INTRODUCTION:

Valsartan N-(I-oxopentyl)-N-[2-(14-tetrazol-5-yl)[ 1,1’– biphenyl]-4-yl] methyl]-L-valine, is an angiotensin II receptor antagonist, produces its BP lowering effects by antagonizing angiotensin II–induced vasoconstriction, aldosterone release and renal reabsorption of sodium¹.

Several high-performance liquid chromatographic (HPLC) methods are available for determination of Valsartan in plasma. All published assay employ native fluorescence of Valsartan and use fluorimetric detection^2-9. The sample preparation involves either liquid-liquid extraction with ethyl acetate², methyl-tert-butyl ether³, and protein precipitation⁹ or solid-phase extraction using C₈^5-7and cyclohexyl^2,7 cartridges. The limit of quantitation in these procedures is 5.0-130.0 ng/mL, run times are typically 10-30 min.

The aim of the present study was to develop a simple, rapid HPLC method for determination of Valsartan in human plasma. The overall speed of analysis was improved by the elimination of tedious preparation of mobile phase, extraction steps and optimization of chromatographic conditions.

MATERIAL AND METHOD:

Chemicals and Reagents:

The working standards of Valsartan and Irbesartan (Internal Standard) were obtained from Lupin Ltd. and Glenmark Pharmaceutical Ltd. respectively, with certificate of analysis.

Acetonitrile and Methanol used was HPLC grade obtained from E-Merck (India) Ltd., Ortho-Phosphoric acid and Tri ethylamine used was of A.R. grade. Methyl tertiary butyl ether (MTBE) was obtained form Riedel Chemicals UK.

Instrumentation:

The HPLC apparatus used for this experiment was JASCO 1500-series equipped with PU-1580 series intelligent pump, JASCO-AS-1550-10 intelligent auto sampler (1-100 µL) and a JASCO FP-920 intelligent Fluorescence detector.

Chromatographic Conditions:

The chromatographic conditions were optimized using stationary phase, Inertsil ODS-3V (250x4.6mm, 5µm). The composition of the mobile phase was (60:40, v/v) consisting of Acetonitrile : Water containing 0.1% Triethylamine. The pH of mobile phase was adjusted to 3.5 with 10% ortho-phosphoric acid. It was important to use the mobile phase with pH < 4, as the higher pH reduced significantly, the intrinsic fluorescence of Valsartan and other angiotensin II receptor antagonists^[6]. The flow rate was set at 1.0 ml/min

Table No.1: Mean Concentration for Calibration Standards in Human Plasma

Nominal Concentration (ng/mL)	Found Concentration (ng/mL)	C.V. (%)	Accuracy (%)
20	20.65	7.391	103.3
25	26.37	3.155	105.5
40	37.41	6.219	93.38
100	94.66	3.441	94.50
500	500.2	4.396	99.87
801	780.9	3.325	97.45
1005	1026	1.940	102.0
1523	1532	2.317	100.6

Mean Correlation : 0.9989 (n = 6)

using fluorescence detector set at 250 nm excitation wavelength and 371 nm emission wavelength.

Preparation of Stock Solutions and Standards:

Stock standard solutions of Valsartan were made by dissolving approximately 100 mg of accurately weighed substance in 100 mL of methanol. Separate solutions were prepared for the calibration curve samples and quality control once. The standard solutions were obtained by serial dilutions of stock solutions with solvent mixture methanol: water (2:8, v/v).

The calibration and quality control plasma samples were prepared by addition of standard solutions to drug-free plasma in volumes not exceeding 0.05% of the plasma volume.¹⁰

Preparation of the Sample:

The samples were stored in the deep freezer at -20⁰C and allowed to thaw at room temperature before processing. To 1 ml plasma, contained in 15 ml centrifuge tubes, fixed aliquots of the internal standard (50 µl of 14 µg/ml solution), Irbesartan were added and tubes were vortexed for 1 min. The solutions were then acidified with 20 µl glacial acetic acid, followed by addition of 5 mL extraction solvent (Methyl tertiary butyl ether). The test tubes were vortexed for 60 sec and centrifuged at 3000 rpm for 10 min. Upper organic layer was transferred to another clean test tube and evaporated to dryness under a stream of nitrogen at 40 ⁰C. The residue was reconstituted in 200 µl mobile phase from which 20 µl aliquot was injected into the HPLC system.

VALIDATION OF THE METHOD:

Linearity, Accuracy and Precision:

Validation of the HPLC method was performed by determining within-day and between-day accuracy-precision under the extraction and analytical conditions as described above. The calibration curve was obtained using eight calibration points, ranging from 20 to 1500 ng/mL.

Accuracy was calculated as a percentage error, whereas precision was expressed as the relative standard deviation of each calculated concentration. Precision was expected to be less than 15% deviation at all concentrations, except for the LLOQ, for which 20% deviation was acceptable.¹⁰ For calibration curve, a linear regression with 1/x weighing factor was used. A correlation of more than 0.99 was desirable for each calibration curve.

Table No.2: Within-day and between-day Accuracy/Precision for Estimation of Valsartan in Plasma

Within-day Accuracy and Precision
Nominal conc. (ng/mL)	Found conc. (ng/mL)	Accuracy (%)	Precision (R.S.D. %)
20 (LLOQ)	20.88	104.3	0.77
60 (LQC)	60.28	100.3	1.19
600 (MQC)	647.3	107.7	3.08
1219 (HQC)	1258	103.3	1.16
Between-day Accuracy and Precision
20 (LLOQ)	21.14	105.7	0.98
60 (LQC)	60.93	101.4	2.94
600 (MQC)	655.2	109.1	4.00
1219 (HQC)	1206	98.92	3.08

Recovery:

Recovery of Valsartan and Internal Standard was evaluated by comparing the mean detector response of different quality control samples post-extracted, with those prepared by adding compound to post-extracted, drug free, plasma at the corresponding concentrations.

Stability:

The stabilities of Valsartan in human plasma was evaluated by analyzing replicates (n=6) of plasma samples at the concentrations of 60, 600 and 1219 ng/mL, which were exposed to different conditions (time and temperature). These results were compared with those obtained for freshly prepared plasma samples. The analytes were considered stable in the biological fluid when 85%-115% of the initial concentrations were found. The short-term stability was determined after the exposure of the spiked samples at 25⁰C for 6.0 hrs and the ready-to-inject samples (after extraction) in the autosampler rack (4⁰C) for 24 hrs. The freeze/thaw stability was evaluated after three complete freeze and thaw cycles on consecutive days.

Plasma Concentration of Valsartan:

Plasma concentration of Valsartan was measured in twelve subjects who volunteered in a clinical study after signing consent forms. The study was approved by the Institutional ethics committee and was submitted to DCG (India).

Following an overnight fast, a single, 80 mg, oral dose of Valsartan was administrated with 250 mL of water and venous blood samples were taken at 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 12.0, 24.0 and 36.0 hours post-dose. Plasma was separated and stored at -20⁰C until analysed for Valsartan. Valsartan is found to be stable for at least 6 months under these conditions.

RESULTS AND DISCUSSION:

Retention times of Valsartan and Internal Standard were 7.2 and 5.6 min., respectively. Representative chromatograms of the drug free human plasma extract and plasma sample obtained from volunteer at 2.0 hrs. after oral ingestion of 80 mg Valsartan are shown in Fig.1 and Fig. 2 respectively. Six separate human plasma were screened for any interfering peaks at the retention time of drug and Internal Standard; absence of such peaks indicates that the method was selective.

FIG. 1 : Typical chromatogram of the drug free Human Plasma

Sensitivity:

The lowest standard on the calibration curve was to be accepted as the lower limit of quantitation, if it complied the acceptance criteria¹⁰ of exhibiting the analyte response five times that of drug free processed plasma. The lower limit of quantification was 20 ng/mL with an acceptable precision and accuracy.

Linearity (Calibration Curves):

The detector response in terms of peak area as obtained for the drug, in the concentration range of 20-1500 ng/mL of plasma. The peak area ratios were found to be linear over the concentration range. For statistical analysis the data was subjected to linear regression least squares fit analysis. The mean (n=6) correlation coefficient was found to be more than 0.99. The results are presented in Table 1.0.

Accuracy and Precision:

The accuracy and precision of the method was evaluated by replicate analysis of spiked quality control samples. The within day data were obtained by replicate analysis of quality control plasma samples (n=6). The between day data were obtained by analyzing the same quality control plasma samples over a period of three weeks. The RSD varied from 0.77-3.08% for within-day precision and 0.98-4.00 for between-day precision. Results of the analysis are reported in Table 2.0.

Recovery:

The mean recovery for Valsartan in human plasma obtained was 81%. The mean recovery for Internal standard obtained was 64%, which was satisfactory for the assay. Internal Standard is structurally related to the drug.

Stability:

Stock solution stability had accuracy value better than 95% throughout the storage period of 6 hours at 23 ± 2^oC. From all stability data the results clearly indicated that the drug remained stable even after three successive Freeze/Thaw cycles, 6 hours at bench top (25⁰C) and 24 hours in an autosampler tray at 4^oC.

FIG. 2: Chromatogram of a plasma sample from volunteer 2.0 hrs. after administration of 80 mg Valsartan

Application to biological samples:

Fig. 3 shows the mean plasma concentrations of Valsartan after single 80 mg oral dose of the drug to 12 healthy subjects. The plasma levels reached their maximum at 2.0 hrs. after the administration and thereafter the plasma level declined with an elimination half-time of 6.0 hrs. These values agree with previously published reports on the Pharmacokinetics of Valsartan¹¹. The mean area under concentration-time curve (AUC) measured from 0 to the last non-zero sampling point was 99.0% of the value of AUC extrapolated from 0 to infinity. In all subject, this value was higher than 90.0%, which indicated a suitability of the analytical method for Pharmacokinetic studies.

CONCLUSION:

The present HPLC method of Valsartan offers advantages over those previously reported, in terms of convenience of using liquid-liquid extraction and use of simple mobile phase. The method has sufficient sensitivity for Pharmacokinetic studies following single oral dose of 80 mg Valsartan.

FIG. 3: The mean plasma concentrations-time profile of Valsartan after single 80 mg oral dose of the drug to 12 healthy volunteers

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Received on 19.02.2009 Modified on 12.04.2009

Research J. Pharm. and Tech.2 (3): July-Sept. 2009,;Page 487-490

Quantitation of Valsartan in Human Plasma by High Performance Liquid Chromatography with Fluorescence Detection and its Application to Bioequivalence Study