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NOVEL SIMULTANEOUS SEPARATION AND
QUANTITATIVE DETERMINATION OF FOUR SARTANS
IN PRESENCE OF HYDROCHLOROTHIAZIDE BY
ISOCRAT...
CONTENTS:
 AIM AND OBJECTIVE
 INTRODUCTION
 DRUGS PROFILE
 LITERATURE SURVEY
 PLAN OF WORK
 MATERIALS AND METHODS
 ...
AIM:
The core AIM of the present study is to develop a novel, rapid,
precise and accurate RP-HPLC method for simultaneous...
Vignan Pharmacy College,Vadlamudi. 4
To develop rapid, sensitive and economical analytical methods
based on spectrophotom...
INTRODUCTION
Vignan Pharmacy College,Vadlamudi. 5
HPLC is a versatile tool for the qualitative and quantitative analysis
...
Vignan Pharmacy College,Vadlamudi. 6
Drug Name : HYDROCHLOROTHIAZIDE
Structure :
IUPAC : 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-
benzothiadiazine-7-sulfonamid...
Drug name : VALSARTAN
Structure :
IUPAC Name :(S)-3-methyl-2-(N-{[2'-(2H-1,2,3,4-tetrazol-5-
yl)biphenyl-4-yl]methyl}penta...
Drug name : LOSARTAN
Structure :
IUPAC Name :(2-butyl-4-chloro-1-{[2'-(1H-tetrazol-5-yl)biphenyl-
4-yl]methyl}-1H-imidazol...
Drug name : OLMESARTAN
Structure :
IUPAC Name : 2,3-dihydroxy-2,2-butenyl-4(1-hydroxy-1-
methylethyl)-2-propyl-1-[p-(o-1H-...
Drug name : TELMISARTAN
Structure :
IUPAC Name : 2-(4-{[4-Methyl-6-(1-methyl-1H-1,3-benzodiazol-
2-yl)-2-propyl-1H-1,3-ben...
Vignan Pharmacy College,Vadlamudi. 12
S.NO AUTHORS TITLE STUDY
JOURNAL
NAME
YEAR,
VOLUME,
ISSUE &
PG.NO
1
Reem Youssef*,
AdnanHbash,
Ahmad Hassan
Development an...
Vignan Pharmacy College,Vadlamudi.
14
S. NO
AUTHORS
TITLE STUDY
JOURNAL
NAME
YEAR,VOL
UME,
ISSUE &
PG.NO
3
T. M.
Kalyankar...
S.NO AUTHORS TITLE STUDY
JOURNAL
NAME
YEAR,VO
LUME,
ISSUE &
PG.NO
5
Lakshmanarao. A,
Bhaskhara Raju .V
Simultaneous
Estima...
PLAN OF WORK:
Vignan Pharmacy College,Vadlamudi. 16
Plan of RP-HPLC technique:
 Literature collection regarding various a...
Vignan Pharmacy College,Vadlamudi.
17
Table 1. Commercial brand names of TELMI, LOS , VAL, IRBE and HYDRO
combinations used for the present study.
Brand name Fo...
Table 2. Materials used in the present study
S.No. Materials Procured from
1. Telmisartan Aristo Pharmaceuticals Pvt.Ltd.,...
Table 3. Instruments used in the present study
S.No. Instrument Name of the company and model
1. HPLC
Shimadzu LC-20AT Pro...
Preparation of reagents and standards:
 Preparation of phosphate buffer pH 3.3
Phosphate buffer with 10 mM was prepared b...
Preparation of sample solution:
A composite of ten Telmisat-H, Zargo-H, Olmecip-H and Valent-H
tablets are prepared by gr...
RESULTS:
Method Development:
 Selection of a common detection wavelength:
 The ultra violet spectrum of four Sartans and...
Vignan Pharmacy College,Vadlamudi. 24
Figure 1. UV overlain spectra of four sartans and HCTZ.
Method optimization:
 Numerous trials were performed to obtain the better separations.
 From all trials, eventually bett...
Figure 2. Typical chromatogram showing the HCTZ, TELM, LOSA, OLME AND VALS
in a synthetic mixture.
Vignan Pharmacy College...
Method validation:
 The developed analytical method was validated in pursuance of the guide
lines of ICH.
SPECIFICITY:
 ...
 The linearity of the method was studied by analyzing different
concentration of the drugs. According to ICH recommendati...
Vignan Pharmacy College,Vadlamudi. 29
Linearity method
HCTZ LOSA TELM OLME VALS
Conc.
μg/mL
Peak area,
mV.s.
Conc.
μg/mL
P...
Table 7. Regression analysis data relating to HCTZ, TELM, LOSA, OLME and VALS
Vignan Pharmacy College,Vadlamudi.
30
Parame...
Figure 3. Standard chromatogram relating to HCTZ
Figure 4. Standard chromatogram relating to TELM
Figure 5. Standard chrom...
Figure 6. Standard chromatogram relating to OLME
Figure 7. Standard chromatogram relating to VALS
Vignan Pharmacy College,...
Figure 8: Standard chromatogram of HCTZ (2.5 μg/ml) and TELM (8 μg/ml)
Figure 9. Standard chromatogram of HCTZ (2.5 μg/ml)...
Vignan Pharmacy College,Vadlamudi. 34
Fig. 11. Standard chromatogram of HCTZ (2.5 µg/ml) and VALS (16 µg/ml)
Vignan Pharmacy College,Vadlamudi. 35
Fig. 12. Sample chromatogram of HCTZ (2.5 µg/mL) and TELM (8 µg/mL)
Fig. 13. Sample ...
Vignan Pharmacy College,Vadlamudi. 36
Fig. 14. Sample chromatogram of HCTZ (2.5 µg/mL) and OLME (8 µg/mL)
Fig. 15. Sample ...
Figure 16. Linearity plot pertaining to
HCTZ
Figure 17. Linearity plot
pertaining to LOSA
Figure 18. Linearity plot pertai...
Figure 20. Linearity plot pertaining to
VALS
Vignan Pharmacy College,Vadlamudi. 38
Figure 19. Linearity plot pertaining to...
Precision:
 The repeatability and intermediate precision experiments were conducted
by determining the intra - day and in...
Vignan Pharmacy College,Vadlamudi. 40
Accuracy/Recovery studies:
 The accuracy of the method was assessed by standard add...
Robustness:
The robustness of developed analytical method was proven by the analysis of HCTZ
with TELM, HCTZ with LOSA, H...
Formulation Telmisat – H
(40mg)
Zargo – H
(50mg)
Olmecip
(40mg)
Valent - H
Composition TELM HCTZ LOSA HCTZ OLME HCTZ VALS ...
Formulation TELMISAT – H
(40mg)
ZARGO – H
(50mg)
OLMECIP – H
(40mg)
VALENT – H
(80 mg)
Robustness, % Assay ± % RSD
Flow ra...
CONCLUSION:
• Statistical analysis lucidly proves that it is feasible for analysis of said ARB’s in their
formulations in ...
REFERENCES:
1. Development and Validation of RP-HPLC Method for the Estimation and
Separation of Valsartan, Losartan and I...
46
Vignan Pharmacy College,Vadlamudi. 47
 We Heart fully thank our guide Dr. P. Ravisankar sir .
I also thank Hetero Labs Limited unit-III, Jeedimetla,
Hyderabad...
49Vignan Pharmacy College,Vadlamudi.
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NOVEL SIMULTANEOUS SEPARATION AND QUANTITATIVE DETERMINATION OF FOUR SARTANS IN PRESENCE OF HYDROCHLOROTHIAZIDE BY ISOCRATIC RP-HPLC

The core AIM of the present study is to develop a novel, rapid, precise and accurate RP-HPLC method for simultaneous separation and quantification of Hydrochlorothiazide along with four sartans Telmisartan(TELM), Losartan(LOSA), Olmesartan(OLME) and Valsartan(VALS).
To develop Novel methods for separation and quantification of all the above said drugs on single chromatographic system without any minor changes in detection wavelength and mobile phase composition.

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NOVEL SIMULTANEOUS SEPARATION AND QUANTITATIVE DETERMINATION OF FOUR SARTANS IN PRESENCE OF HYDROCHLOROTHIAZIDE BY ISOCRATIC RP-HPLC

  1. 1. NOVEL SIMULTANEOUS SEPARATION AND QUANTITATIVE DETERMINATION OF FOUR SARTANS IN PRESENCE OF HYDROCHLOROTHIAZIDE BY ISOCRATIC RP-HPLC Under the guidance of: Dr. Panchumarthy Ravisankar M. Pharm., Ph.D. Professor & HOD Dept. of Pharmaceutical Analysis and Quality Assurance Vignan Pharmacy College Vadlamudi. Presentation by: K. Manjusha (12AB1R0043) V. Laya Sri (12AB1R0044) B. Vijaya Kumar (12AB1R0009) K. Rajyalakshmi (12AB1R0037) K. Avinash kumar (12AB1R0039)
  2. 2. CONTENTS:  AIM AND OBJECTIVE  INTRODUCTION  DRUGS PROFILE  LITERATURE SURVEY  PLAN OF WORK  MATERIALS AND METHODS  RESULTS  CONCLUSION  REFERENCES Vignan Pharmacy College,Vadlamudi. 2
  3. 3. AIM: The core AIM of the present study is to develop a novel, rapid, precise and accurate RP-HPLC method for simultaneous separation and quantification of Hydrochlorothiazide along with four sartans Telmisartan(TELM), Losartan(LOSA), Olmesartan(OLME) and Valsartan(VALS). To develop Novel methods for separation and quantification of all the above said drugs on single chromatographic system without any minor changes in detection wavelength and mobile phase composition. Vignan Pharmacy College,Vadlamudi. 3
  4. 4. Vignan Pharmacy College,Vadlamudi. 4 To develop rapid, sensitive and economical analytical methods based on spectrophotometric & RP-HPLC techniques for estimation and separtion of Hydrochlorothiazide with Telmisartan, Losartan, Olmesartan and Valsartan in pharmaceutical dosage forms.  To develop method with shorter run time and better sensitivity.  Reducing the solvent consumption to make it more eco-friendly. To validate the method for different parameters like Accuracy, Precision, Linearity, specificity, Robustness according to International Conference on Harmonization ICH Q2(R1) guidelines[5] . To apply the developed RP-HPLC method in the analysis of pharmaceutical formulations.
  5. 5. INTRODUCTION Vignan Pharmacy College,Vadlamudi. 5 HPLC is a versatile tool for the qualitative and quantitative analysis of drugs and pharmaceuticals, chemical and biological materials has become indispensable in pharmacokinetics studies.  Of the many instrumental methods available for quantification RP - HPLC is selected as a tool for developing novel analytical procedure.  Method development is done for the products as follows: - New products - Existing products  Alternate methods for existing products are developed for better precision and ruggedness. Method development:
  6. 6. Vignan Pharmacy College,Vadlamudi. 6
  7. 7. Drug Name : HYDROCHLOROTHIAZIDE Structure : IUPAC : 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4- benzothiadiazine-7-sulfonamide Category : Anti hypertensive Molecular Formula : C7H8ClN3O4S2 Molecular Weight : 297.74 g/mol Brand names : Apo-Hydro, Aquazide H, Hydrodiuril, HydroSaluric, Hydrochlorot, Microzide, Esidrex, and Oretic Vignan Pharmacy College,Vadlamudi. 7
  8. 8. Drug name : VALSARTAN Structure : IUPAC Name :(S)-3-methyl-2-(N-{[2'-(2H-1,2,3,4-tetrazol-5- yl)biphenyl-4-yl]methyl}pentanamido)butanoic acid Molecular weight :435.519 g/mol Molecular formula : C24H29N5O3 Brand names : Diovan, Valent -H Vignan Pharmacy College,Vadlamudi. 8
  9. 9. Drug name : LOSARTAN Structure : IUPAC Name :(2-butyl-4-chloro-1-{[2'-(1H-tetrazol-5-yl)biphenyl- 4-yl]methyl}-1H-imidazol-5-yl)methanol Molecular weight :422.91 g/mol Molecular formula :C22H23ClN6O Brand names : Cozaar, Losagard, Zargo-H Vignan Pharmacy College,Vadlamudi. 9
  10. 10. Drug name : OLMESARTAN Structure : IUPAC Name : 2,3-dihydroxy-2,2-butenyl-4(1-hydroxy-1- methylethyl)-2-propyl-1-[p-(o-1H-tetrazol-5- ylphenyl)benzyl]imidazole5-carboxylate2,2-carbonate Molecular weight : 558.59 Molecular formula : C29H30N6O6 Brand names : Olmicep-H, Olmax Vignan Pharmacy College,Vadlamudi. 10 N NN HN Olmesartan N N OH H3C O
  11. 11. Drug name : TELMISARTAN Structure : IUPAC Name : 2-(4-{[4-Methyl-6-(1-methyl-1H-1,3-benzodiazol- 2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl} phenyl)benzoic acid Molecular weight : 514.617 g/mol Molecular formula : C33H30N4O2 Brand names : Telmisat-H, Cresar, Micardis Vignan Pharmacy College,Vadlamudi. 11
  12. 12. Vignan Pharmacy College,Vadlamudi. 12
  13. 13. S.NO AUTHORS TITLE STUDY JOURNAL NAME YEAR, VOLUME, ISSUE & PG.NO 1 Reem Youssef*, AdnanHbash, Ahmad Hassan Development and Validation of RP- HPLC Method for the Estimation and Separation of Valsartan, Losartan and Irbesartan in Bulk and Pharmaceutical Formulation. Separation of 3 sartans using c18 column and its validation according to ICH guidelines. International Journal of Pharmaceutial Sciences Review and Research. (IJPSRR) Jan – Feb 2014, 24(2), 311-314. 2 Ganipisetty Lakshmi Aswini*, D.Dachinamoor thy, J.V.L.N.Seshagi ri Rao A Sensitive RP- HPLC Method Development and Validation for the Simultaneous Estimation of Losartan Potassium and Hydrochlorothiazide . Validation and estimation using mobile phase of acetonitrile and phosphate buffer. International Journal for Pharmaceutica l Research Scholars. (IJPRS) 2014, V-3, I-2, 408-416. Vignan Pharmacy College,Vadlamudi. 13
  14. 14. Vignan Pharmacy College,Vadlamudi. 14 S. NO AUTHORS TITLE STUDY JOURNAL NAME YEAR,VOL UME, ISSUE & PG.NO 3 T. M. Kalyankar*, S. J. Wadher, S. S. Pekamwar N.G. Doiphode Development and Validation of RP- HPLC method for Estimation of Hydrochlorothiazide and Irbesartan in Pharmaceutical Preparation. Analysis using C18 column using methanol as solvent. International Journal of PharmTech Research CODEN (USA): IJPRIF Jan-March 2014, Vol.6, Issue No.1, 330-336. 4 T. Gopala Swamy, K. Nagaraju, A. Lakshmana Rao RP-HPLC Method for the Simultaneous Estimation of Telmisartan and Hydrochlorothiazide in Pharmaceutical Dosage Form. RP-HPLC method was found to be rapid,precise,accurate, Selective. International Journal of Drug Developmen t & Research. (IJDDR) October- December 2011,Vol. 3 ,Issue 4, 362-368.
  15. 15. S.NO AUTHORS TITLE STUDY JOURNAL NAME YEAR,VO LUME, ISSUE & PG.NO 5 Lakshmanarao. A, Bhaskhara Raju .V Simultaneous Estimation Of Valsartan And Hydrochlorothiazide In Tablets By RP- HPLC Method. Column having isocratic mode with mobile phase of acetonitrile and phosphate buffer. IJPIR Jul-sep 2011, Volume 01, Issue-03, 170-174. 6 Hany Mohammed Hafez*, Abdullah Ahmed Elshanawane Quantitative Determination of three Angiotensin-II- receptor Antagonists in Presence of Hydrochlorothiazide by RP-HPLC in their Tablet Preparations. C18 column , phosphate buffer is used. Iranian Journal of Pharmaceuti cal Research. 2013, 12 (4), 635-643. Vignan Pharmacy College,Vadlamudi. 15
  16. 16. PLAN OF WORK: Vignan Pharmacy College,Vadlamudi. 16 Plan of RP-HPLC technique:  Literature collection regarding various aspects like physicochemical properties of the drugs  Procurement of drug reference standards, reagents, solvents, other chemicals and formulations.  Establishment of initial chromatographic conditions.  Optimization of the chromatographic conditions to achieve the system suitability goals and validating the method according to ICH guidelines.  Applying the optimized method for estimation of selected drug candidates in tablets and pharmaceutical dosage forms.
  17. 17. Vignan Pharmacy College,Vadlamudi. 17
  18. 18. Table 1. Commercial brand names of TELMI, LOS , VAL, IRBE and HYDRO combinations used for the present study. Brand name Formulation Labeled amount (mg) Manufacturer Telmisat-H (40 mg) Tablets Telmisartan- 40 mg Hydrochlorothiazide- 12.5 mg Biocon laboratories Zargo-H (50 mg) Tablets Losartan- 50 mg Hydrochlorothiazide- 12.5 mg Biocon laboratories Olmicep-H (40mg) Tablets Olmesartan –40 mg Hydrochlorothiazide – 12.5 mg Cipla Ltd., Mumbai, India. Valent-H (80 mg) Tablets Valsartan- 80 mg Hydrochlorothiazide- 12.5 mg Lupin Laboratories, Hyderabad. Vignan Pharmacy College,Vadlamudi. 18
  19. 19. Table 2. Materials used in the present study S.No. Materials Procured from 1. Telmisartan Aristo Pharmaceuticals Pvt.Ltd., Bombay. 2. Losartan Hetero Labs Ltd., Hyderabad 3. Olmesartan Hetero Labs Ltd., Hyderabad 4. Valsartan Anant Pharmaceuticals, Kamal, Haryana. 5. Acetonitrile HPLC grade Thermo Fisher Scientific India Pvt. Ltd., Mumbai. 6. Water HPLC grade Merck Specialties Pvt. Ltd., Mumbai. 7. Methanol HPLC grade Merck Specialties Pvt. Ltd., Mumbai. 8. Dipotassium hydrogen phosphate Thermo Fisher Scientific India Pvt. Ltd., Mumbai. 9. Potassium dihydrogen phosphate Glaxo Smith Kline Pharmaceuticals Ltd., Mumbai. Vignan Pharmacy College,Vadlamudi. 19
  20. 20. Table 3. Instruments used in the present study S.No. Instrument Name of the company and model 1. HPLC Shimadzu LC-20AT Prominence Liquid Chromatograph with Shimadzu SPD-20A Prominence UV-Vis detector, Welchrom C18 Column (4.6 X 250 mm, 5μm), with Rheodyne manual loop injector (20 μL) and Spinchrom data acquisition software. 2. UV-Vis spectrophotometer UV-Visible Spectrophotometer ELICO – SL - 210 with Spectra treats(TM) software. 3. Weighing balance Essae vibra AJ (0.001g), Essae-Teraoka Ltd. 4. pH meter Elico LI120 pH meter, Elico India Ltd. 5. Ultrasonicator Ultrasonic bath sonicator, PCI ltd., Mumbai. 6. Vacuum pump Single Stage Vacuum Pump. Vignan Pharmacy College,Vadlamudi. 20
  21. 21. Preparation of reagents and standards:  Preparation of phosphate buffer pH 3.3 Phosphate buffer with 10 mM was prepared by dissolving 6.056 g KH2PO4 in 445 mL of HPLC grade water. To this said solution 55 mL of 0.1M H3PO4 was added to adjust the pH 3.3.  Preparation of mobile phase: The above stated prepared phosphate buffer (pH 3.3) acetonitrile were mixed completely in the proportion of 50: 50 v/v and was filtered through 0.45 µm nylon membrane filter and degassed by sonication.  Preparation of standard stock solution: Stock standard solutions containing (1.25, 0.4, 0.5, 0.4, 0.8 mg/mL) of Hydrochlorothiazide, Telmisartan, Losartan, Olmesartan and Valsartan respectively were prepared by dissolving (12.5, 40, 50, 40, 80 mg) of each in methanol in 100 mL volumetric flask respectively. It was then sonicated for 15 minutes and the final volume of solutions was made up to 100 mL with methanol to get stock standard solutions. Vignan Pharmacy College,Vadlamudi. 21
  22. 22. Preparation of sample solution: A composite of ten Telmisat-H, Zargo-H, Olmecip-H and Valent-H tablets are prepared by grinding them to a fine, uniform size powder, triturated using mortar and pestle. After calculating the average tablet weight, amounts of powder equivalent to HCTZ, TELM, LOSA, OLME and VALS respectively are prepared by dissolving (12.5, 40, 50, 40, 80 mg) respectively of each type of tablets were accurately weighed and transferred separately to 100 mL volumetric flasks respectively. Solutions were sonicated for 15 min and the solutions were then filtered through 0.45 µm nylon membrane filters. Aliquots of appropriate volume (10 mL) were transferred to 100 mL calibrated flasks and diluted to volume with mobile phase to furnish the mentioned concentration above. Vignan Pharmacy College,Vadlamudi. 22
  23. 23. RESULTS: Method Development:  Selection of a common detection wavelength:  The ultra violet spectrum of four Sartans and hydrochlorothiazide were scanned individually in the region between 200 - 400 nm. The UV overlain spectra of these five drugs showed that they absorbed at 238 nm. So this wavelength was selected for the determination of sartans. Figure 1 shows UV overlain spectra of four sartans with HCTZ. Vignan Pharmacy College,Vadlamudi. 23
  24. 24. Vignan Pharmacy College,Vadlamudi. 24 Figure 1. UV overlain spectra of four sartans and HCTZ.
  25. 25. Method optimization:  Numerous trials were performed to obtain the better separations.  From all trials, eventually better reproducibility of the results and good resolution, good peak shape, short runtime, minimal peak tailing were well identified when mobile phase consisting of phosphate buffer mixture properly adjusted to pH 3.3, acetonitrile in the proportion of 50:50 v/v with satisfactory results. Table 4. Optimized chromatographic conditions 25Vignan Pharmacy College,Vadlamudi. Parameter Chromatographic conditions for 4 sartans & Hydrochlorothiazide Instrument Shimadzu LC-20AT Prominence liquid chromatograph Column Welchrom C18 column (4.6 X 250 mm, 5 µm) Detector Shimadzu SPD-20A prominence UV-VIS detector Mobile phase 10 mM phosphate buffer (pH 3.3) : Acetonitrile 50:50, v/v Flow rate 1mL/min wave length UV at 238 nm Run time 8 minutes Temperature Ambient temperature (25 0C) Injection volume 20 µL Name of the drugs HCTZ TELM LOSA OLME VALS Retention time (minutes) 3.300 3.780 4.467 5.247 5.570 Th.Pl (Efficiency) 12,312 12,468 14,715 15,250 17,188 Theoretical plates per meter 246247 247368 294308 305005 343756 Resolution - 3.776 4.862 4.931 2.008 Tailing factor 1.194 1.116 1.182 1.160 1.154
  26. 26. Figure 2. Typical chromatogram showing the HCTZ, TELM, LOSA, OLME AND VALS in a synthetic mixture. Vignan Pharmacy College,Vadlamudi. 26
  27. 27. Method validation:  The developed analytical method was validated in pursuance of the guide lines of ICH. SPECIFICITY:  The purpose of this study is to determine the effect of excipients and other additives that are usually present in the formulations. The test results obtained were compared with the results of standard drug. Vignan Pharmacy College,Vadlamudi. 27 Table 5. Results of specificity Result: The present study shows that the ingredients are not interfering with the developed method. Name HCTZ TELM HCTZ LOSA HCTZ OLME HCTZ VALS Mobile phase No peaks No peaks No peaks No peaks No peaks No peaks No peaks No peaks Placebo No peaks No peaks No peaks No peaks No peaks No peaks No peaks No peaks Individual Separate standard solutions. Peak for HCTZ and TELM at 3.300 min. and 3.780 min. respectively. Peak for HCTZ and LOSA at 3.300 min. and 4.467 min. respectively. Peak for HCTZ and OLME at 3.300 min. and 5.247 min. respectively. Peak for HCTZ and VALS at 3.300 min. and 5.570 min. respectively.
  28. 28.  The linearity of the method was studied by analyzing different concentration of the drugs. According to ICH recommendations at least five concentrations are to be used. In this study five Concentrations were chosen, in the ranges of 2.5-12.5, 8-40, 10-50, 8-40, 16-80 µg/mL. Calibration curve is constructed by plotting concentration on X-axis and the peak areas on Y-axis.  The least square analysis method was adopted for achieving slope, intercept and correlation coefficient, regression data values. Vignan Pharmacy College,Vadlamudi. 28
  29. 29. Vignan Pharmacy College,Vadlamudi. 29 Linearity method HCTZ LOSA TELM OLME VALS Conc. μg/mL Peak area, mV.s. Conc. μg/mL Peak area, mV.s. Conc. μg/mL Peak area, mV.s. Conc. μg/mL Peak area, mV.s. Conc. μg/mL Peak area, mV.s. 0 0 0 0 0 0 0 0 0 0 2.5 217.93 10 209.972 8 205.794 8 175.56 16 234.722 5 435.79 20 416.85 16 410.63 16 382.42 32 466.872 7.5 669.786 30 642.32 24 628.47 24 605.81 48 706.7 10 889.52 40 862.97 32 853.23 32 839.64 64 928.68 12.5 1092.803 50 1080.64 40 1068.11 40 1053.92 80 1168.004 Table 6. Results relating to Linearity data
  30. 30. Table 7. Regression analysis data relating to HCTZ, TELM, LOSA, OLME and VALS Vignan Pharmacy College,Vadlamudi. 30 Parameter HCTZ LOSA TELM OLME VALS Detection wavelength(λmax) UV at 238 nm UV at 238 nm UV at 238 nm UV at 238 nm UV at 238 nm Linearity range (µg/mL) 2.5-12.5 µg/mL 8-40 µg/mL 10-50 µg/mL 8-40 µg/mL 16-80 µg/mL Regression equation (Y =aX + b) Y = 88.146 X + 0.0586 Y = 27.099 X - 6.5173 Y = 21.431 X - 8.5685 Y = 13.36 X - 25.10 Y = 14.576 X + 1.0686 Slope(a) 88.146 27.099 20.968 13.366 14.576 Intercept(b) 0.0586 6.5173 8.5685 25.101 1.0686 Standard error of slope (Sa) 0.7592 0.1537 0.24642 0.2900 0.06053 Standard error of intercept (Sb) 1.9488 0.9310 1.49213 1.4277 2.9327 Regression coefficient (R2) 0.9997 0.9998 0.9997 0.9981 0.9999 % Relative standard deviation* 1.1875 2.4993 0.5239 0.104 1.050 Percentage range of errors 0.05 significance level 0.01 significance level 1.02904 1.6138 1.15695 1.8144 0.54992 0.86242 0.12232 0.19183 0.36373 0.57042
  31. 31. Figure 3. Standard chromatogram relating to HCTZ Figure 4. Standard chromatogram relating to TELM Figure 5. Standard chromatogram relating to LOSA Vignan Pharmacy College,Vadlamudi. 31 CHROMATOGRAMS
  32. 32. Figure 6. Standard chromatogram relating to OLME Figure 7. Standard chromatogram relating to VALS Vignan Pharmacy College,Vadlamudi. 32
  33. 33. Figure 8: Standard chromatogram of HCTZ (2.5 μg/ml) and TELM (8 μg/ml) Figure 9. Standard chromatogram of HCTZ (2.5 μg/ml) and LOSA (5 μg/ml). Figure 10. Standard chromatogram of HCTZ (2.5 μg/ml) and OLME (8 μg/ml). Vignan Pharmacy College,Vadlamudi. 33
  34. 34. Vignan Pharmacy College,Vadlamudi. 34 Fig. 11. Standard chromatogram of HCTZ (2.5 µg/ml) and VALS (16 µg/ml)
  35. 35. Vignan Pharmacy College,Vadlamudi. 35 Fig. 12. Sample chromatogram of HCTZ (2.5 µg/mL) and TELM (8 µg/mL) Fig. 13. Sample chromatogram of HCTZ (2.5 µg/mL) and LOSA (5 µg/mL)
  36. 36. Vignan Pharmacy College,Vadlamudi. 36 Fig. 14. Sample chromatogram of HCTZ (2.5 µg/mL) and OLME (8 µg/mL) Fig. 15. Sample chromatogram of HCTZ (2.5 µg/mL) and VAL (16µg/mL)
  37. 37. Figure 16. Linearity plot pertaining to HCTZ Figure 17. Linearity plot pertaining to LOSA Figure 18. Linearity plot pertaining to TELM Vignan Pharmacy College,Vadlamudi. 37 y = 88.146x + 0.0586 R² = 0.9997 0 200 400 600 800 1000 1200 0 5 10 15 peakarea,mV.s Concentration, µg/mL. y = 27.099x - 6.5173 R² = 0.9998 -200 0 200 400 600 800 1000 1200 0 10 20 30 40 50 peakarea,mV.s Concentration, µg/mL. y = 21.431x - 8.0585 R² = 0.9997 -200 0 200 400 600 800 1000 1200 0 20 40 60 peakarea,mV.s Concentration, µg/mL.
  38. 38. Figure 20. Linearity plot pertaining to VALS Vignan Pharmacy College,Vadlamudi. 38 Figure 19. Linearity plot pertaining to OLME y = 13.366x - 25.101 R² = 0.9981 -200 0 200 400 600 800 1000 1200 0 20 40 60 80 100 PeakareamV.S Concentration µg/mL. y = 14.576x + 1.0686 R² = 0.9999 0 200 400 600 800 1000 1200 1400 0 20 40 60 80 100 peakarea,mV.s concentration, µg/mL.
  39. 39. Precision:  The repeatability and intermediate precision experiments were conducted by determining the intra - day and inter - day precision of the method for HCTZ with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with VALS.  The intraday precision was done by repeating the assay thrice for the 3 levels in the same day, under the same experimental conditions Inter - day precision was done by taking over the assay on 3 different days, three times on the each day for the 3 concentration levels respectively. The results of precision study were stated in terms of % RSD which was calculated and results were within the acceptable criteria of NMT 2.0. The results of precision study are presented in table 8. Vignan Pharmacy College,Vadlamudi. 39
  40. 40. Vignan Pharmacy College,Vadlamudi. 40 Accuracy/Recovery studies:  The accuracy of the method was assessed by standard addition method.  Recovery tests were carried out by analyzing mixtures of HCTZ with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with VALS with different compositions. Known amounts of standards drugs were added to a pre-analyzed sample at 3 different levels 80 %, 100 % and 120 % and the mixed standard solutions were analyzed in triplicate at every level as per suggested method. The percent of individual combination of mixtures recovery and % RSD results of accuracy data is shown in table 8.
  41. 41. Robustness: The robustness of developed analytical method was proven by the analysis of HCTZ with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with VALS under different experimental conditions such as making deliberate changes in chromatographic conditions like flow rate (± 0.2 ml/min), detection wavelength (±5 nm) and mobile phase composition (±5 %). All results were well within acceptable limits. LOD and LOQ: Limit of Detection is the lowest concentration in a sample that can be detected, but not necessarily quantified under the stated experimental conditions. Limit of quantification is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy. LOD and LOQ were calculated using following formula LOD = 3.3σ/S and LOQ = 10σ/S, where SD = standard deviation of response (peak area) and S = slope of the calibration curve. The LOD and LOQ results are shown in the following table. Vignan Pharmacy College,Vadlamudi. 41
  42. 42. Formulation Telmisat – H (40mg) Zargo – H (50mg) Olmecip (40mg) Valent - H Composition TELM HCTZ LOSA HCTZ OLME HCTZ VALS HCTZ Linearity (μg/mL) 8 - 40 2.5 – 12.5 5 - 50 2.5 – 12.5 8 – 40 2.5 – 12.5 16 - 80 2.5 – 12.5 LOD & LOQ (μg/mL) 0.570 & 1.889 0.295 & 0.982 0.736 & 2.43 0.299 & 0.986 1.195 & 3.958 0.292 & 0.983 0.116 & 0.352 0.296 & 0.980 Assay ± SD (n = 6) 99.95 ± 1.13 98.16 ± 0.38 99.85 ± 1.16 98.21 ± 0.45 99.90 ± 1.10 99.19 ± 0.16 100.12 ± 0.15 99.59 ± 0.12 Mean % recovery 100.02± 0.68 100.01 ± 0.15 100.06 ± 0.20 99.95 ± 0.09 100.41 ± 0.35 99.93 ± 0.11 100.05± 0.14 99.96 ± 0.15 Precision Intra day (n=6) 0.125 0.131 0.346 0.129 0.435 0.171 0.312 0.213 Inter day (n=3) 0.165 0.321 0.326 0.434 0.657 0.322 0.319 0.435 Table 8. Summary of various validation parameters
  43. 43. Formulation TELMISAT – H (40mg) ZARGO – H (50mg) OLMECIP – H (40mg) VALENT – H (80 mg) Robustness, % Assay ± % RSD Flow rate (± 2 mL/min) 0.8 mL/min 99.81 ± 1.05 97.94 ± 0.46 99.98 ± 1.04 98.93 ± 0.49 98.70 ± 1.02 98.96 ± 0.25 99.91 ± 0.29 100.96 ± 0.23 1.2 mL/min 100.12± 0.97 99.54 ± 0.23 98.76 ± 1.32 98.04 ± 0.36 100.03 ± 1.38 99.97 ± 0.35 100.5 4 ± 0.77 98.74 ± 0.71 Detection wave Length (± 5 nm) 233 nm 99.97 ± 1.46 99.43 ± 0.29 98.42± 1.46 97.95 ± 0.98 100.21 ± 1.37 99.96 ± 0.84 100.4 5 ± 0.97 99.98 ± 0.54 243nm 97.98 ± 1.09 98.04 ± 0.76 100.05 ± 1.03 98.94 ± 1.08 98.79 ± 0.37 98.76 ± 1.09 99.94 ± 1.43 98.47 ± 1.11 Mobile phase compos - ition (± 5%) 45:55 v/v 98.74 ± o.79 99.42 ± 0.99 99.93 ± 1.42 97.92 ± 0.68 98.91 ± 0.94 100.07 ± 0.93 99.01 ± 0.61 98.76 ± 0.37 55:45 v/v 99.96 ± 1.2 97.81 ± 0.27 98.54 ± 0.86 98.86 ± 0.30 98.12 ± 1.57 98.72 ± 1.09 99.49 ± 0.74 100.03 ± 1.18
  44. 44. CONCLUSION: • Statistical analysis lucidly proves that it is feasible for analysis of said ARB’s in their formulations in single run without changing the mobile phase composition and chromatographic conditions. • This method was very fast, cost effective, precise, accurate sensitive, highly efficient and robust than the existing adopted methods. It is noticed that the method is free from interferences of the excipients and additives utilized in the preparation of above stated ARB’s after validation. • This new RP-HPLC method satisfactorily separated all the above said combination drugs with short retention time (eight minutes and elution window of only two minutes), optimum peak shape, good separation and best reproducible results. Hence, it is apt to conclude that it is successfully feasible for the application of routine analysis of said ARB’s agents individually or binary combinations of HCTZ with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with VALS in quality control laboratories for usual analysis. Vignan Pharmacy College,Vadlamudi. 44
  45. 45. REFERENCES: 1. Development and Validation of RP-HPLC Method for the Estimation and Separation of Valsartan, Losartan and Irbesartan in Bulk and Pharmaceutical Formulation, Youssef ,IJPSRR , Jan – Feb 2014, 24(2), 311-314. 2. Quantitative Determination of three Angiotensin-II-receptor Antagonists in Presence of Hydrochlorothiazide by RP-HPLC in their Tablet Preparations. Hany Mohammed Hafez, Abdullah Ahmed, Elshanawane, Lobna Mohammed Abdelaziz, Iranian Journal of Pharmceutical Research, 2013; 12(4): 635–643. 3. RP-HPLC Method for the Simultaneous Estimation of Telmisartan and Hydrochlorothiazide in Pharmaceutical Dosage Form, Gopala Swamy, IJDDR, 2011,Vol. 3 ,Issue 4,362-368. 4. RP-HPLC method for simultaneous estimation of Telmisartan and Hydrochlorothiazide in tablet dosage form. Wankhede SB, Tajne MR, Gupta KR, Wadodkar SG, Indian Journal of Pharmaceutical Sciences, 2007; 298-300. 5. ICH Q2 (R1): Validation of analytical procedures (definitions and terminology) 2005: 9-10. Vignan Pharmacy College,Vadlamudi. 45
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  47. 47. Vignan Pharmacy College,Vadlamudi. 47
  48. 48.  We Heart fully thank our guide Dr. P. Ravisankar sir . I also thank Hetero Labs Limited unit-III, Jeedimetla, Hyderabad for providing gift sample of pure drugs. ACKNOWLEDGEMENT Vignan Pharmacy College,Vadlamudi. 48
  49. 49. 49Vignan Pharmacy College,Vadlamudi.

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