Development and Validation of an Ecofriendly Chemiluminescence Method for the Determination of Citalopram in Pharmaceutical Preparations using Cu 2 +-grafted Oxidized Multiwall Carbon Nanotubes

A novel and sensitive chemiluminescence (CL) method was developed and validated for the determination of citalopram in bulk dosage form and in pharmaceutical preparations. The method is based on the use of Cu-grafted oxidized multiwall carbon nanotubes (Cu/ox-MWCNTs). The weak CL signal arising from the reaction of an alkaline luminol-H2O2 system was significantly enhanced by the addition of citalopram in the presence of ox-MWCNTs/Cu. Furthermore, ox-MWCNTs/Cu exhibited exceptional catalytic activity towards the oxidation of luminol in the luminol-H2O2 CL reaction. Multiwalled carbon nanotubes (MWCNTs) were characterized by scanning electron microscopy (SEM), which also showed the attachment of Cu to MWCNTs. Various factors affecting CL intensity were carefully investigated and optimized for citalopram quantitation. The CL intensity was proportional to citalopram concentration in the range 0.2–8.0 μg mL, with a correlation coefficient of 0.996. The limit of detection (LOD) and limit of quantification were 2.29 × 10 μg mL and 7.64 × 10 μg mL, respectively, and its reproducibility was satisfactory with a relative standard deviation (RSD) of 2.59 % (n = 5). The interference effects of common excipients were studied, and the developed method was effectively applied for the determination of citalopram in pure form and in pharmaceutical preparations. Percentage recoveries were calculated and ranged from 98.67 to 101.46 % for the pure form and from 97.38 to 101.72 % for pharmaceutical preparations.


Introduction
Citalopram (CIT; 1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-1, 3-dihydroisobezofuran-5-carbonitrile) 1 is a second generation selective serotonin reuptake inhibitor, which belongs to the group of drugs known as antidepressants.CIT has broad-spectrum therapeutic activity against depression, anxiety, and obsessive and impulse control disorders. 2,3It has also been reported to reduce the symptoms of diabetic neuropathy and premature ejaculation 4,5 , and may be effective for the treatment of post-stroke pathological crying. 6IT is normally taken as a single daily dose in the morning or evening with or without food.Food does not increase the absorption of CIT but helps to prevent nausea.In the therapeutic dose range, CIT is safe and well tolerated.Recommended doses range from 20 to 40 mg daily, which results in a plasma concentration range from 30-130 ng mL -1 . 7Overdosage may cause drowsiness, vomiting, dizziness, sweating, nausea, coma, confusion, and heartbeat anomalies. 8italopram is absorbed by cytochrome P to its demethylated metabolites, desmethylcitalopram and didesmethylcitalopram.Desmethylcitalopram is the chief metabolite and can also prevent 5-HT reuptake, but is less potent than citalopram. 9Until recently, citalopram was sold as a racemic mixture, consisting of 50 % R-(-)-citalopram and 50 % S-(+)-citalopram, but as the S-(+)-enantiomer has the desired antidepressive effect 10 , it was recently introduced under the generic name escitalopram.
Various analytical techniques have been developed to determine citalopram levels in commercial formulations and biological samples.2][23][24][25][26] Spectrofluorimetric [27][28][29][30] and spectrophotometric [31][32][33] methods have also been reported for the citalopram determination in pharmaceutical formulations.Few CL methods have been recently reported for the determination of citalopram in pharmaceutical preparations and biological samples. 34,35][38][39][40][41] On the other hand, carbon nanotube (CNT) materials are attracting considerable research interest.In particular, in addi- tion to being small sizes, CNTs have huge surface areas, hollow or layered structures, high mechanical strengths, and significant electrical conductivities.3][44][45][46] CNTs are available as singled-walled carbon nanotubes or multiwall carbon nanotubes (MWCNTs) according to the number of layers carbon atom in their walls.Furthermore, when MWCNTs are treated with a mixture of H 2 SO 4 and HNO 3 , carboxyl groups are produced on their walls and some physical properties are changed.][49][50][51][52] In the present study, a CL-based method involving the use of oxidized multiwall carbon nanotubes (ox-MWCNTs) grafted with Cu 2+ was devised to quantify citalopram levels in pharmaceutical preparations.We found that ox-MWCNTs/Cu 2+ enhanced the CL signal arising from the reaction between alkaline luminol and H 2 O 2 , and that CL intensities were further boosted by the presence of citalopram.Based on these observations, we developed a sensitive CL method for the quantification of citalopram in pharmaceutical preparations.Citalopram was found to markedly increase the CL intensity of the luminol-H 2 O 2 -ox-MWCNTs/Cu 2+ system and this enhance was proportional to the concentration of citalopram present.After optimizing experimental parameters, CL intensity was found to be linear to citalopram concentration in the range 0.2-8.0 µg mL -1 with a detection limit of 2.29 × 10 -5 µg mL -1 .

Preparation of Reagents and Sample Solutions
Luminol stock solution (4 × 10 -3 M) was prepared by dissolving 0.035 g of luminol in 4.5 mL of 0.1 M NaOH, diluted to 50 mL with deionized (DI) water, and stored at 4 °C.Hydrogen peroxide solution (4 M) was prepared by diluting 10.213 mL of 30 % H 2 O 2 to 25 mL with DI water.Sodium hydroxide (0.1 M) solution was prepared by dissolving 2.0 g of NaOH in 50 mL DI water.Working solutions were prepared freshly before use from stock solutions by proper dilution with DI water.A standard stock solution of citalopram (250 µg mL -1 ) was prepared by dissolving 0.0125 g of citalopram standard in 2 mL of distilled ethanol, and diluting to 50 mL with DI water.Working solutions of 10 µg mL -1 and 5 µg mL -1 citalopram were prepared daily by diluting proper amounts of the stock solution with DI water.

Synthesis of Oxidized MWCNTs
Ox-MWCNTs were prepared as previously described by Rosca et al. 53,54 Briefly, 0.5 g pristine MWCNTs (p-MWCNTs) were added to 200 mL nitric acid solution in a round-bottomed glass flask.p-MWCNTs were dispersed by sonication for 1 h using an ultrasonic bath (JAC Ultra-sonic 4020p, KODO, South Korea), and the mixture was then refluxed at 120 °C for 12 h under nitrogen.After cooling, the MWCNTs were washed and filtered several times using DI water and a 0.22 µm membrane nitrocellulose filter (Millipore, USA).The MWCNTs so obtained were then dried in an oven at 60 °C for 48 h (Fig. 1).

Instrument
A Hitachi F-4500 spectrofluorometer was used to detect and record CL produced by the luminol-H  system.For CL measurements, the light source of the spectrofluorometer was switched off and only the photomultiplier tube (Model, R 928, Hamamatsu, Japan) was used.The slit width of the emission monochromators was 5 nm and the power supply of the photomultiplier tube used to detect CL signals was 700 V. Light-producing reactions were carried out in a standard 1 × 1 cm quartz cell placed in the spectrofluorometer cell holder.

Analytical Procedure
Analytical procedure consisted of adding the following quantities of equilibrated solutions to the CL reaction cell using a micropipette.Citalopram stock solution to give final concentration of 0.2-8.0 µg mL -1 , 4 × 10 -4 g ox-MWCNTs/Cu 2+ , 400 µL of NaOH solution (8 × 10 -3 mol L -1 ), and 400 µL luminol (4 × 10 -3 mol L -1 ).The kinetic-curve of the CL signal was measured at 430 nm after injecting of 400 µL (4 × 10 -2 mol L -1 ) of H 2 O 2 into cells to initiate the CL reaction.CL intensities (DI) were calculated using DI = I s -I o , where I s and I o are CL signals in the presence and absence of citalopram, respectively.

Application to Pharmaceutical Preparations
Five tablets each containing 20 mg of active citalopram were weighed and weights were averaged.The tablets were then powdered in a mortar.The powder equivalent of 0.0125 g citalopram was dissolved in 2 mL of ethanol, approximately 10 mL of DI water was added, and mixture sonicated for 10 min.The resultant solution was then filtered and diluted to 50 mL with DI water.An appropriate volume of this solution was then diluted with DI water to provide a citalopram concentration in the working range.A certain volume of this solution was then analyzed using the above described procedure and amount of citalopram present per tablet was calculated by the calibration equation.

CL Characterization
A CL emission spectrum of the luminol-H 2 O 2 -citalopram-MWCNTs/Cu 2+ system is presented in Fig. 3.As shown in Fig. 3(a), the luminol-H 2 O 2 system produced a weak CL signal, which was not enhanced by the addition of ox-MWCNTs (Fig. 3(b)).However, the addition of ox-MWCNTs/Cu 2+ to the luminol-H 2 O 2 system augmented the CL signal (Fig 3(c)), and the addition of citalopram markedly enhanced the signals obtained (Fig 3(d)), which exhibited maximum intensity at 430 nm.Furthermore, the CL intensity of the luminol-H 2 O 2citalopram-MWCNTs/Cu 2+ system was proportional to the concentration of citalopram present.

Optimization of the Reagents Concentration
The effect of the amount of MWCNTs/Cu 2+ used was investigated in the range 2-10 × 10 -4 g.Maximum CL intensity of the luminol-H 2 O 2 -citalopram-MWCNTs/Cu 2+ system was achieved at 4 × 10 -4 g (Fig. 4), and thus, this amount was used in further experiments.The effect of sodium hydroxide concentration on the luminol-H 2 O 2 -citalopram-MWCNTs/Cu 2+ system was investigated in the range 2-12 × 10 -3 mol L -1 .CL intensity was found to peak at a NaOH concentration of 8 × 10 -3 mol L -1 (Fig. 5).The

Analytical Features of Merit
Under optimum experimental conditions, the concentration of citalopram and chemiluminescence intensity exhibited a linear relationship in the range 0.2-8.0 µg mL -1 with a correlation coefficient of 0.996-0.9977(Fig. 8a,b).Limit of detection (LOD) was defined as the concentration of citalopram that produced a CL intensity three times the blank standard deviation (LOD = 3 S b /m), whereas limit of quantification (LOQ) was defined as the concentration of the citalopram that produced a CL intensity ten times the blank standard deviation (LOQ = 10 S b /m).Calculated LOD and LOQ values were 2.29 × 10 -5 µg mL -1 and 7.64 × 10 -5 µg mL -1 respectively.The linear regression equation, its slope, intercept, and correlation coefficient, and the relative standard deviation of the response factor are provided in Table 1.The sensitivity of the developed method is compared with other described citalopram methods in Table 2.The table shows that the sensitivity of the devised method is greater than that of any of the previously described methods.

Interference Effects
Interference by common excipients like glucose, starch, fructose, lactose, magnesium stearate, and talc were studied.The interference study was conducted by preparing samples containing fixed amounts of citalopram (0.2 µg mL -1 ) and differ-ent concentrations (5, 10, 20, and 40-fold concentrations) of excipients.No interferences were observed by these common excipients (Fig. 9).

Reliability of the Method
The precision of the proposed method was studied by analyzing pure citalopram and pharmaceutical formulations using three different concentrations of each in triplicate.Results are given in Table 3 for pure citalopram and in Table 4     ) of pure citalopram solution were added to tablet solutions (0.20 µg mL -1 ) and analyzed using the described procedure.
Recoveries were calculated and obtained in the range of 97.38-101.72 % (Table 5), demonstrating the excellent accuracy of the developed method for the determination of citalopram in commercial formulations.

Applicability
The proposed method was fruitfully applied to the determination of citalopram in three different pharmaceutical preparations.The results obtained agreed well with manufacturers' claims (Table 6), indicating that the method can be used to quantify citalopram levels in commercial formulations.

Conclusion
A simple precise accurate and sensitive method was developed for the quantification of citalopram.The method is based on the enhancement of citalopram CL of the luminol-H 2 O 2 -MWCNTs/Cu 2+ system.The method has a wider linear range and lower limits of detection and quantification than other    described methods.In addition, it was successfully applied for the determination of citalopram in pharmaceutical preparations with good recovery and excellent reproducibility.The commonly present excipients in commercial formulations did not interfere with the analysis.Accordingly, we conclude that the developed method is appropriate for the quantification of citalopram in quality control laboratories and industrial fields.

Figure 1
Figure 1 Schematic diagram of the preparation of oxidized MWCNTs and Cu 2+ -grafted MWCNTs.

Figure 9
Figure 9 Effect of excipients on CL intensity.

Table 1
Analytical parameters for the chemiluminescence determination of citalopram.

Table 2
Comparison of the present method with other reported methods for the determination of citalopram.

Table 3
Accuracy and precision of the devised method for the quantification of pure citalopram.
Each result is the averages of separate triplicate analyses; RSD = Relative standard deviation.

Table 4
Evaluation of the accuracy and precision of the devised method for citalopram quantification in pharmaceutical preparations.Each result is the averages of separate triplicate analyses; RSD = Relative standard deviation.

Table 5
Recovery percentages for citalopram in commercial tablet formulations determined by the standard addition method.
Each result is the averages of separate triplicate analyses; RSD = Relative standard deviation.

Table 6
Determination of citalopram active ingredients in pharmaceutical tablet preparations.Each result is the averages of separate triplicate analyses; RSD = Relative standard deviation.