Hptlc Method For Determination Of Fenoverine In Plasma Biology Essay

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Simple, specific, sensitive and precise spectrofluorimetric methods were developed for the estimation of lornoxicam and thiocolchicoside from its dosage form (Formulation VII and VIII) using Jasco F-450 spectrofluorimeter. The proposed method for formulation VII was based on the quantitative quenching effect of lornoxicam on the native fluorescence of eosin in acidic medium due to complex formation. The fluorescence intensity was measured at 545 nm (after excitation at 399 nm). The different experimental parameters affecting the quenching of the native fluorescence of eosin were carefully studied and incorporated into the procedure. Optimization of the reaction conditions like effect of pH, temperature, time, concentration and volume of eosin were studied. The method for formulation VIII was based on the oxidation of thiocolchicoside by cerric ammonium sulphate in sulphuric acid and the resulting fluorescence intensity was measured at 366 nm when excited at 289 nm. The different variables affecting oxidation of thiocolchicoside were studied and optimized.

Bio-Analytical Methods

Methanol/acetonitrile was used as protein denaturants since they are compatible with many solvent systems used in HPTLC analysis. The method involved the addition of an appropriate reagent to the protein containing sample kept in a centrifuge tube. Centrifugation at 3000 rpm for 10 min resulted in a clear supernatant, which was then spotted on the precoated plates.

HPTLC method for the determination of Fenoverine in Plasma

Analysis was performed on 20x10 cm pre-coated silica gel 60GF254 on aluminium sheets. Sample and standard spots were applied as bands by means of an automatic TLC Sampler 4. The separation was achieved using the mobile phase consisting of n-butyl acetate and methanol (8:2, %v/v). The developed plate was scanned at 286 nm by use of a Camag scanning densitometer controlled by Wincats software. The internal standard used was drotaverine.

HPTLC method for the determination of Aceclofenac in Plasma

An external calibration graph method was adopted for the determination of aceclofenac in plasma. Here the stationary phase is similar to that used for fenoverine and mobile phase system consisted of ethyl acetate: methanol: glacial acetic acid (9:1:0.01%v/v/v). The detection was performed at 282 nm. Protein precipitation was achieved with acetonitrile. After centrifugation, the organic phase was evaporated under stream of nitrogen and reconstituted with methanol and analyzed. Both the methods were validated as per ICH guidelines for validation of bio analytical procedures.

Drug displacement Interaction Studies

Step1- Development of validated RP-HPLC method for the estimation of nebivolol/carvedilol in presence of aceclofenac/lornoxicam

Linear graph was prepared for nebivolol and carvedilol in presence of aceclofenac and lornoxicam and the methods were validated in terms of LOD, LOQ, accuracy, precision and robustness.

Step 2- Optimization of nebivolol/carvedilol concentration and its equilibration period

Activated dialysis membrane bags were filled with 5ml solutions of bovine serum albumin (1.5 x 10-4M) and immersed in fixed volume (25 ml) of phosphate buffer containing varying concentration of nebivolol/carvedilol. The system was shaken gently, 1 ml sample was withdrawn at different time intervals and injected into the HPLC system till constant peak area was obtained at 237/285nm. Mobile phase used for nebivolol was 20 mM ammonium acetate and methanol (30:70, v/v) at a flow rate of 1ml/min. Water: acetonitrile (40:60, v/v) at a flow rate of 0.8ml/min was used as the mobile phase for carvedilol.

Step 3- Effect of aceclofenac on nebivolol/carvedilol binding to BSA

Dialysis bags containing 5 ml of 1.5x10-4 M BSA buffer solution was immersed in nine different conical flasks containing 25 ml of nebivolol-aceclofenac mixture. Nebivolol concentration was fixed (1.8 x10-5M ) while aceclofenac was added in increasing concentration. Control and blank were also employed in the experiment. Leakage of BSA was ruled out and a correction factor for bag binding was included in the calculation. Dialysis was carried out and samples were analysed by the newly developed and validated HPLC method. In order to study the effect of aceclofenac/lornoxicam on carvedilol binding to BSA, the above procedure was repeated using carvedilol at a concentration of (1.0824x10-5M) and aceclofenac/lornoxicam in range of 5-40mcg/ml and 1-40mcg/ml respectively.

Validation of the developed methods (3,30).

The method validation was performed according to the ICH guidelines. The various parameters studied were specificity, linearity, detection and quantification limits, precision, accuracy and robustness. In HPLC methods, specificity was confirmed from the resolution. Linearity was tested from low to high concentration of the targeted level of assay concentration for each method for all the selected formulations. The calibration graph was plotted between concentration of each standard analyte against response factor and the range of concentration which fall under linearity with the correlation coefficient more than 0.9900 was considered as linearity range. Precision of the developed methods were confirmed from the repeatability of response during the same day of each study and between the analysis. Accuracy was studied from recovery experiments performed by standard addition method for all the selected formulations. The LOD and LOQ for all the analytes in the selected formulations were determined at signal to noise ratio of 3:1 and 10:1, respectively using series of dilute solutions with known concentrations. Robustness evolution was performed by slightly altering the experimental conditions like mobile phase ratio, flow rate, pH and the resolution was checked.

Robustness in HPTLC was evaluated by making deliberate changes in mobile phase composition, mobile phase volume, chamber saturation time and solvent migration distance (mm).

The developed bio analytical methods were validated in terms of selectivity, accuracy, precision, recovery, linearity and stability of analyte in spiked samples. The selectivity of the method was ensured using blank sample of biological matrix. Accuracy was determined by replicate analysis of samples containing known amounts of the analyte using a minimum of five determinations per concentration. The deviation of the mean from the true value serves as the measure of accuracy. Precision was carried out with respect to intraday and inter day studies as well as repeatability of measurement and repeatability of application using three concentrations in the range and the measurement was taken five times for each concentration. Recovery (extraction efficiency) was determined using the detector response obtained from the unextracted analyte to the extracted one from biological matrix. To establish the linearity and range, a blank sample, zero sample and six standards covering the expected range were employed. The concentration of the standard was plotted against response to get a linear relationship. The stability was evaluated using the stock solution as well as the solution of the analyte in biological matrix.

Observations And Inferences


The present study provides results of stability indicating RP-HPLC methods for five combination drugs (formulation I-V) and a single dosage form (formulation VI). The degradation products were well resolved from the pure drugs with significant differences in the retention time. The degradation studies of formulation I indicated that diacerein was more susceptible to acid, alkaline and neutral hydrolysis, while aceclofenac showed degradation in acid and alkali at 70°C. In formulation II, drotaverine was susceptible to alkali and neutral hydrolysis, oxidation and direct sun light while aceclofenac showed degradation under acid, alkali and neutral hydrolysis at 80°C. Both paracetamol and lornoxicam in formulation III were susceptible to acid and alkali hydrolysis. In the case of formulation IV and V, thiocolchicoside, aceclofenac and lornoxicam were susceptible to acid and alkaline hydrolysis. Formulation VI (fenoverine ) was susceptible to hydrolytic, oxidative and photolytic degradation.


The separations were achieved after the applied spots were developed using the mobile phase in a 10x10 or 20x10 twin trough chamber. Evaluations of developed plates were performed densitometrically by the use of a Camag scanning densitometer controlled by Wincats software. R f value of all drugs (formulation I-V) were investigated.

Spectrophotometric Method

The simultaneous estimation of analytes in combinations for formulation I - IV was carried out by second derivative spectroscopic method. The calibration graphs were constructed between absorbance at the corresponding peak height against concentrations. The results showed that all have good sensitivity at the selected wavelength used. Beer's law concentration for formulation I - IV was found to be 1- 10 and 4-40 µg/ml for diacerein and aceclofenac,4-24 and 5-50 µg/ml for drotaverine and aceclofenac, 1-10 and 4-40 µg/ml for paracetamol and lornoxicam and 4 - 40 and 5- 50 µg/ml for thiocolchicoside and aceclofenac, respectively.


The proposed method was based on the study of the quantitative quenching effect of Lornoxicam (formulation-VII) on the native fluorescence of eosin in acidic medium due to complex formation. The fluorescence intensity was measured at 545 nm (after excitation at 399 nm). The different experimental parameters affecting the quenching of the native fluorescence of eosin were carefully studied and incorporated into the procedure. Under the described conditions, the method is applicable over the concentration range of 2 - 20 mcg/ml with a detection limit of 100 ng/ml. Quantification of thiocolchicoside was done by oxidative spectrofluorimetric method using cerric ammonium sulphate in acid medium. The fluorescence intensity was measured at 366 nm (after excitation at 289 nm) Linearity for thiocolchicoside (formulation-VIII) was found in the range of 1-10 mcg/ml.

Analysis of formulations

All formulations (I -VIII) were analyzed by the respective methods and the amount present in formulation, % label claim and %RSD were calculated. The amount of active ingredients obtained was close to label claim and the % RSD was less than 2.

Bio analytical Methods

The Rf values were 0.42 and 0.61for drotaverine and fenoverine, and 0.71 for aceclofenac. The calibration graph for fenoverine was linear over the range of 100-500 ng/spot while that of aceclofenac was 500 to 1200ng/spot.

In-vitro displacement interactions

The RP-HPLC methods developed for nebivolol and carvedilol in presence of aceclofenac and lornoxicam were simple and precise. Linearity was established between 1-10 µg/ml for both nebivolol and carvedilol with r values greater than 0.99. % RSD was less than 2.

Protein binding attained a steady level at higher concentrations. The percentage binding of nebivolol/carvedilol to BSA at saturation level (1.8x10-5 M /1.0824x10-5 M) was about 88.00%±0.5595 and 81.14%±0.933 respectively. The highest percentage protein binding of nebivolol at saturation level was about 67.90±3.398% (p=0.0024) and 75.19±2.705% (p=0.0017) in presence of aceclofenac and lornoxicam whereas carvedilol showed 80.40%±0.087 and 41.99%±0.48, respectively.

At saturation level, the free concentration of nebivolol bound to BSA was increased from 10.1±--% to 30.2±3.40% in presence of aceclofenac, whereas this increment was from 10.1±-% to 22.91±2.705 in presence of lornoxicam. In the case of carvedilol, the free concentration increased from 17.93±0.0808% to 18.47±0.087% in presence of aceclofenac while this increment was from 17.93±0.0808%to 56.87%±0.48 in presence of lornoxicam.


Linearity was established for all the formulations (I-VI) , nebivolol and carvedilol with a correlation coefficient > 0.998 and the % recovery close to 100 proved the accuracy of the method. All the methods were precise since the %RSD was <2. The active ingredients were well separated from the respective degradants. Peak purity analysis with PDA detector showed the value close to 1 for each peak and the purity angle were less than purity threshold for all the analytes proves that peak of each active ingredient was pure and other degradants were well resolved from the analytes. Robustness evaluation revealed that there was no appreciable change in resolution under deliberate changes in method parameters.

In bio analytical methods, the extraction efficiency and accuracy were found to be good. % CV was less than 15% for all concentrations. The absolute recovery was found to be 80-90 % for fenoverine and aceclofenac.

Summary and Conclusion

Stability indicating RP-HPLC method was developed for five selected fixed dose combinations (formulation I-V) and a single dosage form (formulation VI). Active ingredients were separated from corresponding degradation products under selected chromatographic conditions. Formulation I-V was quantitatively estimated using simple HPTLC method under fixed chromatographic conditions. Derivative spectroscopic methods were developed for simultaneous estimation of drugs in formulation I-IV. Spectrofluorimetric methods were established for the determination of formulation VII and VIII. All the methods developed were validated as per ICH guidelines and applied for respective formulation. Validated bio analytical methods were developed for the estimation of fenoverine and aceclofenac in plasma by HPTLC technique. Drug displacement interaction studies were conducted for two cardiovascular drugs, nebivolol and carvedilol in presence of aceclofenac and lornoxicam after developing validated RP-HPLC methods. The results indicated, the suitability of the stability indicating RP-HPLC methods for the assay of pharmaceutical formulations containing the selected drugs. The current HPTLC methods allow the detection of drugs in nano gram level and are suitable for the quantification of the selected drugs in bulk and formulations. The developed spectrofluorimetric methods were simple, accurate, precise, rapid, highly sensitive and reproducible and are successfully applied to the analysis of commercial tablets and ampoules containing the drugs, and the results were in good agreement with those obtained with reference methods. The two bio analytical methods developed were simple and showed good extraction efficiency and accuracy. The %CV within the limit proves that the method can be applied for the determination of selected drugs in plasma. The RP-HPLC methods developed for the estimation of nebivolol and carvedilol in presence of aceclofenac and lornoxiam were accurate and precise. They were sucessfully applied to drug displacement interaction studies. It was found that aceclofenac displaced nebivolol to a greater extent than lornoxicam while lornoxicam showed a major effect on in-vitro protein binding of carvedilol.