Indian Journal of Chemistry Vol. 58B, July 2019, pp. 855-860

Synthesis and characterization of diclofenac impurity-A for the quality control of diclofenac and its formulation as per international compendiums

Aparna Wadhwa*, Sandhya Verma, Robin Kumar, Puran L Sahu & Abhishek Singh

Reference Standard Division, Indian Pharmacopoeia Commission, Ministry of Health and Family Welfare, Govt. of India, Sector-23, Rajnagar, Ghaziabad 201 002, India

E-mail: waparna90@gmail.com

Received 29 August 2018; accepted (revised) 8 May 2019

Diclofenac is one of the top selling non steroidal anti-inflammatory drug (NSAID). It is available in wide range of dosage form for symptomatic relief of diverse inflammatory conditions. Presence of impurity in drug or drug products is a vital concern now a days for which stringent regulatory guidelines have been implemented to safeguard public health. Under these circumstances, presence of diclofenac impurity-A in diclofenac requires strict quality control to satisfy the specified regulatory limit. Therefore, the objective of the presence study is to synthesize diclofenac impurity-A in the purest form and its subsequent characterization by using a panel of sophisticated analytical techniques to provide as reference standard mentioned in most of the international compendiums including Indian Pharmacopoeia.

Keywords: Diclofenac, diclofenac impurity-A, Indian pharmacopoeia, non steroidal anti-inflammatory drug, symptomatic relief

Diclofenac, is one of the most extensively used nonsteroidal antiinflammatory drugs (NSAID). It is chemically 2-(2,6-dichloranilino) phenylacetic acid and primarily available as the sodium or potassium salt form1,2. It is available in a wide range of dosage forms to combat pain and inflammation for different therapeutic applications3.

Impurities are the organic or inorganic or volatile substance present in the drug or drug products. These are generated either during the manufacturing process of API’s (Active Pharmaceutical Ingredients) or during their storage4. Presence of these impurities in drug substances even in trace amounts can lead to negative impact on the patients in the form of adverse effects. Diclofenac Impurity-A is chemically 1-(2,6-Dichlorophenyl)-1,3-dihydro-2H-indol-2-one and present in diclofenac as an impurity5. With the increase in the worldwide usage of this drug, there is a need to monitor the limit of this impurity as per acceptable limit in the official compendiums. Under these circumstances, we have synthesized this impurity in pure form such that the quality control of this widely used drug can be achieved.

To the best of our knowledge, there is no data available in the literature for the synthesis and characterization of diclofenac Impurity A till date. Hence, the objective of the present study is to synthesize diclofenac Impurity A followed by its characterization by using sophisticated analytical techniques like FT-IR, DSC, TGA, 1H and 13C NMR, LC-MS, CHN analysis, HPLC and GC-HS in order to confirm its identity, property and purity.

Experimental Section

Materials Diclofenac free acid (purity>99%) was received as

gift sample from a renowned manufacturer. Xylene, boric acid, orthophosphoric acid, sodium dihydrogen phosphate (AR grade) and potassium bromide (IR spectroscopy grade) were purchased from Merck (Mumbai, India), whereas petroleum ether, ethyl acetate, o-xylene and N,N dimethyl formamide (GC grade) were purchased from Sigma Aldrich (St. Louis, USA). HPLC grade water having resistivity of 18.2 MΩ cm (Milli Q water purification system) was used throughout the analysis.

Methods

Synthesis of Diclofenac impurity-A A solution of diclofenac free acid (5g, 0.016 mol)

in xylene (50 mL) was mixed with boric acid (1g, 0.016 mol). Then, the reaction mixture was refluxed

List of abbreviations: FT-IR = Fourier Transformer InfraredSpectroscopy; DSC = Differential Scanning Calorimetry; TGA =Thermogravimetric Analyser; NMR = Nuclear Magnetic Resonance;LC-MS = Liquid Chromatography-Mass Spectrometry; GC-HS =Gas Chromatography-Head Space; HPLC = High PerformanceLiquid Chromatography; CHN = Carbon Hydrogen NitrogenAnalyser.

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for 8 h, following which the organic layer was separated and concentrated under reduced pressure. The red coloured product formed was purified by column chromatography using petroleum ether and ethyl acetate (70:30). The product obtained was characterized to confirm its structure, physical or chemical properties and purity by different analytical techniques as follows.

Fourier Transformer Infrared Spectroscopy The FT-IR spectrum of synthesized impurity was

obtained using the FT-IR spectrophotometer instrument (Make: Perkin-Elmer; Model: Frontier Optica with spectrum software). About 5 mg sample was mixed with about 500 mg potassium bromide by triturating in a mortar and then compressed into potassium bromide disk in a hydraulic press. The disk was placed in a cell with an atmosphere of dry nitrogen. The spectrum was collected in the range of 400–4000 cm–1 with an average of 32 scans at spectral resolution of 2 cm–1.

Differential Scanning Calorimetry Differential scanning calorimetry was performed on

the synthesized impurity using DSC instrument (Make: Perkin-Elmer; Model: DSC 6000). 1-2 mg of sample was weighed directly in an aluminium pan and sealed followed by scanning in the temperature range of 30-350°C at a heating rate of 10°C/min under nitrogen atmosphere with the flow rate of 20 mL/min. DSC curves obtained were analyzed with the aid of Pyris software.

Thermo-gravimetric analysis A TGA instrument (Make: Mettler Toledo; Model:

TGA/DSC1) was used and a TGA curve of synthesized impurity was obtained by putting approximately 4-5 mg sample in alumina crucible at a heating rate of 10°C /min in the range of 30–600°C under an atmosphere of nitrogen at the flow rate of 50 mL/min. TGA curve was analyzed with the aid of Stare software.

1H and 13C NMR analysis 1H and 13C NMR spectra were recorded on NMR

system (Make: Agilent Technologies) operating at 500 MHz For 1H spectra, 2-3 mg of sample and for 13C spectra, 15-20 mg of sample was taken in NMR tube and dissolved in suitable quantity of DMSO-d6. Spectra obtained were analyzed with the help of VNMRs-500 software.

LC-MS analysis LC-MS study was performed using liquid

chromatography system coupled with Q-TOF mass

spectrometer (Make: Agilent Technologies; Model: 1200 Infinity series for LC and 6520 Accurate Mass for MS) which was operated in positive modes with ESI source for mass spectrometric detection. Liquid chromatography conditions included an isocratic mobile phase consisting of a mixture of acetonitrile: 0.1% formic acid solution (50:50, %v/v) at a flow rate of 0.3 mL/min with injection volume of 0.1 µL and connected to mass spectrometer through a union. The capillary voltage applied was 3500 V. The gas temperature was set at 300°C, using nitrogen as nebulizing gas and drying gas. Drying gas flow was maintained at 10 L/min, nebulizer pressure 32 psi and fragmentor 75. The mass spectrum was acquired over an m/z range of 50-1000. 2-3 mg of sample was dissolved and diluted with suitable quantity of mobile phase and injected into LC-MS system. The instrument was controlled and the data integration was performed with Mass Hunter software.

CHN analysis Elemental analysis was carried out using CHNS

Elemental analyzer (Make: EuroVector; Model: EA3000). Sample was weighed on electronic weighing balance and put into the sampler of the instrument. Calculations were performed using the areas of peaks using the Callidus software.

HPLC analysis HPLC system (Make: Thermo Fisher Scientific;

Model: Dionex Ultimate 3000) consisted of Quarternary pumps, variable wavelength detector and auto sampler. Chromatographic separation was achieved by using Zorbax eclipse plus C8 (250 mm × 4.6 mm, 5 µm particle size; Agilent Technologies) column, kept at room temperature, with isocratic mobile phase consisting of a solution of 660 mL methanol and 340 mL buffer (0.531 mL of orthophosphoric acid and 1.855 g of sodium dihydrogen phosphate monohydrate in 1000 mL water adjusted to pH 2.5 with OPA) at a flow rate of 1.0 mL/min. Samples were monitored at the wavelength of 254 nm. The chromatographic data were recorded using Chromeleon software. For LC analysis, 20 µL of sample was injected into HPLC system.

Regarding sample preparation for HPLC analysis, system suitability solution was prepared as per IP-2018 (concentration of 5 μg/mL for each diclofenac and synthesized impurity), standard solution (concentration of 500 μg/mL for diclofenac), test solution (concentration of 500 μg/mL for synthesized

WADHWA et al.: DICLOFENAC IMPURITY-A

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impurity) and spiked solution (equal volume of standard and test solution) were prepared in mobile phase. A criterion for system suitability evaluation was resolution between the peaks corresponding to diclofenac and diclofenac impurity-A should be at least 6.5 5. Data was evaluated by % area normalization procedure considering diluent (mobile phase) as blank.

GC-HS analysis Quantification of residual solvents was performed

using gas chromatograph (Make: Agilent Technologies; Model: 7890A) with head space injection system (GC-HS) which was equipped with standard oven for temperature programming, split/split less injection ports and flame ionization detector (FID). Separation was achieved in Elite wax column (30 m × 0.32 mm × 0.5 μm; make: Perkin-Elmer) using nitrogen as carrier gas. Parameters for GC-HS analysis are described as follows: carrier gas flow (1.0 mL/min), hydrogen gas flow (40 mL/min), air gas flow (400 mL/min), injection volume (500 μL), Split ratio (1:1), incubation time (900 s), incubation temperature (90°C), syringe temperature (95°C), injection temperature (200°C), detector temperature (270°C). Temperature programming for analysis was as follows: Initially temperature was maintained at 35°C for 5 min then raised to 80°C at the rate of 2°C/min, and further raised this temperature to 200°C at the rate of 40°C/min, this temperature was maintained for 5 min.

Temperature programming for analysis was as follow: Initially temperature was maintained at 40°C for 10 min and then raised to 130°C at the rate of 8°C/min, then held for 5 min followed by a rise to 240°C at the rate of 35°C /min and then held there for 10 min.

The standard solution was prepared from o-xylene (54.4 μg/mL), pet. ether (124.8 μg/mL) and ethyl acetate (124.8 μg/mL); final concentration of standards were 2170 ppm, 5000 ppm and 5000 ppm respectively. N,N-Dimethyl formamide was used as diluent. The sample was prepared as in duplicate by taking 50 mg of synthesized impurity in head space vial and 2 mL of diluent added. The vial was closed with screw cap properly and analyzed in GC-HS/FID system. Criteria for system suitability evaluation were resolution between each peak should be at least 1.5 and % RSD of replicate injections of standard solution should be not more than 8.0%. Data was evaluated by % area normalization procedure considering diluent as blank using EZChrome Elite software.

Results and Discussion As a part of our investigation in developing a

versatile and efficient method for synthesis of diclofenac impurity A, the present impurity synthesis has been proposed by using commercially available cheap catalyst such as boric acid. It is well known that boric acid can act as a catalyst for intermolecular amide bond formation where it reacts with carboxylic acid to form a mixed anhydride as the actual acylating agent which thereafter couples with amine to form the amide (Figure 1). It was envisaged that boric acid might catalyze intramolecular amide bond formations as well for the affordable synthesis of desired product6,7. Thus, diclofenac free acid was refluxed in p-xylene using catalytic amount of boric acid and gratifyingly, diclofenac Impurity was obtained in excellent yield of 92%. It was a yellowish-orange coloured powder and soluble in methanol, ethyl acetate and petroleum ether. The synthesized product was made to undergo several studies for confirmation of its structure and different properties studied which are discussed below.

In FT-IR spectra, the principal peak obtained at 784 cm–1 may be attributed to C-Cl group (785-540 cm–1) whereas the peak at 1734 cm–1 may be assigned to –C=O stretching in amide group. Though the range of –C=O group for amide stretching lies in the region 1690-1630 cm–1 but due to the presence of electron withdrawing phenyl group as a substituent on nitrogen atom, the frequency shifts to the higher region. The N-H stretching was observed at 3447 cm–1. Therefore, these results appear to confirm the formation of diclofenac impurity-A as indicated by the presence of above mentioned functional groups (Figure 2a).

DSC curve of the product exhibited a shallow broad endothermic effect in the temperature range of 100-150°C as a result of heat of dehydration followed by the sharp endothermic event between 118-120°C with a melting temperature of 119°C. TGA curve exhibited a mass loss of 78.16-115.84% (Figure 2b).

Positive electron spray mass spectra of the synthesized compound have shown an intense [M+H]+ ion peak at m/z 278.0150 (Figure 2c). The diclofenac Impurity-A has the mass (100% abundance) of 277, the mass obtained in LC-MS study corresponds to its molecular mass.

The molecular formula of Diclofenac Impurity-A is C14H9Cl2NO. Anal. Calcd for C14H9Cl2NO: C, 60.02; H, 3.23; N, 5.00. Found: C, 61.22; H, 3.88; N, 5.44%.

858

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INDIAN J. CHEM., SEC B, JULY 2019

860

test solution are presented in Figure 3b. Results show that 17 ppm of petroleum ether and 2327 ppm of ethyl acetate were present in the synthesized impurities which are within limit of 5000 ppm as per ICH guidelines for residual solvents4. The accuracy of the analysis was assured by calculating recovery, which are 81% for petroleum ether and 89% for ethyl acetate. Conclusion

A simple and convenient synthesis method was described for preparation of diclofenac impurity-A. It was synthesized and characterized by FT-IR, DSC, TGA, 1H and 13C NMR, LC-MS, CHN, HPLC and GC-HS. The compound obtained was in sufficiently pure form to be suitable as reference standard with defined potency. Finally, it will be effective for the regulatory agencies to implement strict control of this impurity in wide range of available diclofenac formulations to safeguard public health.

Acknowledgement The authors are grateful to the Indian

Pharmacopoeia Commission, Ministry of Health and Family Welfare, Govt. of India for providing necessary instrumental facilities.

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