Paroxetine is chemically (-)-Trans-4R-(4’-fluorophenyl) -3S-[(3’, 4’methylenedioxyphenoxy) methyl]-piperidine hydrochloride hemihydrate, is a selective serotonin reuptake inhibitor.1 It is an odourless, off white powder, with a melting point of 120°C to 138°C. It is a phenylpiperidine derivative which is not related to the tricyclic or tetracyclic antidepressants chemically. It is used in the treatment of panic disorder, obsessive - compulsive disorder and panic disorder. It inhibits the reuptake of serotonin at the neuronal membrane, enhances serotonergic receptor sites and potentiates 5-HT in the CNS.2
Literature survey reveals that few LC-MS,11-13 Chiral HPLC,15,16 RP-HPLC7-9,14 methods with electrochemical detection, PDA detection were reported for the estimation of biological fluids. Janusz Zukowski et al reported a method for the separation of enantiomers in drug. Barry D. Zusssman et al.,10 conducted investigation for the determination of paroxetine in human plasma using HPLC with fluorescence detection. Massaroti P et al3 reported a selective method for determination of paroxetine in human plasma by LC-MS/MS of ethyl acetate/hexane (50:50% v/v) on C18 column. Vergi -Athanasiou et al.,4 reported a method using acetonitrile/phosphate buffer (pH=3.5) (30:70%v /v) at a flow rate of 1mL/min on Zorbax Eclipse XDB C18 column, using a fluorescence detector for simultaneous determination of paroxetine and its metabolites in plasma. The paroxetine eluted at 10 min and linearity was conducted over a range of 7-200 ng/mL.
N Dominguez et al., conducted bioequivalence studies for the determination of paroxetine in plasma by HPLC on C18 column with fluorescence detector. Later on, Nitasha Agrawal et al.5 investigated a method for the determination of paroxetine in pharmaceutical preparations using HPLC with electrochemical detection. An optimum mobile phase combination of 40% acetonitrile and 0.01 M phosphate buffer to pH 3 with a flow rate of 1 mL/min and electrochemical detection of 0.9V were used, with a linearity range of 0.5-50 ng/mL. Rapid simple HPLC determination of paroxetine in human plasma using 10 mM phosphate buffer/acetonitrile (40:60%v/v) was reported by Jae-Gook et al.6 Hence, the present investigation was aimed to develop a simple, economical and rapid RP-HPLC –PDA method for the estimation of Paroxetine in bulk and its formulations which would be accurate and precise and sensitive. Also the method was developed with LC conditions compatible to MS detection using a mobile phase combination of 10mM ammonium formate/acetonitrile (50:50%v/v).
MATERIALS AND METHODS
Ammonium formate of analytical grade, HPLC Milli-Q water and acetonitrile were used. Paroxetine was a gift sample from Sun pharma. The tablets of paroxetine were obtained from local pharmacy.
Shimadzu HPLC system equipped with Inertsil OSD-3 C18, 250 x 4.6 mm, 5 μ column comprising of auto sampler, a PDA detector and LC solutions software was used. 10 mM ammonium formate was prepared by dissolving 157.6 mg of ammonium formate in 250 mL of HPLC grade water, which is used for mobile phase combination with acetonitrile (50:50%v/v) at a flow rate of 1 mL/min. Detection was carried out at 220 nm and peak purity of paroxetine was also determined.
The method which is developed was validated according to ICH guidelines. 10 mg of pure paroxetine was weighed accurately and transferred into a 10 mL volumetric flask. The content was dissolved by using HPLC grade water, after complete dissolution the volume was made up to the mark by using the same which gives 1000 μg/mL of the drug. From this stock solution a serial dilutions were done in order to obtain a concentration range of 5-25 μg/mL.
Estimation of paroxetine
Ten tablets of paroxetine were weighed accurately; average weight was calculated and powdered well. The powder equivalent to 10 mg of the drug was transferred into a 10 mL calibrated standard flask; 5 mL of HPLC grade water was added. The content of the flask was sonicated for 3 min to dissolve paroxetine and made up to the volume with the same and the resulting mixture was filtered through 0.45μm filter. Subsequent dilution of this solution was made with mobile phase to get concentration of 15μg/mL. This solution (15 μL) was injected six times into the HPLC system. The mean value of peak areas of six determinations was calculated and the drug content in the tablet was quantified.
Limit of detection and limit of quantification
Limit of detection is the lowest amount of analyte in a sample, which can be detected but not necessarily quantified as an exact value.
Where, σ is standard deviation of the intercept and
S is the slope of the calibration curve
Limit of quantification is the lowest amount of analyte in a sample, which can be quantitatively determined.
Precision and accuracy
Repeated injections were used for the determination of intra-day and inter day variability’s. Accuracy was calculated by comparing the standard concentrations with the nominal concentrations.
The HPLC-PDA detector with LC-solution software provides more information regarding the sample composition at more than single wavelength detection. If any impurity or degradation product co-elutes with the paroxetine peak, that can be detected by comparing with the UV spectra. The peak purity analysis was carried out throughout the study.
The standard paroxetine solution was further diluted in 10mL volumetric flask to get various concentrations ranging from 5-25 μg/mL of drug using mobile phase. From this each calibration standard solutions 10 μL was injected into the HPLC system. The chromatograms were recorded. The concentration of the paroxetine in μg/mL is taken in X-axis and peak area of the individual concentrations of calibration standards was taken in Y-axis. The calibration graph was plotted. This is used for the estimation of paroxetine in tablets.
RESULTS AND DISCUSSION
Paroxetine pure drug is soluble in methanol, ethanol and DMSO. Different mobile phase compositions were tried to elute the drug from the column and adequate resolution is achieved with 10 mM ammonium formate and acetonitrile in the ratio of 50:50 % v/v with Intertsil ODS, C18, 250 x 4.6 mm, 5μm column and this solvent system was found to be most suitable for method development and validation. Paroxetine shows maximum absorbance at 220 nm in the proposed method. A typical chromatogram of paroxetine standard solution is shown in Figure 2a and b respectively. The retention time was 4.559 min. The system suitability tests were carried out on freshly prepared standard stock solution. These parameters indicate good sensitivity and selectivity of the developed method.
In the present developed HPLC method, the standard and sample preparation involves simple and rapid extraction procedure and requires less time. A good relationship was obtained in the concentration range of 5-25 μg/mL. Linear regression analysis report is given in Table 1. The proposed method was used to estimate the amount of paroxetine in tablets.
Limit of detection and Limit of quantification
The limit of detection (LOD) and limit of quantification (LOQ) for paroxetine was found to be 0.748 and 2.62 μg/mL, respectively.
Precision and accuracy
Precision of the method was determined by repeatability (intra-day) and intermediate precision (inter-day) was found to be 0.44 and 0.921% respectively. Repeatability refers to the use of the analytical procedure within a laboratory over a short period of time that was evaluated by analyzing six drug solutions, at the final concentration corresponding to 15 μg/mL of paroxetine during the same day. Intermediate precision was assessed by comparing the estimation on different days by different analysts. The paroxetine concentrations were determined and relative standard deviations (%RSD) were calculated. The accuracy of the developed method was carried out by adding the known amount of paroxetine pure drug to placebo solution and subjected to the proposed method. The study was done at 80, 100 and 120% of test concentrations (15 μg/mL) levels.
The proposed method was found to be simple, precise, accurate and rapid for determination of paroxetine from pure form and tablet dosage form. The mobile phase used in this method is simple to prepare and the run time was 7 min, so less time consuming method. The recovery study shows that there is no interference of additives used for the preparation of tablets. Hence, the method can be easily and conveniently applied for routine quality control of paroxetine in its dosage form and can also be used for dissolution studies.
A novel RP-HPLC method is developed and validated for the estimation of paroxetine in pure and dosage forms.
The method is optimized with LC-MS compatible mobile phase, ammonium formate and acetonitrile(1:1) at 1ml/min using intersil ODS column.
The drug is detected at 220nm.
The recovery study shows that there is no interference of additives used for the preparation of tablets.
Hence, the method can be easily and conveniently applied for routine quality control of paroxetine in its dosage form and can also be used for dissolution studies.