Reversed-Phase High-Performance Liquid Chromatographic and Mass Spectrophotometric Methods for Simultaneous Determination of Paracetamol, Aceclofenac and Tramadol in Combined Tablet Dosage Form in Presence of its Degradation Products

 

Govind Sharma¹*, Nitin Bansal² , Dinesh Kumar Jain², Shekhar Verma¹and A. K. Jha¹

¹Faculty of Pharmaceutical Sciences, SSTC, Junwani, Bhilai (CG)

²College of Pharmacy, IPS Academy, Indore [Madhya Pradesh]-452012, India

*Corresponding Author E-mail: govindsharma10aug@gmail.com

ABSTRACT:

Simple, accurate, precise, and sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method for simultaneous estimation of Paracetamol (PCM), Tramadol and Aceclofenac in the presence of its degradation products, have been developed and validated by LC/MS. Linearity of PCM, TRA and ACL was obeyed in concentration range 2-30, 2-14 and 2–35 µg/ml in Acetonitrile: Phosphate buffer (pH 3.4, 20:80 v/v) as the mobile phase at 270 nm, respectively. The detection was carried out using a diode array detector set at 270 nm. Linearity of the LC method was in the concentration range of 2-10 µg/mL for PCM, TRA and ACL respectively. The recoveries were in the range of 99.49 ± 0.74 to 100.08 ± 0.1 for PCM, 99.36 ± 0.73 to 100.05 ± 0.10 for TRA and 98.42 ± 0.96 to 101.04 ± 0.49 for ACL in RP-HPLC. RP-HPLC method has been successfully applied for the analysis of the drugs in a pharmaceutical formulation in the presence of its degradation products. Results of analysis were validated statistically. Degradation study was performed under different conditions using same RP-HPLC method and method was revalidated for simultaneous estimation in presence of their degradation products and confirmed by LC/MS.

 

 

 

INTRODUCTION:

Paracetamol (PCM) is chemically known as N-(4-hydroxyphenyl)acetamide and belongs to the class of compounds known as NSAID,^1-3 and also having antipyretic action. Its action mediates through cyclo-oxygenase-3 inhibition and modulation of central serotonergic pathways.

Aceclofenac (ACL) is chemically known as 2-[2-[2-(2,6-Dichlorophenyl) aminophenyl]acetyl]oxyacetic acid. ACL is phenylacetoxyacetic acid, is a novel non-steroidal anti-inflammatory drug. Aceclofenac inhibited interleukin-1β-induced prostaglandin E2 production by human rheumatoid synovial cells, but had no inhibitory effect on cyclooxygenase-1 or cyclooxygenase-2 activities by itself. Tramadol (TRA) is chemically known as (1R, 2R)-rel-2-[(dimethylamino) methyl]-1-(3-methoxyphenyl) cyclohexanol.

 

RA is a μ-opioid receptor agonist, serotonin releasing agent, norepinephrine reuptake inhibitor, NMDA receptor antagonist, 5-HT2C receptor antagonist (α7)5 nicotinic acetylcholine receptor antagonist, and M1 and M3 muscarinic acetylcholine receptor antagonist. The analgesic action of tramadol has yet to be fully understood, but it is believed to work through modulation of serotonin and norepinephrine in addition to its mild agonist of the μ-opioid receptor. The chemical structures of PCM, TRA and ACL are shown in Figure 1.

 

Figure 1. Chemical structures of PCM, TRA and ACL

Literature survey revealed that the assay of the PCM in pure and dosage forms is official in Indian Pharmacopoeia (1998), British Pharmacopoeia (2004) and USP30-NF25,^1-3 apart from Pharmacopeias several analytical methods have been reported for the determination of PCM in biological fluids and urine,^4–6 including column high-performance liquid chromatography(HPLC)¹⁰, HPLC/MS, and HPLC-NMR.

 

HPLC¹¹ method for determination of ACL from tablet formulation is official in USP30-NF25 and BP (2004). Several analytical methods that have been reported for the determination of TRA in biological fluids and in bulk as well as pharmaceutical formulations  include HPLC, UV absorption spectrophotometry, fluorometry, gas chromatography/MS (GC/MS), and Fourier transform Raman and infrared spectrophotometry.^7-9

 

This paper describes simple, accurate, precise, and sensitive reversed-phase (RP)-HPLC and LC/MS methods for simultaneous determination of PCM, TRA and ACL in presence of its degradation products. The proposed methods were optimized and validated according to International Conference on Harmonization (ICH) guidelines.¹²

 

MATERIAL AND METHODS:

Drugs and Chemicals

Acetonitrile (HPLC grade) and phosphate buffer (AR grade) were purchased from Merck (Mumbai, India) and water (HPLC grade and AR grade) was prepared in institute. All other reagents used were of HPLC grade for RP-HPLC method. Standard bulk drug samples of PCM (99.60% pure) TRA (99.80% pure), and ACL (99.24%) were provided by Ipca laboratories limited (Ratlam, India) as gift sample. The pharmaceutical dosage form used in this study was Zerodol-PT tablets labeled to contain PCM 325 mg, TRA 37.5 and ACL 100 mg/tablet (Ipca lab, Mumbai, India).

 

Instruments

HPLC system consisting of LC 10 AT VP pump equipped with photo diode array detector (Shimadzu, Japan) and Luna C18 (4.6 mm id) column and class M10A software version 1.6 was used. A Rheodyne (Rohnert Park, CA) injector with 20 μL loop was used for injecting the sample. Mass spectrophotometer consisting of MICROMASS ZQ (MS/MS) with photo diode array detector.

 

Chromatographic method (RP-HPLC Method)

In the RP-HPLC method, separation and analysis of PCM, TRA and ACL were carried out on a Luna C18 column (4.6 mm id) with the diode array detector set at 270 nm. Mobile phase consisting of Acetonitrile: Phosphate buffer (pH 3.4, 20:80 v/v; filtered through a 0.2 µm membrane filter, degassed and sonicated) was used with a flow rate of 1mL/min.

 

(a)     Standard stock solutions - Standard stock solutions containing 100 µg/mL PCM, TRA and ACL were prepared by dissolving the pure drugs separately in the mobile phase.

 

(b)     Preparation of the calibration curves - Aliquots of 0.2, 0.4, 0.6, 0.8and 1 mL stock solution of PCM, TRA and ACL were transferred into a series of 10 mL volumetric flasks and the volume was made up to the mark with the mobile phase. Each solution was injected, and chromatogram was recorded. Mean retention times for PCM, TRA and ACL were found to be 4.98, 13.6 and 18.8 min, respectively in Figure 2.

 

The peak area of PCM, TRA and ACL were noted, and respective calibration curves were plotted as peak area against concentration of each drug.

 

(c)     Procedure for analysis of tablet formulation - Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 325 mg PCM, 37.5mg TRA and 100mg ACL weighed and transferred to a 50 mL volumetric flask containing about 35 mL mobile phase, ultrasonicated for 5 min, and the volume was made up to the mark with the mobile phase. The solution was filtered through Whatman (Florham Park, NJ) No. 41 paper, 0.2 mL filtrate was transferred to a 10 mL volumetric flask and the volume was made up to the mark with the mobile phase. Suitable dilution were made. The tablet sample solution was injected, the chromatogram was obtained and the peak areas were recorded. A representative chromatogram is given in Figure 3.

 

 

Figure 2. Chromatogram of PCM (10 µg/mL, retention time 4.98 min); TRA (10 µg/mL, retention time 13.6 min) and ACL (10 µg/mL, retention time 18.8

min)

Figure 3. Chromatograms of PCM, TRA and ACL in degraded state in different medium

 

From the peak area the, drugs concentration of each drug/tablet was estimated from the respective calibration curves.

 

(d)     Recovery studies - To study the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample at 3 different levels: 80, 100, and 120%.

(e)     Precision - Precision of the method was checked by 3 replicate readings at 3 concentration levels of within range expressed as RSD values.

 

 

Figure 4.  Mass Spectra of degraded drugs

Table 1. Regression analysis of calibration curve

Parameters	Drugs
	PCM	TRA	ACL
λmax	270	270	270a
Beer’s law limit, µg/mL	2-35	2-14	2-30
Correlation coefficient	0.9928	0.9914	0.9924
Molar absorptivity	0.094	0.038	0.024
Linear regression equationb
Intercept	0.0041	0.0728	0.0225
Slope	0.919	0.337	0.562
SDc	0.014	0.027	0.061
Detection limit, µg/mL	0.51	0.29	0.114
Quantitation limit, µg/mL	1.56	0.89	0.34

^a    Detection wavelength for HPLC method.

^b    y = mx + c, where y is the absorbance and x is the concentration (µg/mL).

^c    SD = standard deviation.

 

Forced Degradation Studies:

Forced degradation was carried out under acidic, basic, heating and oxidative conditions at controlled environmental conditions. Samples were analyzed by same RP-HPLC and LC-MS for analysis of PARA, TRA and ACL individually, simultaneously in combination as well as in combined tablet dosage form. Acidic conditions consisting of 1 N HCl, Basic conditions consisting of 1N NaOH, Heating at 70^οC and Oxidation consisting of 10 % H₂O_2. All samples were kept under these conditions for 24 hrs.

 

Samples were collected and dilutions of 10μg/ml were prepared in suitable media. Mobile phase used was Acetonitrile: Phosphate buffer (pH 3.4, 20:80 v/v) for RP-HPLC and 0.1 % Formic acid: Acetonitrile (50:50 v/v) for mass spectrophotometer. Mass Spectra of degraded drugs is given in Figure 4.

 

Statistical Analysis: The statistical analysis was performed using Microsoft Excel 2010.

 

Table 2. System suitability parameters for RP-HPLC method

Parameters	Paracetamol Tramadol	Aceclofenac
Calibration range, µg/mL	2-14 2-14	2-14
Theoretical plate number	3747 2100	5186
HETPa	0.0091 0.0043	0.0069
Asymmetric factor	0.17 0.36	0.98
Tailing factor	1.09 1.88	1.43
Capacity factor (k’)	0.48 0.35	3.08
Resolution	4.44 4.00	16.12

^a    HETP = Height equivalent to theoretical plate, cm

 

 

The proposed methods were also evaluated in the assay of commercially available tablets containing PCM, TRA and ACL (Table 3).

 

RESULTS AND DISCUSSION:

For the RP-HPLC method, chromatographic conditions were optimized to achieve the best resolution and peak shape for PCM, TRA and ACL. Different mobile phases containing methanol, acetonitrile, phosphate buffer and water were examined (data not shown), and the mobile phase Acetonitrile: Phosphate buffer (pH 3.4, 20:80 v/v)was selected as optimal for obtaining well-defined and resolved peaks. The optimum wavelength for detection and quantitation was 270 nm, at which the best detector response for all the substances was obtained.

 

 

Table 3. Results of analysis of commercial formulation

RP-HPLC	Label claim, mg/tablet		% claim, estimateda		Standard deviation
	PCM TRA	ACL	PCM      TRA	NAB	PCM     TRA	NAB
	325 37.5	100	99.49 99.61	98.42	0.744 0.56	0.15

^a    Average of 6 determinations.

 

Table 4. Recovery studies of PCM, TRA and ACL

^a    mean ± relative standard deviation (n = 3)

 

Straight line calibration curves were obtained for PCM, TRA and ACL in the spectrophotometric and RP-HPLC methods.

 

Table 1 summarizes the Beer’s law limit, linear regression equation, correlation coefficient, standard deviations (SD), and limit of detection (LOD) and limit of quantitation (LOQ) values for both methods.

 

System suitability parameters for the RP-HPLC method are listed in Table 2.

Six replicate determinations were performed on the accurately weighed amounts of tablets. For PCM, recovery (mean, %, ± SD, n = 6) was found to be 99.49 ± 0.74, 99.53 ± 0.51 and 100.08 ± 0.11. For TRA, recovery was found to be 99.61 ± 0.56, 99.36 ± 0.73 and 100.05 ± 0.10. For ACL, recovery was found to be 98.42 ± 0.96, 99.85 ± 0.63 and 101.04 ± 0.49 (Table 4).

 

Forced degradation study reveals that due to low solubility of aceclofenac in different degradation media, No significant degradation of aceclofenac was observed after degradation due to heat, Acid, Base and Hydrogen peroxide. Tramadol showed different degradation product peaks in Acid and Hydrogen peroxide degradation conditions. No degradation products were found due to degradation of tramadol by base and heat degradation. Paracetamol showed degradation in all degradation media. Maximum degradation was found to be in acidic media. Degradation study was also carried out in combination with different degradation conditions. Aceclofenac showed no peak in combined form. Only Paracetamol and tramadol showed peak for pure drug as well as for degradation products. Mass spectroscopy showed no significant results. Results could not be interpreted successfully due to unavailability of sufficient data.

 

CONCLUSIONS:

The validated RP-HPLC method developed here proved to be simple, fast, accurate, precise, and sensitive. Thus, they can be used for routine analysis of PCM, TRA and ACL in combined tablet dosage form in presence of its degradation products without prior separation.

 

ACKNOWLEDGMENTS:

The authors wish to express their gratitude to Ipca Lab., Ratlam, India, for the samples of pure PCM, ACL and TRA. The authors are also thankful to college of pharmacy, IPS academy, Indore, for providing the necessary facilities and constant encouragement.

 

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Received on 02.09.2011        Modified on 30.09.2011

Accepted on 26.06.2012        © AJRC All right reserved