We studied the developed formulation in rabbits in order to study its in vivo behaviour. Hence we used rabbit plasma.

References:-
1) Lacey, L.F., Keene, O.N., Duquenoy, C., Bye, A., 1994.Evaluation of different indirect measures of rate of drug absorption in comparative pharmacokinetic studies. J.Pharm. Sci. 83, 212–215.
2)Comparison of the lipoprotein profiles obtained from rat, bovine, horse, dog, rabbit and pig serum by a new two-step ultracentrifugal gradient procedure.Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, Volume 84, Issue 1, 1986, Pages 83-89 B. Hollanders, A. Mougin, F. N'Diaye, E. Hentz, X. Aude, A. Girard

We cannot define any tmax when any drug adminstred by iv route becuase drug reach systemic ciruclatiom as soon as it administered and it will give 100% and till now methods available for analysis of valsartan in plasma except HPLC.

References:-
1)L. Gonzalez, J. A. Lopez, R. M. Alonso, R. M. Jimenez Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection.Journal of Chromatography A, Volume 949, Issues 1-2, 8 March 2002, Pages 49-60.
2)Maria del Rosario Brunetto, Yaritza Contreras, Sabrina Clavijo, Dina Torres, Yelitza Delgado, Fernando Ovalles, Carlos Ayala, Máximo Gallignani, Jose Manuel Estela, Victor CerdaMartin.Determination of losartan, telmisartan, and valsartan by direct injection of human urine into a column-switching liquid chromatographic system with fluorescence detection.Journal of Pharmaceutical and Biomedical Analysis, Volume 50, Issue 2, 8 September 2009, Pages 194-199 .

3)Y. Li, Z. Zhao, X. Chen, J. Wang, J. Guo, F. Xiao,HPLC determination of valsartan in human plasma,Yaowu Fenxi Zazhi 20 (6) (2000) 404–406

1)Telmisartan belongs to same class of drug and have structural similarity and so we used as the internal standard in the HPLC analysis.
2)To get precise result and to avoid errors. we go for internal standard for the analysis of valsartan.
3)We used different mobile phases and they increased the runtime more than 20minutes.Iwill try for the next method in order to decrease the mobile phase runtime and thank for ur suggestion

References :-
1) E. Satana, S. Altinay, N.G. Goger, S.A. Ozkan, Z. Senturk,Simultaneous determination of valsartan and hydrochlorothiazidein tablets by first-derivative ultraviolet spectrophotometry and LC, J. Pharm. Biomed. Anal. 25(5–6) (2001) 1009–1013.

2) L. Gonzales, R.M. Alonso, R.M. Jimenez, High-performance liquid-chromatographic method for the screening angiotensin II receptor antagonists in human urine, Chromatographia 52 (11–12) (2000) 735–740.

3) A.Sioufi, F. Marfil, J. Godbillon, Automated determination of an angiotensin II receptor antagonist, CGP 48933, in plasma by high-performance liquid chromatography,J. Liq. Chromatogr. 17 (10) (1994) 2179–2186.

4)Y. Li, Z. Zhao, X. Chen, J. Wang, J. Guo, F. Xiao,HPLC determination of valsartan in human plasma,Yaowu Fenxi Zazhi 20 (6) (2000) 404–406.

1)Rats cannot be used for oral administration of tablets or capsules. Hence we used Rabbits. And can be easily handled when compared to other animals(1).

2)Freeze thaw cycle is one of the parameter useful to determine the stability of the product and it is the process of freezing and thawing, and it is necessary to show that the measured analyte concentration does not change after the sample has been re-frozen and subsequently re-analysed.

In this present work, we stored at -70degrees, then thawed at room temperature and analysed. Again stored at -70oc like this upto (3 cycles) we performed(2).

3)UPLC means Ultra Performance Liquid Chromatography. By using smaller particles, speed and peak capacity (number of peaks resolved per unit time in gradient separations) can be extended to new limits, termed Ultra Performance Liquid Chromatography. The technology takes full advantage of chromatographic principles to run separations using columns packed with smaller particles and/or higher flow rates for increased speed,with superior resolution and sensitivity(3).
UPLC provided advantages over HPLC regarding higher therotical plates, the higher peak capacity and the highest separation quality. With UPLC increased resolution in shorter run times can generate more information faster without sacrifices(4).

UPLC can also be used to significantly improve the success of the drug discovery process. Drug discovery is heavily dependant upon the early prediction of metabolic fate and interactions of drug candidate molecules. To prevent “poor” candidates from progressing through the discovery process, factors such as metabolic stability, toxic metabolite production, p450 inhibition, and induction are all routinely monitored.

References:-
1)Analysis and pharmacokinetics of bulaquine and its major metabolite primaquine in rabbits using an LC-UV method— a pilot study Journal of Pharmaceutical and Biomedical Analysis, Volume 32, Issue 1, 24 April 2003, Pages 141-150 .Jawahar Lal, Nitin Mehrotra, Ram Chandra Gupta.
2)B. A. Kozikowski, T. M. Burt, D. A. Tirey, L. E. Williams, B. R. Kuzmak, D. T.Stanton, K. L. Morand, and S. L. Nelson,The effect of freeze/thaw cycles on the stability of compounds in DMSO,J.Biomol.Screen.8(2003), 210–215.
3) Jerkovich, A.D, LoBrutto,R., and Vivilecchia, R.V., The Use of Acquity UPLC™ in Pharmaceutical Development, published in LC-GC,( 2005 ).
4)Novakova, L., Matyosva, l., and Solich , p. (2005). Advantages of application of UPLC in pharmaceutical analysis .Talanta,68,909-918.

1) EDTA is used to prevent coagulation of blood while collecting the blood samples from marginal vein of rabbit. Because rabbit blood clots relatively quickly at room
temperature and to avoid artifactural changes associated with clot formation.
2)Yes, promising results will obtain when HPLC is used along with MS but in our study we found HPLC is sufficient.

References:-

1. Harcourt-Brown F. Textbook of Rabbit Medicine.Woburn, MA: Butterworth Heinemann, 2002.

2. Benson KG, Paul-Murphy J. Clinical pathology of the domestic rabbit. Vet Clin North Am: Exotic AnimPract 1999;3(2):539-552.

3. Murray MJ. Rabbit and ferret sampling and artefact considerations. In Fudge AM (ed): Laboratory Medicine: Avian and Exotic Pets. Philadelphia: WB Saunders, 2000, pp 265-268.

4. McLaughlin RM, Fish RE. Clinical biochemistry and hematology. In Manning PJ, Ringler DH, Newcomer CE (eds). The Biology of the Laboratory Rabbit, 2nd ed. San Diego:Academic Press, 1994, pp 111-127.

Mobile phase pH varied at 2, 3, and 4 keeping the composition of ACN: KH2PO4 buffer (10 mM) 40:60 and flow rate of 1.0 mL/min fixed. There was no difference in retention time (Rt) between the pH 2.0 and pH 4.0. At pH 3.0 there was good separation between valsartan andInternal standard( IS), Rt was found to be 10.8 ± 0.3 and 16.9 ± 0.2 min respectively.
We did not try for other mobile phases for the valsartan analysis. Based on available literature on valsartan we selected ACN: KH2PO4 buffer as mobile phase.
References :-

1) E. Satana, S. Altinay, N.G. Goger, S.A. Ozkan, Z. Senturk,Simultaneous determination of valsartan and hydrochlorothiazidein tablets by first-derivative ultraviolet spectrophotometry and LC, J. Pharm. Biomed. Anal. 25(5–6) (2001) 1009–1013.

2) L. Gonzales, R.M. Alonso, R.M. Jimenez, High-performance liquid-chromatographic method for the screening angiotensin II receptor antagonists in human urine, Chromatographia 52 (11–12) (2000) 735–740.

3)Y. Li, Z. Zhao, X. Chen, J. Wang, J. Guo, F. Xiao,HPLC determination of valsartan in human plasma,Yaowu Fenxi Zazhi 20 (6) (2000) 404–406.

Specificity/selectivity test was performed by running blank plasma from different rabbits, mobile phase mixture without adding any drug or Internal Standard (IS) to confirm absence of any peak at drug and Internal Standard( IS) Retention time.

References :-
1) E. Satana, S. Altinay, N.G. Goger, S.A. Ozkan, Z. Senturk,Simultaneous determination of valsartan and hydrochlorothiazidein tablets by first-derivative ultraviolet spectrophotometry and LC, J. Pharm. Biomed. Anal. 25(5–6) (2001) 1009–1013.

2)Y. Li, Z. Zhao, X. Chen, J. Wang, J. Guo, F. Xiao,HPLC determination of valsartan in human plasma,Yaowu Fenxi Zazhi 20 (6) (2000) 404–406.

1) System suitability parameters include theoretical plates and retention time, asymmetry and HETEP,Resolution , Capacity Factor. In order to achieve an optimum separation, following conditions were studied: (i) Mobile phase pH varied at 2, 3, and 4 keeping the composition of ACN: KH2PO4 buffer (10 mM) 40:60 and flow rate of 1.0 mL/min fixed. (ii) Mobile phase composition varied at 50:50, 40:60, and 30:70 with pH and flow rate kept constant at 3 and 1.0 mL/min, respectively. (iii) Flow rate was varied (0.8, 1.0, and 1.2 mL/min) with mobile phase composition and pH maintained at 40:60 and 3, respectively (iv) The phosphate buffer (pH 3.0) was prepared in different strengths such as 10, 20 and 30 mM and chromatograms were recorded with mobile phase composition, flow rate and pH maintained at 40:60, 1 mL/min and 3, respectively. The effects of different level of all these three factors were systematically addressed on system suitability parameters such as resolution, theoretical plates, retention time (Rt), capacity factor, asymmetry, and HETP of valsartan and telmisartan.
All values were found to be within the limits in the optimized method.

System suitability parameter Acceptance criteria
Peak asymmetry (10%) Should not be more than 2.0
Capacity factor (k1) Should not be less than 2.0
Theoretical plates/meter Should not be less than 2000
%CV of 6 injection %RSD should be less than 1.0

2)Administered orally using feeding cannula.
3)Formulations were administered intact and care was taken to reach stomach without any obstructions and after administration water was given to avoid spitting.
4) Randomized,open and Parallel design is followed for bioavailability studies.
5)PK solution software was used to calculate Phamacokinetic parameters and independent sample T-test using SPSS software to compare the parameters between groups.

References :-
1)Y. Li, Z. Zhao, X. Chen, J. Wang, J. Guo, F. Xiao,HPLC determination of valsartan in human plasma,Yaowu Fenxi Zazhi 20 (6) (2000) 404–406.

2)E.Francotte, A. Davatz, P. Richert, Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester, J. Chromatogr. B 686 (1996) 77–83.

3)G. Carlucci, V. di Carlo, P. Mazzeo, Simultaneous determination of valsartan and hydrochlorothiazide in tablets by high-performance liquid chromatography, Anal. Lett. 33 (12) (2000) 2491–2500.

4)M.A. Kane, N. Chen, S. Sparks, J.L. Napoli, Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS, Biochem. J. 388 (2005) 363–369.

5) Benz JR, Black HR, Graff A, et al. Valsartan and hydrochlorothiazide in patients with essential hypertension. A multiple dose, double-blind, placebo controlled trial comparing combination therapy with monotherapy. J Hum Hypertens. 1998;12:861–866.
6) G. Carlucci, V. di Carlo, P. Mazzeo, Simultaneous determination of valsartan and hydrochlorothiazide in tablets by high-performance liquid chromatography, Anal. Lett. 33 (12) (2000) 2491–2500.

7)Tatar, S., & Saglik, S. (2002). Comparision of UV- and second derivative spectrophotometric and HPLC methods for the determination of valsartan in pharmaceutical formulation. J. Pharm. Biomed. Anal., 30, 371–375

Yes, in this method of development for the quantification of valsartan we used uv detector and simple liqid extaction method to extract the drug from plasma

References :-
1)Y. Li, Z. Zhao, X. Chen, J. Wang, J. Guo, F. Xiao,HPLC determination of valsartan in human plasma,Yaowu Fenxi Zazhi 20 (6) (2000) 404–406

2)G. Carlucci, V. di Carlo, P. Mazzeo, Simultaneous determination of valsartan and hydrochlorothiazide in tablets by high-performance liquid chromatography, Anal. Lett. 33 (12) (2000) 2491–2500.

3) Benz JR, Black HR, Graff A, et al. Valsartan and hydrochlorothiazide in patients with essential hypertension. A multiple dose, double-blind, placebo controlled trial comparing combination therapy with monotherapy. J Hum Hypertens. 1998;12:861–866.

4)Tatar, S., & Saglik, S. (2002). Comparision of UV- and second derivative spectrophotometric and HPLC methods for the determination of valsartan in pharmaceutical formulation. J. Pharm. Biomed. Anal., 30, 371–375.

The various detectors often used in HPLC may be categorized into three major heads, namely
(i) Bulk-property detectors : They specifically measure the difference in some physical property of
the solute present in the mobile-phase in comparison to the individual mobile-phase.
(a) Refractive-index detectors, and
(b) Conductivity detectors.

(ii) Solute-property detectors. They critically respond to a particular physical or chemical characteristic
of the solute which should be ideally and absolutely independent of the mobile-phase being used. But complete independence of the mobile-phase is hardly to be seen, however, signal discrimination is good enough to enable distinctly measurable experimental procedures with solvent changes, such as : gradient-elution.
The solute-property detectors include :
(a) UV-detectors, and
(b) Fluorescence Detectors.

(iii) Multipurpose detectors : Besides, providing a high degree of sensitivity* together with a broadlinear-response-attainable range, invariably a particular situation critically demands detectors of
more selective nature that could be accomplished by using ‘multipurpose detectors’, such as : “Perkin-Elmer ‘3D’ System”that combines UV absorption, fluorescence and conductometric detection.

(iv) Electrochemical detectors : ‘Electrochemical detector’ in HPLC usually refers to either
amperometric or coulometric detectors, that specifically measure the current associated with the
reduction or oxidation of solutes. As only a narrow spectrum of compounds undergo electrochemical
oxidation, such detectors are quite selective ; and this selectivity may be further enhanced by monitoring
the potential applied to the detector so as to differentiate between various electroactive species. Naturally, electrochemical detection essentially makes use of conducting mobile phases, for instance : inorganic salts or mixtures of water with water-miscible organic solvents.

References:-
 D. Wickam, in A Practical Guide to HPLC Detection, D. Parriott, ed., Academic Press, San Diego, CA, 1993, p. 67.

 Dorschel, C.A., Ekmanis, J.L., Oberholtzer, J.E., Warren, F.V. and Bidlingmeyer, B.A. (1989) LC detectors:evaluation and practical implications of linearity. Analytical Chemistry, 61, 951–968.

 Fielden, P.R. (1992) Recent developments in LC detector technology. Journal of Chromatographic Science, 30, 45–52.

 L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Non-ionic samples: Reversed- andnormal phase HPLC, in Practical HPLC Method Development, 2nd ed., Wiley,New York, 1997, pp. 266–289.

HPLC is routinely used for both qualitative and quantitative analyses of environmental,pharmaceutical, industrial, forensic, clinical, and consumer product samples.
 Determination of impurities in the compounds.
 Useful in pharmacokinetic studies
 Isolation of natural pharmaceutically active comounds like alkaloids and glycosides etc.
 Assay of drugs like doxorubicin, rifampicin etc

 Separation of Inorganic compounds, Enantiomers.

 Useful for identification and characterization of proteins, enzymes, nucleicacids and vitamins etc.

 Estimation of release kinetics of drugs.

References:-

1) C. Marcel, J. Alain, Normal-Phase Liquid Chromatography, in E. Katz, R. Eksteen,
P. Schoenmakers, and N. Miller (ed.), Handbook of HPLC, Marcel Dekker, NewYork, 1998, pp. 325–363.

2) P. Jandera, Comparison of various modes and phase systems for analytical HPLC
in K. Valko (ed.), Handbook of Analytical Separations, Separation Methods in Drug Synthesis and Purification,Vol. 1, Elsevier, New York, 2000, pp. 1–71.

3)R. LoBrutto, in J. Cazes (ed.), Encyclopedia of Chromatography, Marcel Dekker,
New York, 2001.

4) L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Non-ionic samples: Reversed- and
normal phase HPLC, in Practical HPLC Method Development, 2nd ed., Wiley,New York, 1997, pp. 266–289

HPLC methods using flurometric detector for bioanalytical and UV method for analytical. No, we cannot use the same method for the quantification of all AT1 receptor antagonists we have to modify the method as evident from Telmisartan(internal standard) retention time .

Refereces :-
1) Sevgi Tatar, Serap Saglik Comparison of UV- and second derivative-spectrophotometric and LC methods for the determination of valsartan in pharmaceutical formulation.Journal of Pharmaceutical and Biomedical Analysis, Volume 30, Issue 2, 5 September 2002, Pages 371-375.

2) Eric Francotte, Alexander Davatz, Paul Richert .Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester Journal of Chromatography B: Biomedical Sciences and Applications, Volume 686, Issue 1, 8 November 1996, Pages 77-83.
3) M. Rizzo, D. Ventrice, F. Casale, G.F. Caselli, F. Makovec Pharmacokinetic study of a new angiotensin-AT1 antagonist by HPLC.Journal of Pharmaceutical and Biomedical Analysis, Volume 48, Issue 2, 29 September 2008, Pages 422-427.
4) M. Rizzo, D. Ventrice, F. Monforte, S. Procopio, G. De Sarro, M. Anzini, A. Cappelli, F. Makovec Sensitive SPE–HPLC method to determine a novel angiotensin-AT1 antagonist in biological samples.Journal ofPharmaceutical and Biomedical Analysis, Volume 35, Issue 2, 16 April 2004, Pages 321-329.

Freeze thaw cycle is one of the parameter useful to determine the stability of the product and it is the process of freezing and thawing, and it is necessary to show that the measured analyte concentration does not change after the sample has been re-frozen and subsequently re-analysed.
In this present work, we stored at -70degees , then thawed at room temperature and analysed. Again stored at -70oc like this upto (3 cycles) we performed.

References :-

1)B. A. Kozikowski, T. M. Burt, D. A. Tirey, L. E. Williams, B. R. Kuzmak, D. T.Stanton, K. L. Morand, and S. L. Nelson, The effect of freeze/thaw cycles on the stability of compounds in DMSO, J. Biomol. Screen. 8 (2003), 210–215.

No other analytical methods used for this purpose.

References:-
1)Y. Li, Z. Zhao, X. Chen, J. Wang, J. Guo, F. Xiao,HPLC determination of valsartan in human plasma,Yaowu Fenxi Zazhi 20 (6) (2000) 404–406.
2) Tatar, S., & Saglik, S. (2002). Comparision of UV- and second derivative spectrophotometric and HPLC methods for the determination of valsartan in pharmaceutical formulation. J. Pharm. Biomed. Anal., 30, 371–375.

1) Extraction of drug from plasma is the critical step to be taken into consideration and developed method should be sensitive and specific.
2) This method development is same as analytical method.
3) System suitability parameters are same as analytical method.

References:-
1) Tatar, S., & Saglik, S. (2002). Comparision of UV- and second derivative spectrophotometric and HPLC methods for the determination of valsartan in pharmaceutical formulation. J. Pharm. Biomed. Anal., 30, 371–375.
2) Y. Li, Z. Zhao, X. Chen, J. Wang, J. Guo, F. Xiao,HPLC determination of valsartan in human plasma,Yaowu Fenxi Zazhi 20 (6) (2000) 404–406.
3) J. Macek, J. Klıma, P. Ptacek, J.Chromatogr. B. 832 (2006) 169–172.