detailed simultaneous estimation method as well as validation parameters for gliclazide and rosiglitazone by reversed phase HPLC methods

1. SIMULTANEOUS ESTIMATION AND VALIDATION
FOR GLICLAZIDE
& ROSIGLITAZONE IN TABLET DOSAGE FORM BY
RPHPLC
METHOD
Prepared by;
Yachita rajwadwala
M.pharm(q.a.)

2. CONTENTS
Abstract 3
Introduction 5
Materials and methods 6
Preparations 8
Validation 11
Results and discussion 17
Conclusion 24
Reference 25
2

3. ABSTRACT
A simple, economic, accurate reverse phase isocratic
RPHPLC method was developed for the Simultanious
estimation of Gliclazide 80mg & Rosiglitazone 2mg in Tablet
dosage form. The quantification was carried out using
Symmetry C18 (4.6 x 100mm, 5μm, Make: XTerra) with UV
detected at 229 nm. The elution was achieved isocratically
with a mobile phase comprises of mixture of buffer 500 mL
(50%) and 500 mL of Acetonitrile HPLC (50%). The flow rate
was 0.8ml/min. The procedure was validated as per ICH
rules for Accuracy, Precision, Detection limit, Linearity,
Reproducibility and Quantitation limit. The linearity
concentration range was 20-100ppm for Gliclazide and 10-
50ppm for Rosiglitazone with the correlation coefficient of
0.999.
3

4. The percentage recovery for Gliclazide & Rosiglitazone was
found to be 100.6% and 101.2% respectively. Limit of
detection values were found to be 0.002mcg/ml and
0.001mcg/ml respectively for Gliclazide & Rosiglitazone.
Limit of quantitation values were found to be 0.009mcg/ml
and 0.006mcg/ml respectively for Gliclazide & Rosiglitazone.
The method has been successfully used to analyze
commercial solid dosage containing Gliclazide &
Rosiglitazone with good recoveries and proved to be robust.
 Key Words: Gliclazide, Rosiglitazone, Tablets, HPLC,
Validation.
4

5. INTRODUCTION
 Gliclazide is chemically 1-(3-Azabicyclo [3.3.0] oct-3-yl)-3-
tosylurea.
Mechanism of action: It acts by increasing the sensitivity
of the beta cells of islets of langerhans.
 Rosiglitazone is chemically 5-(4-(2-(methyl (pyridine-2-
yl) amino) ethoxy)benzyl) thiazolidine-2, 4-dione.
Mechanism of action: It acts by activation of the
intracellular receptor class of the peroxisome activated
receptors.
 Literature surveys reveal that only a few methods have
been reported for this combination.
 Aim: to develop a high degree of accurate and precise
HPLC method for the simultaneous determination of
rosiglitazone and gliclazide in pure and pharmaceutical
dosage forms. 5

6. MATERIALS AND METHODS
 Gliclazide (GLI) and rosiglitazone (ROS) were obtained as
generous gift sample from Dr. Reddy’s Laboratory Limited
(Hyderabad, India).
 A commercial sample ROSINORM-G (tablet containing
gliclazide 80mg & rosiglitazone 2mg) were purchased from
local pharmacy store and used within their shelf-life period.
 The HPLC grade aceto nitrile and water from Rankem (New
Delhi, India) and all other chemicals used were of
pharmaceutical or analytical grade from Rankem.
 HPLC grade water was prepared using Millipore purification
system.
 The HPLC system consisted of a LC-20AT VP Shimadzu
liquid chromatography (Japan) equipped with diode array
detector (SPD-M10 AVP). 6

7.  Manual injections were made using a Rheodyne Injectable
valve (20 μl loop).
 The detector wavelength was set at 229nm.
 The chromatographic separations were performed at
ambient temperature on a Symmetry C18 (4.6 x 100mm,
5μm, Make: XTerra).
 The mobile phase was a mixture of buffer 500 mL (50%)
and 500 mL of Acetonitrile HPLC (50%), filtered and
degassed prior to use, and flowing at the rate of 0.8ml/min
and run time is 8 minutes.
 Buffer was prepared by dissolving 7.0g of potassium
dihydrogen orthophosphate in 1000ml of water, adjusted the
pH to 3.0 with orthophosphoric acid and filtered through
0.45 μ or filter porosity membrane filter.
 The data were collected and analyzed with software in a
computer system. 7

8. PREPARATIONS
Stock solution:
 A stock solution of ROS (100 μg/mL) and GLI (1000 μg/mL)
were prepared by accurately weighed 10 mg of ROS and 100
mg of GLI working standards into two separate 100 mL
volumetric flasks containing 50 mL of diluent and volume
was made up to the mark with diluent, respectively.
 The stock solution is protected from light using aluminum
foil.
 Aliquots of the standard stock solutions of ROS and GLI were
transferred using A-grade bulb pipettes into 100 mL
volumetric flasks and solutions were made up to the mark
with mobile phase to give the final concentrations of 0.5-2.5
μg/mL and 20-100 μg/mL, respectively.
8

9.  To determine the content of ROS & GLI in tablets (label
claim: 2 mg ROS & 80mg GLI), 20 tablets were taken and
contents were weighed and mixed.
 An aliquot of powder equivalent to the weight of one tablet
was accurately weighed and transferred to 100 mL
volumetric flask and was dissolved in 50 mL of diluent and
volume was made up to the mark with diluent.
 The flask was sonicated for 30 min to affect complete
dissolution.
 The solution was filtered through a 0.45 μm filter under
vacuum filtration.
 Suitable aliquot of the filtered solution was transferred into
a 100 mL volumetric flask and made up to the volume with
mobile phase to get concentrations of ROS and GLI were
1.5μg/mL and 60μg/mL, respectively. 9

10.  The experiments were performed six times under the
chromatographic conditions described above.
 The peak areas were measured at 229 nm and
concentrations in the sample were determined by
comparing the peak areas of sample with that of the
standards.
10

11. VALIDATION
 The described method has been validated for the assay of
ROS & GLI using following parameters....
 Precision
 Accuracy
 Linearity
 Robustness
 Limit of detection
 limit of quantitation
11

12.  Precision
 Precision was studied to find out variations in the test
methods of ROS & GLI on the same day and on different day
by using different make column of same dimensions
(Ruggedness).
 The standard solution was injected for five times and
measured the area for all five injections in HPLC. Precision
and Ruggedness were done on the same day and the
differenct day respectively and the %RSD was calculated for
each.
 accuracy
 The accuracy of the method was shown by analyzing
model mixtures which were obtained by adding known
amount of ROS & GLI to pharmaceutical preparation.
12

13. Data for precision
Concentration
(32mcg/ml)
Precision
Precision Intermediate
Precision (Ruggedness)
GLI ROS GLI ROS
Peak area
2711498 1709846 2712520 1709085
2720476 1713220 2720112 1713452
2723970 1712125 2722942 1714415
2727862 1713452 2720442 1712440
2736663 1710258 2726423 1712224
Mean 2724094 1711780 2720488 1712323
Standard deviation 9273 1661.8 5120.52 2009.92
%RSD 0.34 0.1 0.19 0.12
13

14. DATA FOR ACCURACY
Drug
%Concent
ration
(at
specificati
on
Level)
Area
Amount
Added
(mg)
Amount
Found
(mg)
%
Recovery
Mean
Recovery
Gliclazide
50% 1441240 21 21 100%
100.60%100% 2761715 40.2 40.24 100.10%
150% 4336588 62.2 63.2 101.60%
rosigitazo
ne
50% 930766 10.7 10.89 101.80%
101.20%100% 1771665 20.5 20.73 101.10%
150% 2665830 31 31.19 100.60% 14

15.  The model mixtures contained 50, 100 and 150% of ROS &
GLI compared to the labeled drug amount.
 After injected the standard solution, Accuracy - 50%,
Accuracy -100% and Accuracy -150% solutions, the Amount
found, Amount added for GLI & ROS, individual recovery and
mean recovery values were calculated.
 linearity
 The linearity of the method was shown by analyzing model
mixtures of concentration range from 20 to 100ppm for GLI
and 10 to 50ppm for ROS.
 After Injection of each level into the chromatographic
system, peak area was measured.
 A graph of peak area versus concentration (on X-axis
concentration and on Y-axis Peak area) was plotted. 15

16. 16
 Robustness
 As part of the Robustness, deliberate change in the Flow rate and
Mobile Phase composition were made to evaluate the impact on
the method.
 The flow rate was varied at 0.6 ml/min to 1.0 ml/min. The Organic
compostion in the Mobile phase was varied from 45% to 55%.
 Limit of detection and limit of quantitation
 Limit of detection and limit of quantitation were calculated by the
method which was a common approach which is to compare
measured signals from samples with known low concentrations of
analyte with those of blank samples; the minimum concentration
at which the analyte can be reliably detected is established.
 Ratio of Signal Obtained from LOD or LOQ solution (S) and Average
Baseline Noise obtained from Blank (N) was calculated for both
the drugs.

17. RESULTS AND DISCUSSION
 A reverse – phase isocratic procedure is proposed as a
suitable method for the analysis of ROS & GLI in tablets. A
mixture of mixture of buffer 500 mL (50%) and 500 mL of
Acetonitrile HPLC (50%) at a flow rate of 0.8ml/min was
found to be an appropriate mobile phase allowing adequate
and rapid separation of analyte.
 The retention time was found to be 4.3 and 6.3 for GLI and
ROS respectively. The percentage of purity of ROS & GLI in
tablet dosage form is 100.5 and 98.7.
 System suitability for the ROS, Tailing factor Obtained from
the standard injection was 0.9 and Theoretical Plates
Obtained from the standard injection was 2559.
 System suitability for the GLI, Tailing factor Obtained from
the standard injection was 1.0 and Theoretical Plates
Obtained from the standard injection was 2696.
17

18. 18

19.  As shown in the Fig. substances were eluted forming well
shaped symmetrical single peaks, well removed from the
solvent front.
 The precision of the HPLC system was determined using the
%RSD of the peak areas for five injections of the standard
solution of ROS and GLI.
 Precision data were present in Table. The %RSD was less
than 2.
 In order to verify the accuracy of the described method,
recovery studies were carried out by analyzing model
mixtures of ROS and GLI.
 The recovery of ROS and GLI was evaluated from 50 to 150%
of the labeled tablet.
 The mean percentage recoveries were found to be 101.2%
and 100.6% for ROS and GLI respectively. Accuracy data
were present in Table. 19

20. 20

21. 21

22.  For quantitative application a linear calibration curve was
obtained over the concentration range from 20 to 100ppm
for GLI and 10 to 50ppm for ROS.
 The parameters of the calibration graph for ROS and GLI
were y = 89777x – 34024 and y = 69516x – 9569 respectively
where x is concentration and y is peak area; correlation
coefficient for ROS and GLI 0.9985 and 0.9995.
 Percentage curve fitting for ROS and GLI was found to be
99.85% and 99.95% respectively. Calibration curve present
in Figure 2 and 3.
 The results of robustness indicate that the variation in flow
rate affected the method significantly.
 The method is robust only in less flow condition. Even
variation in organic composition in the mobile phase affected
the method significantly. 22

23.  Hence it indicates that the method is not robust even by
change in the flow rate ±10% and change in the Mobile phase
±10% for ROS and GLI.
 Limit of detection values were found to be 0.002mcg/ml and
0.001mcg/ml respectively for GLI & ROS. S/N ratio values for
LOD were found to be 3.14 and 3.33 for GLI & ROS
respectively.
 Limit of quantitation values were found to be 0.009mcg/ml
and 0.006mcg/ml respectively for GLI & ROS. S/N ratio values
for LOQ were found to be 9.39 and 9.2 for GLI & ROS
respectively.
23

24. CONCLUSION
 The presented method is precise, sensitive and accurate.
The advantages of proposed method are its short analysis
time and a simple procedure for sample preparation.
 The satisfying recoveries and low coefficient of variation
confirmed the suitability of proposed method for the
routine analysis of mixtures of GLI & ROS in
pharmaceuticals.
24

25. REFERENCE
 Indian drug review, Jan- March, 2007, 81
 Remington, The science and practice of pharmacy, 20th
edition, Lippincott Williams & Wilkins, USA, 2000, 1526.
 Tripathi K D, Essential of medical pharmacology 6th ed.,
Jaypee brothers medical Publishers (p) ltd, New Delhi, 234-
267.
 Wiwin F K, Henny C and Gunawan I, J Liq Chrom Relat Tech.,
2003, 26(7), 1059-1067.
 Kesarin B, Worapot S and Leena S, J pharm biomed anal.,
2006, 41(1),158-164.
 Kazue T, Kenji F, Tomoyuki M and Hiroyuki N, J chromatogr
A, 1998, 812(1-2), 205-211.
 Satoru K, Sadao I, Yasuhiro M, Tokuji S and Shizuo N,
Biochem pharmacol., 1995, 50, 1645-1650. 25

26.  Loyd L, Snyder R, Josen J. Kirkland and Joseph L, Practical
hplc method development 2nd ed., Mc.Clavrckes,London,
2004, 101.
 Beckett A H and Stenlake J B, Practical pharmaceutical
chemistry, Part -2, 4th ed., CBS, Publishers and distributors,
2004, 138.
 Robert M Silverstein, Spectrophotometric identification of
organic compounds; 6th ed., John
 Willey & sons inc., 1996,119.
 James A Howell, Hand book of instrumental techniques for
analytical chemistry, 1997, 211.
 Frank settle, Hand book of instrumental techniques for
analytical chemistry, 1st ed., Pearson education (Singapore)
Pte. Ltd., 2004, 165.
26

27.  P.D. Sethi, HPLC quantitative analysis of pharmaceutical
formulation, CBS publication and distributors; 2008, 11-160.
 Alfonso R. Gennaro. Remington: The Science andPractice of
Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams
& Wilkins, Philadelphia 2000, Volume 1, 603-620.
 International conference on Harmonization, draft guideline
on validation procedure, definition and terminology federal
register, 1995, 60: 11260.
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28. Thank you
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