Vol 3, Isuue 1, 2013.
Authot Name et al.
ISSN NO: 2231-6876
Title of the Article (14 pt Bold Font Times New Roman)
First Author1, Second Author2 (12 pt Bold)
1
(Department, College/ University Name, Country Name) (10 Italic)
(Department, College/ University Name, Country Name) (10 Italic)
2
Abstract (12 Bold)
Abstract Text (Font size 12 pt)
Corresponding Auther (12 pt Italic)
Name, affiliation, email ID, Contact (10 pt Italic)
Introduction (14 Bold)
Pineapple (Ananas comosus, Bromeliaceae) and Pomegranate (Punica granatum, Punicaceae) are consumed
around the world and has been used as traditional medicine for a variety of therapeutic purposes[1, 2]. Both the
pineapple root and fruit may be eaten or applied topically as an anti-inflammatory or as a proteolytic agent
while Pomegranate fruit shows potential antioxidant activity such as inhibition of low density lipoprotein
oxidation and decrease in cardiovascular diseases.Based on these findings the fruits have high demand which
allows for possible drug fruit interaction[3, 4]. Although, GLM has been shown to undergo hepatic oxidative
biotransformation via CYP450 system [5, 6] and its metabolism also has been reported using CYP specific
species of seven CYP2C9 variants found in Japanese subjects, oxidative biotransformation by in vitro studies
using HLM has not been demonstrated [8]. Glimepiride is a widely used third-generation sulfonylurea suitable
for once daily administration in treatment of type 2 diabetes mellitus. It is completely absorbed after oral
administration [9] and is eliminated mainly via metabolism by cytochrome P450 (CYP) 2C9. The oral
bioavailability of glimepiride is close to 100%[10, 11]. (Font size 12 pt)
Materials and Methods (14 Bold)
Pure drug samples of ATV and FEN was kindly gifted by Sun Pharmaceutical, Delhi and EZET was kindly
gifted by ALEMBIC Pharmaceutical Ltd, Delhi.
Experimental/ Methodology
The mobile phase was prepared by mixing methanol and 0.05M phosphate buffer (pH-6.3 adjusted with sodium
hydroxide and filtered through 0.20 μm nylon filter paper) in a ratio of (85:15)% v/v and degassed [12-15].
Table 1: Construction of Calibration set (Tables only with horizontal lines)
S. No.
ATV
(μg ml-1)
EZET
(μg ml-1)
FEN
(μg ml-1)
1
2
3
4
5
6
4
4
4
4
3
3
5
5
5
5
2
3
8
16
24
32
16
16
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Vol 3, Isuue 1, 2013.
Authot Name et al.
ISSN NO: 2231-6876
Results and discussion
The developed HPLC method has been applied for the simultaneous determination of ATV, EZET and FEN.
The chromatographic method depends on reversed phase separation using Hypersil BDS C8 column (250X4.6
mm i.d, 5 , particle size). The method has been optimized after studying the effect of different parameters on
the separation. The mobile phase was chosen after several trials with methanol and buffer solutions in various
proportions and at different pH. The studies suggested that a mobile phase consisting of methanol and 0.05M
phosphate buffer (pH-6.3 adjusted with sodium hydroxide) in the ratio of (85:15)% v/v was found to give good
peak shape, optimum retention and resolution of ATV, EZET and FEN on above said column at ambient
temperature.
Figure 2: HPLC chromatogram of 20 µl injection of tablet sample containing 112μg ml -1 of FEN and 7 μg ml-1
of ATV and EZET.
Method validation
Table 4: Results of system suitability parameters for the proposed HPLC method for the determination of ATV,
EZET and FEN
No.
1.
2.
3.
4.
5.
6.
Parameters
Theoretical plates per meter
% RSD
Theoretical plates per column
% RSD
Capacity factor
Selectivity(α)
Symmetry factor/Tailing factor
Resolution
ATV
92794
0.45
4640
0.64
2.85
2.544
1.257
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Data obtained
EZET
85084
0.85
4254
0.24
1.12
1.272
1.122
4.417
FEN
118137
0.14
5907
0.65
1.65
2.152
0.873
4.431
Vol 3, Isuue 1, 2013.
Authot Name et al.
ISSN NO: 2231-6876
For chemometric spectrophotometry
Figure 3 shows the overlain zero-order absorption spectra of ATV, EZET and FEN and their ternary mixture
solution in methanol in the 200-400 nm absorption region.
Figure 3: overlain zero-order absorption spectra of 2 µg ml-1 ATV, 2 µg ml-1 EZET and 32 µg ml-1 FEN and
their ternary mixture (containing 2 µg ml-1 ATV, 2 µg ml-1 EZET and 32 µg ml-1 FEN) solution in methanol.
Conclusion
RP-HPLC techniques are generally used for separation and determination of components in final
pharmaceutical preparations and are superior with regard to identification and specificity. However, the
chemometric methods are less expensive by comparison and do not require sophisticated instrumentation nor
any prior separation step. The proposed chemometric-assisted spectrophotometric methods are applicable,
prompt, and specific for the simultaneous determination of ATV, EZET and FEN in their synthetic mixtures and
commercial pharmaceutical tablets. The results obtained were compared with the proposed RP-HPLC method
and good coincidence in the means of recovery was observed as there was no significant difference between the
methods compared. The three proposed methods were accurate, precise with good reproducibility and
sensitivity; hence can be used for the routine analysis of ATV, EZET and FEN in their combined
pharmaceutical formulations.
Authors’ Statements
Competing Interests
The authors declare no conflict of interest.
References
1. Remington, The science and practice of pharmacy. Lippincott Williams & Wilkins, 2006, 1368.
2. Chaudhari BG, Patel NM, Shah PB, Modi KP, Development and Validation of a HPTLC method for the
simultaneous estimation of Atorvastatin Calcium and Ezetimibe, Indian J Pharm Sci. 2006, 68(6), 793-796.
3. Jamshidi A, Nateghi AR, HPTLC Determination of Atorvastatin in Plasma. Chromatographia. 2007, 65,
763-766.
4. Shirkhedkar AA, Surana SJ, Development and validation of a reversed-phase high-performance thin-layer
chromatography-densitometric method for determination of atorvastatin calcium in bulk drug and tablets. J
AOAC Int. 2010, 93(3), 798-803.
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Vol 3, Isuue 1, 2013.
Authot Name et al.
ISSN NO: 2231-6876
5. Farahani H, Norouzi P, Beheshti A, Sobhi HR, Dinarvand R, Ganjali MR, Quantitation of atorvastatin in
human plasma using directly suspended acceptor droplet in liquid–liquid–liquid microextraction and highperformance liquid chromatography-ultraviolet detection. Talanta 2009, 80, 1001–1006.
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dosage forms by high-performance liquid chromatography and high-performance thin layer
chromatography. J AOAC Int. 2010, 93(5):1450-1457.
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