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3.0
Chapter- III
Spectrophotometric and RP-HPLC
methods for the determination of
CEFDINIR
3.1
Chap
ter-III
Cefdinir is chemically known as 8-[2-(2-amino-1,3-thiazol-4-
yl)-1-hydroxy-2- nitroso-ethenyl] amino- 4-ethenyl-7-oxo- 2-thia-6-
azabicyclo [4.2.0]oct-4-ene-5-carboxylic acid, a semi-synthetic, broad-
spectrum antibiotic in the third generation of the cephalosporin class. It
is used to control for common bacterial infections of the ear, sinus,
throat, and skin. Therapeutic uses of cefdinir include otitis media, soft
tissue infections, and respiratory tract infections, including sinusitis,
strep throat, community-acquired pneumonia and acute exacerbations
of bronchitis.
Side effects of cefdinir include diarrhea, vaginal infections or
inflammation, nausea, headache, and abdominal pain.
3.2
Chap
ter-III
DRUG PROFILE
Fig.1.3.1.Molecular structure of cefdinir
Systematic (IUPAC) name
8-[2-(2-amino-1,3-thiazol-4-yl)-1-hydroxy-2-nitroso-
ethenyl]amino-4-ethenyl-7-oxo-2-thia-6-
azabicyclo[4.2.0]oct-4-ene-5-carboxylic acid
Formula C14H13N5O5S2
Mol. mass 395.416 g/mol
3.3
Chap
ter-III
Table-I.3.1 : List of important brand names of CEFDINIR
formulations
Brand
Name Formulation Strength Manufacturer
CEFDIEL Tablet 300mg
RanbaxyA-41,
Industrial
Area Sahibzada Ajit
Singh Nagar,
Mohali - 160 071
KEFNIR Capsule 300mg
Glenmark B / 2,
Mahalakshmi
Chamber, Desai
Road,Bhulabhai Desai
Road,Mumbai
RTIST Capsule 500mg
Lupin
laboratory,Bandra
Kurla
complex,Mumbai-
51Mumbai
Very few spectrophotometric methods for the estimation of cefdinir
were reported. The selectivity and sensitivity of the visible
spectrophotometric method depends only on the nature of chemical
reaction involved in the color development. Reagents like 3-methyl-2-
benzothiazolinone hydrazone (MBTH) Folin Ciocalteu reagent (FCR),
p-Dimethyl amino benzaldehyde (PDAB), 4-Amino Phenazone and 1,
10-phenanthroline were used as chromogenic agents (chapter-I).
Several liquid chromatographic techniques for the estimation of
cefdinir were reported1-20
. A mixture of Ammonium formate and
methanol, phosphate buffer and methanol, methanol and water-formic
3.4
Chap
ter-III
acid were reported as mobile phases. In all these reported methods
separation was achieved on a inertsil C18 HPLC column. Ammonium
hydrogen phosphate and acetonitrile was used as mobile phase to
determine cefdinir in human plasma.
Catechol or Pyro catechol 1, 2 dihydroxy benzene (or p-amino
acetophenone, AAP) and sodium per iodate ( chapter-II) were used as
reagents in this chapter for the spectrophotometric determination of
cefdinir.
3.5
Chap
ter-III
Spectrophotometric method for the
determination of Cefdinir using
Catechol and Sodium metaperiodate
Experimental
Results and Discussion
3.6
Chap
ter-III
EXPERIMENTAL
Preparation of Solutions
Catechol : 0.1% solution was prepared by dissolving 0.1 g of
catechol sample (A.R .grade SDFCL Mumbai) in 100 mL of distilled
water.
Sodium meta per iodate, NaIO4 : 2.1392 g of NaIO4 (AR grade Hi
Media laboratories Mumbai-66) was dissolved in distilled water and
the total volume was brought to 1 Lt (0.01M) in a standard volumetric
flask.
Standard solution of cefdinir (in dosage form)
RTIST – DT – 100 mg (Lupin Laboratories, Mumbai) was prepared
by dissolving 100 mg of drug sample in 100mL of distilled water.
Working solutions of drug sample (100 g / mL) were prepared by
diluting aliquots of the stock solutions with distilled water.
Instrumentation.
Spectral measurements and absorbance readings were made on
Elico SL 177 double beam Spectrophotometer.
pH measurements were carried out using Elico pH metre LI 615.
3.7
Chap
ter-III
Absorbance curves.
In order to ascertain the optimum wave lengths (λmax) of the
colored species formed on mixing cefdinir with suitable reagents in
appropriate pH medium exhibiting maximum absorbance, the
absorption spectrum was scanned on a spectrophotometer in the range
400 – 670 nm against the reagent blank using the proposed procedure
under experimental conditions (Table – II.3.1) and the results are
graphically presented in Fig 2.3.1.
Fig.1.3.1. Standard curve of cefdinir
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
400 450 500
A
B
S
O
R
B
A
N
C
E
WAVE LENGTH nm
3.8
Chap
ter-III
Establishment of optimum conditions
Concentration of Reagents:
The optimum conditions were established in each case basing
on the development of maximum color and stability and the results are
presented in Table – II.3.1 Among the various oxidizing agents tried,
IO4- is the best one, followed by H2O2. The other oxidizing agents
such as IO3-, Fe(III), MnO4
-, ocl
-, Fe(CN)6
3- are inferior. The
efficiency of the oxidizing agent depends upon its relative reactive
tendency towards reactants, (drug, catechol) products (indo-dyes) and
also on the behavior of its reduced form. The formation of colored
species of same λmax in the case of cefdinir with each pair of reagents
(Catechol – IO4- or AAP-IO4
-) suggests that the indo dye formed with
both compounds is the same. However for operational feasibilities only
catechol-IO4- related results are presented although experiments were
conducted with AAP-IO4- reagent also.
Order of addition of reagents.
The suitable order of addition of reactants for getting maximum
absorbance and stability has been found to be, cefdinir solution,
oxidizing agent and catechol. The order of addition of reactants
influences in color development. Any delay in adding catechol to
3.9
Chap
ter-III
oxidant causes considerable decrease in absorbance depending upon
the nature of oxidant.
These studies reveal that the oxidant is capable of oxidizing cefdinir or
catechol under chosen pH conditions.
Effect of temperature:
All experiments and absorbance measurements were carried out
at laboratory temperature (280 + 3
0 C). At low temperature (< 20
0c) the
stability of the colored species is less.
Effect of solvent:
A mixture of 15ml of buffer, requisite concentrations of
Cefdinir, oxidizing agent and catechol were placed in a separating
funnel and was diluted to 25mL with distilled water. After keeping it
for some time, for allowing the reaction to complete, 10mL of
chloroform or n-butanol (if insoluble in chloroform) was added to the
separating funnel and the contents were shaken well for 2 min. and
left for 10 min. to get clear separation of two phases. It was noticed
that the colored species formed in the case of Cefdinir with the reagent
(Catechol- IO4-) is extractable in butanol but not into chloroform. The
absorbance of the organic phase was measured at appropriate wave
length against a reagent blank. As solvent extraction did not give any
additional advantage, it was excluded in further investigations. The
studies on the influence of other water miscible (polar) solvents such
3.10
Chap
ter-III
as acetonitrile, methanol, t-butyl alcohol, or acetone instead of water
revealed that the aqueous medium was the best one for maximum color
development.
Effect of Buffer:
Potassium acid phthalate buffer (3.4-4.0), and aqueous media without
usage of any buffer were found to be suitable in the determination of
cefdinir with pairs of reagents catechol—IO4-, or A.A.P—IO4
-. The
variation in λmax, of indo dyes formed from cefdinir and catechol,(or
AAP) under two different pH conditions is furnished under Table-
II.2.1 for the purpose of comparison of cefdinir—(neutral or anionic
form ) with catechol / or its oxidized form, o-benzoquinone forming
indo-dyes as products.
Stability of Color:
The influence of time for maximum color development and
stability of the colored species of cefdinir with superior reagents were
studied and the results are incorporated in Table-II.3.1
3.11
Chap
ter-III
Table—II.3.1 : Experimental Conditions
pH Catechol NalO4
Time for
max.
color
development
Stability
of colour
min.
max
nm
4.0±0.4 1ml 1ml 5min 120 460
3.4±0.4 1ml 1ml 3min 100 460
Optical Characteristics
Adherence to Beer’s law.
In order to test whether the cefdinir-catechol-IO4-(or AAP- IO4
- )
system adheres to Beer’s law, the absorbance at λmax of a set of
solutions (25 mL) containing varying amounts of cefdinir,15 mL of
buffer solutions, specified concentrations of catechol, (or AAP) and
oxidizing agent (Table-II.3.1) were measured against blank on
spectrophotometer. The linearity of the plot between absorbance and
the concentration range specified in Table-II.3.1 shows that the color
system obeys Beer’s law, Fig.2.3.1. Beer’s limits, molar absorptivity,
optimum photometric range and Sandal’s sensitivity values of the
method in the case of cefdinir were calculated and results are
incorporated in Table-III.3.1
3.12
Chap
ter-III
Fig.2.3.1 Beer’s Law plot for cefdinir
Table-III.3.1 Optical characteristics
Reagent
Beer’ Law
Range
µg/25 ml
MolarAbsor
ptivity
Lt/mol/cm
Sandell’s
Sensitivity
µg/cm2/0.01
absorbance
units
Optimum
Photo
metric
Range
µg/25 ml
Catechol
-IO4-
Cefdinir
50 – 250 4.5 X 103
0.029 100—257
In view of all the observations it is felt that the following procedure for
the spectrophotometric assay of cefdinir using catechol, (or AAP) and
oxidizing agent will be highly suitable for routine analysis.
0
0.1
0.2
0.3
0.4
0.5
050
100150
200250
Ab
sorb
ance
Concentration µg
3.13
Chap
ter-III
Assay procedure:
For Cefdinir using Catechol- IO4- :
15 mL of buffer solution, 0.4 –5 mL of aliquots of cefdinir
solution, 1mL of IO4-, 1mL of catechol (or AAP) were successively
placed in a 25 ml volumetric flask, so as to make total volume of 25
mL. The absorbance of colored species was measured at 460 nm
between 5—70 min. against corresponding reagent blank prepared in a
similar manner. The amount of cefdinir was read from calibration
curve prepared with the standard solution under identical conditions.
Precision and Accuracy.
The precision and accuracy of the method in the determination
of cefdinir, were tested by measuring the absorbance of six replicates,
each containing approximately ¾ of the Beer’s law limit
concentrations. The percentage relative standard deviations and
confidence limits (0.05 and 0.01 levels) in methods are presented in
Table-IV.3.1
3.14
Chap
ter-III
Table.IV.3.1.Precision and Accuracy
*Average of six samples
The accuracy of the method was determined by taking known
different amounts (within Beer’s law range) of cefdinir and estimating
these amounts with the proposed methods. The results are incorporated
in Table-IV.5.1. The accuracy of the method was further tested in
injections with proposed and reported methods. The results of these
estimations are incorporated in Table-V.5.1.
Table –V.3.1.Analysis of Formulations—Recovery Experiments
Sample
Labeled
Amount
mg
Mean of %
amount found
% Recovery
Experiments
Reported
method
Proposed
method
Amount
added %Recovery
Cefdinir
injection
200 197.2 197.7 0.250 99.2
Cefdinir
injection
200 196.8 197.4 0.200 98.9
Cefdinir
Amount of
Drug *
% Error % R.S.D
% Range of Error
Taken
mg
Found
mg
95%
Confidence
Limit
99%
Confidence
Limit
Catechol-
IO4-
reagent
and
cefdiir
0.25 0.247 1.174 2.657 ±1.6 ±2.92
0.15 0.148 1.3 1.26 ±1.32 ±2.92
3.15
Chap
ter-III
RESULTS AND DISCUSSION
As mentioned on page 3.8, AAP-IO4- is behaving in a similar manner
like catechol-IO4-
in case of λmax, of course with a negligible
improvement in color intensity. However the mechanism of color
formation is different in case of catechol-IO4-
and AAP-IO4- reagent
systems.
Based on the results furnished in Tables II.3.1 – V.3.1 it can be
inferred that the method proposed for the spectrophotometric
determination of cefdinir is simple, rapid, sensitive and specific with
reasonable precision and accuracy. The methods have been extended to
determine cefdinir different pharmaceutical preparations.
The proposed method appears to be superior to many of the reported
methods7-9
and so they can be employed in routine determinations.
Catechol is readily oxidisable by sodium meta per iodate to form
o-benzoquinone. Cefdinir by virtue of its strong electron donating
group, (-NH2) oxidative coupling reaction takes place with electron
deficient o-benzo quinone to form indo dye.
3.16
Chap
ter-III
OH
OHO
O
NaIO4
CEFDINIR
O
OH
OH
N
S
CH2
O
NH
O
N
SN
N
O
Fig.4.3.1 Formation of indo-dye.
As AAP contains electron withdrawing group,- CO-CH3 in para
position to aromatic amine, AAP-IO4- can successfully be used for the
estimation of cefdinir.
The failure of Resorcinol (or Pyrogellol) to develop color with
all the proposed pairs of reagents may be due to the less reactive nature
of its oxidative product, m-benzo quinone, and so it does not undergo
coupling reaction giving indo dye.
Conclusion.
Thus the proposed spectrophotometric method is found to be simple,
economic, sensitive, with reasonable precision and accuracy for the
estimation of cefdinir in bulk and pharmaceutical forms.
3.17
Chap
ter-III
Development and Validation of
Cefdinir by RP-HPLC Method
Experimental
Results and Discussion
3.18
Chap
ter-III
EXPERIMENTAL
Materials and Methods.
Instrumentation.
The author attempted to develop a liquid chromatographic
method for the quantitative estimation of cefdinir. A Schimadzu
HPLC equipped with a Luna C18 column (250 nm X 4.6nm,5µ) an LC
20 AD pump and a SPD 20 AD UV- Visible detector was employed in
this study. Chromatographic analysis and data acquision was
monitored by using Spinchrome software. A 20 µL Hamilton syringe
was used for sample injection. Degassing of the mobile phase was
done by using a spectra lab. DGA 20A3 Ultra sonic bath sonicator. A
Shimadzu electronic balance was used for weighing the materials. The
reference samples of cefdinir was supplied by Venus Remedies
Limited ,India and the branded formulations of cefdinir, (Cefdiel and
RTIST) were used.
Chemicals and Solvents.
Methanol – HPLC grade (Merck, Worli, Mumbai ) ortho-
phosphoric acid HPLC grade (SD chemicals, Mumbai),Tetra hydro
furan (THF: Merck, Worli, Mumbai ), Aceto nitrile (Merck, Worli,
Mumbai ) were used.
3.19
Chap
ter-III
Preparation of Mobile phase and stock solutions.
Mobile phase is a mixture of 5% tetra hydro furan (THF), 15%
methyl alcohol,40% acetonitrile and 40% (0.1%) ortho phosphoric acid
(OPA) which was prepared by mixing 5 mL of THF, 15 mL of methyl
alcohol,40 mL of acetonitrile and 40 mL of OPA in one Liter flask.
This mixture was used as a diluent for preparing working standard
solutions of the drug.
About 100mg of cefdinir was weighed accurately and
transferred into a 100 ml volumetric flask containing 20 ml of mobile
phase. The solution was sonicated for 20 min. and then the volume was
made up with a further quantity of mobile phase to get 1 mg/ml
solution. This solution was suitably diluted with mobile phase to get a
working standard solution of 100µg/ml of cefdinir.
Optimization of Chromatographic Conditions.
Method Development: A systematic study was followed for developing
the method and optimization of chromatographic conditions. This was
carried out by varying one parameter keeping the other conditions
constant at particular point of time.
Column: A non polar C18 column was chosen as the stationary phase
for this study.
3.20
Chap
ter-III
Mobile Phase: In order to get sharp peak and base line separation of
the components, the author has carried out a number of experiments by
varying the commonly used solvents with different compositions and
its flow rates. In order to establish ideal separation of the drug under
isocratic conditions mixtures of commonly used solvents like water,
methanol, acetonitrile, o-phosphoric acid with or without different
buffers, in different combinations were tested as mobile phase on a C18
stationary phase.
A mixture of 5% tetra hydro furan (THF), 15% methyl
alcohol,40% acetonitrile and 40% (0.1%) ortho phosphoric acid (OPA)
was proved to be the most suitable of all the combinations since the
chromatographic peaks obtained were well defined, resolved and free
from tailing. A flow rate of 1.0 mL/min mobile phase was found to be
suitable in the studied range of 0.5—1.5 mL/min.
Wave length: The spectra of diluted solutions of cefdinir in methanol
were recorded on UV spectrophotometer. The peaks of maximum
absorbance wavelengths were observed. The spectra of cefdinir
showed a balanced wavelength at 284 nm.
Retention Time: Under the above optimized conditions a retention
time of 5.1 min was obtained for cefdinir. A typical model
3.21
Chap
ter-III
chromatogram showing the separation of cefdinir is presented in Fig
1.3.2
After a thorough study of the various parameters the following
optimized conditions mentioned in Table-I.3.2 were followed for the
determination of cefdinir in bulk samples and pharmaceutical
formulations.
Fig.1.3.2 Chromatogram of cefdinir.
3.22
Chap
ter-III
Table.1.3.2 HPLC Report.
• CONCENTRATION--5mg/mL
• RETENTION TIME (R.T) 5.1min
• AREA-- 798418
• THEORETICAL PLATES--3408
• WAVE LENGTH--284 nm
• MOBILE PHASE-THF (5%),ACN
(40%),MeOH(15%),0.1% OPA
(40%) (v/v)
• COLUMN--C18
• FLOW RATE-1.0 mL/min
• RUN TIME--10 min
• pH-- 3.3
• LINEARITY RANGE-100-500µg
H.P.L.C Report
A.P.I.Cefdinir
3.23
Chap
ter-III
Linearity and Construction of Calibration Curve
The quantitative determination of the drug was accomplished by
the external standard method. The mobile phase was filtered through a
0.45µ membrane filter before use. The flow rate of the mobile phase
was adjusted to 1.0 mL/min. The column was equilibrated with mobile
phase for at least 30 min. prior to injection of the drug solution. The
column temperature was maintained at 25±10C through out the study.
Linearity of the peak area response was determined by taking six
replicates at seven concentration points. Working solutions of cefdinir
are prepared by diluting 10mL volumetric flasks with mobile phase. 20
microliters of the dilution was injected six times into the column. The
drug in the eluents was monitored at 284 nm and corresponding
chromatograms ware obtained.
The mean peak areas were noted from the chromatograms and a
plot of concentrations over the peak areas was constructed. The
regression of the plot was computed by least square method. The
linearity was found to be in the range of 100—500 µg/ml between the
concentration of cefdinir and peak area response. This regression
equation was later used to estimate the amount of cefdinir in
pharmaceutical dosage forms. The linearity was shown in Fig 2.3.2 and
the linearity data and statistical parameters for linearity plot are
reported in Table II.3.2 and Fig 2.3.2
3.24
Chap
ter-III
Fig 2.3.2: Linearity of Cefdinir
Table-II.3.2. Linearity of cefdinir
Table III.3.2 Regression Characteristics of the linearity plot of cefdinir
0
200000
400000
600000
800000
0100 200
300400
500
AR
EA
CONCENTRATIONµg
CONCENTRATION AREA
100µg/mL 204562.7
200µg/mL 352580.9
300µg/mL 484083.5
400µg/mL 635797.9
500µg/mL 798418.8
PARAMETER VALUE
Linearity Range(µg/ml) 100-500
Slope(a) 1470929.2
Intercept(b) 0.36
Correlation Coefficient 0.9993
Regression Equation Y=1470929.2x+0.36
3.25
Chap
ter-III
VALIDATION OF THE PROPOSED METHOD
The method was validated in compliance with guidelines of
International Conference on Harmonization (ICH). The following
parameters were determined for validation.
Specificity:
The specificity of the method was assessed by comparing the
chromatograms obtained from the drug with the most commonly used
excipients mixture with those obtained from the blank solution. The
blank solution was prepared by mixing the excipients in the mobile
phase without the drug. The drug to excipient ratio used was similar to
that in the commercial formulations. The commonly used excipients in
formulations like lactose, microcrystalline cellulose, ethyl cellulose,
hydroxyl propyl methyl cellulose, magnesium stearate and colloidal
silicon di oxide were used for the study. The mixtures were filtered
through 0.45µ membrane filter before injection. An observation of
chromatograms indicates absence of excipients peaks near the drug
peak in the study runtime. This indicates that the method is specific.
Precision:
Precision is the degree of repeatability of an analytical method under
normal operational conditions. The precision of the method was
studied in terms of repeatability in intra-day assay and inter-day assay
3.26
Chap
ter-III
(intermediate precision). Method repeatability was studied by repeating
the assay three times in the same day for intra-day precision, and
intermediate precision was studied by repeating the assay on three
different days, three times each day (inter day precision).The intra day
and inter day variation for determination of cefdinir were carried out at
four different concentrations. %RSD values are presented in the Table-
IV.3.2 show that the method provides acceptable (<2) intra day and
inter day variation.
Table-IV.3.2 Intra and Inter-day Precision
Concentrat
ion of
Cefdinir
µg/mL
Intra-Day Precision Inter-Day Precision
Mean
amount
found
n=3
%
Amount
found
% RSD
Mean
amount
found
n=3
%
Amount
found
%RSD
50 49.78 98.9 1.64 50.02 100.1 1.62
100 99.5 101.25 0.82 99.92 99.8 0.81
150 149.95 99.91 0.54 149.52 99.2 0.55
200 200.55 100.68 0.40 199.65 99.56 0.41
Accuracy:
Accuracy of the method is evaluated by standard addition method. An
amount of the pure drug at three different concentrations in its solution
3.27
Chap
ter-III
has been added to the pre analyzed working standard solution of the
drug. The sample solutions were analyzed in triplicate at each level as
per the proposed method. The percent individual recovery and %RSD
for recovery at each level are calculated. The results are tabulated
(Table-V.3.2). A mean recovery of 99.55 - 99.96 has been obtained
which indicates the accuracy of method.
Table-V.3.2 Accuracy Data
Amount
taken
µg
Amount
found
µg
Percent
%Recovery
Mean
Recovery
100 99.43 99.53
99.55
100 99.41 99.41
100 99.73 99.73
300 299.65 99.88
99.96 300 300.2 100.006
300 300.05 100.016
500 499.85 99.97
99.96 500 499.61 99.92
500 500.02 100.004
Robustness:
A study was conducted to determine the effect of deliberate variations
in the optimized chromatographic condition of the mobile phase, flow
rate, and the pH of the mobile phase. The effect of these changes on
the system suitability parameters like tailing factors, the number of
3.28
Chap
ter-III
theoretical plates, and on assay was studied. A single condition was
carried at a time keeping all other parameters constant. The results
were found to be within the allowed limits indicating that the method is
robust.
Variation in composition of mobile phase: The effect of variation in
percent organic content in mobile phase was evaluated by changing the
composition of organic component in the mobile phase. The tailing
factor and the number of theoretical plates showed a little change with
change in mobile phase composition. The values are presented in
Table-VI.3.2
Variations in flow rates: A study was conducted to determine the
effect of variation in flow rate. The system suitability parameters were
evaluated at 0.9 mL/min and 1.1 mL/min. The results were within the
acceptance criteria. Hence the allowable variation in flow rate is 0.9
mL/min. to 1.1mL/min.
3.29
Chap
ter-III
Table – VI.3.2 Results of Robustness Study
Variation of Mobile Phase Chromatographic Parameters
Tailing
factor
Theoretical
plates %Assay
THF ACN MeOH OPA
5 45 10 40 1.58 3490 99.85
5 40 20 35 1.47 3502 99.76
10 40 15 35 1.6 3570 99.32
Stability of the analytical solution: A study to establish bench to top
stability of the drug solution was performed. A freshly prepared
working standard solution (100µg/mL of the drug) was analyzed
immediately at different time intervals. The tailing factor theoretical
plates, and the difference in percent assay at different time intervals
were calculated and the results are given in Table-VII.3.2. A maximum
difference of 0.32 % in the assay at the end of 24 hours was observed.
The difference in percent assay meets the acceptance standard. The
above study concludes that the standard drug solution is stable for
twenty four hours on bench top.
Table - VII.3.2 Stability of Standard solution.
Time in
hours
CEFEPIME
%Assay % Difference
Initial 99.58
6 99.43 0.15
12 99.38 0.2
24 99.26 0.32
3.30
Chap
ter-III
Limit of Detection and Limit of Quantification.
Limit of detection (LOD) is defined as the lowest concentration
of analyte that gives a measurable response. LOD is determined based
on signal to noise ratio (S/N) of three times typically for HPLC
methods.
LOD = 3.3X S.D of y intercept÷ Slope of Calibration curve
The limit of quantification (LOQ) is defined as the lowest
concentration that can be quantified reliably with a specified level of
accuracy and precision. It is the lowest concentration at which the
precision expressed by RSD of less than 2%.
LOQ = 10X S.D of y intercept÷ Slope of Calibration Curve
In this study the analyte response is 10 times greater than the
noise response. For this study six replicates of the analyte at lowest
concentration in the calibration range were measured and quantified.
The LOD and LOQ of Cefdinir obtained by the proposed method were
5 and 20 µg/mL respectively.(Table-VIII.3.2)
Table-VIII.3.2 LOD and LOQ of Cefdinir
Parameter Value (µg/mL)
LOD 5
LOQ 20
3.31
Chap
ter-III
System Precision and System Suitability: System precision and
system suitability studies were carried out by injecting six replicates of
the working standard solution. The % RSD for the peak areas obtained
was calculated. The data presented in Table IX.3.2 reveals that %RSD
is <2 and establishes reproducible performance of the instrument. The
system suitability parameters are presented in Table- XI.3.2.
Table- IX.3.2 System Precision
ESTIMATION OF THE DRUG FROM DOSAGE FORMS.
Since satisfactory results are obtained with the method
developed for the assay of cefdinir, the author has attempted its
applicability for the estimation of the drug in its formulations.
Injection
Number Peak Area Theoretical Plates
1 488960.9 3245
2 470118.8 3480
3 468755.8 3478
4 466523.1 3522
5 475804.0 3408
6 467828.4 3468
Mean 472998.5 ---
SD 7720.351 ----
%RSD 1.7 ----
3.32
Chap
ter-III
Ten tablets of cefdinir were weighed and powered into uniform
size in a mortar. An average weight of a tablet was calculated from this
powder. An accurately weighed portion from this powder equivalent to
100mg of cefdinir was transferred to 100mL volumetric flask
containing 20 mL of mobile phase. The contents of the flask were
sonicated for about 20 min. for complete solubility of the drug and the
volume was made up to 100 mL with water. Then the mixture was
filtered through 0.45µ membrane filter. 4mL of above solution was
taken into a separate 100mL volumetric flask and made up to the
volume with mobile phase and mixed well. The above solution (20µL)
was then injected six times into the column. The mean peak area of the
drug was calculated and the drug content in the formulation was
calculated by the regression equation of the method. The results of the
recovery are tabulated. The percent recovery was reported in Table-
X.3.2
Table – X.3.2.Analysis of formulations & Recovery experiments
Sample
Labeled
Amount
mg
Amount
found
mg
%Recovery
CEPIME 500 499.6 99.92
MEGAPIME 500 495.75 99.15
3.33
Chap
ter-III
RESULTS AND DISCUSSION
The present study was a humble presentation of the author in
developing a sensitive, precise and accurate HPLC method for the
analysis of cefdinir in bulk drug and pharmaceutical dosage forms. In
order to effect analysis of the component peaks, tetra hydro furan
(THF), methyl alcohol, acetonitrile and (0.1%) ortho phosphoric acid
(OPA) in different combinations were tested as mobile phase on a C18
stationary phase. A mixture of tetra hydro furan (THF), methyl
alcohol, acetonitrile and (0.1%) ortho phosphoric acid (OPA) in a
proportion of 5:15:40:40 (v/v) was proved to be the most suitable of
all combinations since the chromatographic peaks were better defined
and resolved and almost free from tailing. The retention time obtained
for cefdinir was 5.1 min.
Each of the samples was injected six times and the same
retention times were obtained in all cases. The peak areas of cefdinir
were reproducible as indicated by low coefficient of variation. A good
linear relationship (r= 0.9993) was observed between the concentration
of cefdinir and the respective peak areas. The regression curve was
constructed by linear regression fitting and its mathematical expression
was Y=1470929.2x+0.36 where Y gives peak area and x is the
concentration of the drug. The regression characteristics are presented
in Table II.3.2.When cefdinir solutions containing 50,100,150,200
3.34
Chap
ter-III
µg/mL were analyzed by the proposed method for finding out intra and
inter day variations, low % RSD was observed. High recovery values
obtained from the dosage form by the proposed method indicates that
the method is accurate. The absence of additional peaks indicates non
interference of common excipients used in the tablets.
The drug content in tablets was quantified using the proposed
analytical method. The tablets were found to contain an average of
99.53% of the labeled amount of the drug. The deliberate changes in
the method have not much affected the peak tailing theoretical plates
and percent assay. This indicates that the present method is robust. The
lowest values of LOD and LOQ obtained by the proposed method
indicate the method is sensitive. The standard solution of the drug was
stable up to 24 hours as the difference in percent assay is within
acceptable limit.
System suitability parameters were studied with six replicates
standard solution of the drug and the calculated parameters are within
the acceptance criteria. The tailing factor and the number of theoretical
plates are in the acceptable limits.
Conclusion
Hence the author concludes that the proposed HPLC method is
sensitive and reproducible for the analysis of cefdinir in
pharmaceutical dosage forms with short analysis time.
3.35
Chap
ter-III
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