Indo American Journal of Pharmaceutical Research, 2015
ISSN NO: 2231-6876
A NEW STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS
ESTIMATION OF GEMCITABINE AND CLARITHROMYCIN IN TARCEVA TABLETS
B. Koteswara Rao1, K.R. Manjula2, M. Nageswara Rao3, K.Suresh Babu4, C. Rambabu1*
1
Acharya Nagarjuna University, A.P, India.
Y.V.N.R Govt Degree College, Kaikaluru, A.P, India.
3
P.V.P.Siddhartha College of Engineering, Vijayawada, A.P, India
4
Satavahana College,Vijayawada,A.P,India.
2
ARTICLE INFO
Article history
Received 30/04/2015
Available online
09/05/2015
Keywords
Gemcitabine,
Clarithromycin,
Simultaneous Determination,
Degradation
Studies,
Assay,
Stability Indicating RP-HPLC.
ABSTRACT
A simple, accurate, specific and rapid stability indicating RP-HPLC method was developed
for the simultaneous estimation of Gemcitabine and Clarithromycin in combined dosage
form. A Novapack symmetry, C18, 150mm x3.9mm, 5µ column was used for the complete
separation of the drugs. A mixture of potassium dihydrogenorthophosphate and dipotassium
hydrogen orthophosphate in dilute phosphoric acid at a pH of 3.5 was used as buffer solution
and a 55:45 ratio mixture of buffer and acetonitrile was used as mobile phase ( diluent ) .The
chromatograms were taken at a flow rate of 1.0 mL/min, temperature of 30 0 C and detection
wave length of 212 nm with isocratic elusion . The retention time for Gemcitabine standard
(sample) was 2.373 min and for Clarithromycin standard (sample) was 5.995min respectively.
The linearity range for Gemcitabine was 18.75 -112.50 µg/mL and for Clarithromycin 12.5 75 µg/mL The recovery of Gemcitabine was in the range 99.86 – 99.95 % and for
Clarithromycin 99.94 – 99.97 %. Gemcitabine degraded from 15.45% to 37.01% and
Clarithromycin degraded from 9.51% to 37.46% under varied stress conditions. The present
stability indicating RP-HPLC method can be used for the accurate, precise, and rapid
simultaneous determination of Gemcitabine and Clarithromycin in the combined dosage
form. No co eluting peaks were obsereved with main peaks and the method is specific for the
estimation of both the drugs in presence of their degradation products.
Copy right © 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Please cite this article in press as B. Koteswara Rao et al. A New Stability Indicating RP-HPLC Method for The Simultaneous
Estimation of Gemcitabine and Clarithromycin in Tarceva Tablets. Indo American Journal of Pharm Research.2015:5(04).
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Corresponding author
Prof. Chintala Rambabu
Prinicipal, University College of Sciences,
Acharya Nagarjuna University,
Nagarjuna Nagar-522510, Guntur District,
Andhra Pradesh, India,
[email protected]
9949838299
Vol 5, Issue 04, 2015.
B. Koteswara Rao et al.
ISSN NO: 2231-6876
INTRODUCTION
Chemically Gemcitabine was 21 –Deoxy-21, 21-difluoro cytidine and its molecular formula is C9H11F2N3O4, molecular weight
is 263.198. The Chemical structure of Gemcitabine was shown in Fig.1a. It was a pyrimidine analog. Gemcitabine was active against
solid tumors. It was used in the treatment of cancers of breast, kidney, pancreas, biliary tract and lung bladder either singly or in
combination with other cytotoxic agents. In testicular and ovarian tumors and lymphomas Gemcitabine response rates were
satisfactory. After uptake of Gemcitabine into the cells, it will be converted to its active metabolite Gemcitabine triphosphate via
nucleoside transporters. In the conversion of Gemcitabine to its monophosphate, diphosphate and triphosphate, several enzymes were
involved via successive phosphorylation. Gemcitabine triphosphate will be incorporated into DNA, blocking DNA polymerase and it
also inhibits the ribonucleotide reductase resulting in the decrease of the Deoxyribonucleotide pool necessary for DNA synthesis, there
by contributing to the anti-tumour effect. As per the literature, Gemcitabine was determined individually by RP-HPLC, LC-MS/MS
methods in pure and formulations and in blood plasma[1-19] . It was also simultaneously determined with Capecitabine HCl by RPHPLC methods [20,21]. Simultaneous methods one with Irinotecan in pharmaceutical formulation by RP-HPLC [22], and another with
Gemcitabine squalence by LC tandem mass spectrometry in human plasma were reported.[23]
Clarithromycin was chemically 6-methoxy Erythromycin and it was derived from Erythromycin. Molecular formula is C 38
H69 NO13 and molecular weight is 747.953. The structure of Clarithromycin was shown in Fig.1b. Clarithromycin inhibits bacterial
protein synthesis by binding to the bacterial 50 S ribosomal subunit. It was currently being evaluated for the treatment of some
refractory infections in AIDS patients.
Fig 1a: Structure of Gemcitabine.
Fig 1b: Structure of Clarithromycin.
Literature survey revealed that Clarithromycin was determined by RP-HPLC methods [24-30] in various dosage forms.
Simultaneous methods with other compounds by HPLC methods were also reported [31-34].
Even though many methods were available in literature for the determination of
Gemcitabine and Clarithromycin
individually and along with other compounds simultaneously, no method was reported for the simultaneous determination of
Gemcitabine and Clarithromycin in combined form in the literature and hence forms the study. The presently developed stability
indicating RP-HPLC method was a simple, precise, rapid, specific and accurate for the simultaneous determination of Gemcitabine
and Clarithromycin. These drugs are present in TARCEVA tablets in which 150 mg of Gemcitabine and 100 mg of Clarithromycin .
MATERIALS AND METHODS
Reagents and Chemicals
Reference standard sample of Gemcitabine was provided by Hetero drugs , Hyderabad and Clarithromycin was gifted by
Gladcare formulations, Hyderabad. All chemicals used in the method development and validation were Merck brand and purchased
from Bharath Scientifics, Hyderabad . Ultrapure water was used for the preparation of the solutions.
Mobile Phase
3.24 gm of KH2PO4 and 0.6 gm of K2HPO4 were mixed in 100 mL flask and adjust pH to 3.5 with dilute orthophosphoric
acid which was the buffer solution. Buffer and acetonitrile solution were mixed in the ratio of 55:45. It was filtered through a 0.45 µ
membrane and degassed after 15 minutes and was used as mobile phase(diluent).
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Preparation of standard solution : 5mL of the stock solution was taken in 100 mLvolumetric flask, some diluent was added, sonicated to dissolve , degassed
and made up to the mark by the diluent. This solution was used as the standard solution for linearity, precision and accuracy studies.
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Preparation of standard stock solution
Exactly 150 mg of Gemcitabine and 100mg of Clarithromycin were weighed separately and transferred into a 100 mL
volumetric flask , 50 mL diluent was added. Sonicated to dissolve ,degassed, filtered through filter paper and made upto the mark by
the diluent.
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Preparation of sample solution :Nearly 20 TARCEVA tablets were grind into powder in mortar and pestile. Powder quantitatively equivalent to 150mg of
Gemcitabine and 100 mg of Clarithromycin was taken into 100 mL volumetric flask and 50 mL of diluent was added. The solution
was sonicated to dissolve, degassed and filtered through filter paper. Now the solution was made up to the mark by the diluent.
Preparation of Placebo :2 gm of placebo solution was weighed and transferred to 100 mL volumetric flask and 50 mL mobile phase was added.
Sonicated for 5 min and diluted up to the mark, filtered and filtrate was used as placebo.
Instrumentation :A waters HPLC 2 2695 series consisting of 4 pump auto sampler with 5 racks, each has 24 vials holding capacity with
temperature control was used. Auto injector has capacity to inject 5 - 500
solutions, U.V-visible detector with P.D.A. Thermostat
column compartment connected has a capacity to maintain 8 0C to 600 C column temperature. Waters (alliance) HPLC system was
equipped with empower software – 2 software.
Chromatographic Conditions :Depending on the structure of the constituent drugs, the chemical compounds present in the formulation, molecular weight of
the drugs, acidity / basicity of the compounds, and sample solubility, the present chromatographic method was developed for the
simultaneous determination of Gemcitabine and Clarithromycin in combined dosage form.
Detector Selection
UV absorption of Gemcitabine and Clarithromycin were recorded along with mobile phase. A 200 – 400 nm UV detector
was used . Upon several trials 212 nm wave length was selected for the detection of the drugs. At this wave lengths both the drugs
had good absorbance.
Mobile Phase
The drugs under consideration are polar and they are soluble in polar solvents. Different mixture of organic solvents and
aqueous buffers were selected. By changing the composition of the mobile phase, acceptable resolution was obtained. A mixture of
KH2PO4 and K2HPO4 with pH =3.5 was selected as buffer solution and a 55:45 ratio mixture of buffer and acetonitrile was selected as
mobile phase which was also used as diluent. The isocratic elusion method was preferred.
Type of the Column and Packing Material
To achieve number of theoretical plates, peak asymmetry and resolution columns of different carbon chain lengths were used.
A Novapack symmetry, C18, 150 mm x 3.9 mm, 5
column was selected.
Method Development
By changing chromatographic conditions like the column, mobile phase, composition of mobile phase, flow rate of the
mobile phase, injection volume, run time, column temperature and detection wave lengths one after the other by keeping others
constant, different trials were made for getting optimum chromatographic conditions . After so many trials the chromatograms were
Method Validation: As per the ICH guidelines, the aim of the method validation is that the developed method was suitable for the planned
purpose. The present method was validated for specificity, accuracy and selectivity.
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Fig 2:Overlain UV Spectra of Gemcitabine and Clarithromycin.
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recorded with Novapack symmetry, C18, 150mm x3.9 mm, 5
column at 300 C and at flow rate of 1.0 mL /min. A mixture of KH 2
PO4, K2HPO4 dissolved in dil. Orthophosphoric acid at pH =3.5 was used as buffer and a 55:45 mixture of buffer and acetonitrile was
used for isocratic elusion at a detection wave length of 212 nm. The Overlain Spectra of Gemcitabine and Clarithromvcin was shown
in fig-2
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Linearity: Linearity of the method was tested, by recording the chromatograms of six solutions separately. Separate calibration curves
were constructed for Gemcitabine and Clarithromycin. The linearity range of Gemcitabine is 18.75-112.5 g /mL. The linearity range
of Clarithromycin is 12.5-75 µg /mL . The Calibration curves were shown in fib -3a for Gemcitabine and fig -3b for Clarithormyicn
The Chromatograms of linear solution were shown in fig-3c to fig -3h. The amounts of drugs present were shown in table -1 .The
results of linearity studies of the drugs were shown in Table – 2.
Fig 3a: Linearity plot of gemcitabime.
Fig 3d: Linearity Chromatogram
of 50% Standard Solution.
Fig 3e: Linearity Chromatogram
of 75% Standard Solution
Fig 3f: Linearity Chromatogram
of 100% Standard Solution.
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Fig 3c: Linearity Chromatogram
of 25% Standard Solution
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Fig 3b Linearity plot of clarithromycin.
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B. Koteswara Rao et al.
Fig 3g: Linearity Chromatogram
of 125% Standard Solution
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Fig 3h: Linearity Chromatogram
of 150% Standard Solution.
Table 1: Amounts of drugs present in the linear solutions.
S.no
1
2
3
4
5
6
% of
linear
solution
25
50
75
100
125
150
vol of Final volume amount of drugs present (µ g/ml)
stock
Gemcitabin
Clarithromycin
solution mL
1.25
100
18.75
12.50
2. 5
100
37.5
25.00
3.75
100
56.75
37.50
5
100
75.0
50.00
6.25
100
93.75
62.50
7.5
100
112.50
75.00
Table 2: Results of Linearity.
S.no
1
2
Name of the Drug Linearity
Concentration
Range
Gemcitabin
18.75µ g /mL -112.5µg/mL
Clarithromycin 12.5µ g /mL -75µg/mL
Regression
Equation
y=106x-56210
y=33537x-31599
Correlation
Coefficient
0.998
0.999
Specificity :The standard and sample solutions of Gemcitabine and Clarithromycin were chromatogrammed and no additional peaks
were observed in the chromatogram of the sample. It means that the excipients may not interfere with the drug peaks . The
chromatograms of standard and sample were observed and it was concluded that the present method is specific . The Chromatograms
of blank, sample and standard were shown in fig -4a, 4b and 4c The results of specificity was shown in table 3.
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Fig 4a:Chromatogram of blank.
Fig 4b: Chromatogram of Sample
Fig 4c : Chromatogram of Standard
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Table 3: Results of Specificity Studies.
S.no
Name of the Sample
1
2
3
4
Blank
Mixed Standard
Placebo
Sample
Gemcitabin
RT
AREA
2.373 3978951
2.375 3989320
Clarithromycin
RT
AREA
5.995 1298602
5.996 1296677
LOD & LOQ :The standard solutions were injected into the HPLC system and the LOD and LOQ of the present method were calculated.
LOD is the lowest concentration of the analyte that was determined by the method developed ( 3.3
/s) and LOQ is the lowest
concentration that was estimated by the method developed ( 10
/s). LOD and LOQ for Gemcitabine were 5.262µg/mL,
15.95µg/mL and for Clarithromycin 61.77µg/mL, 187.18µg/mL respectively. The Results of LOD and LOQ were shown in table -4
= standard deviation of the intercept of calibration curve ,
S = slope of the calibration curve
Table 4: Results of LOD and LOQ.
S.no
1
2
Name of the Drug
Gemcitabine
Clarithromycin
Slope
10 6
33537
SD
1594709.04
627736.486
LOD
5.262
61.77
LOQ
15.95
187.18
SD: Standard Deviation
LOD: Limit of Detection
LOQ: Limit of Quantification
Precision :System precision ( Repeatability):It is determined by assaying six different concentrated solution of Gemcitabine and Clarithromycin mixture by the
present RP HPLC method on the same day under identical experimental conditions. The results of System precision were
shown in table - 5
Table 5: Results of System Precision(Repeatability).
S.no
Property
1
2
3
Mean
SD
%RSD
Gemcitabine
RT
AREA
2.363 4136530
0.0068 27129.40
0.288 0.656
Clarithromycin
RT
AREA
5.980 1315829
0.0250 10359.07
0.419 0.787
Method precision :The method precision is determined by assaying six solutions of the mixture of Gemcitabine and Clarithromycin by the same
method on different days. The results of Method precision were shown in table -6
Table 6: Results of Method Precision.
1
2
3
Mean
SD
%RSD
Gemcitabine
RT
AREA
2.370 3977454
0.0019 10272.79
0.079 0.258
Clarithromycin
RT
AREA
5.998 1297571
0.0017 1598.28
0.028 0.123
Accuracy :The accuracy of the method now developed is studied by the recovery of the API spiked with the pre-analysed sample at
three different levels i.e., 80% , 100% , 120% with respect to precision concentration . The amounts of API’s Spiked were shown in
table -7 and the results of Accuracy were shown in table -8
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Property
Page
S.no
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Table 7: Amounts of API’s Spiked.
S.no
Name of the Drug
1
2
Gemcitabine
Clarithromycin
Level of the Solution
80%
100%
120%
120mg
150mg
180mg
80mg
100mg
120mg
Standard Solution (100%)
150mg
100mg
Table 8: Results of Recovery Studies.
S.
no
1
2
Amount
present in
the
Standard
(mg)
Drug
Gemcitabine
150mg
Clarithromycin
100mg
Recovery
Level
Amount
Added
(mg)
Amount
Recovered
(mg)
%
Recovery
80%
100%
120%
80%
100%
120%
120
150
180
80
100
120
119.836
149.923
179.442
79.975
99.940
119.867
99.86
99.96
99.69
99.97
99.94
99.89
Ruggedness :Under the identical experimental conditions six solutions of the sample were analyzed by different persons with different
instruments. The ruggedness data was shown in table 9. The RSD of ruggedness is less than 2 and the method was rugged. The results
of Ruggedness were shown in table -9.
Table 9: Results of Ruggedness.
S.no
Property
1
2
3
Mean
SD
%RSD
1
2
3
Mean
SD
%RSD
Gemcitabine
RT
AREA
DAY 1
2.366 3982770
0.0015 576.2
0.065 0.014
DAY 2
2.367 3983659
0.0021 2431.0145
0.090
0.061
Clarithromycin
RT
AREA
5.996
0.0015
0.025
1297641
829.10
0.064
5.985
0.0016
0.027
1296492
165.4638
0.013
Robustness : The robustness of a method is a measure of remain unaffected by the deliberate changes made during the determination, like
change in mobile phase composition, flow rate, temperature etc.,
The RSD of flow-1, flow-2, temp-1, temp-2 are less than 2, hence the method was robust. The results of Robustness were shown in
table – 10.
Table 10: Results of Robustness.
Clarithromycin
RT
AREA
6.387 1669446
5.420 1405266
6.439
1510422
5.489
1583855
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Robustness Flow-1
Robustness Flow-2
Temperature-1
Temperature-2
Gemcitabine
RT
AREA
2.56 5185425
2.179 4347307
2.356 4738302
2.349 4869445
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Property
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Assay of sample :
5mL of the standard stock solution was transferred into a 100 mL volumetric flask and some diluent was added and made up
to the volume by the diluent. Take sample equivalent to 150 mg of Gemcitabine and 100mg of Clarithromycin into a 100mL
volumetric flask, some diluent was added , sonicated to dissolve, degassed, made up to the mark with the diluent . Filtered the
solution through the filter paper. 5 mL of the filtrate was transferred into a 100mL volumetric flask and made up to the mark by the
diluent. Injected 20 µL of blank , standard and sample solutions into the HPLC system and chromatograms were recorded . The
results of Assay determination & Tarceva tablets were shown in table -11& 12
Table 11: Results of Assay determination.
S.no
1
2
Name of the Drug
Gemcitabine
Clarithromycin
Dosage
150 mg
100 mg
% of Assay
99.88
99.90
Table 12: Assay Results of Tarceva Tablets.
S.no
1
Name of the Tablet Name of the
Dosage(mg)
Drug
Tarceva
Gemcitabine
150
Clarithromycin
100
Amount
Found(mg)
149.78
99.89
%Assay
99.85
99.89
Degradation studies :
The stability of the drugs is estimated under different stress conditions by the degradation studies.
Acid hydrolysis :Exactly 150mg of Gemcitabine and 100mg of Clarithromycin were transferred into a 250mL RB flask and 100mL of freshly
prepared 0.1 N HCl was added. Kept it for 12 hours. After 12 hours the solution was filtered through filter paper and neutralized the
solution with NaOH. 5mL of the filtrate was diluted to 100mL by the addition of diluent, 20 µL of the solution was injected and
chromatogram was recorded.
Base hydrolysis : Exactly 150mg of Gemcitabine and 100mg of Clarithromycin were transferred into a 250mL RB flask and 100mL of freshly
prepared 0.1 N NaOH solution was added. Kept it for 12 hours. After 12 hours, the solution was filtered through filter paper and
neutralized the solution with HCl solution. 5mL of the filtrate was diluted to 100mL by the addition of diluent. 20 µL of the solution
was injected and chromatogram was recorded.
Thermal Degradation : Exactly 150mg of Gemcitabine and 100mg of Clarithromycin were transferred into a 250mL volumetric flask and seal the
lid. Place the volumetric flask in a oven at temperature of 100 0 C for 12 hours . After 12 hours it was cooled and 50 mL diluent was
added , sonicated to dissolve and made up to the mark by the diluent. The above solution was filtered and 5mL of filtrate was diluted
to 100mL by the diluent . 20 µ L of the solution was injected and chromatogram was recorded.
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Degradation by UV exposure : Exactly 150mg of Gemcitabine and 100mg of Clarithromycin were transferred on to a dry and clean Petridish and kept it in
UV cabinet for 12 hours . After that 50mL of the diluent was added, sonicated to dissolve, degassed and filtered through filter paper .
5mL of the filtrate was diluted to 100 mL by diluent. 20 µL of the solution was injected into the HPLC system and chromatogram was
recorded .
The Cromatograms of degradation studies were shown in Fig-5a to 5e and the results of degradation were shown in table -13.
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Oxidative degradation by Hydrogen Peroxide : Exactly 150mg of Gemcitabine and 100mg of Clarithromycin were transferred into a 250ml RB flask and 100mL of freshly
prepared 1% H2O2 solution was added . leave it for 12 hours and after that the solution was filtered through the filter paper . 5mL of
the filtrate was diluted to 100mL by the diluent. Injected 20 µL of the solution into the HPLC system and chromatogram was
recorded.
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Fig 5a: Degradation Chromatogram
in acid hydrolysis
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Fig 5b: Degradation Chromatogram in
base hydrolysis.
Fig 5c: Degradation Chromatogram in Oxidation
Fig 5d: Degradation Chromatogram on heat.
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Fig 5e: Degradation Chromatogram in UV exposure.
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Table 13: Results of Degradation.
S.no
Drug
1
Gemcitabine
2
Clarithromycin
1
Gemcitabine
2
Clarithromycin
1
Gemcitabine
2
Clarithromycin
1
Gemcitabine
2
Clarithromycin
1
Gemcitabine
2
Clarithromycin
Type
Peak Area
Acid Hydrolysis
Degraded API
2507362
Standard
3975536
62.82
Degraded API
811610
Standard
1297721
62.44
Base Hydrolysis
Degraded API
3144548
Standard
3975536
78.79
Degraded API
1020585
Standard
1297721
78.52
Oxidative Degradation
Degraded API
3265897
Standard
3975536
81.83
Degraded API
159987
Standard
1297721
81.55
Heat Degradation
Degraded API
3263923
Standard
3975536
81.78
Degraded API
1059228
Standard
1297721
81.50
UV Degradation
Degraded API 3367570
Standard
3975536
84.38
Degraded API 1174903
Standard
1297721
90.39
Found Assay
% Assay
99.83
37.01
99.90
37.46
99.83
21.04
99.90
21.38
99.83
18.00
99.90
18.35
99.83
18.05
99.90
18.40
99.83
15.45
99.90
9.51
% Degradation
It can be concluded from the above results that Gemcitabine was degraded more in Acid Hydrolysis and least in Oxidation
with Hydrogen peroxide, Clarithromycin degraded more in Acid Hydrolysis and least in UV Exposure.
Study of dosage form : Each TARCEVA tablet contains 150mg of Gemcitabine and 100mg of Clarithromycin. 20 tablets were finely grinded to
powder in a mortar and pestile. Sample equivalent to 150 mg Gemcitabine and 100mg of Clarithromycin was taken in a 100mL
volumetric flask and 50mL of diluent was added, sonicated to dissolve, degassed and made up to the mark by the diluent . The
solution was filtered through filter paper and 5 mL of the filtrate was diluted to 100mL with the diluent. 20 µL of the solution was
injected into HPLC system and chromatogram was recorded.
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specific. From the regression analysis slope and standard deviation were calculated and the limit of detection (LOD) (3 /s), limit of
quantification (LOQ) (10 /s) were calculated. In the study of system precision, statistical parameters such as mean, standard
deviation and % RSD for six replicates on retention time and peak area were calculated and % RSD values of peak area were found
0.656 and 0.787 for Gemcitabine and Clarithromycin. In the method precision % RSD value of peak areas were found to be 0.25 and
0.123 for Gemcitabine and Clarithromycin. The % of RSD values were less than 2, hence the method was precise. Accuracy studies
were made by standard addition of API”s at three concentrations, i.e., 80 % , 100 %, 120 % of the precision concentration , and
determination of % recovery method.
The % recovery of Gemcitabine was 99.69 to 99.96% and that of Clarithromycin was 99.89 to 99.97 % . As per results the
method was accurate. In the ruggedness studies, the same sample was analyzed in two different days by the same method and found
that the present method was not affected by carrying out the analysis in different days. Hence the method was rugged. The robustness
of a method is the measure of the capacity to be remained unaffected by small deliberate changes made in the method parameters like
flow rate, temperature, polarity of the solvent, composition of the mobile phase etc., . By the changing the flow rate, oven temperature
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RESULTS AND DISCUSSION
In the linearity studies, the response of the detector was determined by injecting the six solutions of concentration 25 % , 50
% , 75 % , 100% , 125 % and 150 % with respect to precision concentration into HPLC system and chromatograms were recorded .
Each of the solution was injected in duplicate for reproducible response. The calibration curves for both the drugs were plotted
separately as concentration versus peak area. The linear concentration range for Gemcitabine was 18.75 µ g/ mL to 112.5 µg/mL and
for Clarithromycin, it was 12.5 - to 75µg/ mL. The correlation coefficient, intercept were evaluated for both the drugs by statistical
study. The correlation coefficient for Gemcitabine was 0.998 and for Clarithromycin it was 0.999, which are acceptable.
By chromatogramming solutions of blank , mixed standard, placebo and sample solutions, it is observed that no additional
peaks were observed and complete resolution of the two drugs takes place in presence of inactive excipients and hence the method is
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of the method and results were obtained, it was concluded that the small changes in flow rate and oven temperature did not affect the
method significantly. As per the results the method was robust. The % of Assay was calculated by using peak area of standard, sample
and average weight of standard and sample and their concentrations. The Assay of Gemcitabine was 99.88 % and Clarithromycin was
99.90%. The Assay of Gemcitabine and Clarithromycin in formulation Tarceva were 99.85 % and 99.89 % respectively. The stability
of the drugs were studied under different stress conditions and found that the drugs are stable. The % degradation of Gemcitabine was
found in the range 18.00% to 37.01% and that of Clarithromycin in the range 9.51% to 37.46% respectively.
CONCLUSIONS
The presently developed stability indicating RP – HPLC method was simple, specific, accurate, and rapid isocratic method
for the simultaneous determination of Gemcitabine and Clarithromycin in dosage form . In the literature no method was reported for
the simultaneous determination of Gemcitabine and Clarithromycin . Hence the present method must be used for the simultaneous
determination of Gemcitabine and Clarithromycin in the quality control laboratories. It can be concluded that there are no other co
eluting peaks with the main peaks indicating that the method is specific for the estimation of both the drugs in presence of their
degradation products.
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ACKNOWLEDGEMENTS
The authors are thankful to Hetero drugs Ltd., Hyderabad for the donation of Gemcitabine and to Gladcare Formulations,
Hyderabad, for the donation of Clarithromycin standard samples and to Bio-Leo labs for providing facilities to do the research work .
One of the authors Bandla Koteswara Rao expresses his thanks to Acharya Nagarjuna University for providing registration for Ph.D
program.
Vol 5, Issue 04, 2015.
B. Koteswara Rao et al.
ISSN NO: 2231-6876
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