms are recorded by injecting standard and test solutions of the drug in to
the column. The system suitable parameters are evaluated and found within the limits.
The developed method is simple, rapid, precise, accurate and linear. The results are
given in Table-3.01(a) and Table-3.01(b); P: 183. A plot is drawn between
Chapter-III Levofloxacin Part-A: HPLC-Method
180
concentration and the instrument response and found to be linear with good
correlation coefficient(r=0.9996) (Table-3.03, P: 184; Fig.3.03 (a), P: 182). Precision
and accuracy of the developed method are expressed in %RSD and % of recovery of
the active pharmaceutical ingredient. Low %RSD values 0.813 and 0.668 and high
%recovery 100.45%, 99.77% and 100.74%corresponding to 75% ,100% and 125%
spike levels indicate that the method is highly precise and accurate (Table-3.04, P:
184). Pharmaceutical formulations are analyzed by the developed method by
estimating the amount of drug recovered by standard addition method.
A graph is drawn between the amount of drug added and the amount of drug
recovered, the plot is linear and regression equation is given by y=1.010x+0.0727with
r2 =
0.9997(Table-3.05; P: 185). A study is conducted between two analysts, two
different systems, and two columns and compared the results. The system suitability
parameters are evaluated as per the test method on both the systems, columns and for
analysts and found to be within limits. The average % assay and relative standard
deviation are within the limits (Table-3.06(a)-Table-3.06(c), P: 185-186). The
change in system suitability parameters are evaluated by studying the effect of change
chromatographic parameters and found to be acceptable (Table-3.07, P: 187).
3.07 Conclusion: The developed method for the determination of Levofloxacin is
optimized and validated using the test method and found to be linear, precise,
accurate, rugged and robust. The developed RP-HPLC method is cheap, economical
and completed within short time. The developed method can be successfully applied
for the routine analysis of levofloxacin in bulk pharmaceutical formulations.
Chapter-III Levofloxacin Part-A: HPLC-Method
181
Fig.3.01Chromatogram of Levofloxacin (Standard)
Fig.3.02 Chromatogram of Levofloxacin (Formulation)
Chapter-III Levofloxacin Part-A: HPLC-Method
182
Linearity of detector responce to the concentration of the Levofloxacin
y = 6796.7x + 4253.8
R2 = 0.9996
0.0E+00
3.0E+05
6.0E+05
9.0E+05
1.2E+06
1.5E+06
0 50 100 150
Concentration of Levofloxacinin µg/ml
Dete
cto
r R
esp
on
ce
Fig.3.03 (a) Linearity of the detector response
Linearity of the test Methody = 1.0099x - 0.0727
R2 = 0.9997
0.0
5.0
10.0
15.0
20.0
0.0 5.0 10.0 15.0 20.0
Weight of Drug added in µg/ml
Weig
ht
of
dru
g r
eco
vered
in
µg
/ml
Fig. 3.03(b) Linearity of the test Method
Chapter-III Levofloxacin Part-A: HPLC-Method
183
Table-3.01(a): System Suitability
System suitability parameters Observed value Acceptance criteria
USP tailing factor 1.752 NMT2.0
Number of theoretical plates 3764 NLT2000
RSD(six replicate measurements) 1.089 NMT 2.0%
Table-3.01(b): System Precision
Injection ID Average Area Statistical parameters Value of the parameters
1 689881 Mean 690857
2 691864
3 687963 SD 1736
4 690795
5 691846 %RSD 0.251
6 692793
Table-3.02: Linearity of detector response
Injection
ID
Concentration
μg/ml
Average
Area Regression Parameter
1 3.13 172470 Slope 55013
2 6.25 344941 Intercept 2507
3 9.38 517411 Correlation Coefficient 0.9996
4 12.50 689881 LOD µg/ml 0.341
5 15.63 862351 LOQ µg/ml 1.137
6 18.75 1011000
Chapter-III Levofloxacin Part-A: HPLC-Method
184
Table-3.03: Precision of the test method
Percent of Assay Labeled Amount
Sample No. Labeled Amount 250mg/Tablet Labeled Amount 500mg/tablet
Amount Found %Recovery Amount Found %Recovery
1 253.30 101.32 498.40 99.68
2 254.00 101.60 499.35 99.87
3 249.65 99.86 497.75 99.55
4 249.45 99.78 503.60 100.72
5 252.00 100.80 503.35 100.67
6 249.58 99.83 497.25 99.45
Mean 251.33 100.53 499.95 99.99
SD 2.044 0.818 2.820 0.564
%RSD 0.813 0.813 0.564 0.564
Table-3.04: Accuracy of the method
Spike
level
Sample
ID
Amount
Added(μg/ml)
Amount
Found(μg/ml)
% of
Recovery Statistical Analysis
75% 1 9.38 9.51 101.37 Mean 100.52
2 9.38 9.35 99.72 SDV 0.828
3 9.38 9.42 100.45 %RSD 0.824
100% 1 12.50 12.68 101.40 Mean 100.38
2 12.50 12.50 99.96 SDV 0.891
3 12.50 12.47 99.77 %RSD 0.888
125% 1 15.63 15.45 98.88 Mean 100.08
2 15.63 15.73 100.64 SDV 1.047
3 15.63 15.75 100.74 %RSD 1.047
Chapter-III Levofloxacin Part-A: HPLC-Method
185
Table-3.05: Linearity of test method
Spike
level (%)
Drug
added(mg)
Drug
Recovered(mg) Regression parameters
25 3.13 3.00 Slope 1.010
50 6.25 6.22
75 9.38 9.59 Intercept 0.0727
100 12.50 12.53
125 15.63 15.66 Correlation Coefficient 0.9997
150 18.75 18.84
Table -3.06(a) Intra Day and Inter Day precision
Percent of assay of Levofloxacin formulation
Sample No. Day-1 Day-2
1 98.86 100.9
2 99.92 100.8
3 99.82 99.75
4 100.5 99.85
5 100.9 99.53
6 99.85 100.4
Mean 99.98 100.21
SD 0.695 0.577
%RSD 0.695 0.576
Comparison of the precision with method precision
F=1.384,t=1.955 F=2.011,t=1.381
Comparison of precision between two analysts
F=1.451 t=0.572
Chapter-III Levofloxacin Part-A: HPLC-Method
186
Table -3.06(b) System to System Variation
Percent of assay of Levofloxacin formulation
Sample No. Systemt-1 System-2
1 99.99 101.02
2 100.6 100.94
3 99.94 99.84
4 100.35 99.73
5 99.62 100.32
6 100.84 99.78
Mean 100.22 100.27
SD 0.455 0.588
%RSD 0.454 0.587
Comparison of the precision with method precision
F=3.226,t=1.649 F=1.933,t=1.076
Comparison of precision between two analysts
F=1.671 t=0.190
Table -3.06(c) Column to Column Variation
Percent of assay of Levofloxacin formulation
Sample No. Column-1 Column-2
1 99.88 100.36
2 99.84 100.69
3 100.88 99.94
4 99.93 99.85
5 101.04 100.75
6 99.85 99.94
Mean 100.24 100.25
SD 0.563 0.402
%RSD 0.562 0.401
Comparison of the precision with method precision
F=2.108,t=1.275 F=4.142,T=1.676
Comparison of precision between two analysts
F=1.961 t=0.043
Chapter-III Levofloxacin Part-A: HPLC-Method
187
Table 3.07: Study of Robustness
Variable Variation USP Plate Count USP Tailing %RSD
Standard -------- 3764 1.752 0.000
Flow Rate 0.9ml/min. 3491 1.639 1.100
1.1 ml/min. 3871 1.674 0.790
Wavelength 300nm 3175 1.352 0.921
290 nm 3769 1.726 0.721
Temperature 28oC 3879 1.598 1.040
32oC 3582 1.526 0.974
%Mobile
Phase
70%:10%:20% 3498 1.498 0.737
70%:15%:15% 3762 1.521 1.694
Buffer pH 2.8 3751 1.427 1.436
3.2 3462 1.519 1.489
Acceptance Criteria USP Tailing Factor not more than 2.0, Theoretical Plates
not less than 2000
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
188
DETERMINATION OF LEVOFLOXACIN BY VISIBLE
SPECTROPHOTOMETRY
3.08 Introduction
The functional groups that are present in levofloxacin are not fully exploit
hence the author has made some attempts to develop new spectrophotometric methods
for the determination of levofloxacin in pure and formulations. The developed
methods have better sensitivity, selectivity, precision and accuracy.
3.09(i) Experimental
UV-Visible Spectrophotometer: ElicoSL159 model, 2nm high resolution, double
beam, 1cm length quartz coated optics and wavelength range190-1100nm instrument
is used for all the spectral measurements.
3.09(ii) Preparation of solutions
3.09(ii) (a) Standard Solution of Levofloxacin
The stock solution of Levofloxacin is freshly prepared by transferring
accurately weighed 100mg of Levofloxacin into 100ml volumetric flask, dissolving,
and then made up to the mark with double distilled water. Working standard solution
200μg/ml and 250μg/ml are prepared and used in the reactions M2(a), M3, M18 and
M1(a), M1(d), M5(c), M12, M14, M17 respectively.
3.09(ii) (b) Preparation of reagents
Analytical grade chemicals and reagents are used in the preparation of
solutions and all the solutions are prepared in double distilled water.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
189
Method M1(a) and M1(d)
Aqueous solutions of ARS (0.2%, 5.49 x 10-3
M), BTB (0.1%, 1.60 x 10-3
M),
TPooo (0.2%, 5.71 x 10-3
M) and HCl solution (0.1M) are prepared in the same way
as described under NTT in Chapter – II, P: 81-82.
Method M2(a)
MB solution (Fluka; 0.2%, w/v 6.25x10-3
M): Prepared by dissolving 200mg of MB in
100ml of distilled water.
Buffer solution pH 9.8 NH4OH – NH4Cl: 7.0gms of NH4Cl and 6.8ml of ammonia
solution are dissolved in 100ml of distilled water and pH is adjusted to 9.8.
Method – M3
Fe (III) solution (Wilson labs; 0.054%, 3.33x10-3
M): Prepared by dissolving 54mg
of anhydrous Ferric chloride in 100ml of distilled water
O-PHEN solution (E.Merck, 0.2%, 1.10x10-2
M): Prepared by dissolving 200mg of
o-phenanthroline in 100ml of distilled water with warming.
O-phosphoric acid solution (Qualigens, 2.0x10-2
M): Prepared by mixing 1.27ml of
o-phosphoric acid with 100ml of distilled water. 10.0ml of this stock solution is
further diluted to 100ml with distilled water
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
190
Method M5(c)
Aqueous solutions of MBTH (0.2%, 8.56 x 10-3
M) and Ce (IV) (1%, 9.35 x 10-
3M), NaIO4 solution (0.2%, 9.35 x 10
-3M), AcOH solution (Qualigens; 2.3 M) are
prepared in the same way as described under NTT in Chapter – II P: 83.
Method M12
NaIO4 solution (Loba; 0.855%, 4.00x10-2
M): Prepared by dissolving 855mg of
NaIO4 in 100ml of 0.3M HCl.
PHH (Loba; 4.0%, 6.90x10-2
M): Prepared by dissolving 4.0g of Phenyl Hydrazine
Hydrochloride (PHH) in 100ml of distilled water and filtered.
K3Fe(CN)6 solution (Sd-fine;.2.0%,6.00x10-2
M): Prepared by dissolving 2.0g of
K3Fe(CN)6 in 100ml distilled water.
NaOH solution (BDH, 0.4%, 0.1M): Prepared by dissolving 400mgs of NaOH to
100ml distilled water and standardized.
Method M14
CTC solution (E.Merck; 2.86x 10-1
M): Prepared by dissolving 7.25g of cobaltous
nitrate and 3.8g of ammonium thiocyanate in 100ml of distilled water.
Buffer solution (pH 2.0): Prepared by mixing 306ml of trisodium citrate (0.1M) with
694ml of HCI (0.1M) and the pH of the solution were adjusted to 2.0.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
191
Method M17
Aqueous solutions of PA (0.1%, 4.36 x 10-3
M) and Buffer pH – 9.8 are
prepared in the same way as described under NTT in Chapter – II, P: 84.
Method M18
4-AP Solution (BDH; 0.5%, 2.45 x 10-2
M): 500mg of 4-AP is accurately weighed
and dissolve din 100ml of methanol
3.09(iii) Procedures proposed for the assay of Levofloxacin
After a systematic and detailed study of the various parameters involved, the
following procedures are proposed for the assay of levofloxacin in bulk and
pharmaceutical formulations.
Method – M1(a) and M1(d)
Into a series of 125ml separating funnels containing aliquots of standard LEF
solution 8.3-50.0μg/ml and 16.7-100.0μg/ml, for methods M1(a) and M1(d) respectively,
6.0ml of 0.1M HCI solution and 1.0ml of 0.2%ARSdye solution for M1(a) and 2.0ml
0.2%TP ooo dye solution for M1(d) are added successively. The total volume of aqueous
phase in each separating funnel is adjusted to 15ml with distilled water. To each
separating funnel 10ml of chloroform is added and the contents are shaken for 2min. The
two phases are allowed to separate and the absorbance of the separated chloroform layer
is measured at max (415nm for ARS; and 515nm for TPooo) against a similar reagent
blank. The amount of levofloxacin is deduced from the calibration curve ARS and TPooo
(Fig.3.13 – Fig.3.14, P: 207).
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
192
Method M2(a)
Aliquots of standard drug solution (LEF) 13.3-80.0μg/ml and 1.0ml of pH 9.8
buffer solution are placed separately in a series of 125ml separating funnels. 0.5ml of
MB is added and the total volume of aqueous phase in each funnel is adjusted to
10.0ml with distilled water. Then 10.0mlof chloroform is added in each separating
funnel and the contents are shaken for 2min and allowed to separate. The organic
layer is collected through cotton plug and the absorbance is measured immediately at
650nm against a reagent blank. All the colored species are stable for 2 hours. The
amount of drug in a sample solution is obtained from the Beer’s Lambert plot
(Fig.3.15, P: 207).
Method – M3
Into a series of 25ml calibrated tubes aliquots of standard Levofloxacin
solution (20.0-120.0g/ml) are transferred and then 3.0ml (3.33 x 10-3
M) of Fe (III),
2.0ml of (1.01 x 10-2
M) o-phenanthroline are added successively. The total volume in
each tube is brought to 10.0ml with distilled water. The tubes are kept on a boiling
water bath for 30min. The tubes are removed and cooled to room temperature. 2.0ml
of (2.16 x 10-2
M) o-phosphoric acid is added and volume in each tube is made up to
the mark with distilled water. The absorbance of the colored complex solution is
measured after 5 min at 510nm against a reagent blank. The content of the drug is
computed from the appropriate calibration graph (Fig.3.16, P: 208)
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
193
Method – M5(c)
Aliquots of standard LEF solution (10.0-60.0g/ml) are transferred into a series of
25ml calibrated tubes. Then 1.0ml (9.35 x 10-3
M) of NaIO4 solution,1.0ml of acetic
acid solution are and the total volume is adjusted to 10.0 ml and kept in a water bath
for 45min. The solutions are cooled suddenly. After that 1.0ml (8.56 x 10-3
M) of
MBTH solution is added and kept aside for 10min. The volume is made up to the
mark with distilled water. The absorbance is measured at 610nm against a similar
reagent blank. The amount of LEF is computed from its calibration graph. (Fig.3.17,
P: 208).
Method-M12
Different aliquots of standard LEF solution (10.0-60.0g/ml) are transferred
into a series of 25ml-calibrated tubes. Then 1.0ml of NaIO4 solution is added to each
tube and the volume made up to 5ml with distilled water. After keeping the tubes in a
hot water bath for 5min., 1.5ml of PHH solution and 1.0ml of K3Fe(CN)6 solutions
are added successively and shaken well. The tubes are kept in ice water for
5min.Later 5.0ml of HCl is added and the solution in each tube is made up to 25ml
with ethanol. The absorbance is measured after 15min. at 430nm against reagent
blank. The amount of LEF is computed from its calibration graph (Fig.3.18, P: 208).
Method - M14
Into a series of 125ml separating funnels, aliquots of standard Levofloxacin
solution (5.0-30.0g/ml) are taken and 2.0ml of buffer (pH 2.0) and 5.0ml (2.5x10-
1M) of CTC solutions are added. The total volume of aqueous phase in each
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
194
separating funnel is adjusted to 15.0ml with distilled water. To each separating funnel,
10.0ml of nitrobenzene is added and the contents are shaken for 2 min. The two
phases are allowed to separate and the absorbance of the separated nitrobenzene layer
is measured at 660nm against a similar reagent blank. The amount of LEF is
computed from its calibration graph (Fig. 3.19, P: 208).
Method M17
Into a series of 50 ml separating funnels containing aliquots of drug (8.3 –
50.0μg/ml) solutions, 2 ml of buffer of pH 9.8 and 2.0 ml of 0.1% picric acid
solutions are added successively. The total volume of aqueous phase in each
separating funnel is adjusted to 15 ml with distilled water. To each separating funnel
10 ml of chloroform is added and the contents are shaken for 2 min. The two phases
are allowed to separate and the absorbance of the separated chloroform layer is
measured at 415nm against a reagent blank prepared under similar conditions. The
amount of drug is deduced from the calibration graph (Fig.3.20, P: 209).
Method: M18
Delivered aliquots of standard LEF solution (10.0 – 60.0g/ml) into a series of
10ml calibrated tubes, then 3.0ml (5.83 x 10-3
M) of 4-AP is added to each tube and
kept aside for 15min. Later the solution in each tube is made up to 10ml with methanol.
The absorbance is measured at 440nm against the reagent blank. The amount of LEF is
computed from its calibration graph. (Fig.3.21, P: 209).
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
195
3.09 (iii) Optimum Conditions
Optimum conditions for the maximum color development and maximum
stability of the proposed methods (M1(a), M1(d), M2(a), M3, M5(c), M12, M14,
M17 and M18 are established by varying the parameters one at a time, keeping the
others fixed and observing the effect produced on the absorbance of the colored
species. The optimum conditions thus established are incorporated in recommended
procedures.
Method - M1(a) and M1(d)
The Procedure to establish optimum conditions for these methods is as same as
described in Chapter II of NTT Table 2.08(a), P: 97.
Method – M2(a)
The author performed controlled in pediments by measuring absorbance at
max 655nm of a series of solutions varying one and fixing the other parameter in
order to fix the optimum conditions. Various parameters such as type of acid for
buffer, concentration of acid, concentration of dye or choice of organic solvent, ratio
of organic phase to aqueous phase, shaking time, temperature, intensity and stability
of the colored species in organic phase are investigated and the results are recorded in
Table 3.08 (a), P: 200.
Method – M3
In order to establish optimum conditions necessary for rapid and quantitative
formation of the colored complex with maximum stability and sensitivity, the author
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
196
has performed control experiments by varying one and fixing the other parameters,
such as effect of pH of the buffer solution, volume of buffer solution, volume of Fe
(III) and o-phenanthroline solution, temperature, time of heating, order of addition of
reagents and nature of solvents for final dilution. The optimum conditions are
incorporated in Table 3.08(b), P: 201.
Method –M5(c)
The optimum conditions established for method M5(c) are found to be same as
described in Chapter II of NTT Table 2.08(d) and Table 2.08(e) P:100-101.
Method M12
In developing this method, the effect of various parameters likes strength and
volume of NaIO4, time and temperature required for oxidation, volume and strength
of reagents such as PHH and K3Fe(CN)6], volume of HCl, solvent for final dilution in
developing color of maximum stability and intensity are studied. The results are
incorporated in Table 3.08(c), P: 202.
Method - M14
The optimum conditions incorporated in the recommended procedure are
ascertained by performing systematic investigations. The volume of cobalt
thiocyanate, volume of buffer solution, solvent for the extraction of colored species,
time required for maximum color development and stability period of the colored
species are studied and the results are incorporated in Table 3.08(d), P: 203.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
197
Method – M17
The optimum conditions established for methods M17 are found to be same as
described in Chapter II of NTT Table 3.08(i), P: 105.
Method: M18
The optimum conditions are established basing on the study of the effects of
various parameters such as volume of (2.54 x 10-2
M) 4-AP solution, volume of
solvents solution used initially and subsequently for final dilution and the stability of
colored species after final dilution, measuring absorbance at 430nm. The optimum
conditions developed and actual conditions chosen for the procedure are recorded in
Table 3.08(e), P: 204.
3.10 Validation of the methods
3.10 (i) Linearity
Concentration of the drug is plotted against absorbance and is found to be
passing linearly through the origin. Slope, intercept and correlation coefficient are
evaluated and found within the limits and are presented in Table 3.10(a) – Table
3.10(b), P: 212.
3.10 (ii) Precision
The precision of the developed method is ascertained from the absorbance
values obtained by actual determination of six replicates of a fixed amount of LEF in
total solution. The percent relative standard deviation and percent range of error (at
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
198
0.05confidence limits) are calculated for the proposed methods and represented in
Table 3.11(a) – Table 3.11(b), P: 213.
3.10(iii) Accuracy
To determine the accuracy three different concentrations of bulk samples of
LEF within the Beer’s law limits are taken and analyzed by the proposed method.
The results are recorded in Table 3.12(a) – Table 3.12(i), P: 213-217.
3.10(iv) Limit of Detection and Limit of Quantification
The limits of detection and quantification are calculated from the standard
deviation of intercept and slope of the absorbance measurements obtained for a series
of solutions and are presented in Table 3.10(a) – Table 3.10(b), P: 212.
3.11 Assay of pharmaceutical formulations
An accurately weighed portion of powdered tablets equivalent to 100mg of
LEF is dissolved in 20ml of methanol (MeOH), shaken well and filtered, the filtrate
is diluted to 100ml with MeOH to get 1mg/ml of drug in formulations and then
25.0ml, 20.0ml and 10.0ml of this solution are further diluted to 100ml to obtain
working standard solutions of 250μg/ml, 200μg/ml and 100μg/ml respectively.
Commercial formulations containing Levofloxacin are successfully analyzed by the
proposed methods. The values obtained by the proposed and reference methods for
the formulations are compared statistically with F-test and t-test and found to be not
different significantly. Present of recoveries are determined by adding standard drug
to preanalysed formulations. The results of the recovery experiments by the
proposed methods are also listed in Table 3.13(a) – Table 3.13(e), P: 218-222.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
199
3.12 Results and Discussions
3.12 (i) Spectral Characteristics
In order to ascertain the optimum wavelength of maximum absorption (max) of the
colored species, specified amounts of the drug are taken and color is developed separately
by following the above mentioned procedures. The absorption spectra are scanned on a
spectrophotometer in the wavelength region of 370 to 900nm against similar reagent blank.
The results are graphically represented in Fig.3.04 – Fig.3.12, P: 205-207.
3.12(ii) Optical Characteristics
Absorbences at maximum wavelength of a set of six standard solutions
containing different amounts of levofloxacin and specified of amounts of reagents are
recorded against the corresponding reagent blanks. In order to test whether the
colored species formed in above methods adhere to Beer’s law, a plot of concentration
against absorbance is drawn and it is found to be linear. Beer’s law limits, molar
absorptivity, Sandell’s sensitivity and optimum photometric range in each method are
calculated. The values are presented in Table 3.09 (a) – Table 3.09(b), P: 212. The
optimum photometric range of the proposed methods is determined from the Ringbom
plots Fig.3.22-Fig.3.30, P: 209-211.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
200
Table 3.08(a)
Optimum conditions established for Method M2(a) for LEF Parameter Optimum range Conditions in
procedure
Remarks
max (nm)
650 - 660 650 --
Effect of buffer on color development 9.0 - 10.0
pH-9.8
Variations of the pH less than 6.0 and greater than11.0 resulted in low
absorbance values
Volume of buffer required for maximum
intensity of color (ml)
0.5 - 1.5
1.0
Optimum volume of 1.0ml of buffer is sufficient for maximum color
development
Effect of volume of dye MB
1.0 - 5.0
1.0
1.0ml of MB dye is necessary for covering the broad range of beer’s law
limits
Choice of organic solvent for extraction of
colored complex
Chloroform
Chloroform
Water immiscible solvents tested for the extraction of the colored
complex into organic phase include chlorobenzene, dichloro methane,
CCl4, C6H6 and butanol. CHCl3 is preferred for its selective extraction of
the colored drug-dye complex from the aqueous phase.
Effect of the ratio of organic to aqueous
phase on extraction
1:1
1:1
The extraction of the colored species in to Chloroform layer is in
complete when the ratio of chloroform to aqueous phase is more than the
specified ratio in each case
Effect of shaking time (min)
1 – 5
2
Constant absorbance values are obtained for the shaking period of 1-5
min.
Effect of temperature on the colored species
Laboratory -
Temperature
(285)
Laboratory-
Temperature
(285)
At low temperature (<200C) and at high temperature (>35
0C) the
extraction of the colored species is found to be improper and the stability
of the colored species is found to be very less.
Stability of the colored species
Immediate to
60 min
10 min
The colored species after separation from organic phase is stable for 60
min, after wards the absorbance gradually decreases.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
201
Table 3.08(b)
Optimum conditions established for Method M3 for LEF
Parameter Optimum range Conditions in
procedure
Remarks
max (nm) 490-510 510
Effect of volume of 3.33x10-3
M of Fe(III)
solution on color development
1.0-4.0ml
3.0ml
Variation of volume below and above of this range gave erratic results.
Effect of volume of 1.01x10-2
M of
O-PHEN on color development 1.5 –3.0ml 2.0ml
Variation of volume below and above of this range gave erratic results.
Effect of temperature on colored species
90-990C
Boiling Water
Bath.
It is found that boiling water bath is necessary for uniform temperature
and maximum color development. Below this temperature the intensity
of the colored complex is weak.
Effect of heating time
25-40min.
30min.
Below 25min. the colored complex is not completely formed.
Effect of volume of 2.16x10-2
M of o-
phosphoric acid.
1.0-3.0ml
2.0ml
To complex excess of Fe (III) ions, a minimum of 1.0 ml of o-
phosphoric acid is required.
Effect of order of addition of reagents on
color development
LEF, Fe (III)
solution, o-Phen
before heating
and phosphoric
acid after heating.
LEF, Fe (III)
solution and o-Phen
before heating and
phosphoric acid after
heating.
Interchanging the order of LEF, Fe (III) solution and o- Phen has no
effect on absorbance of the colored species.
Nature of solvent for final dilution.
Distilled Water
Distilled Water
Other water miscible solvents, like acetonitrile, methanol, ethanol,
acetone and 1, 4-dioxan did not enhance the intensity of the final colored
product.
Stability of the colored species after final
dilution.
5-60 min
5 min
The absorbance of the colored product decreased slowly beyond 60 min.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
202
Table 3.08(c)
Optimum conditions established in Method M12 for LEF
Parameter
Optimum range
Conditions in
procedure
Remarks
max (nm) 510-550
430
Volume of (4.0×10-2
M) NaIO4 solution 0.5-2.0ml
1.0ml
Beyond the upper and lower limits low absorbance is observed either in
lower or upper Beer’s law limits.
Time and temperature.
25-35min. at
Laboratory
temperature
30min. at
Laboratory
temperature
30min. is necessary to complete oxidation.
Volume of (2.76×10-1
M) PHH solution. 0.5-2.5ml 1.5ml
1.5ml of PHH solution is found necessary for color development.
Volume of Hexacyano ferrate (III)
(3.02x10-3
M) solution 0.5-2.5ml 2.0ml
Addition of <0.5ml results in erratic values especially in upper region of
Beer’s law limits.
Time and temperature prior to the addition
of Conc.HCl.
5min.
0-50C
5min. in ice bath. Minimum cooling for 5min. in ice bath has been found to be necessary.
Addition of Conc.HCl.
4-6ml
5ml
4.0ml of Conc. HCl is found necessary for maximum color
development.
Solvent for final dilution Ethanol Ethanol Ethanol is found to be the best solvent for final dilution.
Stability period after final dilution. Immediate-
30min. 15min.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
203
Table 3.08(d)
Optimum conditions established in Method M14for LEF
Parameter Optimum range Conditions in
procedure
Remarks
max (nm) 610 - 630 660
Effect of buffer pH on color.
pH - 2.0
Variation of pH of the buffer beyond the upper and lower limits resulted
in low absorbance values.
Volume of buffer required for maximum
intensity of color.
1.5 - 2.5ml
2.0ml
2.0 ml of buffer of pH 2.0 is found to be necessary in aqueous phase for
maximum color development in nitrobenzene layer.
Effect of volume of (2.86 x 10-1
M) of CTC
solution required for complex formation.
4.0 – 6.0ml
5.0 ml
5.0 ml of CTC is found to be necessary for complex formation and to
cover broad range of Beer’s law limits. No added advantage is observed
with more than 5.0ml.
Solvent for extraction of complex.
Nitrobenzene
Nitrobenzene
Among the various solvents tried for extraction (dichloromethane,
chloroform, chlorobenzene and nitrobenzene), nitrobenzene is found to
be better due to higher sensitivity.
Effect of the ratio of organic to aqueous
phase on extraction.
1:1,1:2,2:3
2:3
A ratio of 2:3 organic to aqueous phases is required for the efficient
extraction of the complex.
Stability period.
5 - 60 min
5 min
The intensity the colored species begins to decrease slowly after 60 min.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
204
Table 3.08(e)
Optimum conditions established in method M18 for LEF
Parameter
Optimum
range
Conditions in
procedure
Remarks
max (nm)
400 - 470
440
Effect of volume (2.45 x 10-2
M) of 4-AP
in MeOH and waiting time. 2 - 4ml, 15min 3.0ml ,15min
3ml of 4-AP and 15min waiting time are preferred for covering
broad range in Beer’s law limits.
Solvent for final dilution. Methanol Methanol MeOH has been found to be suitable for final dilution to give
better absorbance values.
Stability period after final dilution. Immediate -
40min 10min
After 40min the absorbance of colored species diminish slowly
with time.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
205
Method M1(a):ARS+CHCl3
[LEF]=9.00x10-5
M,[ARS]=3.66x10-4
M
[HCl]=4.00x10-2
M
0.000
0.030
0.060
0.090
0.120
0.150
0.180
375 400 425 450 475 500
Wavelength nm
Ab
so
rba
nc
e
BlankTest
Fig. 3.04 Absorption Spectra of LEF
with ARS M1(a)
Method M1(d):TPooo+CHCl3
[LEF]=9.00x10-5M,
[TPooo]=3.81x10-4M,[HCl]=4.00x10-2M
0.000
0.100
0.200
0.300
475 525 575Wavelength nm
Ab
so
rba
nc
e
Ox.DyeTestAq. Dye
Fig. 3.05 Absorption Spectra of LEF
with TPooo M1(c)
MethodM2(a):MB+CHCl3
[LEF]=7.20x10-5
M
[MB]=4.17x10-4
M,Buffer of pH=9.8
0.000
0.100
0.200
0.300
550 600 650 700 750Wavelength nm
Ab
so
rba
nc
e
Blank
Test
Fig. 3.06Absorption Spectra of LEF
with MB M2(a)
Method M3:Fe(III)+O-PHEN
[LEF]=1.08x10-4
M, [Fe(III)]=3.99x10-4
M
[O-PHEN]=8.07x10-4
M
[O-PHOS]=1.73x10-3
M
0.000
0.030
0.060
0.090
0.120
0.150
0.180
400 450 500 550 600Wavelength nm
Ab
so
rba
nc
e
Blank
Test
Fig. 3.07 Absorption Spectra of LEF
with Fe(III)+O-PHEN M3
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
206
Method M5(c):MBTH+NaIO4+AcOH
[LEF]=5.40x10-5M,[MBTH]=3.42x10-4M
[NaIO4]=3.74x10-4M,[AcOH]=1.28x10-1M
0.000
0.050
0.100
0.150
0.200
500 550 600 650 700Wavelength nm
Ab
so
rba
nc
e
BlankTest
Fig. 3.08 Absorption Spectra of LEF
with MBTH+NaIO4+AcOH M5(a)
MethodM12:PHH+NaIO4+[Fe(CN)6]
[LEF]=5.40x10-5M, [PHH]=1.66x10-3
[Fe(CN)6]-3=2.42x10-4M,[HCl]=2.40M
0.000
0.050
0.100
0.150
0.200
375 400 425 450 475
Wavelength nm
Ab
so
rba
nc
e
Blank
Test
Fig. 3.09 Absorption Spectra of LEF
with PHH+NaIO4+[Fe(CN)6]-3
M12
Method
M14:CTC+Buffer+Nitrobenzene
[LEF]=5.40x10-5M,[CTC]=5.71x10-2M
Buffer of pH=2.0
0.000
0.100
0.200
0.300
550 600 650 700 750Wavelength nm
Ab
so
rba
nc
e
Blnk
Test
Fig. 3.10 Absorption Spectra of LEF
with CTC +Buffer+Nitrobenzene M14
Method M17:PA+CHCl3
[LEF]=4.50x10-5
M,[PA]=5.82x10-4
M
[HCl]=4.00x10-2
M
0.000
0.100
0.200
0.300
375 400 425 450 475
Wavelength nm
Ab
so
rba
nc
e
Blank
Test
Fig. 3.11 Absorption Spectra of LEF
with PA M17
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
207
Method M18:4- AP
[LEF]=1.08x10-4
M
[4-AP]=2.45x10-2
M
0.000
0.100
0.200
0.300
0.400
380 400 420 440 460 480 500
Wavelength nm
Ab
so
rba
nc
e
Blank
Test
Fig. 3.12 Absorption Spectra of LEF
with 4-AP M18
Method M1(a):ARS+CHCl3
[LEF]=2.25x10-5M - 1.35x10-4M
[ARS]=3.66x10-4M
[HCl]=4.0x10-2M
0.000
0.200
0.400
0.600
0 10 20 30 40 50 60
Weight of the Drug in μg/ml
Ab
so
rba
nc
e
Fig.3.13 Beer’s Law Plot of LEF with
ARS M1(a)
Method M1(d):TPooo+CHCl3
[LEF]=4.50x10-5M - 2.70x10-4M
[TPooo]=3.81x10-4M, [HCl]=4.0x10-2M
0.000
0.250
0.500
0.750
0 25 50 75 100
Weight of the Drug in μg/ml
Ab
so
rba
nc
e
Fig.3.14 Beer’s Law Plot of LEF with
TPooo M1(d)
Method M2(a):MB+Buffer+CHCl3
[LEF]=3.6x10-5M - 2.16x10-4M
[MB]=4.17x10-4M,Buffer of pH=9.8
0.000
0.300
0.600
0.900
0 20 40 60 80 100
Weight of the Drug in μg/ml
Ab
so
rba
nc
e
Fig.3.15 Beer’s Law Plot of LEF with
MB M2(a)
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
208
Method M3:Fe(III)+O-PHEN
[LEF]=5.40x10-5M - 3.24x10-4M
[Fe(III)]=3.99x10-4M,
[O-PHEN]=8.07x10-4M
[O-PHOS]=1.73x10-3M
0.000
0.200
0.400
0.600
0 40 80 120
Weight of the Drug in μg/ml
Ab
so
rba
nc
e
Fig.3.16 Beer’s Law Plot of LEF with
Fe(III)+O-PHEN M3
Method M5(c):MBTH+NaIO4+AcOH
[LEF]=2.70x10-5M - 1.62x10-4M
[MBTH]=3.42x10-4M,
[NaIO4]=3.74x10-4M
[AcOH]=1.28x10-1M
0.000
0.250
0.500
0.750
0 20 40 60
Weight of the Drug in μg/mlA
bs
orb
an
ce
Fig.3.17 Beer’s Law Plot of LEF with
MBTH+NaIO4+AcOH M5(c)
MethodM12:PHH+NaIO4+[Fe(CN)6]-3
[LEF]=2.70x10-5M - 1.62x10-4M
[PHH]=1.66x10-2M,[NaIO4]=1.60X10-3M
[[Fe(CN)6]-3]=2.42X10-4M
0.000
0.200
0.400
0.600
0.800
0 20 40 60
Weight of the Drug in μg/ml
Ab
so
rba
nc
e
Fig.3.18 Beer’s Law Plot of LEF with
PHH+NaIO4+[Fe(CN)6]-3
M12
MethodM14:CTC+Buffer+NB
[LEF]=1.35x10-5M - 8.10x10-5M
[CTC]=5.71x10-2M,Buffer of pH=2.0
0.000
0.100
0.200
0.300
0.400
0.500
0 10 20 30
Weight of the Drug in μg/ml
Ab
so
rba
nc
e
Fig.3.19 Beer’s Law Plot of LEF with
CTC+Buffer+Nitrobenzene M14
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
209
Method M17:PA+CHCl3
[LEF]=2.25x10-5M - 1.35x10-4M
[PA]=5.82x10-4M,[HCl]=4.0x10-2M
0.000
0.300
0.600
0.900
0 20 40 60
Weight of the Drug in μg/ml
Ab
so
rba
nc
e
Fig.3.20 Beer’s Law Plot of LEF with
PA M17
Method M18 4-AP
[LEF]=2.25x10-5
M - 1.35x10-4
M
[4-AP]=2.45x10-2
M
0.000
0.200
0.400
0.600
0 25 50 75
Weight of the Drug in μg/ml
Ab
so
rba
nc
e
Fig.3.21 Beer’s Law Plot of LEF with 4-
AP M18
Method M1(a) :ARS+CHCl30
30
60
90
0.50 1.00 1.50 2.00
LOG(Concentration in μg/ml)
%T
ran
sm
ita
nc
e
Fig.3.22 Ringbom Plot of LEF with ARS
M1(a)
Method M1(d):TPooo+CHCl30
30
60
90
1.00 1.25 1.50 1.75 2.00
LOG(Concentration in μg/ml)
%T
ran
sm
ita
nc
e
Fig.3.23 Ringbom Plot of LEF with
TPooo M1(d)
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
210
Method M2(a):MB+CHCl3
0
20
40
60
80
1.00 1.50 2.00
LOG(Concentration in μg/ml)
%T
ran
sm
ita
nc
e
Fig.3.24 Ringbom Plot of LEF with MB
M2(a)
Method M3:Fe(III)+O-PHEN
30
60
90
1.00 1.50 2.00 2.50
LOG(Concentration in μg/ml)
%T
ran
sm
ita
nc
e
Fig.3.25 Ringbom Plot of LEF with
Fe(III)+O-PHEN M3
Method M5(c):MBTH+NaIO4+AcOH
0
30
60
90
0.40 0.80 1.20 1.60
LOG(Concentration in μg/ml)
%T
ran
sm
ita
nc
e
Fig.3.26 Ringbom Plot of LEF with
MBTH+NaIO4+AcOH M5(c)
Method M12:PHH+NaIO4+[Fe(CN)6]-3
0
20
40
60
80
0.50 1.00 1.50 2.00
LOG(Concentration in μg/ml)
%T
ran
sm
ita
nc
e
Fig.3.27 Ringbom Plot of LEF with
PHH+NaIO4+[Fe(CN)6]-3
M12
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
211
Method M14:CTC+Buffer+NB20
40
60
80
100
0.50 0.75 1.00 1.25 1.50
LOG(Concentration in μg/ml)
%T
ran
sm
ita
nc
e
Fig.3.28 Ringbom Plot of LEF with
CTC+Buffer+Nitrobenzene M14
Method M17:PA+CHCl30
20
40
60
80
0.50 1.00 1.50 2.00
LOG(Concentration in μg/ml)
%T
ran
sm
ita
nc
e
Fig.3.29 Ringbom Plot of LEF with PA
M17
Method M18:4-AP
25
50
75
100
0.50 1.00 1.50 2.00 2.50
Concentration of the Drug in μg/ml
%T
ran
sm
ita
nc
e
Fig.3.30 Ringbom Plot of LEF with 4-AP
M18
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
212
Table-3.09(a): Optical Characteristics of the proposed methods for LEF
Name of the Parameter M1(a) M1(d) M2(a) M3 M5(c)
Maximum Wavelength λmax 415 nm 515 nm 650 nm 510 nm 610 nm
Beer's Law Limits µg/ml 8.3-50.0 16.7-100.0 13.3-80.0 20.0-120.0 10.0-60.0
Optimum Photometric Range µg/ml 16.7-41.7 33.3-83.3 26.7-66.7 40.0-100.0 20.0-40.0
Sandell's Sensitivity µg/cm2 / 0.001 Abs 8.59E-02 1.52E-01 9.46E-02 2.60E-01 9.62E-02
Molar Absorptivity lt/mole/cm 3.85E+03 2.36E+03 3.64E+03 1.62E+03 3.55E+03
Table-3.09(b): Optical Characteristics of the proposed methods for LEF
Name of the Parameter M12 M14 M17 M18
Maximum Wavelength λmax 430 nm 660 nm 415nm 440
Beer's Law Limits µg/ml 10.0-60.0 5.0-30.0 8.3-50.0 10.0-60.0
Optimum Photometric Range µg/ml 20.0-50.0 10.0-25.0 17.7-41.7 10.0-50.0
Sandell's Sensitivity µg/cm2 / 0.001 Abs 8.33E-02 7.46E-02 6.27E-02 0.1031
Molar Absorptivity lt/mole/cm 3.91E+03 5.01E+03 5.93E+03 3.21E+03
Table-3.10(a): Linear least square regression analysis
Name of the Parameter M1(a) M1(d) M2(a) M3 M5(c)
Slope (b) 1.04E-02 6.38E-03 9.82E-03 4.38E-03 9.57E-03
Intercept(a) 2.27E-03 6.00E-04 -5.33E-04 -1.05E-02 6.00E-03
Standard Deviation on Slope(Sb) 1.45E-04 2.95E-05 1.35E-04 4.94E-05 8.72E-05
Standard Deviation on Intercept(Sa) 4.69E-03 1.91E-03 7.02E-03 3.85E-03 3.40E-03
Correlation coefficient (r ) 0.9994 0.9999 0.9994 0.9999 0.9998
Limit of Detection (LOD) µg/ml 1.362 0.906 2.150 2.646 1.066
Limit of Quantification (LOQ) µg/ml 4.525 3.006 7.158 8.802 3.552
Table-3.10(b): Linear least square regression analysis
Name of the Parameter M12 M14 M17 M18
Slope (b) 1.06E-02 1.35E-02 1.60E-02 8.65E-03
Intercept(a) 1.60E-03 -4.00E-04 6.33E-03 2.27E-03
Standard Deviation on Slope(Sb) 1.73E-04 4.47E-05 2.00E-04 1.21E-04
Standard Deviation on Intercept(Sa) 6.74E-03 8.69E-04 6.48E-03 4.69E-03
Correlation coefficient (r ) 0.9991 0.9999 0.9995 0.9994
Limit of Detection (LOD) µg/ml 1.922 0.197 1.214 1.627
Limit of Quantification (LOQ) µg/ml 6.394 0.643 4.053 5.423
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
213
Table 3.11(a) Precision of the proposed methods
Statistical Parameter Value M1(a) M1(d) M2(a) M3 M5(c)
Concentration (μg/ml) 33.33 66.67 53.33 80.00 40.00
Mean( of six replicates) (μg/ml) 33.26 66.56 53.25 79.81 40.05
Standard Deviation (s) 0.252 0.280 0.304 0.533 0.191
%Relative Standard Deviation(%RSD) 0.763 0.436 0.553 0.676 0.484
0.05 level confidence limit µg/ml 0.415 0.474 0.495 0.884 0.326
Table 3.11(b) Precision of the proposed methods
Statistical Parameter Value M12 M14 M17 M18
Concentration (μg/ml) 40.00 20.00 33.33 40.00
Mean( of six replicates) (μg/ml) 39.95 19.95 33.32 39.93
Standard Deviation (s) 0.213 0.208 0.185 0.251
%Relative Standard Deviation(%RSD) 0.525 0.985 0.552 0.629
0.05 level confidence limit µg/ml 0.343 0.327 0.304 0.413
Table-3.12(a): Accuracy of the proposed method M1 (a)
Sample
ID
Amount
Taken µg/ml
Amount
Found µg/ml
Percent of
Recovery
Statistical analysis
1 25.00 25.10 100.41 Mean 100.16
2 25.00 25.07 100.29 SD 0.340
3 25.00 24.94 99.77 %RSD 0.340
1 33.33 33.58 100.75 Mean 100.53
2 33.33 33.72 101.17 SD 0.774
3 33.33 33.22 99.67 %RSD 0.770
1 41.66 42.07 100.98 Mean 100.06
2 41.66 41.23 98.96 SD 1.020
3 41.66 41.76 100.23 %RSD 1.019
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
214
Table-3.12(b): Accuracy of the proposed method M1(d)
Sample
ID
Amount taken
µg/ml
Amount found
µg/ml
Percent of
Recovery
Statistical analysis
1 50.00 49.21 98.42 Mean 99.922
2 50.00 49.74 99.48 SD 1.773
3 50.00 50.94 101.87 %RSD 1.774
1 66.67 66.94 100.40 Mean 99.830
2 66.67 65.99 98.98 SD 0.751
3 66.67 66.74 100.10 %RSD 0.752
1 83.34 84.21 101.05 Mean 100.251
2 83.34 82.96 99.55 SD 0.754
3 83.34 83.47 100.16 %RSD 0.752
Table-3.12(c): Accuracy of the proposed methodM2(a)
Sample
ID
Amount taken
µg/ml
Amount found
µg/ml
Percent of
Recovery
Statistical analysis
1 40.00 40.95 102.38 Mean 101.448
2 40.00 39.92 99.81 SD 1.426
3 40.00 40.86 102.16 %RSD 1.406
1 53.33 53.99 101.24 Mean 100.444
2 53.33 52.96 99.31 SD 1.011
3 53.33 53.75 100.79 %RSD 1.006
1 66.66 67.05 100.58 Mean 99.986
2 66.66 66.94 100.42 SD 0.892
3 66.66 65.97 98.96 %RSD 0.892
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
215
Table-3.12(d): Accuracy of the proposed Method M3
Sample
ID
Amount taken
µg/ml
Amount found
µg/ml
Percent of
Recovery
Statistical analysis
1 60.00 61.22 102.03 Mean 100.139
2 60.00 59.72 99.53 SD 1.676
3 60.00 59.31 98.85 %RSD 1.674
1 80.00 81.42 101.78 Mean 100.629
2 80.00 79.16 98.95 SD 1.486
3 80.00 80.93 101.16 %RSD 1.477
1 100.00 101.31 101.31 Mean 100.067
2 100.00 98.96 98.96 SD 1.181
3 100.00 99.93 99.93 %RSD 1.180
Table-3.12(e): Accuracy of the proposed method M5(c)
Sample
ID
Amount taken
µg/ml
Amount found
µg/ml
Percent of
Recovery
Statistical analysis
1 30.00 30.71 102.37 Mean 100.956
2 30.00 30.41 101.37 SD 1.655
3 30.00 29.74 99.13 %RSD 1.640
1 40.00 40.77 101.93 Mean 100.500
2 40.00 40.32 100.80 SD 1.596
3 40.00 39.51 98.78 %RSD 1.588
1 50.00 50.73 101.46 Mean 99.793
2 50.00 49.21 98.42 SD 1.541
3 50.00 49.75 99.50 %RSD 1.544
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
216
Table-3.12(f): Accuracy of the proposed method M12
Sample
ID
Amount taken
µg/ml
Amount found
µg/ml
Percent of
Recovery
Statistical analysis
1 30.00 30.07 100.23 Mean 99.820
2 30.00 29.95 99.83 SD 0.413
3 30.00 29.82 99.40 %RSD 0.414
1 40.00 40.74 101.85 Mean 100.534
2 40.00 39.85 99.63 SD 1.167
3 40.00 40.05 100.13 %RSD 1.161
1 50.00 50.21 100.42 Mean 99.587
2 50.00 49.66 99.32 SD 0.737
3 50.00 49.51 99.02 %RSD 0.740
Table-3.12(g): Accuracy of the proposed method M14
Sample
ID
Amount taken
µg/ml
Amount found
µg/ml
Percent of
Recovery
Statistical analysis
1 15.00 15.30 102.00 Mean 101.333
2 15.00 14.98 99.87 SD 1.272
3 15.00 15.32 102.13 %RSD 1.255
1 20.00 19.94 99.70 Mean 100.083
2 20.00 20.08 100.40 SD 0.355
3 20.00 20.03 100.15 %RSD 0.354
1 25.00 25.08 100.30 Mean 99.833
2 25.00 24.95 99.80 SD 0.451
3 25.00 24.85 99.40 %RSD 0.452
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
217
Table-3.12(h): Accuracy of the proposed method M17
Sample
ID
Amount taken
µg/ml
Amount found
µg/ml
Percent of
Recovery
Statistical analysis
1 25.00 24.85 99.41 Mean 99.837
2 25.00 24.98 99.93 SD 0.389
3 25.00 25.04 100.17 %RSD 0.389
1 33.33 33.14 99.43 Mean 100.350
2 33.33 33.52 100.57 SD 0.832
3 33.33 33.68 101.05 %RSD 0.829
1 41.66 41.17 98.82 Mean 100.026
2 41.66 41.98 100.76 SD 1.055
3 41.66 41.87 100.50 %RSD 1.054
Table-3.12(i): Accuracy of the proposed method M18
Sample
ID
Amount taken
µg/ml
Amount found
µg/ml
Percent of
Recovery
Statistical analysis
1 30.00 29.75 99.17 Mean 100.233
2 30.00 30.31 101.03 SD 0.961
3 30.00 30.15 100.50 %RSD 0.959
1 40.00 39.85 99.63 Mean 100.817
2 40.00 40.62 101.55 SD 1.041
3 40.00 40.51 101.28 %RSD 1.033
1 50.00 50.46 100.92 Mean 99.827
2 50.00 49.77 99.54 SD 0.982
3 50.00 49.51 99.02 %RSD 0.984
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
218
Table3.13 (a): Assay of Formulations of Levofloxacin; Reference Method [235]
Sample
Amount
Taken
(mg/tablet)
Amount found in
proposed methods*
Percent of Recovery
Ref.Method Proposed methods**
M1(a) M1(d) M1(a) M1(d)
Levoflox 250 Mean 250.69 252.14 %REC 100.42 100.28 100.86
SD ±2.131 ±2.312 %RSD ±0.86 ±0.851 ±0.916
F-test 1.026 1.147
t-test 0.561 1.601
Leon 250 Mean 250.05 250.95 %REC 100.08 100.02 100.038
SD ±2.074 ±2.841 %RSD ±0.5 ±0.829 ±1.132
F-test 1.083 1.731
t-test 0.042 0.578
Levoflox 500 Mean 499.67 498.94 %REC 100.42 99.93 100.036
SD ±3.814 ±3.869 %RSD ±0.86 ±0.763 ±0.284
F-test 1.282 1.245
t-test 0.151 0.474
Leon 500 Mean 500.51 503.26 %REC 100.08 100.10 100.65
SD ±2.984 ±2.96 %RSD ±0.5 ±0.596 ±0.589
F-test 2.094 2.116
t-test 0.296 1.900
*Average of six determinations are considered, AVG=Average, SD=Standard
deviation, F=F-test value, t=t-test value; Theoretical values at 0.05 level of
confidence limit F=5.19, t=1.833.
**%REC=% of Recovery, %RSD=%of Relative standard deviation; 250.0 and
500.0mg formulations (Average of six determinations)
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
219
Table3.13 (b): Assay of Formulations of Levofloxacin, Reference Method [235]
Sample
Amount
Taken
(mg/tablet)
Amount found in
proposed methods*
Percent of Recovery
Ref.Method Proposed methods**
M2(a) M3 M2(a) M3
Levoflox 250 Mean 249.65 251.31 %REC 100.42 99.86 100.52
SD ±1.984 ±2.136 %RSD ±0.86 ±0.794 ±0.849
F-test 1.184 1.021
t-test 0.305 1.061
Leon 250 Mean 24.94 248.94 %REC 100.08 99.58 99.58
SD ±1.975 ±2.136 %RSD ±0.5 ±0.793 ±0.858
F-test 0.1.195 1.022
t-test 0.929 0.859
Levoflox 500 Mean 502.21 502.36 %REC 100.42 100.44 100.47
SD ±3.853 ±2.537 %RSD ±0.86 ±0.0.767 ±0.505
F-test 1.255 2.896
t-test 0.992 1.609
Leon 500 Mean 495.82 500.78 %REC 100.08 99.16 100.16
SD ±3.741 ±2.946 %RSD ±0.5 ±0.754 ±0.588
F-test 1.332 2.147
t-test 1.933 0.458
*Average of six determinations are considered, AVG=Average, SD=Standard deviation,
F=F-test value, t=t-test value; Theoretical values at 0.05 level of confidence limit F=5.19,
t=1.833.
**%REC=% of Recovery, %RSD=%of Relative standard deviation; 250.0 and
500.0mg formulations (Average of six determinations)
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
220
Table 3.13 (c): Assay of Formulations of Levofloxacin, Reference Method [235]
Sample
Amount
Taken
(mg/tablet)
Amount found in
proposed methods*
Percent of Recovery
Ref.Method Proposed methods**
M5(c) M12 M5(c) M12
Levoflox 250 Mean 250.08 248.69 %REC 100.42 100.33 99.48
SD ±1.683 ±1.924 %RSD ±0.86 ±0.671 ±0.773
F-test 1.645 1.259
t-test 0.843 1.174
Leon 250 Mean 248.57 251.02 %REC 100.08 99.43 100.41
SD ±2.617 ±2.154 %RSD ±0.5 ±1.052 ±0.858
F-test 1.469 1.004
t-test 0.945 0.821
Levoflox 500 Mean 502.21 496.98 %REC 100.42 100.062 99.40
SD ±2.859 ±4.142 %RSD ±0.86 ±0.480 ±0.833
F-test 2.281 1.087
t-test 1.337 1.261
Leon 500 Mean 499.43 501.53 %REC 100.08 99.89 100.31
SD ±3.754 ±1.994 %RSD ±0.5 ±0.751 ±0.397
F-test 1.323 0.4.689
t-test 0.265 0.265
*Average of six determinations are considered, AVG=Average, SD=Standard deviation,
F=F-test value, t=t-test value; Theoretical values at 0.05 level of confidence limit F=5.19,
t=1.833.
**%REC=% of Recovery, %RSD=%of Relative standard deviation; 250.0 and 500.0mg
formulations (Average of six determinations)
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
221
Table 3.13 (d): Assay of Formulations of Levofloxacin, Reference Method [235]
Sample
Amount
Taken
(mg/tablet)
Amount found in
proposed methods*
Percent of Recovery
Ref.Method Proposed methods**
M14 M17 M14 M17
Levoflox 250 Mean 251.08 248.56 %REC 100.42 100.43 99.42
SD ±1.376 ±2.142 %RSD ±0.86 ±0.548 ±0.862
F-test 2.462 1.016
t-test 1.353 1.163
Leon 250 Mean 248.95 251.24 %REC 100.08 99.58 100.50
SD ±2.164 ±1.794 %RSD ±0.5 ±0.869 ±0.714
F-test 1.005 1.448
t-test 0.839 1.196
Levoflox 500 Mean 497.99 501.37 %REC 100.42 99.60 100.27
SD ±3.868 ±2.794 %RSD ±0.86 ±0.776 ±0.557
F-test 1.246 2.388
t-test 0899 0.848
Leon 500 Mean 502.11 498.79 %REC 100.08 100.42 99.76
SD ±2.415 ±2.995 %RSD ±0.5 ±0.481 ±0.601
F-test 3.196 2.078
t-test 1.512 0.699
*Average of six determinations are considered, AVG=Average, SD=Standard deviation,
F=F-test value, t=t-test value; Theoretical values at 0.05 level of confidence limit F=5.19,
t=1.833.
**%REC=% of Recovery, %RSD=%of Relative standard deviation; 250.0 and 500.0mg
formulations (Average of six determinations)
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
222
Table 3.13 (e): Assay of Formulations of Levofloxacin, Reference Method [235]
Sample
Amount
Taken
(mg/tablet)
Amount found in
proposed method*
Percent of Recovery
Ref.Method Proposed method**
M18 M18
Levoflox 250 Mean 249.57 %REC 100.42 99.83
SD ±1.859 %RSD ±0.86 ±0.745
F-test 1.348
t-test 0.400
Leon 250 Mean 251.31 %REC 100.08 100.52
SD ±2.125 %RSD ±0.5 ±0.845
F-test 1.032
t-test 1.066
Levoflox 500 Mean 499.57 %REC 100.42 99.91
SD ±3.513 %RSD ±0.86 ±703
F-test 1.511
t-test 0.212
Leon 500 Mean 500.68 %REC 100.08 100.14
SD ±3.152 %RSD ±0.5 ±0.629
F-test 1.876
t-test 0.373
*Average of six determinations are considered, AVG=Average, SD=Standard deviation,
F=F-test value, t=t-test value; Theoretical values at 0.05 level of confidence limit F=5.19,
t=1.833.
**%REC=% of Recovery, %RSD=%of Relative standard deviation; 250.0 and 500.0mg
formulations (Average of six determinations)
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
223
3.13 Nature of the colored species
The important functional groups that are responsible for the color development
in Levofloxacin are substituted quinoline, substituted pyrazine, carboxylic acid and
carbonyl (keto) group. Based on the reviews concerning the reagents used for the
development of color by exploiting appropriate functional moieties in Levofloxacin,
an attempt has been made to indicate the nature of colored species in each of the
proposed methods.
Tertiary nitrogen atom of pyrazine is responsible for the development of ion-
ion association complex formation with acid dyes such as Alizarin Red-S (ARS) and
Tropaeolin ooo (TPooo) in method M1(a) and method M1(d) respectively; Another
extractive method is developed with basic dye MB which forms ion-ion association
complex with carboxylic acid functional group of the drug in method M2(a). Ortho-
position of substituted quinoline is favorable for electrophlic substitution reactions in
oxidative coupling reaction in method M5(c). The tertiary nitrogen in pyrazine is also
responsible for ion-ion association complex with PA in basic buffer medium method
M17. 4 – amino phenazone (4- AP) gave a colored hydrazone with keto groups of the
drug M18 . Fe (III) converts into a Fe (II) due to oxidation of the drug and reduced
form of iron is detected by the usual reagent o-phenanthroline methodM3. The
colored species formed is the coordination complex of the drug (electron donor) and
the central atom of cobalt thiocyanite, which is extractable into nitrobenzene from
aqueous solution method M14.
Methods - M1(a) and M1(d)
Tertiary nitrogen atom of substituted pyrazine ring is protonated in acidic
medium. The protonated drug molecule is associated with anion of the acidic dyes
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
224
such as ARS and TP ooo and behaves as a single unit being held together by
electrostatic force of attraction which is extractable into chloroform from aqueous
phase. The scheme of the colored product is given below.
N
O
F
O
OH
O
HN
NH N
O
F
O
OH
O
HNH
N
Protonation
Levofloxacin Cation of the Drug
Anion of ARSN
O
F
O OH
O
H
HN N
O
O
-O3S
HON
O
F O
OHO
H
NH
N
Cation of the DrugColored Product
Anion of TPooo
N
O
F O
OHO
H
NH
N
OH
N
N
O3S
N
O
F O
OHO
H
NH
N
Cation of the Drug
Colored Product
Scheme 3.01
Method - M2(a)
Levofloxacin possesses carboxylic acid in substituted quinoline ring which is
responsible for color formation in ion association complex with basic dye methylene
blue which is extractable into chloroform. The carboxylate anion of LEF is expected to
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
225
attract the oppositely charged part of methylene blue and behaves as a single unit being
held together by electrostatic attraction. The scheme of the reaction is given below
N
O
F
O
OH
O
HN
N
N
O
F
O
O
O
H
N
N + HBasic Buffer
LevofloxacinAnion of Levofloxacin
N
O
F O
OO
H
N
N
NS
CH
3
N
N+
CH
3
CH3CH3
Cation of the Dye
+ N
O
F
O
O
O
HN
N
N
SCH3
NN+
CH3
CH3
CH3
Anion of Levofloxacin Colored Product
Scheme 3.02
Method M3
Levofloxcin is oxidized in the presence of oxidizing agent Fe (III) which is
reduced into Fe (II).The reduced form of iron can be easily converted into colored
product by using o-phenanthroline. The reduction product is tris complex of Fe (II),
well known as ferroin.
Fe(III) + Levofloxacin Fe(II) + Levofloxacin
Reduction
Oxidation
Unreacted Fe(III)
Unreacted Fe(III) + O-Phosphoric Acid Fe(III)-O-Phosphoric Acid Complex
+
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
226
N
N
3 + Fe(II)
NN
N
N N
NFe(II)
Colored ComplexO-PHEN
Scheme 3.03
Method –M5(c)
Under the reaction conditions, MBTH on oxidation in the presence of oxidant
loses two electrons and one proton forming and electrophilic intermediate, which is the
active coupling species that reacts with the coupler levofloxacin by electrophillic attack
on ortho position of the most nucleophilic site on cyclic ring substituted quinoline.
N
S
CH3
N-NH2
MBTH
N
S
CH3
N NH Ce(IV)
Electrophilic intermediate
-2e , H
/ NaIO4+AcOH
N
S
CH3
N
Electrophilic intermediate
NHN
S
CH3
N NH
Electrophilic intermediate
N
O
F
O
OH
O
HN
N
N
S
CH3
N NH
Electrophilic intermediate
N
O
F
O
OH
O
HN
NN
S
CH3
NNH
Colored Product
Scheme 3.04
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
227
Method M12
In this method aldehyde formed from epoxide portion of levofloxacin through
oxidation, is first converted into its phenyl hydrazone, then a red color is developed
under the action of the oxidizing agent [(hexacyanoferrate (III)] in acid medium. The
colored species formation may be represented as under scheme.
N
O
F
O
HO
O
HN
N
H2O2
N
O
F
O
HO
O
HN
NOHO
N
O
FO
HO O
H
N
N
OH
Levofloxacin
Epoxide
N
NH2
H
.HCl
+
H
CO
HN
N C
H
N
NH2
H
.HCl
[Fe(CN)6]-3
N NCl
I
II
I + II
HN
N C
H
NNFormazine Dye
Scheme 3.05
Method – M14
Formation of the colored complex is obtained when LEF is treated with CTC
due to the presence of tertiary amine group. The colored species formed is the
coordination complex of the drug (electron donor) and the central atom of cobalt
thiocyanite, which is extractable into nitrobenzene from aqueous solution.
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
228
CNS
NCS
SCN
Co
SCN
N
O
F
O
OH
O
H
N
N
N
O
F
O
HO
O
H
N
N
Scheme 3.06
Method M17
In basic medium picric acid immediately loses the acidic proton, to produce a
stable anionic species. Levofloxacin possesses a tertiary nitrogen atom in substituted
Pyrazine ring and it immediately accepts the proton thus the drug molecule behaves as
a cation. The cation ion of the drug and the negative ion of the dye form a stable ion-
ion associated complex which is extractable into chloroform solvent. The scheme of
the colored product is represented in scheme
OH
NO2
NO2
O2N
O
NO2
NO2
O2N + HBasic Buffer
Stable Anion of Picric AcidPicric Acid
N
O
F
O
OH
O
HN
N + H NH
O
F
O
OH
O
HN
NProtonation
Levofloxacin Protonated Cation
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
229
NH
O
FO
OHO
H
N
N O
NO2
NO2
O2N+
NH
O
F
O
OH
O
HN
N
O
NO2
NO2
O2N
Ion-Ion Associated ComplexCation of the Drug Anion of the reagent
Scheme 3.07
Method: M18
4 – Amino phenazone (4- AP) gave a colored hydrazone with keto group of
the drug Levofloxacin due to condensation. The colored species formation may be
represented as under scheme.
CH3
CH3H2N
O
C6H5
-H2O
Condensation
4-AP
N
O
FO
HO O
H
N
N
+
CH3H3C
O
C6H5
N
FO
HO O
H
N
N
N
Scheme 3.08
3.14 Conclusions
The proposed methods exploit the various functional groups that are present in
Levofloxacin. These methods are simple, sensitive and reliable and can be used for
routine determination of LEF in bulk and pharmaceutical formulations. Statistical
Chapter – III Levofloxacin Part-B: Visible Spectrophotometry
230
analysis of the results shows that the proposed procedures have good precision and
accuracy. Results of the analysis of pharmaceutical formulations reveal that the
proposed methods are suitable for their analysis with virtually no interference of the
usual additives present in pharmaceutical formulations. The descending order of
sensitivity (εmax) and maximum wavelength (λmax) among the proposed methods are
shown below.
The descending order of sensitivity (εmax) among the proposed methods is shown
below.
M17›M14›M12›M1(a) ›M2(a) ›M5(c) ›M18›M1(d) ›M3
The descending order maximum wavelength (λmax) among the proposed methods is
shown below.
M14›M2(a)› M5(c) ›M1(d) ›M3›M12›M18›M1(a)›M17
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
231
ESTIMATION OF LEVOFLOXACIN BY DERIVATIVE
SPECTROPHOTOMETRY
3.15Introduction
The derivatization of spectra can lead to more accurate determination of the
wavelengths of broad peak maxima, of peaks which appear only as shoulders, as
well as the isolation of small peaks from interfering large background absorption.
The influence of an impurity on the absorption spectrum of a substance can be
eliminated by considering derivative curves. The main advantage of Derivative
spectrophotometry is background correction, multicomponent analysis with better
selectivity which is not possible in normal spectrophotometry.
For quantitative purpose, peak heights are usually measured for the long-
wave peak satellite of the second derivative curves or the short – wave peak satellite
of the same curves. Single or multi-components can be analyzed in the presence of
broad, interfering background matrix absorption. Such measurements are of
particular importance in clinical, biological, biochemical and food laboratories. An
extensive literature survey reveals that a few derivative spectrophotometric methods
are reported to determine the amount of Levofloxacin. The objective of the present
work is to develop simple, rapid and economical Second Order Derivative
Spectrophotometric methods for determination of levofloxacin in bulk and
formulations.
3.16(i) Experimental
UV-Visible Spectrophotometer: ElicoSL159 model, 2nm high resolution, double
beam, 1cm length quartz coated optics; Wavelength range190-1100nm; High
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
232
stability, linearity, and precision instrument is used for all the spectral
measurements.
3.16 (ii) Preparation of stock, working standard and reagent solutions
Stock solution of levofloxacin is freshly prepared by transferring accurately
weighed 100mg of levofloxacin into 100ml volumetric flask and dissolved in triply
distilled water and made up to the mark. Then working standard solutions 250μg/ml
for methods M2(a), M5(a) and M15; 200μg/ml for method M3 are prepared by
transferring 25.0ml and 20.0ml of the stock solution into two 100ml standard flask
and made up to the mark with triple distilled water.
Preparation of reagents
Method M2(a)
MB solution (Fluka; 0.2%, w/v 6.25x10-3
M): Prepared by dissolving 200mg of
MB in 100ml of distilled water.
NH4OH – NH4Cl Buffer solution: 7.0gms of NH4Cl and 6.8ml of liquid ammonia
solution are mixed and diluted to 100ml with distilled water and pH is adjusted to
9.8
Method – M3
Fe (III) solution (Wilson labs; 0.054%, 3.33x10-3
M): Prepared by dissolving
54mg of anhydrous Ferric chloride in 100ml of distilled water.
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
233
O-PHEN solution (E.Merck, 0.2%, 1.10x10-2
M): Prepared by dissolving 200mg
of o-phenanthroline in 100ml of distilled water with warming
O-phosphoric acid solution (Qualigens, 2.0x10-2
M): Prepared by mixing 1.27ml
of o-phosphoric acid with 100ml of distilled water. 10.0ml of this stock solution is
further diluted to 100ml with distilled water.
Method M5(a)
MBTH Solution (Fluka; 0.2%, 8.56 x 10-3
M): 200mg of 3-Methyl-2-
BenzaThiazolinone Hydrazone (MBTH) is accurately weighed, transferred into
100ml volumetric flask, made up to the mark with distilled water
Ce(IV)solution(Merck); 1%, 9.35 x 10-3
M): 1% Ce(IV) solution is prepared by
dissolving accurately weighed 1.0g of potassium dichromate in 100ml of distilled
water
Method – M15
p-CA solution (Sd-fine; 0.1%, 4.785x10-3
M: Prepared by dissolving 100mg of p-
chloranilic acid initially in 20ml of isopropyl alcohol followed by dilution to 100ml
with chloroform.
3.16 (iii) Procedures of the proposed methods
Method M2(a)
Aliquots of standard drug solution of levofloxacin 6.7 – 106.7 μg/ml are
added into a series of 25ml tubes. 1.0ml buffer solution of pH 9.8 is placed
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
234
separately, and then 0.5ml of MB is added. The total volume of aqueous phase in
each funnel is adjusted to 10.0ml with distilled water. Then 10.0mlof chloroform is
added in each separating funnel and the contents are shaken for 2min and allowed to
separate. The organic layer is collected through cotton plug into another tube. The
zero order, first derivative and second derivative spectra are recorded using the
prepared solutions against reagent blank (Fig.3.31, Fig.3.35, Fig.3.39; P: 238-240).
The second derivative amplittiud values are plotted against concentration to
construct the calibration curves (Fig.3.43; P : 241).
Method – M3
Aliquots (10.0-160.0g/ml) of standard LEF solution are transferred into a
series of 25ml calibrated tubes and then solutions of 3.0ml (3.33 x 10-3
M) of Fe
(III), 2.0ml of (1.01 x 10-2
M) o-phenanthroline are added successively. The total
volume in each tube is brought to 10.0ml with distilled water. The tubes are kept on
a boiling water bath for 30min. The tubes are removed and cooled to room
temperature. 2.0ml of (2.16 x 10-2
M) o-phosphoric acid is added and volume in each
tube is made up to the mark with distilled water. The absorption spectra, first
derivative and second derivative spectra are recorded using the prepared solutions
against reagent blank (Fig.3.32, Fig.3.36, Fig.3.40; P: 238-240). The values of the
second derivative amplitude are obtained using five different concentrations
(Fig.3.44; P: 241).
Method – M5(a)
Aliquots of standard LEF solution (2.5 – 40.0g/ml) are transferred into a
series of 25ml calibrated tubes. Then 0.5ml (8.56 x 10-3
M) of MBTH solution is
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
235
added and kept aside for 5min. After that 2.0ml (1.58 x 10-2
M) of ceric ammonium
sulphate is added and kept aside for 10min. The volume is made up to the mark
with distilled water. Zero order, first and second derivative spectra are recorded
against reagent blank (Fig.3.33, Fig.3.37, Fig.3.41; P: 238-240). Calibration plot is
constructed by plotting the values of second derivative amplitude against
concentration of the drug (Fig.3.45; P: 241).
Method – M15
Into a series of 10ml calibrated tubes containing aliquots of standard LEF
solution (6.25 – 100.0g/ml), 2.0ml of chloranilic acid (4.785 x 10-3
M) is added and
kept aside for 30 min at lab temperature. The volume in each tube is made up to the
mark with chloroform. The spectra of the extracted organic layer are recorded
against reagent blank (Fig.3.34, Fig.3.38, Fig.42; P: 238-240). The values of the
second derivative amplitude are obtained for five different concentrations, the
values of are plotted against concentration to construct the calibration curve
(Fig.3.46; P: 241).
3.17 Method Development
The zero-order (0D) spectra of levofloxacin are showed in Fig.3.31 –Fig.
3.34; P: 238.The zero-crossing points are assigned from the 1D and
2D spectra of
the proposed methods are shown Fig3.35- Fig.3.42; P: 239-240. The 1D zero-
crossing point of levofloxacin for the proposed methods are found at 640nm for
method M2(a), 517nm for method M3, , 640nm for method M5(a), and 525 nm
for method M15 (Fig.3.35-Fig.3.38; P: 239 ) respectively; and 2D zero-crossing
point of LEF for the proposed methods are found to be 600nm and 680nm for
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
236
method M2a, 470nm, 530nm for method M3; 610nm and 670 nm for method
M5(a) and 485nm and 567 nm for method M15 (Fig3.39-Fig3.42; P:240)
respectively. The selection of the optimal wavelength is based on the fact that the
absolute value of the total derivative spectrum at the selected wavelength has the
best linear response to the analyte concentration. Second derivative amplitudes are
measured at wavelength 650nm, 510nm, 640nm and 540nm for the methods M2(a),
M3, M5(a) and M15respectively.
3.18. Method Validation
Linearity and Range: The calibration curves are constructed by plotting the 2D
value against concentration at the zero-crossing wavelengths. The linearity of the
calibration curves and the adherence of the method to Beer’s law are validated by
the high value of the correlation coefficient and the value of intercept on ordinate
which is close to zero. The calibration plots are linear in the range of 6.7 –
106.7μg/ml (M2a); 10.0-160.0 μg/ml for (M3), 2.0-16.0 μg/ml (M5a) and 6.2 –
100.0 μg/ml (M15) (Fig.3.43 – Fig 3.46; P: 241). The 2D spectra of levofloxacin
showed better resolution and linearity hence chosen for the determination of
levofloxacin. The correlation coefficient of the standard curves (n = 5) for all the
methods is greater than 0.999.
Precision: One of the most common statistical terms employed to express precision
is the standard deviation. The precision of each proposed method is determined for
five replicates of a constant amount of LEF in total sample solution. The standard
deviation and percent relative standard deviation are calculated for the proposed
methods and are presented in Table 3.15, P: 242.
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
237
Accuracy: To determine the accuracy of each proposed method, different amounts
of samples (75%, 100%, and 125%) of levolfoxacin within the linearity limits are
taken and analyzed by the proposed methods. The results (percent error) are
recorded in Table 3.16(a)-Table 3.16(d), P: 242-244.
LOD and LOQ: The limit of detection and limit of quantitation are calculated and
the results are presented in Table 3.14, P: 242.
3.19Results and Discussion
Spectrophotometric parameters including derivative order, wavelength and
Δλ values should be optimized to obtain sensitivity and reproducibility. In this study
second-derivative technique (2D) traced with Δλ=2.0 nm is used. The plots drawn
between second derivative amplitude and concentration of the drug are linear
(Fig.3.43-Fig.3.46, P: 241). Calculated parameters such as linearity limits, slope
intercept, correlation coefficient, LOD and LOQ are presented in Table: 3.14, P:
242. Correlation coefficient values are found to be more than 0.999. This indicates
that there is good correlation between the second derivative amplitude and the
concentration of the drug in the developed method. Low percent of relative standard
deviation values 0.437, 0.171, 0.240 and 0.292 for the methods M2(a), M5(a), M7
and M15 indicate that these methods are precise ( Table: 3.15, P: 242). Mean
percent of recovery for the four methods is found in between 99.43% to 100.57% in
three concentration levels within the linearity limits reveals that the methods are
highly accurate
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
238
Zero Order Spectra of LEF with
MB,CHCl3
0.000
0.250
0.500
0.750
1.000
550 650 750
Wavelength nm
Ab
so
rba
nc
e4.17μg/ml8.33μg/ml16.67μg/ml33.33μg/ml66.67μg/ml
Fig.3.31 Absorption Spectra of LEF with
MB M2(a)
Zero Order Spectra of LEF with
Fe(III),O-PHEN
0.000
0.250
0.500
0.750
400 450 500 550 600 650
Wavelength nm
Ab
so
rba
nc
e
10μg/ml20μg/ml40μg/ml80μg/ml160μg/ml
Fig.3.32 Absorption Spectra of LEF
with MB M3
Zero Order Spectra of LEF with
MBTH,Ce(IV)
0.000
0.200
0.400
0.600
0.800
550 650 750
Wavelength nm
Ab
so
rba
nc
e
2.5μg/ml5.0μg/ml10.0μg/ml20.0μg/ml40.0μg/ml
Fig.3.33 Absorption Spectra of LEF with
MBTH+ Ce(IV) M5(a)
Zero Order Spectra of LEF
with p-CA,CHCl3
0.000
0.250
0.500
0.750
450 500 550 600 650
Wavelength nm
Ab
so
rba
nc
e
6.25μg/ml12.5μg/ml25.0μg/ml50.0μg/ml100μg/ml
Fig.3.34 Absorption Spectra of LEF
with p-CA M15
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
239
First Derivative Spectra of LEF
with MB, CHCl3
-1.5E-02
-1.0E-02
-5.0E-03
0.0E+00
5.0E-03
1.0E-02
1.5E-02
500 600 700 800
Wavelength nm
ΔA
/Δλ
4.17μg/ml8.33μg/ml16.67μg/ml33.33μg/ml66.67μg/ml
Fig.3.35 First Derivative Spectra of
LEF with MB M2(a)
First Derivative Spectra of LEF with
Fe(III),O-PHEN
-1.E-02
-8.E-03
-6.E-03
-4.E-03
-2.E-03
0.E+00
2.E-03
4.E-03
6.E-03
8.E-03
375 425 475 525 575 625
Wavelength nm
ΔA
/Δλ
10μg/ml20μg/ml40μg/ml80μg/ml160μg/ml
Fig.3.36 First Derivative Spectra of
LEF with MB M3
First Derivative Spectra of LEF with
MBTH,Ce(IV)
-1.E-02
-9.E-03
-6.E-03
-3.E-03
0.E+00
3.E-03
6.E-03
9.E-03
500 600 700 800
Wavelength nm
ΔA
/Δλ
2.5μg/ml5.0μg/ml10.0μg/ml20.0μg/ml40.0μg/ml
Fig.3.37 First Derivative Spectra of
LEF with MBTH+ Ce(IV) M5(a)
First Derivative Spectra of LEF
with p-CA,CHCl3
-2.E-02
-1.E-02
-5.E-03
0.E+00
5.E-03
1.E-02
2.E-02
425 475 525 575 625
Wavelength nm
ΔA
/Δλ
6.25μg/ml12.50μg/ml25.0μg/ml50.0μg/ml100μg/ml
Fig.3.38 First Derivative Spectra of
LEF with p-CA M15
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
240
Second Derivative Spectra of LEF
with MB,CHCl3
-6.0E-04
-4.0E-04
-2.0E-04
0.0E+00
2.0E-04
4.0E-04
500 600 700 800
Wavelength nm
Δ2A
/Δ2λ
4.17μg/ml8.33μg/ml16.67μg/ml33.33μg/ml66.67μg/ml
Fig.3.39 Second Derivative Spectra of
LEF with MB M2(a)
Second DerivativeSpectra of LEF
with Fe(III),O-PHEN
-4.0E-04
-3.0E-04
-2.0E-04
-1.0E-04
0.0E+00
1.0E-04
2.0E-04
3.0E-04
375 425 475 525 575 625
Wavelength nm
Δ2A
/Δ2λ
10μg/ml20μg/ml40μg/ml80μg/ml160μg/ml
Fig.3.40 Second Derivative Spectra of
LEF with Fe(III)+O-PHEN M3
Second Derivative Spectra of LEF
with MBTH,Ce(IV)
-6.E-03
-4.E-03
-2.E-03
0.E+00
2.E-03
4.E-03
500 600 700 800
Wavelength nm
Δ2A
/Δ2λ
2.5μg/ml5.0μg/ml10.0μg/ml20.0μg/ml40.0μg/ml
Fig.3.41 Second Derivative Spectra of
LEF with MBTH+Ce(IV) M5(a)
Second Derivative Spectra of LEF
with p-CA,CHCl3
-4.E-03
-3.E-03
-2.E-03
-1.E-03
0.E+00
1.E-03
2.E-03
3.E-03
425 475 525 575 625
Wavelength nm
Δ2A
/Δ2λ
6.25μg/ml12.50μg/ml25.0μg/ml50.0μg/ml100μg/ml
Fig.3.42 Second Derivative Spectra of
LEF with p-CA M15
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
241
Plot of Second Derivative
Amplitudes aginst Concentration
(MB,CHCl3)
0.0E+00
1.0E-04
2.0E-04
3.0E-04
4.0E-04
5.0E-04
0 40 80 120
Concentration in μg/ml
Am
plitu
de
Fig.3.43 Calibration plot of LEF with
MB Method M2(a)
Plot of Second Derivtive
Amplitudes against Concentration
(Fe(III),O-PHEN)
0.0E+00
1.0E-04
2.0E-04
3.0E-04
4.0E-04
0 50 100 150 200
Concentration in μg/ml
Am
plitu
de
Fig.3.44 Calibration plot of LEF with
Fe(III)+O-PHEN Method M3
Plot of Second Derivative
Amplitudes aginst Concentrtion
(MBTH,Ce(IV)
0.0E+00
2.0E-03
4.0E-03
6.0E-03
0 10 20 30 40
Concentration in μg/ml
Am
plitu
de
Fig.3.45Calibration plot of LEF with
MBTH +Ce(III) Method M5(a)
Plot of the Second Derivative
Amplitudes against Concentration
(p-CA,CHCl3)
0.0E+00
1.0E-03
2.0E-03
3.0E-03
0 30 60 90 120
Concentration in μg/ml
Am
plitu
de
Fig.3.46 Calibration plot of LEF with p-
CA Method M15
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
242
Table 3.14 Regression parameters for the proposed methods
S.No Name of the Parameter M2(a) M3 M5(a) M15
1 Linearity Limits µg/ml 6.7-106.7 10.0-160.0 2.5-40.0 6.25-100.0
2 Slope (b) 3.65E-06 2.16E-06 1.28E-04 3.00E-05
3 Intercept(a) 2.08E-06 -7.33E-07 7.17E-05 -2.03E-05
4 Correlation Coefficient ( r ) 0.9999 0.9998 0.9997 0.9998
5 Limit of Detection (LOD) µg/ml 0.345 4.665 1.471 1.554
6 Limit of Quantification (LOQ) µg/ml 1.149 15.551 4.904 5.180
Table 3.15 Precision of the proposed methods
S.No Name of the Parameter M2(a) M3 M5(a) M15
1 Amount Taken (µg/ml) 33.33 80.00 50.00 20.00
2 Mean (n=5)( µg/ml) 33.29 79.97 49.96 19.99
3 Standard Deviation (S) 0.145 0.137 0.05 0.146
4 %Relative Standard Deviation 0.437 0.171 0.240 0.292
5 0.05 level confidence limit 0.253 0.238 0.084 0.254
6 %Recovery 99.87 99.96 99.93 99.99
Table-3.16(a): Accuracy of the method M2(a)
Concentration-1 Concentration-2 Concentration-3
Taken Found %Recovery Taken Found %Recovery Taken Found %Recovery
24.99 25.32 101.29 33.33 33.65 100.96 41.66 42.06 100.95
24.99 24.87 99.49 33.33 33.78 101.35 41.66 41.45 99.49
24.99 24.97 99.89 33.33 33.11 99.33 41.66 41.26 99.03
24.99 25.31 101.25 33.33 33.12 99.36 41.66 42.18 101.24
24.99 25.21 100.85 33.33 33.77 101.32 41.66 41.39 99.34
24.99 25.32 101.29 33.33 33.65 100.96 41.66 42.06 100.95
Mean 25.13 100.55 33.486 100.46 41.66 100.01
SD 0.205 0.342 0.420
%RSD 0.815 1.022 1.010
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
243
Table-3.16(b): Accuracy of the method M3
Concentration-1 Concentration-2 Concentration-3
Taken Found %Recovery Taken Found %Recovery Taken Found %Recovery
60 59.63 99.38 80 80.56 100.70 100 98.96 98.96
60 60.42 100.70 80 79.63 99.53 100 99.79 99.79
60 60.13 100.22 80 79.16 98.95 100 101.85 101.85
60 59.75 99.58 80 80.47 100.58 100 101.45 101.45
60 59.44 99.07 80 80.84 101.05 100 100.75 100.75
60 59.63 99.38 80 80.56 100.70 100 98.96 98.96
Mean 59.87 99.79 80.13 100.16 100.56 100.56
SD 0.396 0.706 1.19
%RSD 0.662 0.881 1.18
Table-3.16(c): Accuracy of the method M5(a)
Concentration-1 Concentration-2 Concentration-3
Taken Found %Recovery Taken Found %Recovery Taken Found %Recovery
15 14.93 99.53 20 19.64 98.20 25 25.45 101.80
15 15.22 101.47 20 19.52 97.60 25 24.87 99.48
15 15.11 100.73 20 20.32 101.60 25 24.98 99.92
15 14.97 99.80 20 20.21 101.05 25 24.81 99.24
15 14.89 99.26 20 19.74 98.70 25 25.61 102.44
15 14.93 99.53 20 19.64 98.20 25 25.45 101.80
Mean 15.02 100.16 19.88 99.43 25.14 100.57
SD 0.137 0.356 0.362
%RSD 0.915 1.794 1.439
Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty
244
Table-3.16(d): Accuracy of the method M15
Concentration-1 Concentration-2 Concentration-3
Taken Found %Recovery Taken Found %Recovery Taken Found %Recovery
37.5 37.88 101.01 50 49.84 99.68 62.5 62.96 100.73
37.5 37.54 100.11 50 49.61 99.22 62.5 62.41 99.85
37.5 37.23 99.28 50 50.71 101.42 62.5 62.11 99.37
37.5 37.89 101.04 50 50.41 100.82 62.5 62.86 100.57
37.5 37.91 101.09 50 50.53 101.06 62.5 62.32 99.71
37.5 37.88 101.01 50 49.84 99.68 62.5 62.96 100.73
Mean 37.69 100.50 50.22 100.44 62.53 100.05
SD 0.299 0.471 0.363
%RSD 0.794 0.938 0.581
3.20 Conclusion
The developed methods are simple, rapid, precise, reliable, sensitive,
reproducible and economical. These methods could be readily applied to routine
quality control analysis of levofloxacin by ordinary laboratories.