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Good manufacturing practices in drug industry demand a zero defective approach in the analytical field. This necessarily means development of reliable, competent, precise, sensitive and selective methods for the analysis of drug substances and drug products. End-product testing of pharmaceuticals relies only upon appropriate analytical tests to demonstrate the quality of medicinal material. Also, quality control is the last line of defence for the protection of human health by satisfying the required prerequisites like purity, strength, stability efficiency and safety of dosage forms1. Oxidation, thermal degradation, acid/base hydrolysis and photolysis of drug molecules pose a serious stability problem and can cause a major halt in pharmaceutical development. The only meaningful evaluation of the stability of a drug is to use a stability-indicating method, an analytical method distinguishing the intact molecule from the degradation products2. The parent drug stability test guideline Q1A(R2) issued by the International Conference on Harmonization (ICH) suggests that stress studies should be carried out on a drug to establish its inherent stability characteristics, leading to identification of degradation products and, hence, supporting the suitability of the proposed analytical procedure. It also recommends that analytical procedures for testing the stability of samples should be stability-indicating3. Modern instrumental methods of analysis are extremely sensitive, providing precise, sufficiently reliable, rapid and detailed information from small samples of material. They are now in widespread use in product development, quality control, quality assurance, stability studies and pharmacokinetic evaluations1.
A review of literature over the last five years has indicated that the most popular front-line separation techniques are high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC). The HPLC, particularly reversed-phase HPLC, offer a multitude of advantages over other methods for the separation of components of a complex mixture and is currently the most suitable method for quantitative analysis. It provides high reliability, precision and accuracy in quantification and can be performed on a fully automated instrumentation. HPLC provides excellent reproducibility and is applicable to a wide array of compounds by judicious choice of instrumental and experimental conditions. They are also official in most of the pharmacopoeias for determining content uniformity, purity profile, assay values and dissolution rates in number of monographs4. HPLC technique finds its application not only in the quantification of active pharmaceutical ingredients, but may be effectively used for drug-drug interaction studies because of its capacity for high resolution and wide range of sensitivity.
HPTLC is a well established analytical method with precise instruments for sample application and chromatogram evaluation. The HPTLC method offers several advantages over liquid chromatographic methods such as the possibility of simultaneous analysis of sample and standard on the same plate, short system equilibrium time, and large sample capacity, multiple/repeated scanning of chromatograms, minimum solution consumption, short run time and no prior treatment for solvents like filtration and degassing5.
Among the most widely used analytical methods are those based on UV- spectrophotometric techniques, due to both experimental rapidity and simplicity. Derivative spectrophotometry which involves the conversion of a normal spectrum to its first, second or higher order derivative spectrum possesses the added advantages of enhanced resolution and bandwidth discrimination and the later increases with increasing derivative order. Second derivative spectra are one of the most frequently employed derivative orders for quantitative purposes.
Fluorescence is the intrinsic property of structurally rigid molecules. Such molecules emit fluorescence light that is used to quantify them. Non-fluorogenic moieties shall be estimated by either converting to fluorogenic or by indirect spectrofluorimetric techniques. The outstanding advantage of fluorescence analysis is its sensitivity and in this respect it is considered to be superior to absorption spectrophotometry6.
Analytical methods must be of demonstrably high quality to ensure confidence in results and be able to provide reliable data. Analytical method validation is the process of demonstrating that analytical procedures are suitable for their intended use. Primary objective of analytical method validation is to provide a high degree of assurance that the specified method consistently provides accurate test results which evaluate a product against its defined specification and quality attributes. During the validation, data are collected to show that the developed method meets requirements for accuracy, precision, specificity, detection limit, linearity, range, and robustness2,7.
Antipsychotic agents (neuroleptics) encompass a large number of prescription medications of diverse chemical groups used for the treatment of schizophrenia and allied psychiatric disorders. Most of them are potent drugs, available in smaller doses8, hence demanding analytical techniques which are highly sensitive, selective and reliable for the separation, identification, quantification and to a variety of practical applications.
The demand of developing newer analytical techniques arises when (a) there are no official pharmacopoeial methods available (b) reported methods are tedious and time consuming (c) no stability indicating methods reported (d) no reported methods for fixed dose combination but for single drugs and (e) lower sensitive methods.
AIMS AND OBJECTVES
In the current study, few antipsychotic drugs and their combinations which include zotepine, iloperidone, paliperidone, levosulpiride, amoxapine, pimozide, levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide were selected for the method development and validation. These drugs are approved for the treatment of schizophrenia and other psychiatric disorders.
Extensive literature survey revealed that a gas-liquid chromatographic method involving time consuming multiple extraction steps9 and a GC-MS method using solid phase extraction and evaporation processes10 were reported for the quatification of zotepine.
Iloperidone has been determined from human plasma by a LC-MS method11.Though there is a report on stability indicating assay of iloperidone in bulk and formulation by RP-HPLC method, it involved tedious mobile phase preparation and the specified linear range was limited within 0.1-0.6 µg/ml of iloperidone12.
A LC-MS-MS method has been described for the determination of paliperidone in human plasma13. The reported RP-HPLC method in tablet formulation14 was laborious and the quantification limit of paliperidone by this method was 1 mcg/ml.
The lack of simple and sensitive methods for the assay of levosulpiride, amoxapine and pimozide in the literature arises the need to develop validated analytical techniques for these drugs in formulations
There have been numerous reports describing various methods for the quantification of rabeprazole individually and in combination with other drugs15-17. However, there was no published data that allows simultaneous determination of levosulpiride and rabeprazole in combined dosage form.
Several methods have been reported for the estimation of olanzapine18-21 and fluoxetine22 in biological fluids. Quantification of olanzapine and fluoxetine individually in pharmaceutical dosage forms using RP-HPLC and HPTLC methods have been described in the literature23-25. Simple HPTLC methods for the estimation of fluoxetine in combination with paroxetine26 and alprazolam27 are available. However, RP-HPLC methods for the simultaneous estimation of olanzapine and fluoxetine described in the literature28,29 used either a tedious procedure or demonstrated narrow linear range and lower sensitivity.
A variety of assays for the analysis of trifluoperazine based on HPTLC30 RP-HPLC31,32 and UV-spectrophotometry33 were reported. A stability indicating RP-HPLC method for the quantification of multicomponent formulation containing trifluoperazine hydrochloride, trihexyphenidyl hydrochloride andi chlorpromazine hydrochloride has been reported34. The RP-HPLC method35 established for the binary mixture of trifluoperazine HCl and chlordiazepoxide using methanol:water (97:03, v/v) was simple but the specified range of linearity was limited within 0.1-1 µg/ml for trifluoperazine and 0.5-5 µg/ml for chlordiazepoxide.
Therefore, the primary objective of the study was to develop and validate simple, rapid, and sensitive instrumental methods for the quantitative estimation of selected antipsychotic drugs in formulations as per ICH recommendations. The following were the specific goals of the study:
To develop validated RP-HPLC methods for the quantification of selected antipsychotic drugs such as zotepine, iloperidone, paliperidone, levosulpiride, amoxapine and pimozide in bulk and single dosage form and levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide in their combined dosage form.
To develop validated stability indicating HPTLC methods for the estimation of zotepine, iloperidone, levosulpiride and pimozide and also to separate and quatify levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide simultaneously in fixed dose combinations by validated HPTLC techniques
To establish validated spectrofluorimetric methods for the quantification of zotepine, paliperidone and levosulpiride in bulk and formulations.
To develop validated UV-spectrophotometric method for the quantification of zotepine and iloperidone and second derivative spectrophotometric methods for the estimation of paliperidone, levosulpiride, amoxapine and pimozide and combined dosage forms containing levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide.
MATERIALS AND METHODS
MATERIALS: Reference standards of zotepine, iloperidone, paliperidone, levosulpiride, amoxapine, pimozide, levosulpiride, rabeprazole, olanzapine, fluoxetine, trifluoperazine, and chlordiazepoxide (all having assigned purity >99%) were used for the study. Solvents like methanol and acetonitrile were of HPLC grade. All other chemicals and reagents were analytical grade. Formulations containing the selected antipsychotic drugs were purchased from local pharmacy.
METHODS: The various instrumental methods developed and validated in the current study were based on high performance liquid chromatography, high performance thin layer chromatography, spectrofluorimetry and UV spectrophotometry.
A Shimadzu LCMS-2010EV system equipped with a binary gradient pump, degasser (DGU-20A3), and a variable wavelength programmable PDA detector (SPD-M20A) with auto sampler system (SIL-20AC) was used to perform chromatography under ambient conditions. The instrumentation was controlled by Shimadzu LCMS Solution Software. Chromatographic separations of the analyte and internal standard were achieved on a LiChrospher® RP-18 HPLC column (5 μ particle size and 25 cm - 4.6 mm internal diameter).
Most suitable detection wavelengths were determined prior to chromatographic method development by obtaining the UV spectra of drug solutions prepared in suitable solvents. The mobile phases were prepared freshly, filtered and sonicated for 30 minutes prior to use in order to deaerate the mobile phases. The column was equilibrated for 30-40 min with mobile phase prior to injection of the analyte. The volume of injection was 20 µL. The system suitability parameters that are critical to the analytical separation and quantification were carefully evaluated throughout the study. Various factors like cost of solvent system, time for analysis and peak shape were considered while fixing the composition of mobile phase and flow rate. The separation of analyte and internal standard were evaluated in different proportions of the chosen mobile phase, and for each condition, retention factor (k) and resolution (Rs) were considered. Various stages in RP-HPLC method development included fixing of initial chromatographic conditions, optimizing separations and validation as per ICH recommendations-Q2(R1)7 followed by assay of formulations using the validated method.
For the RP-HPLC methods of zotepine, levosulpiride and pimozide, the suitable internal standard was aceclofenac while paracetamol was used as the internal standard for paliperidone and iloperidone. Diclofenac sodium served as the internal standard for amoxapine. Organic phase in the mobile phase consisted of either methanol, acetonitrile or a mixture of both in a prefixed ratio. The aqueous phase for chromatographic separation consisted of trifluoroacetic acid (0.1-0.5% v/v) for zotepine, iloperidone and amoxapine while varying strengths of ammonium acetate buffer was used for paliperidone and pimozide. To separate levosulpiride from internal standard, 5 mM ammonium formate was used as the aqueous buffer. Different strengths of ammonium acetate and ammonium formate with methanol, acetonitrile or both in various ratios were selected as the optimized mobile phase for achieving chromatographic resolution of levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide. The pH of mobile phase was adjusted using 0.2% v/v triethyl amine or 0.1% v/v ortho-phosphoric acid when necessary. The mobile phase was delivered at a rate of 1ml/min for all drugs and combinations except for paliperidone where the flow rate was set at 0.7 ml/min.
The developed RP-HPLC methods were applied to the assay of dosage forms of the selected drugs. The experiments were conducted in six replicates. The RP-HPLC method developed for pimozide was also applied to in-vitro displacement interaction studies between pimozide and commonly used NSAIDs like aceclofenac, diclofenac sodium and lornoxicam. The method involved preliminary studies for the optimization of drug concentration, establishing equilibration period for protein binding followed by displacement interaction studies using dialysis membranes by following the principles of equilibrium dialysis36. Phosphate buffer of pH 7.4 was employed in the study to simulate in-vivo conditions.
Samples were spotted as bands (6 mm wide and 6 mm apart) by means of a CAMAG (Muttenz, Switzerland) Linomat 5 sample applicator equipped with a 100 µL syringe (Hamilton, Bonaduz, Switzerland) on a 10 x 10 cm/20 x 10 cm aluminium sheet pre-coated with silica gel 60F254 of 250 µm thickness (E. Merck, Darmstadt, Germany). The plates were prewashed with methanol and activated at 110°C for 5 minutes prior to chromatographic analysis. Linear ascending development was performed using a twin-trough glass chamber (CAMAG). Densitometric scanning was done using CAMAG TLC Scanner 3 in the reflectance mode. The scanner was controlled by winCATS software (Version 1.2.6). The radiation source used was deuterium lamp which emits a continuous UV spectrum between 200-400 nm.
Various steps involved in the HPTLC method development was sample preparation, sample application, chromatographic development, detection of spots, scanning and documentation of chromatoplate. Effect of experimental variables such as mobile phase composition, chamber saturation time, plate equilibration time, band width of the spot and solvent front on the Rf value of the drugs were evaluated. Chamber saturation time of 10 to 20 min was tried. Plate equilibration time was also optimized to get reproducible results in HPTLC as it helps to av
oid secondary solvent fronts. The mobile phase was taken in one side of a twin trough chamber and spotted plates on the other side. The plates were kept for equilibrium with the vapour phase for 5-30 minutes. In order to determine the effect of solvent front, the plates were developed with a distance of solvent front ranging between 7.0 to 9.0 cm. Band width was optimized through several trials in order to obtain good peak shape and reproducibility of Rf values. After development, densitometric evaluations were carried out in order to understand the effect of aforementioned variables over the peak shape and Rf values of the selected drug and thus to fix the initial chromatographic conditions. Various mobile phase compositions were tried to achieve good separation between levosulpiride and rabeprazole, trifluoperazine and chlordiazepoxide as well as olanzapine and fluoxetine.
Stability indicating high performance thin layer chromatographic method was developed for zotepine, iloperidone, levosulpiride and pimozide. Forced degradation studies were carried out using four samples, viz., 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. Acid/base induced degradation was carried out by refluxing at 60-80°C in 0.01-1N hydrochloric acid/sodium hydroxide. Oxidative degradation was performed using 3-30% hydrogen peroxide. For photodegradation studies, an appropriate amount of drug was spread evenly in a petridish and was exposed to day light for a specific period of time. Thermal degradation behaviour was studied by subjecting the drug to 60-80°C in a temperature controlled oven. Periodically, aliquots of samples were withdrawn for analysis of the parent molecule and degradation products.
All spectrofluorimetric measurements were performed using a Jasco FP-750 spectrofluorimeter equipped with a xenon discharge lamp ( 150 W), a 1 cm2 glass cell, monochromators (holographic grating with 1200 lines/mm modified Rowland mount), detector (silicon photodiode for excitation monochromator and photomultiplier for emission monochromator ), and a recorder. The spectrofluorimeter was interfaced with a computer controlled by Spectra manager software (version 1.2).
Highly sensitive indirect spectrofluorimetric methods were developed for zotepine, paliperidone and levosulpiride. The first step involved in the spectrofluorimetric analysis was the selection of excitation and emission wavelength. Keeping the emission wavelength constant, the excitation spectrum was measured in the spectral measurement mode of the instrument. Similarly, the emission spectrum was again measured with the fixed excitation wavelength. The methods for zotepine and paliperidone involved the base-catalysed condensation of mixed anhydrides of organic acids (citric acid/acetic anhydride) where the tertiary amino group in zotepine and paliperidone acts as the basic catalyst and the resulting product produced intense fluorescence. Influence of experimental and instrumental parameters over the fluorescence intensity was initially evaluated in order to optimize the analysis.
The spectrofluorimetric method developed for levosulpiride was based on the oxidation of levosulpiride using cerium (IV) in presence of sulphuric acid and monitoring the fluorescence of the formed cerium (III) at prefixed excitation and emission wavelengths. All experimental variables affecting the reaction conditions such as cerium (IV) concentration, sulphuric acid concentration, heating time, temperature and diluting solvents as well as instrumental variables like band width and response time were studied carefully during the method development phase of levosulpiride.
Jasco V-630 UV/VIS Spectrophotometer with Spectra Manager (Version 2) as the control software was used. UV absorption of the reference and sample solution were recorded in 1 cm quartz cells in the scan range of 200 to 400 nm at a scan speed of 400 nm/min and fixed band width of 1.5 nm. The reference or sample solution was transferred into a spectrophotometer cell and spectral measurements were carried out at prefixed λmax against a blank solution. Second order derivative spectra were produced by processing the spectrophotometer output.
Standard and sample solutions of zotepine, iloperidone, paliperidone, levosulpiride and the drug combinations of levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide were prepared in methanol while acetonitrile was used as the solvent for amoxapine and pimozide.
The various instrumental methods developed for the estimation of the selected antipsychotic drugs were validated in accordance with ICH guidelines7 for linearity and range, specificity, LOD, LOQ, accuracy, precision and robustness. Concentration and response in terms of peak area, absorbance or fluorescence intensity was subjected to least square linear regression analysis for the calculation of the calibration equation and correlation coefficient, y-intercept, and slope. The LOD and LOQ of the methods were determined from the standard deviation of y-intercept of regression line and slope of the calibration curve. Accuracy of the method was evaluated through recovery studies performed by standard addition method at three different levels (50, 100 and 150%) and the samples were analyzed by the corresponding method. The repeatability and intermediate precision of the method were established by intra- and inter-day precision studies. Robustness of all the developed methods was confirmed through the evaluation of response after introducing slight deliberate changes in the experimental conditions. Stability studies for all the selected drugs in solution were carried out under room temperature and refrigeration for 24 hours and 5 days respectively. To confirm the suitability of the various analytical methods developed, statistical evaluation was carried out by applying student's t test and one way ANOVA.
Application of the validated instrumental methods for the assay of selected antipsychotic drugs in formulations
Commercially available formulations of iloperidone, paliperidone, levosulpiride, amoxapine, pimozide and fixed dose combinations of levosulpiride-rebeprazole, trifluoperazine-chlordiazepoxide and olanzapine-fluoxetine were estimated by the proposed methods. For the assay of formulations, 20 tablets were weighed, average weight was calculated, finely powdered, homogenized and a portion of the powder mass equivalent to one tablet was weighed accurately, transferred into a volumetric flask, dissolved in suitable solvent, sonicated for atleast 20 minutes to aid complete dissolution and then filtered through whatman filter paper. The volume was made up with suitable solvents and analyzed by the respective method and the amount present in the formulations was calculated.
OBSERVATIONS AND INFERENCES
Various mobile phase systems tried initially resulted in peak tailing, fronting, split peaks, or inadequate resolution. The RP-HPLC methods developed for the selected antipsychotic drugs and combinations were well retained on the fixed mobile phase system with ideal peak characteristics.
The RP-HPLC methods of zotepine, iloperidone, paliperidone, levosulpiride, amoxapine and pimozide have the advantage of using an internal standard which compensates for any error that may occur due to baseline drift, fluctuations in the response of the detector and other run to run variations, thus improving precision and accuracy. The selected compounds met all the criteria to be used as an internal standard. Under the optimized conditions, they were well resolved from analyte peaks (Rs > 2) with good peak shape (tailing factor < 1.5). They were stable during the analysis and were readily available. For all the developed methods, including simultaneous estimation of levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide, the overall run time was less than 10 minutes.
The chromatographic separation conditions used for the quantification of pimozide by RP-HPLC method was also helpful to achieve satisfactory resolution between pimozide and the selected NSAIDs - aceclofenac/diclofenac sodium/lornoxicam. Hence, the study was extended to infer about in-vitro displacement interaction between pimozide and aforementioned NSAIDs. At the optimized experimental conditions, aceclofenac was found to displace pimozide from its protein binding sites than diclofenac and lornoxicam.
Zotepine, paliperidone, levosulpiride and amoxapine were quantitatively estimated using a calibration graph prepared in the concentration range of 1-5µg/ml. The RP-HPLC method of paliperidone and pimozide were linear over the concentration range 1-10 µg/ml and 2-20 µg/ml respectively. Regression analysis showed a linear relationship for the peak area responses over the concentration range of 1-10 µg/ml for levosulpiride and 2-20 µg/ml for rabeprazole. The RP-HPLC method for olanzapine and fluoxetine demonstrated linearity within the concentration range 2-20 µg/ml and 4-40 µg/ml respectively. A linear correlation was observed for trifluoperazine and chlordiazepoxide over the concentration range of 0.5-5µg/ml and 1-10 µg/ml respectively. Linear regression analysis of the data yielded correlation coefficient greater than 0.99 over the established linear range. Method precision studies using six homogeneous samples from the same batch produced RSD values less than 2%. The coefficient of variation values of inter-day precision were less than 2%. Accuracy investigation by recovery calculations at different concentration levels yielded results close to 100%. All the methods developed were found to be specific as none of the excipient interfered with the analyte of interest. The LOD and LOQ values obtained demonstrated the methods were sufficiently sensitive. The methods were robust enough to withstand slight changes in the experimental conditions. There were no significant changes in the retention time, peak symmetry or resolution of peaks of interest during these experiments.
Validity of the analytical procedure and resolution between the peaks of interest were ascertained through system suitability studies. The capacity factor (k') obtained for the IS and drug peak indicated that the peaks were well resolved with respect to the void volume. The tailing factor (T) <1.5 were indicative of adequate peak symmetry. The resolution (Rs) for the principle peak and internal standard was found to be >2 signifying good separation. Theoretical plate number (N) > 2000 demonstrated good column efficiency. Solutions of samples were found to be reasonably stable for the purpose of analysis at room temperature for 24 hours and under refrigeration upto 5 days.
The optimized chromatographic conditions of HPTLC produced well defined compact spots and symmetric peaks for all the analytes of interest. Stability indicating HPTLC methods were developed for zotepine, iloperidone, levosulpiride and pimozide. The drug combinations such as levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide were simultaneously estimated by newly developed HPTLC methods. Several initial trials with various mobile phase compositions and other chromatographic conditions showed the drug that has not moved from the site of application, a spot that has moved along with the solvent front, a less dense spot or asymmetric peaks.
Reproducible Rf value at 0.29 were obtained for zotepine with a mobile phase composed of toluene: methanol (8:2 v/v). Stress studies showed that the drug is susceptible to oxidation and acid hydrolysis. Degradants showed significantly different Rf values from that of the analyte. Densitometric analysis was performed at 265 nm in the reflectance mode. A good linear relationship was observed between concentration and response over 20-80 ng/spot.
The stability indicating HPTLC method of iloperidone demonstrated excellent linear relationship between peak area and concentration from 160 to 320 ng/spot. Chromatogram of the drug showed a symmetrical peak at Rf 0.36. Forced degradation studies confirmed that iloperidone is susceptible to acid and alkaline hydrolysis while it is stable to all other stress conditions.
Stress studies of levosulpiride showed satisfactory resolution between the analyte and degradant peaks. Since the method could effectively separate the drug from its degradation products, it can be used as a stability indicating method. Standard graph of pimozide was prepared in the concentration range 80 to 360 ng/spot. The method was highly accurate and precise over the linearity range.
Pimozide was subjected to acid and alkali hydrolysis, oxidation, photolysis and thermal degradation and the resulting solutions were evaluated densitometrically using a mobile phase composed of toluene:acetone:ammonia (5:5:0.1 v/v). The drug was found to undergo degradation in acid and base while the chromatogram of samples subjected to photolysis and thermal degradation showed no additional peaks. The spots of degradation products and pimozide were well resolved.
The HPTLC method adapted for simultaneous estimation of levosulpiride and rabeprazole showed well resolved peaks of the drugs when developed with a mobile phase composed of toluene:ethyl acetate:methanol:ammonia (2:4:4:0.1v/v/v/v). Densitometric scanning was done at 260 nm.
The optimized mobile phase for the simultaneous estimation of olanzapine and fluoxetine by HPTLC consisted of a mixture of acetone:methanol:butanol (5:2:1 v/v/v) The Rf values of the two drugs were found to differ significantly. The peaks were detected at 225 nm. Linearity was found to be in the range of 150-1350 ng/spot for olanzapine and 300-2700 ng/spot for fluoxetine respectively.
The densitogram obtained in a mixture of acetone:methanol:ammonia (7:3:0.05 v/v/v) produced sharper peaks of trifluoperazine and chlordiazepoxide with adequate resolution and acceptable Rf values. The most suitable detection wavelength was 260nm. Both the drugs showed linearity in the concentration range of 100-500 ng/spot.
Linear regression analysis showed acceptable correlation coefficients of determination (r2 > 0.99). The method was found to be precise with low RSD values. Recovery studies demonstrated excellent accuracy of the method. Repeatability of sample application, repeatability of measurement, intra- and inter-day precision of all the developed HPTLC methods were observed to be within the acceptable range. The methods were also robust as the Rf values and peak symmetry were not altered due to minor changes introduced in the experimental conditions.
Indirect spectrofluorimetric methods were developed and validated for zotepine, iloperidone and paliperidone. Initially, a series of experiments were conducted under fixed wavelength measurement mode in order to establish the optimum analytical conditions. The solvents which showed maximum fluorescence intensity under the optimized conditions were chosen for further studies.
For zotepine and paliperidone, the condensation products formed were measured spectrofluorimetrically at 665 nm after excitation at 332 nm for zotepine and 330 nm for paliperidone. The response was linear over the concentration range of 0.5-2.5 µg/ml and 0.5-3 µg/ml for zotepine and paliperidone respectively. The optimized methods were found to be highly sensitive, accurate and precise for the quantification of zotepine and paliperidone.
In the spectrofluorimetric estimation of levosulpiride, the fluorescence intensity of the cerium (III) formed due to oxidation of the drug was monitored at λex = 278 nm and λem=364 nm. Concentration of cerium (IV), sulphuric acid, heating time, temperature and diluting solvents showed significant effect on fluorescence intensity. Linearity was obtained in the concentration range 5-25 ng/ml.
The spectrofluorimetric methods developed were highly sensitive as evidenced by their low LOD and LOQ values. The methods were also selective since the additives showed no interference. Excellent accuracy of the methods was indicated through recovery studies by standard addition method. The relative standard deviation for intra- and inter-day precision studies was found to be less than 2%, indicating good repeatability of the developed spectrofluorimetric methods.
UV Spectrophotometric Methods
Simple, rapid and reliable UV spectrophotometric methods were developed for the quantification of zotepine and iloperidone while most accurate and reliable second order derivative spectroscopic methods were developed for the quantification of paliperidone, levosulpiride, amoxapine and pimozide, and the combinations of levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide in pharmaceutical dosage forms. Methanol or acetonitrile was used to prepare standard and sample solutions. The drug substances showed good solubility and stability in the selected solvents. Also, maximum sensitivity was observed with minimal interferences at the selected detection wavelength of analytes.
The calibration graphs generated showed linear relationship in the concentration range of 2.5-25 μg/ml and 2 to 20 µg /ml at 265 nm and 229 nm for zotepine and iloperidone respectively. At the λmax 237 nm, linearity for paliperidone was obtained in the concentration range of 10 to 50 μg/ml while it was observed from 2.5-25 µg/ml for levosulpiride at 235 nm. Linearity of amoxapine and pimozide were found to be within 4-20 µg/ml at 256 nm and 10-80 μg/ml at 285 nm respectively. In simultaneous estimation, calibration graphs were linear in the range of 10 to 50 μg/ml and 10-45 μg/ml for levosulpiride and rabeprazole, 5 to 25 μg/ml and 20 to 180 μg/ml for olanzapine and fluoxetine; 10-90 μg/ml and 10-100 μg/ml for trifluoperazine and chlordiazepoxide respectively. Validation of these methods as per ICH guidelines showed correlation coefficients (r2) greater than 0.99. High recovery (close to 100%) and low relative standard deviations were observed for all the developed methods.
Application of the validated instrumental methods for the assay of selected antipsychotic drugs in formulations
The various instrumental methods developed and validated in the current study were effectively applied for the assay of commercial formulations containing the selected antipsychotic drugs. The relative standard deviations for the assay of selected drugs in formulations were less than 2%, thus representing the true drug content accurately. All the developed methods established good agreement between the assay results and label claim of the tablet formulation. No interference was observed due to the presence of excipients in the formulations. Statistical analysis showed no significant differences between the results obtained.
SUMMARY AND CONCLUSION
Antipsychotic drugs, especially the newer generations, which are more potent and administered in lower doses create increasing demands on the sensitivity of analysis. In the current study, various instrumental techniques based on RP-HPLC, HPTLC, UV-spectrophotometry and spectrofluorimetry were developed for the estimation of antipsychotic drugs such as zotepine, iloperidone, paliperidone, levosulpiride, amoxapine and pimozide and the combination of levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide. These methods were validated in accordance with ICH guidelines Q2(R1) for the validation of analytical procedures.
The optimized mobile phase in HPLC techniques gave acceptable k' value, adequate resolution, short run time and symmetric peaks. The internal standards chosen in the current study have met all the necessary criteria. The retention time lower than 10 minutes grants speed to the routine analysis for all the selected drugs with better accuracy and selectivity. All the validation parameters were found to be within the acceptance criteria. The high correlation coefficient obtained from the linear regression analysis was indicative of the good linear relationship between concentration and responses. The RSD values lower than 2% in all the validated techniques confirmed method precision. Mean recovery close to 100% demonstrated the accuracy of the methods. Robustness evaluation of the methods has shown fairly constant response over a variety of minor changes in the experimental conditions. The study also explored the application of RP-HPLC method for the in-vitro interaction studies by inferring about the displacement interactions of pimozide, a typical antipsychotic with commonly co-administered NSAIDs like aceclofenac, diclofenac sodium and lornoxicam.
The presented HPTLC methods could provide highly selective quantitative stability indicating methods for zotepine, iloperidone, levosulpiride and pimozide in presence of their degradation products. It was observed that the degradants were well resolved from the analyte peaks. Validation the methods were performed by following the ICH guidelines Q2(R1) for the validation of analytical procedures. The HPTLC methods developed for simultaneous estimation of levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide were highly sensitive, selective, accurate, precise, reproducible and specific. The method could minimize the cost of reagents and time of analysis.
The validated spectrofluorimetric methods developed for the assay of zotepine, paliperidone and levosulpiride were simple, rapid, inexpensive and selective. They were also found to be superior in sensitivity compared to other methods. They can be considered as good alternatives to the high cost HPLC methods.
The UV-spectrophotometric methods developed in the present study can be easily applied for quantification purpose due to the simplicity and rapidity of the methods. Improved spectral resolution was achieved, especially with the second order derivative methods. These techniques can be considered a promising, simpler, faster, direct and relatively less expensive alternative for the determination of active drug content in pharmaceutical formulations with sufficient reliability and may be used as alternative methods when advanced instruments like HPLC are not available for routine quantification purpose.
Stability of drug solutions in bench top as well as refrigerated conditions were ascertained for adequate duration and were found to be reasonably stable. The results of drug content determination by all the developed methods were in good agreement with the label claim declared in the respective formulations. The data generated were subjected to t-test and one way ANOVA in order to understand if there is significant difference between the developed methods and reported methods.
The various instrumental methods developed in the current study have been well validated as per ICH recommendations. The RP-HPLC methods developed were highly precise and accurate. The most critical system suitability parameters were carefully evaluated throughout the chromatographic analysis. The validated stability indicating HPTLC methods of zotepine, iloperidone, levosulpiride and pimozide were simple, sensitive and cost effective. HPTLC methods developed for the simultaneous estimation of levosulpiride-rabeprazole, olanzapine-fluoxetine and trifluoperazine-chlordiazepoxide were highly sensitive and accurate over a wide linear range. The UV spectrophotometric methods developed were found to be reliable and rapid while the spectrofluorimetric methods were observed to be extremely sensitive. Statistical evaluations further strengthened the validity of all the methods developed. The results observed in the current study indicated that the introduced methods can be classified as highly selective and sensitive methods. These merits suggest the use of these newly developed methods in routine quality control analysis without interference of commonly encountered dosage form additives.