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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