Quality Control Plays Vital Role Determining Safety Drugs Biology Essay

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Development of validated UV-Visible Spectroscopic, Spectrofluorimetric, HPLC and HPTLC methods for Certain Selected Drugs in Formulations and Biological Fluids.

Introduction

As the technology is developing, a number of new drugs are launched in the market and it becomes necessary to develop newer analytical methods. It is necessary to do if, no analytical methods are available for a drug in official books, no literature reveals methods for the determination of drugs, analytical method available only for a single drug in combined dosage form and no methods reported for the estimation of drugs in biological fluids.

Quality control plays a vital role in determining safety and efficacy of drugs. Highly specific and sensitive analytical technique holds the key to the design, development, standardization and quality control of medicinal products. It is important that the analytical procedure proposed for a particular active ingredient or its dosage form should be systematically evaluated so as to demonstrate that the method is scientifically sound under the condition in which it is to be applied (1,2). Method validation is the process of documenting or proving that an analytical method provides analytical data acceptable for the intended use (3)

Chromatographic and spectroscopic methods are widely used instrumental techniques for the analysis of pharmaceuticals. HPLC is a versatile separation technique and is official in most of the Pharmacopoeias for determining content uniformity, purity profile, assay values and dissolution rates in unlimited number of monographs (4). It offers variety of stationary phases, which allows a great diversity of selective interactions and more possibilities for separation. Sample recovery is easy in HPLC (5). It is specific, highly sensitive, precise and readily adaptable to accurate quantitive determinations. RP-HPLC is more convenient, widely used for assay and impurity analysis for pharmaceutical quality control. It is rugged than other forms of liquid chromatographic techniques and more likely to result in a satisfactory final separation (6).

The International Conference on Harmonization (ICH) guideline entitled "Stability testing of New Drug Substances and Products" (Q1A) requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance(7).

HPTLC is the most simple separation technique available to the analyst. It is cost effective, environment friendly and less time consuming. Solvent consumption is less when compared to HPLC. Sample clean up is not required. Other advantages of HPTLC include simultaneous processing of sample and standards, better analytical precision and accuracy, and less need for internal standard. In this, no prior treatment for solvent like filtration and degassing are required. There is no interference from previous analysis since fresh stationary phase and mobile phase are used for each analysis. Sample applied in the form of bands results in better separation and significantly less matrix effects. A major advantage in HPTLC technique is that the standards, formulations and recovery samples can be run simultaneously (4).

Methods of measuring drugs in biological media are more important nowadays. Problems related to bioavailability and bioequivalence, new drug development, drug abuse, clinical pharmacokinetics and drug research are highly dependent on bio analytical methods (8)

The technique of derivative spectroscopy is useful in eliminating matrix interference in the assay of many medicinal substances. Hence, the specificity of detection is more in derivative spectroscopy. Generally, first and third derivatives are used to fix the accurate λ max. The second and fourth order derivatives are widely used for quantitative determination. These spectra are sharper than original band but of the same height; its sign alternates with increasing order. It is clear that resolution is improved in the even-order spectra (9). For quantitative measurements, peak heights (measured in mm) of the long-wave peak (DL) satellite of second order derivative curve are usually measured. This technique is useful in the analysis of two or more components in a single formulation without prior separation and leads to better spectral isolation (10). Major advantages of second derivative spectroscopy include the possibility of recognizing absorption bands when two or more absorption bands are overlapping at the same wavelength or at slightly different wavelengths. The quantitative analysis becomes simpler in presence of background absorption since a linear relationship with good correlation can be drawn between the derivative value and the concentration (9).

The selectivity of spectrofluorimetric method is greater than absorption methods because both the emission and excitation spectra can be obtained as single spectrum. Luminescence measurements are more sensitive than absorption methods. In fluorescence method, concentration is directly related to fluorescence intensity, which can be measured at right angle to the incident radiation (5).

Several popular fixed dose combinations are available in Indian pharmaceutical market, which have flourished in the last few years. Fixed dose combinations have two or more drugs at a fixed ratio in a single dosage form. (11). NSAIDs such as aceclofenac and lornoxicam are commonly available in combination with antispasmodics like drotaverine, thiocolchicoside and analgesics like diacerein and paracetamol. Fenoverine and thiocolchicoside are novel antispasmodic drugs. An extensive literature review reveals that there are limited methods reported for the analysis of the above mentioned fixed dose combinations and fenoverine ,lornoxicam and thiocolchicoside in single dosage form. Further, drug displacement interaction studies also necessitate accurate and sensitive analytical techniques. Nebivolol and carvedilol are cardiovascular drugs used for treating hypertension and heart failure. NSAIDs like aceclofenac and lornoxicam are widely prescribed. NSAIDs are highly bound to plasma albumin and displace several concomitantly administered drugs. Literature reported that nebivolol and carvedilol are also highly bound to plasma albumin. But the effect of aceclofenac and lornoxicam on protein binding of nebivolol and carvedilol are not yet reported. Therefore, there is an unmet need to develop validated chromatographic, spectroscopic and bioanalytical methods for the assay of selected single and fixed dose combinations and for in-vitro drug displacement interactions.

Aim and Objectives:

An extensive literature review reveals that there are limited methods reported for the analysis of the above mentioned fixed dose combinations and in single dosage forms. The available bio analytical methods for fenoverine and aceclofenac are time consuming and involve more sophisticated instruments. Hence, it is in need for developing bio analytical methods for these drugs using HPTLC technique, now a day's which is more popular. Further, drug displacement interaction studies for the effect of aceclofenac and lornoxicam on protein binding of nebivolol and carvedilol are not yet reported.

Therefore, the aim of the present study is to develop validated chromatographic, spectroscopic and bio analytical methods for the assay of selected single and fixed dose combinations and in-vitro drug displacement interactions for nebivolol and carvedilol with aceclofenac and lornoxicam. The objective of the wok was as follows:

1.To develop validated spectroscopic and chromatographic methods for certain selected formulations ( formulation I- VIII)

Diacerein and aceclofenac (formulation I)

Drotaverine and aceclofenac (formulation II)

Paracetamol and lornoxicam (formulation III)

Thiocolchicoside and aceclofenac (formulation IV)

Thiocolchicoside and lornoxicam (formulation V)

Fenoverine (formulation VI)

Lornoxicam (formulation VII)

Thiocolchicoside (formulation VIII)

2. To develop validated bio-analytical methods using HPTLC technique for two selected drugs.

Fenoverine

Aceclofenac

3.To develop validated RP-HPLC methods for nebivolol and carvedilol and its application to in vitro drug displacement interaction studies as follows:

Nebivolol with aceclofenac and lornoxicam

Carvedilol with aceclofenac and lornoxicam

Literature Review

Santosh et al (2010) reported a validated HPTLC method for the determination of diacerein and aceclofenac in pharmaceutical dosageform by solid-liquid extraction method. Paracetamol was used as the internal standard. Ethyl acetate: methanol: glacial acetic acid in the ratio of (12: 0.5: 0.2 v/v/v) was used as the mobile phase.

Sarika et al (2010) developed a simultaneous UV spectrophotometric method for the determination of diacerein and aceclofenac in tablets by simultaneous equation method. The selected wavelengths were 258 and 274 nm. Linearity was found to be 1-10 and 5-40 mcg/ml for diacerein and aceclofenac.

Gopal et al (2010) reported a RP-HPLC method for the simultaneous determination of diacerein and aceclofenac in tablet dosage form using 0.01 M potassium dihydrogen phosphate and acetonitrile 60:40 (v/v) at 280 nm. The retention time was found to be 3.61 and 6.28 min, respectively. RSD was less than 2%.

Vivek et al (2009) developed three different UV spectroscopic methods for the simultaneous estimation of drotaverin and aceclofenac in combined dosage form. The method involved solving simultaneous equation, absorbance ratio method and First Order Derivative Spectroscopy. Linearity was found to be 10-50 for both the drugs.

Vishnu et al (2010) proposed a RP-HPLC method with PDA detector for the simultaneous determination of drotaverine HCl and aceclofenac in tablet dosage form. Methanol: tetrahydrofuran: acetate buffer (68:12:20 v/v) at a flow rate of 1.0 mL min-1 was used to achieve separation. Temperature of the column was maintained at 50 °C. Linearity was found to be in the range of 1 - 150 and 1.25 - 187.5 μg mL-1 for Drotaverine HCl and Aceclofenac respectively.

Bhavsar et al (2010) reported a spectrophotometric method for the simultaneous estimation of paracetamol and lornoxicam by solving simultaneous equation using 0.1 N NaOH as solvent. Linearity was found to be 5-30 and 2-10 mcg/ml at 257 and 287 nm for paracetamol and lornoxicam, respectively.

Kiran et al (2009) demonstrated a stability indicating RP-HPLC method for lornoxicam in its dosage form. The separation was achieved in presence of degradation products using 0.05%trifluoroacetic acid with acetonitrile in the ratio of 70:30 at 295 nm.

Venumadhav et al (2010) established two colorimetric methods for the estimation of lornoxicam in bulk and dosage form using ferric chloride in presence of 2,2' bipyridine and bathophenanthroline. The linearity was found to be in the range of 3-20 and 2-10 µg/ml.

Arvind et al (2011) reported a stability indicating RP -HPLC method for the estimation of thiocolchicoside in capsules. Separation was achieved using C18 column (250mm Ã- 4mm, 5μm) with a mobile phase composed of acetonitrile: water (70:30) at a flow rate was 1.0 mL min-1 . Linearity was established in the range of 0-10μg/ml and correlation coefficient as 0.9996.

Sohan et al (2010) developed spectrophotometric and Chromatographic method for the simultaneous estimation of thiocolchicoside and aceclofenac in fixed dose combination. Linearity was found to be 4-36 μg/ml for thiocolchicoside and aceclofenac using spectrophotometric technique. Acetonitrile: water: 0.025M pot.dihydrogen orthophosphate buffer in the ratio of 70:10:20 % v/v/v was used as mobile phase for HPLC method. pH was adjusted to 3 using orthophosphoric acid

Bhavsar et al (2010) reported a RP-HPLC technique the simultaneous estimation of lornoxicam and thiocolchicoside from tablets. Resolution was obtained using a Phosphate buffer and methanol in the ratio of 45:55at a flow rate of 1.5ml/min. Detection was carried out at 290 nm.

Sasmita et al (2010) reported four different UV spectrophotometric methods for the determination of thiocolchicoside in formulations. All methods obeyed beer's law concentration in the range of 2.5-50 μg/ml

Hu OY et al (----- ) reported the determination of fenoveine in capsules and plasma by RP-HPLC method. A Nucleosil 5-micron CN column was used as the stationary phase. Mobile phase composed of acetonitrile:0.1 M ammonium acetate (60:40) was used as the mobile phase and detection was performed at 254 nm. Linearity was established in the range of 24.6 to 147.6 mcg/ml of fenoverine.

Suresh et al (2008) reported HPLCmethod with UV detection for the estimation of fenoverine in human serum. The extracting solvent used was dichloromethane and chlorpromazine hydrochloride was used as internal standard (I.S). Acetonitrile, water, and 0.375% v/v triethylamine ( 41:59 v/v) at a flow rate of 1 mL min-1 was employed to achieve the separation. The method was linear in the range of 5 to 2000 ng mL-1.

Sahoo et al (2009) proposed RP-HPLC method for the estimation of nebivolol in tablet dosage form using Hypersil ODS C18 column as stationary phase and methanol-water in the ratio of 80:20 as mobile phase. Detection was carried out at 282 nm. The linearity was found between 1-400 gm/ml using the internal standard chlorzoxazone. LOD was found to be 0.0779 gm/ml.

Patel et al (2006) developed two simple chromatographic techniques for the determination of carvedilol. The stationary phase and mobile phase used in RP-HPLC method were Lichrospher 100 C-18, 5 µm column consisting of 200Ã-4.6 mm and 50 mM KH 2 PO 4 buffer (pH 3.0±0.1): acetonitrile: methanol (60:50:10 v/v/v) at a flow rate of 1ml/min. In HPTLC, precoated silica gel 60F 254 and ethyl acetate: toluene: methanol (1:4:3.5 v/v/v) were used for the separation. The linearity was found to be 1-35 µg/ml for HPLC and 50-300 ng/spot for HPTLC.

Alam et al (2008) employed equilibrium dialysis method for the in-vitro displacement interaction study of amlodipine and arseinic to bovine serum albumin. Free concentration of amlodipine was determined spectrophotometrically at a wavelength of 238 nm. It was found that amlodipine was slowly displaced from its binding site by arsenic.

Mohiuddin et al (2009) demonstrated in-vitro displacement interaction of gliclazide and metformin with caffeine by equilibrium dialysis method. Unbound concentrations were measured using UV spectrophotometer at 273nm. It was reported that both gliclazide and metformine displaces caffeine from its binding site, resulting in increase in the free concentration of caffeine in plasma.

Materials And Methods

Chromatographic Methods

HPLC

HPLC methods were developed for simultaneous estimation of five different formulations (formulation I-V) and a single dosage forms (formulation VI). Since all the selected analytes are polar in nature, RP-HPLC technique was employed. Prepacked RP-18 column (250Ã-4.6 mm, 5 µm particle size) and HPLC - Cartridge RP-18 column (250Ã-4 mm, 5 µm particle size) were used as the stationary phase. RP-18 columns are generally efficient, stable and reproducible because of the solvents used. Organic phase was selected on the basis of retention time and shape of the peak obtained. Mobile phase was selected on the basis of solubility and stability of the analytes. For improved separation, different solvent strength, solvent type, solvent composition and varying pH were tried. The ratio of the mobile phase was chosen in order to get acceptable k values and retention time.

Water: acetonitrile (45: 55, v/v) was used as the mobile phase for formulation I. For formulation II and IV, 0.1%trifloro acetic acid:acetonitrile (45:55,v/v) was used. Methanol: 10mM ammonium acetate in the ratio of 50:50,v/v was used for formulation III and V. In the case of formulation VI, methanol, acetonitrile and 10mM ammonium formate (70:10:20, v/v/v) was used.

Forced degradation studies were conducted under different stress conditions like hydrolysis, oxidation, dry heat and photolysis. Dry heat and photolytic degradation of drug product were carried out in solid state. For each study, four samples were prepared: the blank solution stored under normal condition, the blank subjected to stress in the same manner as the drug solution, zero time sample containing the drug which was stored under normal conditions and the drug solution subjected to stress treatment.

A Camag high performance thin layer chromatography system comprising of Linnomat V automatic sample applicator, Hamilton Syringe, Camag TLC Scanner-3, Camag Win CAT software with stationary phase precoated silica gel 60F 254 and mobile phase consisting of ethyl acetate: toluene: methanol (1:4:3.5 v/v/v).

HPTLC

HPTLC methods were developed for simultaneous estimation of five selected combinations (formulation I-V). A Camag high performance thin layer chromatography system comprising of Linnomat V automatic sample applicator, Hamilton Syringe, Camag TLC Scanner-3, Camag Win CAT software with stationary phase precoated silica gel 60F 254 were used. Chemical properties of analytes and the sorbent layer were taken into consideration while fixing the mobile phase. Mobile phase was selected by controlled process of trial and error. Mobile phase containing n- hexane- ethyl acetate-dioxane-ammonia, (5.0: 2.2: 1.8:0.02v/v/v) was used in formulation I. A mixture of methanol-ethyl acetate-glacial acetic acid in the ratio of 1:9:0.01v/v/v and 2:8:0.01v/v/v were used as the mobile phase for formulation II and IV. Toluene, n-propanol and ammonia (7:3:0.2) was used for formulation III while Toluene: ethanol: ammonia (6:4:0.1) was the suitable mobile phase for formulation V. The classical method of linear ascending development in a mobile phase vapor-saturated, covered twin trough chamber was used for the development. The separated compounds were detected under UV light. Densitometric quantitative analysis was done by UV scanning of fluorescence-quenched zones in the absorbance-reflectance mode. In the present study, external standardization was used for quantification with a calibration curve generated from a series of standards covering the full concentration range of analysis.

Spectroscopic Methods

UV-Spectroscopic Method

Derivative spectroscopic technique is useful in analysis of two or more components in a single formulation without prior separation and leads to better spectral isolation. Here normal spectra were converted to second order derivative spectra. For establishing the optimum parameters for the estimation of formulation I - IV, suitable concentrations of standard stock solutions were prepared using methanol. Absorption maximum and Beer's law concentration at λ max of selected drugs were established. Linear graph of second derivative spectra was prepared using the long-wave peak (DL) height covering the wavelength range ±1nm of individual λ max of each drug selected versus concentration of the corresponding drug. By comparison with the calibration plots of individual components, the concentrations of unknown drugs were deduced. The proposed methods can be applied successfully to selected formulations in their combined dosage forms.

Spectrofluorimetric Method

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 (Snider).

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 (Website).

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

HPLC

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.

HPTLC

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.

Spectrofluorimetry

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.

Validation

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.

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