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Liquid Chromatographic Method for Simultaneous Determination of Citalopram with NSAIDs in Bulk Drug, Pharmaceutical Formulation and Human Serum.

Byline: Saeeda Nadir Ali, Nida Naz, Amtul Qayoom and Shumaila Akram

Summary: A high performance liquid chromatographic method was developed and validated to simultaneously quantify citalopram with piroxicam, celecoxib and diclofenac sodium. Chromatographic analysis was performed at ambient temperature using Shimadzu Shim-pack CLC-ODS (M) 25M column linked to a UV-visible detector adjusted at 230 nm, employing 80:20 (v/v) methanol: water (pH 3.5) as mobile phase with flow rate 1.0 mL min-1. Validation was performed in the ranges 0.6-20, 0.9-28, 0.6-20 and 1.0-32 ug mL-1 with lowest level corresponding to detection limit 16.45, 23.33, 27.66 and 14.44 ng mL-1 respectively. With-in the day precision ranged from 0.14-1.67% and between-day precision from 0.40-1.50%, accuracies were 99.61-100.86%. The analytes were successfully detected without any observable interference in pharmaceutical formulation and human serum samples demonstrating effectiveness of method.

Keywords: Citalopram, Piroxicam, Celecoxib, Diclofenac sodium, HPLC-UV.

Introduction

Multiple medical problems are often managed together e.g. antibiotics are prescribed to heal infection; however Non-steroidal anti-inflammatory drugs (NSAIDs) are also given to get pain relief [1]. Similarly patients suffering from tuberculosis and diabetes are cured at the same time [2]. Since a patient simultaneously takes medicines for multiple medical reasons, this is interesting to develop a method for simultaneous determination of co-administered drugs in biological samples. There are several reported analytical methods for simultaneous determination of different drugs [3]- [6]. In past, we have also reported simultaneous determination of alprazolam with antihistamines [7] and ACE inhibitors [8].

Number of people including children and adults are mainly influenced by psychotic disorders which may lead to intensify suicidal risk, psychosocial impairment as well as depression, eating disorders, delinquency, chronicity and drug and alcohol abuse. Although, many clinical treatments for depression have been evidenced but Selective Serotonin Reuptake Inhibitor (SSRIs) by reason of fewer adverse side-effect profile is considered to be better tolerated and safer than classical tricyclic anti-depressants (TCA) [9]. SSRIs are effective against depression as well as anxiety [10]. Citalopram, a highly selective SSRI, offers a broad spectrum of therapeutic action against depression, anxiety, obsessional and impulse control syndromes [11]. Moreover, it acts by averting the endorsement of a neurotransmitter called serotonin via nerve cells [12].

Previously reported methods for determination of citalopram are potentiometric determination [13], adsorptive square wave voltammetry (ASWV) [14], chemiluminescence [15], spectrophotometry [16], spectrofluorometry [17], micellar electro kinetic chromatographic method [18], HPTLC [19], LC-MS/MS [20] and HPLC [21]. NSAIDs are clinically prescribed worldwide to provide relief against pain, fever, inflammation [22]. It shows efficacy by inhibiting the activity of cyclooxygenase-1(COX-1) and cyclooxygenase-2(COX-2) enzymes in the body and thus prevents synthesis of prostaglandins [23]. Moreover, risk of arising Alzheimer's disease can be diminished by consuming NSAIDs for extended period [24]. Piroxicam, celecoxib and diclofenac sodium are clinically prescribed NSAIDs for osteoarthritis and rheumatoid arthritis as well as to treat severe pain, ankylosing spondylitis and acute pain in musculoskeletal condition and serious gout [25-27].

Literature survey reveals several methods for determination of NSAIDs including GC-MS [28], RP-HPLC [29], GC [30], HPLC-DAD [31], capillary electrophoresis [32], HILIC-MS/MS [33] and spectrofluorometry [34].

Risk of upper gastrointestinal bleeding has been associated with SSRIs [35] and it can be increased when SSRIs are taken with NSAIDs [36]. Main objective of this study is the advancement of validated HPLC method for simultaneous determination of citalopram with reported NSAIDs (Fig. 1). ICH guidelines have been followed for validation [37]. Furthermore, reported method is applicable successfully for quantification of citalopram and NSAIDs in active pharmaceutical ingredient, dosage formulation and human serum without showing any interference of excipients or unwanted serum components.

Experimental

Reagents and Chemicals

Citalopram (> 98% purity) was acquired from Eros Pharmaceutical Pvt. Ltd. Pakistan. Standards of piroxicam, celecoxib and diclofenac sodium were obtained from Pfizer Pakistan Ltd, Getz Pharma Pakistan (Pvt.) Ltd. and Sami pharmaceuticals (Pvt.) Ltd respectively. Pakistan with purity over 99.1%. Pharmaceuticals formulation Pramcit (20 mg), Feleden flesh (20 mg), Celbex (100 mg) and Dicloran (50 mg) were purchased from local pharmacy of Karachi in the form of tablets or capsules. High performance liquid chromatographic grade methanol, acetonitrile and o-phosphoric acid purchased from Merck (Darmstadt, Germany). Ultra-pure water was used during the course of entire analysis.

Instrumentation

Liquid chromatographic analysis was performed on Shimadzu Corporation (Japan) chromatograph equipped with online degasser (DGU-20A3/20A5), two solvent delivery modules (LC-20AT), a SIL-20AHT/20ACHT UFLC auto sampler coupled with UV/VIS detector (SPD-20A/20AV). All the data was acquired with Shimadzu communication bus module (CBM-20A) and processed with LC solution GPC software version 1.25.

Chromatographic Conditions

Chromatographic separation was achieved on Shimadzu Shim-pack CLC-ODS (M) 25M (4.6 mm i.d x 25 cm) column. Gradient elution was performed with (A) 80% methanol and (B) 20% water (pH 3.5 modified by o-phosphoric acid) at flow rate of 1.0 mL min-1 after filtration through 0.45 um pore size. All the studied analytes were simultaneously determined at isosbestic point at 230 nm.

Preparation of stock and calibration standards solutions 1000 ug mL-1 stock standard solutions of citalopram, piroxicam, celecoxib and diclofenac sodium were prepared by dissolving 100 mg of individual drug in 100 mL diluent i.e. 80:20 v/v methanol-water. These solutions were kept at -20C until analysis. Each stock standard solution was then diluted with diluent in 25 mL volumetric flask to prepare working standard solutions. Working standards for liquid chromatographic assay were prepared by serial dilution of stock standard solutions with 80:20 v/v methanol-water diluent to level up six concentrations in the range 0.6-20, 0.9-28, 0.6-20 and 1.0-32 ug mL-1 for citalopram, piroxicam, celecoxib and diclofenac sodium respectively. The solutions were introduced to auto sampler after micro filtration through 0.45 um millipore filter paper. All the solutions were prepared once and analyzed daily for with-in day and in-between day precision.

Assay for Commercial Formulation

For the assay of drugs in dosage form, ten tablets of each drug were accurately weighed and crushed into fine powder. The amount of powder equivalent to 100 mg of citalopram and 100 mg of NSAIDs (piroxicam, celecoxib and diclofenac sodium) were transferred to 100 mL volumetric flask separately and dissolved fully in diluent by sonication in an ultra-sonic bath for 15 min and volumes were made up to mark to bring final concentration 1000 ug mL-1. The resulting solutions were filtered through 0.45 um pore size filter to remove undissolved excipients and finally diluted to the desired concentration for analysis of drug content.

Assay for Drug Serum Solution

Sample of blood, collected from a healthy donor at Fatmid Foundation Karachi was transferred to a sterile EDTA glass tubes followed by centrifugation at 1600 x g for 10 min at 4AoC to separate plasma. Into 1 mL portion of plasma, 9.0 mL acetonitrile was mixed, vortexed for 1 min and centrifuged at 10,000 rpm for 10 min. Clear serum solution obtained was fortified with standard solutions of citalopram, piroxicam, celecoxib and diclofenac sodium in required concentrations for their assay.

Method validation

Validation procedure was followed as stated by guidelines of ICH Q2 (R1) [37] comprising system suitability, linearity, precision, robustness, accuracy, limit of quantitation and detection limit. Column efficiency was investigated by performing system suitability test including parameters like retention time (tR), tailing factor (T), theoretical plates (N) and resolution (Res). Calibration curves were obtained to evaluate linearity and regression characteristics, which involves correlation coefficient, slope, standard error, intercept and standard error estimate. Accuracy of method was estimated from recovery of pure drug in dosage formulation. Precision in terms of inter-day and intra-day was evaluated as %RSD at six different concentrations of each standard analyte. Values of LOD and LOQ were reported as 3 and 10 times signal/noise ratio to baseline of peak response. Flow rate, pH, wavelength and mobile phase composition were altered deliberately to verify method feasibility.

Result and Discussion

Method Development and Optimization

Method development has great importance for advancement of different industrial products; it has also been widely used in pharmaceutical industry to monitor the drug at every step of quality control and quality assurance. This work describes a new method for simultaneous determination of citalopram with NSAIDs (piroxicam, celecoxib and diclofenac sodium), its validation and application in pharmaceutical formulation and human serum.

For optimization, a variety of operating conditions were rationally evaluated. Different mobile phase compositions were tested at first, including (1) acetonitrile and water and (2) methanol and water. Comparison data confirmed that the best separation with finest peak shape was achieved with methanol and water. Both mobile phases were tried in various ratios in the gradient elution increasing the organic solvent content from 50% to 90%. It has been observed that in reversed-phase chromatography, organic solvent content lessens the static retention and reduce the retention time of analyte. Acetonitrile-water system eluted all targets in short analysis time, however resolution was not appropriate. Thus the condition of 80% methanol and 20% water was maintained throughout the analysis at flow rate 1.0 mL min-1. In addition, to stabilize the retention and selectivity, it is imperative to regulate pH in reversed phase liquid chromatographic system.

For this purpose, pH of water (used for gradient elution) was adjusted with o-phosphoric acid and scanned between pH 2 to 4. Well separated, narrow peaks with symmetry suggested pH 3.5 +- 0.2 is appropriate for the proposed method. Furthermore, to best select the detector wavelength, each drug was scanned in the region 200-800 nm on Shimadzu-1800 UV-visible detector and compared. The isosbestic point 230 nm was found to be satisfactory (Fig. 2), permitting the detection of all the studied analytes with adequate sensitivity confirming the applicability of the developed method on commercial formulation (Fig. 3) and human serum (Fig. 4).

Method validation

The developed method was validated for system suitability, specificity, linearity, repeatability, reproducibility, recovery, limits of detection (LOD) and quantification (LOQ) and robustness following guidelines given by International Conference on Hormonization (ICH) [37].

System suitability test

System suitability tests were performed on each day of method validation by ten replicate analysis of standard in order to ratify the column efficiency and resolution of the chromatographic system (Fig. 5a, 5b and 5c). The number of theoretical plates [greater than or equal to] 2000 and tailing factor a$? 2 confirms the suitability of method (Table-1).

Table-1: System suitability of method.

###Drugs###tRa###Nb###Tc###Resd

###Citalopram###2.3###2510###1.533###0.00

###Piroxicam###4.4###4244###1.326###9.619

###Celecoxib###5.4###5192###1.203###3.834

Diclofenac sodium###7.2###5809###1.178###4.884

Table-2: Regression characteristics.

###Parameters###Citalopram###Piroxicam###Celecoxib###Diclofenac sodium

###Slope###1843.8###2200.1###3795.8###2257.3

###Linearity (ug mL-1)###0.6-20###0.9-28###0.6-20###1.0-32

###Intercept###1665.2###5579###2428.9###2927

###Correlation coefficient###0.9999###0.9988###0.9983###0.9984

###Standard error###0.1312###0.4769###0.3558###0.6585

###Standard error estimate###0.2036###0.8954###0.6934###1.2808

###LOD (ng mL-1)###16.45###23.33###27.66###14.44

###LOQ (ug mL-1)###0.04986###0.07071###0.08383###0.04386

Linearity

A linear regression model using least squares regression was applied to evaluate linearity. Excellent linearity was observed over the examined range within an overall correlation coefficient 0.998. The typical regression equations for calibration curves (n=6) of citalopram and NSAIDs along with standard error and standard error estimate are given in Table-2.

Precision

Data for precision is summarized in Table-3. For repeatability, six replicates in the linearity ranging 0.6-20, 0.9-28, 0.6-20 and 1.0-32 ug mL-1 for citalopram, piroxicam, celecoxib and diclofenac sodium respectively, were injected to chromatograph under the same operational conditions. The RSD values were found to be in the range 0.31-1.6, 0.23-0.92, 0.32-1.13, and 0.14-1.67% for citalopram, piroxicam, celecoxib and diclofenac sodium respectively for interday precision.

Intermediate precision study was extended to two consecutive days of method validation at mentioned concentration levels. Intra-day precision was determined to be 0.43-1.2, 0.67-1.03, 0.33-1.50, and 0.61-1.31% RSD for citalopram, piroxicam, celecoxib and diclofenac sodium respectively. All the obtained values fit to the acceptance criteria assuring the repeatability and reproducibility of method.

Accuracy

Recovery experiments were performed to estimate accuracy of the developed method. It was calculated by reviewing the recovery values at three concentration levels of each bulk drug in the range of 1.25-5.0, 1.6-7.0, 1.25-5.0 and 2.0-4.0 ug mL-1 for citalopram, piroxicam, celecoxib and diclofenac sodium respectively in pharmaceutical formulation and human serum. Recovery values were found to be 99.64-100.86%, 99.61-100.03%, 99.86-100.54% and 99.93 -100.36% for citalopram, piroxicam, celecoxib and diclofenac sodium respectively. Table 4 represents good %recovery values of each drug in pharmaceutical formulation and in human serum (Fig. 4a and 4b).

Specificity

The specificity of method was assessed by observing the absence of interfering peaks at the retention times of analytes. Chromatograms were recorded for pure solvent, reference standard, pharmaceutical formulation, blank serum solution and spiked serum sample, and it was observed that there was no endogenous interference at the retention time of studied analyte and chromatograms for all the injected solutions exhibited good specificity (Fig. 6).

Table-3: Precision of method.

###Citalopram###Piroxicam###Celecoxib###Diclofenac sodium

Conc###Conc###%RSDb###Conc###Conc

###%RSDa###%RSDb###%RSDa###%RSDa###%RSDb###%RSDa###%RSDb

ug mL-1###ug mL-1

###ug mL-1###ug mL-1

###20###0.31###1.09###28###0.23###0.90###20###0.32###0.46###32###0.34###0.67

###10###1.60###1.20###14###0.92###0.72###10###0.84###0.33###16###0.14###0.61

###5###0.16###0.43###7###0.45###0.67###5###0.86###1.33###8.0###1.47###0.51

###2.5###1.49###0.50###3.5###0.61###1.03###2.5###0.83###0.40###4.0###1.67###1.31

###1.25###0.57###1.40###1.6###0.36###0.71###1.25###0.96###0.52###2.0###0.68###0.78

###0.6###0.88###1.11###0.9###0.80###1.02###0.6###1.13###1.50###1.0###0.85###0.94

###Human Serum

###Citalopram###Piroxicam###Celecoxib###Diclofenac sodium

###Conc.###Conc.###Conc.###Conc.

###%RSD###%RSD###%RSD###%RSD

###ug mL-1###ug mL-1###ug mL-1###ug mL-1

###10###1.43###14###1.73###10###0.78###16###0.67

###5###0.09###7###1.50###5###0.36###8.0###1.61

###2.5###1.69###3.5###0.97###2.5###1.56###4.0###0.26

Table-4: Recovery of method.

###PramcitA(r)(20 mg)###Feldene(10 mg)###Celbex(100 mg)###Dicloran(50 mg)

###Conc.###Conc.###Conc.###Conc.

###%Rec###%Error###%Rec###%Error###%Rec###%Error###%Rec###%Error

ug mL-1###ug mL-1###ug mL-1###ug mL-1

###10###99.64###-0.36###14###100.03###0.03###10###100.05###0.05###16###99.93###-0.07

###5###100.86###0.85###7###99.68###-0.32###5###99.86###-0.14###8.0###100.28###0.28

###2.5###99.73###-0.27###3.5###99.61###-0.39###2.5###100.54###0.54###4.0###100.36###0.35

###Human Serum

###Citalopram###Piroxicam###Celecoxib###Diclofenac sod

###Conc.###Conc.###Conc.###Conc.

###%Rec###%Error###%Rec###%Error###%Rec###%Error###%Rec###%Error

###ug mL-1###ug mL-1###ug mL-1###ug mL-1

###10###98.54###-1.486###14###99.43###-0.570###10###100.87###0.860###16###98.54###-1.486

###5###98.63###-1.390###7###98.45###-1.575###5###99.89###-0.114###8.0###98.08###-1.961

###2.5###101.07###1.060###3.5###98.25###-1.782###2.5###99.47###-0.531###4.0###100.79###0.786

Table-5: Robustness of the proposed method.

###Citalopram###Piroxicam###Celecoxib###Diclofenac sod

###Parameters###T###N###T###N###T###N###T###N

###0.9###1.764###2425###1.500###4042###1.352###4895###1.356###5597

###Flow rate (mL min-1)###1.0###1.714###2285###1.429###3974###1.294###4858###1.243###5495

###1.1###1.692###2126###1.401###3828###1.274###4788###1.221###5367

###228###1.756###2226###1.509###3800###1.366###4640###1.321###5255

###Wave length (nm)###230###1.766###2234###1.500###3831###1.366###4692###1.319###5327

###232###1.757###2233###1.509###3853###1.363###4713###1.313###5362

###3.4###1.942###1979###1.520###3485###1.309###4498###1.202###5281

###pH###3.5###1.647###2429###1.443###4092###1.312###5079###1.256###5704

###3.6###1.832###2129###1.404###3534###1.304###4512###1.198###5239

###78:22###1.775###2222###1.711###3746###1.286###4322###1.270###3476

###Mobile phase###80:20###1.746###2283###1.324###3790###1.365###5831###1.283###6377

###82:18###1.748###2268###1.463###3919###1.351###5252###1.306###6445

Sensitivity

Regression data was used to find standard deviation and slope to evaluate the sensitivity of method. The limits of detection (LOD) and quantitation (LOQ) are the concentration resulting in a signal-to-noise ratio greater than or equal to three and ten respectively. The LOD of citalopram and studied NSAIDs i.e. piroxicam, celecoxib and diclofenac sodium were found to be 16.45, 23.33, 27.66 and 14.44 ng ml-1 and 0.04986, 0.07071, 0.08383 and 0.04386 ug mL-1 respectively. Table 2 represents the detailed LOD and LOQ data for each analyte.

Robustness

Robustness study was estimated by considering effect of changes in the operating parameters of the established analytical method which involved variation in pH of solvent, composition of mobile phase, flow rate range and wavelength. Robustness data confirmed the reliability of the proposed method throughout normal practice. It was observed that the method stood up to deliberate changes made in chromatographic parameters. Table 5 shows robustness data.

Assay in pharmaceutical formulation

The validated LC method was found to be accurate and applicable for the simultaneous determination of citalopram (Pramcit 20 mg) with piroxicam (Feldene 10 mg), celecoxib (Celbex 100 mg) and diclofenac sodium (Dicloran 50 mg) in commercial formulations showing good separation with high resolution for all the analytes at the isosbestic point. Proposed method exhibited good percent recoveries and %RSD values (Table 3 and 4). Furthermore, resulted chromatograms confirmed elution of analytes was not interrupted by any excipient in the drug formulation. Hence, developed method is reliable and sensitive for the simultaneous determination of citalopram with piroxicam, celecoxib and diclofenac sodium in commercial formulation without any interference of unwanted species.

Assay in human serum

Proposed method for simultaneous determination and quantification of citalopram with piroxicam, celecoxib and diclofenac sodium in human serum was assessed for routine analysis. Results showed good separation and percent recoveries within the satisfactory limits (Table 3 and 4). Any interference of endogenous components of serum was not detected. Therefore, it is confirmed that the validated LC method is appropriate for simultaneous determination of citalopram with piroxicam, celecoxib and diclofenac sodium in human serum and applicable for routine and clinical analysis.

Conclusion

The assay defined herein for simultaneous liquid chromatographic method with UV detection determination of citalopram with NSAIDs in bulk drug, pharmaceutical formulation and human serum. The method was found to be reliable, sensitive, specific and robust and showed satisfactory linearity and accuracy for all the studied analytes. Based on validation and applicability, it is concluded that the method is well suited and meets the need for routine analysis of studied analytes as well as for laboratory applications.

Acknowledgement

The authors highly acknowledge the support of Prof. Dr. Saqib Anjum, Chairman, Department of Chemistry and Dr. Zahoor-ul-Hassan Awan, Assistant Professor, Department of Chemical Engineering, NED University of Engineering and Technology, Karachi. Authors acknowledge Higher Education Commission, Pakistan for SRGP research grant (21-250/SRGP/RandD/HEC/2014).

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Title Annotation:non-steroidal anti-inflammatory drugs
Author:Ali, Saeeda Nadir; Naz, Nida; Qayoom, Amtul; Akram, Shumaila
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Dec 31, 2017
Words:5081
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