Download Analytical method development for pharmaceutical

“DEVELOPMENT OF NEW ANALYTICAL METHODS AND THEIR VALIDATION
FOR THE DETERMINATION OF PANTOPRAZOLE SODIUM AND
NORFLOXACIN HYDROCHLORIDE IN BULK AND MARKETED
FORMULATIONS”
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE
BY
DEVADAS SHWETHA
M.Pharm-I
Under The Guidance of
Dr. E.V.S. SUBRAHMANYAM. M.PHARM,Ph.D
DEPARTMENT OF QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
2012-2014
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE – II
REGISTRATION OF SUBJECT FOR DISSERTATION
1.0
NAME AND ADDRESS OF
DEVADAS SHWETHA
THE CANDIDATE
1st YEAR M. PHARM,
DEPT. OF QUALITY ASSURANCE
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL,POST PARENGIPETE,
MANGALORE -574143
2.0
NAME OF THE INSTITUTE
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, PARENGIPETE (POST)
MANGALORE-574143
3.0
COURSE OF STUDY &
MASTER OF PHARMACY
SUBJECT
(QUALITY ASSURANCE)
4.0
DATE OF ADMISSION
26TH MAY, 2012
5.0
TITLE OF THE TOPIC:
“DEVELOPMENT OF NEW ANALYTICAL METHODS AND THEIR
VALIDATION FOR THE DETERMINATION OF PANTOPRAZOLE
SODIUM AND NORFLOXACIN HYDROCHLORIDE IN BULK AND
MARKETED FORMULATIONS”
2
6.0
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for study:
Analytical method development for pharmaceutical formulations:
Analytical methods are required to characterize drug substances and drug
products composition during all phases of pharmaceutical development. Development
of methods to achieve the final goal of ensuring the quality of drug substances and
drug products must be implemented in conjunction with an understanding of the
chemical behavior and physicochemical properties of the drug substance. These
determinations require highly sophisticated instruments and methods like HPLC,
HPTLC, Gas Chromatography and Spectrophotometer etc.
Extensive literature survey reveals that several analytical methods have
been reported for the estimation of Pantoprazole sodium and Norfloxacin
hydrochloride in pharmaceutical dosage form which includes Spectrophotometric
methods, HPLC and RP-HPLC.
Hence there is a need for the development of newer, simple, sensitive,
rapid, accurate and reproducible analytical methods for the routine estimation of
Pantoprazole sodium and Norfloxacin hydrochloride in bulk and pharmaceutical
dosage form.
6.2 Basic criteria for new method development of drug analysis:

The drug or drug combination may not be official in any pharmacopoeias.

A proper analytical procedure for the drug may not be available in the
literature due to patent regulations.

Analytical methods may not be available for the drug in the form of a
formulation due to the interference caused by the formulation excipients.

Analytical methods for a drug in combination with other drugs may not be
available.

The existing analytical procedures may require expensive reagents and
solvents. It may also involve cumbersome extraction and separation procedures
and these may not be reliable.
3
Analytical method development provides the support to track the quality of the
product from batch to batch. Estimation can be performed by the following two
methods:

Titrimetric methods and

Instrumental methods.

Spectrophotometric Methods

Chromatographic Methods
Methods for analyzing drugs in dosage forms can be developed, provided one
has knowledge about the nature of the sample, its molecular weight, polarity, ionic
character and the solubility parameter. Method development involves considerable
trial and error procedures. The most difficult problem usually is where to start, what
type of column is worth trying with what kind of mobile phase.

The following is a suggested method development scheme for a typical HPLCUV related substance method.
1. To define the goals for method development (e.g., what is the intended use of
the method?), and to understand the chemistry of the analytes and the drug
product.
2. To develop preliminary HPLC conditions to achieve minimally acceptable
separations. These HPLC conditions will be used for all subsequent method
development experiments.
3. To develop a suitable sample preparation scheme for the drug product.
4. To determine an appropriate standardization method and the use of relative
response factors in calculations.
5. To identify the “drawbacks” of the method and optimize the method through
experimental design. Understand the method performance with different
conditions, different instrument set ups and different samples.
6. To complete method validation according to ICH guidelines as mentioned in
Q2 (R1).
4
6.3 DRUG PROFILE OF PANTOPRAZOLE SODIUM:1,2,3
DRUG CATEGORY : Antiulcer, Proton pump inhibitors (PPI’s)
CHEMICAL STRUCTURE:
PANTOPRAZOLE SODIUM
IUPAC NAME: sodium 5- (difluoromethoxy) - 2 – [[(3, 4-dimethoxy-2-pyridinyl)
methyl] sulfinyl]-1H-benzimidazole sesquihydrate
EMPIRICAL FORMULA: C16H14F2N3NaO4S,11/2 H2O
MOLECULAR WEIGHT: 432.4
SOLUBILITY: Freely soluble in water and very slightly soluble in phosphate buffer
pH 7.4 and practically insoluble in n-hexane.
BIOAVAILABILITY: 77%
PROTEIN BINDING: 98%
HALF-LIFE: 1 hour
EXCRETION: Renal
PHYSICAL STATE: White to off white powder.
PHARMACOLOGY3
Pantoprazole sodium is a newer H+ K+ ATPase inhibitor. It is more acid
stable and has higher bioavailability than omeprazole. It is available for i.v.
administration, particularly employed in bleeding peptic ulcer and for prophylaxis of
5
acute stress ulcers. It is used for short-term treatment of erosion and ulceration of the
oesophagus caused by gastroesophageal reflux disease. Initial treatment is generally of
eight weeks' duration, after which another eight week course of treatment may be
considered if necessary. It can be used as a maintenance therapy for long term use
after initial response is obtained.
Pantoprazole is metabolized in the liver by the cytochrome P450 system.
Metabolism mainly consists of demethylation by CYP2C19 followed by sulfation.
Another metabolic pathway is oxidation by CYP3A4. Their metabolites are not
thought to have any pharmacological significance. It is relatively free of drug
interactions; however, it may alter the absorption of other medications that depend on
the amount of acid in the stomach, such as ketoconazole or digoxin. Generally inactive
at acidic pH of stomach, thus it is usually given with a pro kinetic drug.
MECHANISM OF ACTION:4
Pantoprazole is a proton pump inhibitor (PPI) that suppresses the final step in
gastric acid production by forming a covalent bond to two sites of the (H+,K+ )ATPase enzyme system at the secretory surface of the gastric parietal cell. This effect
is dose related and leads to inhibition of both basal and stimulated gastric acid
secretion irrespective of the stimulus.
ADVERSE EFFECTS:3
Antacid preparations such as pantoprazole by suppressing acid mediated break
down of proteins, leads to an elevated risk of developing food and drug
allergies. This happens due to undigested proteins then passing into the
gastrointestinal tract where sensitization occurs.
Common effects are:

Gastrointestinal: abdominal pain (3% ), diarrhoea (4% ), flatulence (4% )

Neurologic: headache (5% )
Serious effects are:

Gastrointestinal: atrophic gastritis, clostridium difficile diarrhoea

Hematologic: thrombocytopenia (less than 1% )

Immunologic: stevens-johnson syndrome, toxic epidermal necrolysis
6

Musculoskeletal: disorder of muscle, fracture of bone, osteoporosis, hip
fracture, rhabdomyolysis

Renal: interstitial nephritis, acute (rare).
6.4 REVIEW OF LITERATURE:
A great deal of work has been done by the scientist about the current application
and future possibilities for altering the drug activities and evaluation with new method
development by instrumental methods.

Kumar RS, Pamireddy P, Emmadi CS, Sereya K5 carried out simultaneous
determination of naproxen sodium (NAP) and pantoprazole sodium (PAN) in
bulk
and
pharmaceutical
dosage
form
by
validated
ultra-violet
spectrophotometric method. The spectrophotometric method involves the
simultaneous equation method at 232.0 nm and 291.0 nm over the
concentration range of 10μg/ml for both by using 0.1M NaOH as solvent. The
method was validated for linearity, precision, sensitivity, and specificity. The
calibration curves were linear over the range of 2-10 μg/ml for both NAP and
PAN, with significant high value of correlation coefficient (>0.995 for both
drugs). The percentage recovery value for NAP was 100.03% and for PAN
was 100.2%.

Reddy BPK, Reddy YR, Ramachandran D6 carried out determination of
pantoprazole sodium and lansoprazole in individual dosage form tablets by RP
HPLC using single mobile phase. The compounds were well separated an
isocratically on a C18 column [Use Inertsil C18, 5m , 150 mm x 4.6 mm]
utilizing a mobile phase consisting of acetonitrile: phosphate buffer (60:40,
v/v, pH 7.0) at a flow rate of 1.0 ml/min with UV detection at 230 nm. The
retention time of pantoprazole sodium and lansoprazole was found to be 2.017
min and 2.538 min. The procedure was validated for linearity (Correlation
coefficient = 0.999). The study showed that reversed-phase liquid
chromatography is sensitive and selective for the determination of
pantoprazole sodium and lansoprazole using single mobile phase.
7

Kakde RB, Gedam SN, Chaudhary NK, Barsagade AG, Kale DL,
Kasture AV7 carried out three-wavelength spectrophotometric method for
simultaneous estimation of pantoprazole(PAN) and domperidone(DOM) in
pharmaceutical preparations. The absorbance value at 331nm was used for the
estimation of PAN where DOM showed zero absorbance. The absorbance
value for DOM was estimated by taking difference of absorbance at two
wavelengths 284 nm and 364.5nm. This method obeyed Beer’s law in the
concentration range of 10-50 μg/ml for PAN, DOM and their mixture. The
results of analysis have been validated statistically and recovery studies
confirmed the accuracy of the proposed method. The method was found to be
simple, rapid and accurate. Hence can be used for routine simultaneous
estimation of these drugs in formulations.

Cass QB, Degani ALG, Cassiano NM, Pedrazolli
enantiomeric
determination
of
pantoprazole
in
JJ8
human
carried out
plasma
by
multidimensional high-performance liquid chromatography. Multidimensional
HPLC is a powerful tool for the analysis of samples of a high degree of
complexity. This work reports the use of multidimensional HPLC by coupling
a RAM column with a chiral polysaccharide column to the analysis of
pantoprazole in human plasma by direct injection. The enantiomers from the
plasma samples were separated with high resolution on a tris (3,5dimethoxyphenylcarbamate) of amylose phase after clean-up by a RAM BSA
octyl column. Water was used as solvent for the first 5 min in a flow-rate of
1.0 ml/min for the elution of the plasmatic proteins and then acetonitrile–water
(35:65 v/v) for the transfer and analysis of pantoprazole enantiomers, which
were detected by UV at 285 nm. Analysis time was 28 min with no time spent
on sample preparation. A good linear relationship was obtained in the
concentration range of 0.20 to 1.5 μg/ml for each enantiomer. Inter and intraday precision and accuracy were determined by one low (0.24 μg/ml), one
medium (0.70 μg/ml) and one high (1.3 μg/ml) plasma concentration and gave
a C.V. varying from 1.80 to 8.43% and accuracy from 86 to 92%. Recoveries
of pantoprazole enantiomers were in the range of 93.7–101.2%.
8

Erk N9 studied differential pulse anodic voltammetric determination of
pantoprazole in pharmaceutical dosage forms and human plasma using glassy
carbon electrode. The best voltammetric response was reached for a glassy
carbon electrode in Britton–Robinson buffer solution of pH 5.0 submitted to a
scan rate of 20.0 mV s−1 and pulse amplitude of 50.0 mV. This
electroanalytical procedure was able to determine pantoprazole in the
concentration range 6.0 × 10−6 –8.0 × 10−4 M. Precision and accuracy of the
developed method was checked with recovery studies. The limit of detection
and limit of quantitation were found to be 4.0 × 10−7 and 9.0 × 10−7 M,
respectively. Rapidity, precision, and good selectivity were also found for the
determination of pantoprazole in pharmaceutical dosage forms and human
plasma. For comparative purposes high-performance liquid chromatography
with a diode array and UV/VIS detection at 290.0 nm determination also was
developed.

Thanikachalam S, Rajappan M, Kannappan V10 studied stability-indicating
HPLC method for simultaneous determination of pantoprazole and
domperidone from their combination drug product. The proposed HPLC
method utilizes Phenomenex® Gemini C18 column (150 mm × 4.6 mm i.d., 5
μm) and mobile phase consisting of methanol-acetonitrile-20mM dipotassium
hydrogen phosphate and phosphoric acid buffer pH 7.0 (20:33:47, v/v/v) at a
flow rate of 1.19 mL min−1. Quantitation was achieved with UV detection at
285 nm based on peak area with linear calibration curves at concentration
ranges 0.5–5.0 μg mL−1 for domperidone and 1.0–10 μg mL−1 for pantoprazole
(R 2 > 0.999 for both drugs). The method was validated in terms of accuracy,
precision, linearity, limits of detection, limits of quantitation and robustness.
This method has been successively applied to pharmaceutical formulation and
no interference from the tablet excipients was found. Domperidone,
pantoprazole and their combination drug product were exposed to acid, base
and neutral hydrolysis, oxidation, dry heat and photolytic stress conditions and
the stressed samples were analyzed by the proposed method.
9

Raman NVVSS, Reddy KR, Prasad AVSS, Ramkrishna K11 carried out
validated chromatographic methods for the determination of process related
toxic impurities in pantoprazole sodium. A GC–MS method for the
simultaneous determination of two process related toxic impurities viz. 2(chloromethyl)-3, 4-dimethoxypyridine hydrochloride (CDP) and dimethyl
sulfate (DMS) and RP-LC for the routine determination of CDP in
pantoprazole sodium (PPS) are presented. In GC–MS, a temperature gradient
program was performed on a capillary DB-624 column (60 m × 0.32 mm × 1.8
μm). LC analysis of CDP was done on a Novaflex C18 (250 × 4.6 mm, 5 μm)
column using mobile phase containing buffer (0.02 M potassium dihydrogen
phosphate and 0.0025 M di potassium hydrogen phosphate) and acetonitrile in
46:54 v/v ratio. The flow rate was 1.0 mL min−1 and the elution was monitored
at 220 nm. Both methods were validated as per International Conference on
Harmonization (ICH) guidelines. GC–MS is able to quantitate up to 3.0 ppm of
CDP and DMS whereas with RP-LC up to 9.0 ppm of CDP could be
quantitated.

Jagatiya V, Patel J12 carried out simultaneous estimation of cinitapride and
pantoprazole sodium by Rp-Hplc in their marketed formulation. A reversedphase C-18 column (250 mm × 4.60 mm i.d., particle size 5 μm) with mobile
phase consisting of acetonitrile: water: triethylamine (80:20:0.05) was used.
The flow rate was 1.2 ml/min and effluents were monitored at 260nm. The
retention times of cinitapride and pantoprazole sodium were found to be 5.26 ±
0.10 min and 1.72 ± 0.10 min, respectively. The method was validated in terms
of linearity, range, accuracy, and precision, limit of detection (LOD) and limit
of quantitation (LOQ). The method showed good linearity in the range of 1228 μgm/ml for cinitapride and 24-56 μgm/ml for pantoprazole sodium. The
percentage recoveries of cinitapride and pantoprazole sodium were found to be
between 99.52 - 99.85% and 99.38 – 99.74 % respectively. The percentage
RSD for the method precision was found to be less than 2%.
10
6.5
DRUG PROFILE OF NORFLOXACIN HYDROCHLORIDE13,14
DRUG CATEGORY: Antibacterial
CHEMICAL STRUCTURE:
.HCl
NORFLOXACIN HYDROCHLORIDE
IUPAC NAME: 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3carboxylic acid hydrochloride
EMPIRICAL FORMULA: C16H18FN303.HCl
MOLECULAR WEIGHT: 355.79
SOLUBILITY: Freely soluble in water and in glacial acetic acid.
Bioavailability: 30-40%
PROTEIN BINDING: 10-15%
HALF-LIFE: 3-4 hours
EXCRETION: Renal and Fecal
PHYSICAL STATE: A white to pale yellow crystalline powder.
PHARMACOLOGY:14
Norfloxacin is a synthetic chemotherapeutic antibacterial agent occasionally
used to treat common as well as complicated urinary tract infections and genital tract
infections. It is also good for bacterial diarrhoeas. It is not recommended for
respiratory and other systemic infections, particularly where gram positive cocci are
involved. It is sold under various brand names with the most common being Noroxin
and in form of ophthalmic solutions it is known as Chibroxin. It is a first generation
synthetic fluoroquinolone (quinolone) developed by Kyorin Seiyaku K.K. (Kyorin).
11
Chibroxin (ophthalmic) is approved for use in children older than one year of age.
Norfloxacin was first patented in 1979. Kyorin granted Merck & Company,
Inc., an exclusive license, to import and distribute norfloxacin under the brand name
Noroxin. The U.S. Food and Drug Administration (FDA) approved Noroxin for
distribution in the United States on October 31, 1986. Since the approval of Noroxin
in 1986, there have been numerous upgrades to the warning sections of the package
inserts, as well as recent restrictions placed upon the use of Noroxin to treat urinary
tract infections (UTIs).
MECHANISM OF ACTION:15
Norfloxacin is a broad-spectrum antibiotic that is active against both
Gram-positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase, a
type II topoisomerase, and topoisomerase IV, enzymes necessary to separate bacterial
DNA, thereby inhibiting cell division. It inhibits the enzyme bacterial DNA gyrase,
which nicks double-stranded DNA, introduces negative supercoils and then reseals the
nicked ends. This is necessary to prevent excessive positive supercoiling of the strands
when they separate to permit replication or transcription. The DNA gyrase consists of
two A and two B subunits. The A subunit carries out nicking of DNA, B subunit
introduces negative supercoils and then the A subunit reseals the strand. Norfloxacin
bind to A subunit with high affinity and interfere with its strand cutting and resealing
function. Recent evidence indicates that in gram positive bacteria the major target of
norfloxacin action is a similar enzyme topoisomerase IV which nicks and separates
daughter DNA strands after DNA replication .Greater affinity for topoisomerase IV
may confer higher potency against gram-positive bacteria. The bacteriocidal action
probably results from digestion of DNA by exonucleases whose production is signaled
by the damaged DNA.
In place of DNA gyrase or topisomerase IV, the mammalian cells possess
an enzyme topisomerase II (that also removes positive supercoils which has very low
affinity for norfloxacin hydrochloride. Hence the low toxicity to host cells.
12
ADVERSE EFFECTS: 16

Diarrhoea

Seizure (convulsions)

Confusion, hallucinations, tremors, feeling restless, unusual thoughts or
behavior, feeling light headed

Severe dizziness, fainting, fast or pounding heartbeats

Sudden pain, bruising, etc.
6.6 REVIEW OF LITERATURE:
A great deal of work has been done by the scientist about the current
application and future possibilities for altering the drug activities and evaluation
with new method development by instrumental methods:

Pant M, Dadare K, Khatri NC17 have reported the application of UV
spectrophotometric methods for simultaneous estimation of norfloxacin and
tinidazole in bulk and tablet dosage forms. Two simple, accurate and
reproducible spectrophotometric methods have been developed for the
simultaneous estimation of norfloxacin and tinidazole in pharmaceutical
dosage forms. The first method involves determination using the vierodt’s
method (simultaneous equation method); the sampling wavelengths selected
are 273 nm and 319 nm over the concentration ranges of 2.5-20μg/mL and 540 μg/mL for norfloxacin and tinidazole respectively. The second method
involves determination using the multicomponent mode method; the sampling
wavelengths selected are 273 nm and 319 nm over the concentration ranges of
2.5-20μg/mL and 5-40 μg/mL for norfloxacin and tinidazole respectively.

Wankhede SB, Prakash A, Kumari B, Chittange SS18 have reported
simultaneous spectrophotometric estimation of norfloxacin and ornidazole in
tablet dosage form. Three simple, accurate and economical methods have been
13
developed for the estimation of norfloxacin and ornidazole in tablet dosage
form. First method is based on the simultaneous equations, wavelengths
selected for analysis were 273.0 nm (λmax of norfloxacin) and 318.5 nm
(λmax of ornidazole), respectively, in 0.1N NaOH. Second method is Qanalysis method, based on absorbance ratio at two selected wavelengths 297.0
nm (iso-absorptive point) and 318.5 nm (λmax of ornidazole). Third method is
first order derivative spectroscopy using 297.5 nm (zero cross for norfloxacin)
and 264.0 nm (zero cross for ornidazole). The linearity was obtained in the
concentration range of 4-20 μg/ml and 5-25 μg/ml for norfloxacin and
ornidazole, respectively.

Maheshwari RK19 carried out application of hydrotropic solubilization
phenomenon in spectrophotometric estimation of norfloxacin in tablets.
Hydrotropic solubilization technique is one of the techniques used to increase
the aqueous solubilities of poorly water soluble drugs. In the present
investigation hydrotropic solution of urea (8 M) has been employed as
solubilizing agent to solubilize poorly water-soluble drug, norfloxacin from
fine powder of its tablet dosage forms for spectrophotometric determination in
ultraviolet region. Norfloxacin shows maximum absorbance at 272 nm. Beer’s
law was obeyed in the concentration range of 2-14 μg / ml. Results of analysis
were validated statistically and by recovery studies.

Sebaiy MM, El-Shanawany AA, El-Adl SM, Abdel-Aziz LM and Hashem
HA20 carried out rapid Rp-Hplc method for simultaneous estimation of
norfloxacin and tinidazole in tablet dosage form. Separation was carried out on
a Chromolith® Performance RP-18e (100 x 4.6 mm) using a mobile phase of
MeOH: 0.025M KH2PO4 adjusted to pH 3 using ortho-phosphoric acid
(20:80, v/v) at ambient temperature. The flow rate was 4 ml/min and maximum
absorption was measured at 290 nm. The standard curve was linear in the
concentration range of 1-80 μg/mL for both drugs. The retention time of
tinidazole and norfloxacin was noted to be 1.2 and 1.6 minutes respectively,
indicating shorter analysis time.
14

El-Walily AFM, Belal SF, Bakry RS21 carried out spectrophotometric and
spectrofluorimetric estimation of ciprofloxacin and norfloxacin by ternary
complex formation with eosin and palladium (II). Both methods are based on
the formation of a ternary complex between palladium (II), eosin and the
fluoroquinolone in the presence of methyl cellulose, as surfactant.
Spectrophotometrically, under the optimum conditions, the ternary complexes
showed an absorption maximum at 545 nm, with apparent molar absorptivities
of 3.4 × 104 and 2.7 × 104 mol−1 cm−1 and sandell's sensitivities of 1.01 × 10−2
and 1.12 × 10−2 μg cm−2 for ciprofloxacin and norfloxacin, respectively. The
solution of the ternary complex obeyed Beer's law in the concentration range
3–10 μg ml−1 for both quinolones.

Rege PV, Sathe PA, Salvi VS, Trivedi ST22 carried out simultaneous
determination of norfloxacin and tinidazole in combined drug formulation by a
simple electroanalytical technique. In present study, a successful attempt has
been made to develop a simple method for the simultaneous determination of
norfloxacin and tinidazole using linear sweep voltammetry (LSV) technique.
Quantification of norfloxacin and tinidazole was done in Britton-Robinson
buffer of pH 6.5 using 0.1M KCl as a supporting electrolye. Both norfloxacin
and tinidazole exhibit reduction cathodic peak in given pH with peak potential
at -1.40V for norfloxacin and -0.48V for tinidazole. 0.1N HCl was used as
solvent for the analysis. The parameters used for the method validation are
linearity, accuracy, precision, robustness, ruggedness, loss on drying and loss
on quantification.

El-Brashy AM, El-Sayed MM, El-Sepai FA23 carried out spectrophotometric
and atomic absorption spectroscopic determination of some fluoroquinolone
antibacterials by ion-pair complex formation with bismuth (III) tetraiodide.
These methods have been developed for the determination of levofloxacin (I),
norfloxacin (II) and ciprofloxacin (III) in pure form, tablet formulations and
spiked human urine. The methods are based on the formation of ion-pair
associates between the drugs and the inorganic complex, bismuth (III)
15
tetraiodide. The reaction occurs in acidic medium to form orange-red ion-pair
associates which are instantaneously precipitated. The formed precipitates are
then filtered off and the residual unreacted metal complex in the filtrate is
determined either spectrophotometrically at 453 nm or by AAS at 223.1 nm.
The methods permit the determination of the three studied drugs in the range of
5-80 g mL-1.

El-Khateeb SZ, Razek SA, Amer MM24 carried out Stability-indicating
methods for the spectrophotometric determination of norfloxacin. Two
spectrophotometric procedures for the selective determination of norfloxacin
(NF) in the presence of its decarboxylated degradant are described. The first
depends upon measurement of the pH-induced absorbance difference (delta A)
of the drug solution between 0.1 N HCl and 0.1 N NaOH at 280 nm. The
second involves chelation of the intact drug with iron (II) in acetate buffer
solution (pH 5.7 +/- 0.1) to form a yellow-coloured chelate which absorbs at
358 nm. The two procedures are applied successfully for the determination of
the intact drug both in pure form and in tablet form. The two methods retain
their accuracy in the presence of up to 62% and 76% degradants, respectively.
6.7 OBJECTIVES OF THE STUDY:
In the proposed work, attempt shall be made :
 To develop a new method for estimation of Pantoprazole sodium.
 To develop a new method for estimation of Norfloxacin hydrochloride.
 To apply validated method for the estimation of Pantoprazole sodium and
Norfloxacin hydrochloride in pharmaceutical formulation.
 To develop a validated method according to ICH guidelines.
16
7.0
7.1 MATERIALS AND METHODS:
 Drug: Pantoprazole sodium and Norfloxacin hydrochloride
 Reagents:

Ferric chloride

Folin ciocaltaeau

Potassium dichromate

Bromothymol blue

Indigo carmine

3-methyl-1,2-benzothiazoline hydrazone (MBTH) etc.
Method development:
 All experiments will be carried out in the Department of Quality Assurance.
Srinivas college of Pharmacy, Valachil, Mangalore.
 Pure sample of
Pantoprazole sodium and Norfloxacin hydrochloride will be
procured from Industries involved in bulk manufacture of this drug.
 Dosage formulation will be procured from local market.
 The methods will be developed and validated in Q.A. lab of Srinivas college of
Pharmacy.
 The methods will be first developed, then Validated as per ICH guidelines,
then the method will be applied to the formulations.
 UV spectrophotometer Shimadzu-UV1700 with spectral band width of 2nm
and 10nm and matched quartz shall be used for measuring absorbance for
Pantoprazole sodium and Norfloxacin hydrochloride solutions.
 HPLC instrument JASCO ISOCRATIC HPLC-2000 SYSTEM with C18
column shall be used.
 Colorimeter instruments Systronics Spectrophotometer 166 shall be used.
17
7.2 SOURCES OF DATA:
 References from library – Srinivas College of Pharmacy, Valachil,
Mangalore.
 www.pharmainfo.net.
 www.google.com
 www.sciencedirect.com
 www.rxlist.com
 www.pubmed.com
 www.medline.com
 www.wikipedia.com
7.3 Does the study require any investigation to be conducted on patients or
animals?
No
7.4 Has the ethical clearance been obtained from your institution in case of 7.3?
Not applicable
18
8.0
LIST OF REFERENCES
1) Indian pharmacopoeia 2010; vol 3:1865
2) http://rxlist.com/protonix-drug.htm
3) en.wikipedia.org/wiki/Pantoprazole
4) www.drugbank.ca/drug/Pantoprazole(DBoo213)
5)
Kumar RS, Pamireddy P, Emmadi CS, Koneru S. Simultaneous
determination of naproxen sodium and pantoprazole sodium in bulk and
pharmaceutical dosage form by validated ultra- violet spectrophotometric
method. Int J Chem Anal Sci 2012; 3(10): 1569-72
6) Reddy BPK, Reddy YR, Ramachandran D. Determination of pantoprazole
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9.0
SIGNATURE OF THE CANDIDATE
(DEVADAS SHWETHA)
The candidate is working under my direct supervision
10.0
REMARKS OF THE GUIDE
in laboratory of Srinivas College of Pharmacy,
Mangalore-574143
DR. E.V.S. SUBRAHMANYAM,
10.1
NAME AND DESIGNATION OF GUIDE
PROFESSOR AND HEAD OF DEPARTMENT,
DEPARTMENT OF QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY
10.2
SIGNATURE OF GUIDE
DR. E.V.S. SUBRAHMANYAM,
PROFESSOR AND HEAD OF DEPARTMENT,
11.0
HEAD OF THE DEPARTMENT
DEPARTMENT OF QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY
11.1
SIGNATURE OF THE HOD
12.0
REMARKS OF THE PRINCIPAL
RECOMMENDED AND FORWARDED FOR
FAVOURABLE CONSIDERATIONS.
12.1
SIGNATURE OF THE PRINCIPAL
DR.RAMAKRISHNA SHABARAYA A.
PRINCIPAL AND DIRECTOR,
HEAD OF DEPARTMENT,
DEPARTMENT OF PHARMACEUTICS
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, MANGALORE.
22
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