HPTLC for the Simultaneous Determination of Paracetamol and Lornoxicam in Pharmaceutical Dosage Form

 

Lakshmi Sivasubramanian¹*, T. Tintu² and K.S. Lakshmi²

¹Department of Pharmaceutical Analysis, Bharat Institute of Technology (Pharmacy), Mangalpally (V), Ibrahimpatnam (M), RR district, Hyderabad – 501 510, Andhra Pradesh

²Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRM University, Kattankulathur – 603 203, Tamil Nadu.

ABSTRACT:

The present work reports a method for the simultaneous determination of Paracetamol and Lornoxicam in binary mixture. The method was based on HPTLC separation of the two drugs followed by densitometric measurements of their spots at 254nm. The separation was carried out using Ethyl acetate: Methanol: Chloroform: Triethylamine in the ratio of 9:0.5:0.5:0.2 v/v/v/v as mobile phase on Merck HPTLC aluminium sheets of silica gel 60 GF254. The linearity was in the range of 0.5-2.5 µg/spot for both the drugs. Proposed method has been successfully applied to pharmaceutical formulation. No chromatographic interference from the tablet excipients was found. The method was validated in terms of precision, robustness, recovery and limits of detection and quantitation.

 

 

INTRODUCTION:

Paracetamol [Figure 1] and Lornoxicam [Figure 2] are available in tablet dosage form. Chemically Paracetamol (PAR) is N acetyl P-aminophenol. It has antipyretic and analgesic activity. Lornoxicam is (3E) -6- chloro-3-[hydroxy(pyridine-2-ylamino)methylene]2-methyl-2,3-dihydro-4H-thieno [2,3-e] [1,2] thiazin-4-one 1,1-dioxide. It has non-steriodal anti-inflammatory activity. Paracetamol is official in IP^[1], BP^[2] and USP ^[3], while Lornoxicam is not official in any Pharmacopoeia, but listed in the Merck Index ^[4]. Literature survey reveals many analytical methods for determination of Paracetamol such as UV Spectrophotometry^[5], HPLC ^{[6], [7], [8], [9], [10], [11]} and capillary electrophoresis ^[12] methods from pharmaceutical preparations. Few analytical methods for determination of Lornoxicam using UV Spectroscopy^{[13], [14]} HPLC^{[15], [16]} and polarography^[17] in plasma and pharmaceutical formulation have been reported.

 

Figure 1: Structure of Paracetamol

 

The present work presents a new method for simultaneous determination of Paracetamol and Lornoxicam in tablets using HPTLC densitometry. The method is simple, reduces the duration of the analysis and suitable for routine determination of two drugs.

 

Figure 2: Structure of Lornoxicam

 

EXPERIMENTAL:

Materials

Pharmaceutical grade of Paracetamol and Lornoxicam were kindly supplied as a gift sample by Burgeon Pharma Ltd, Chennai, India used without further purification and certified to contain 99.81% (w/w) and 99.72% (w/w) respectively on dried basis. All chemicals and reagents used were of HPLC grade and were purchased from Merck Chemicals, India.

 

Instrumentation and Chromatographic Conditions:

The samples were spotted in the form of bands of width 6mm with a Camag 100 µl sample (Hamilton Bonaduz, Switzerland) syringe on precoated silicagel aluminium plate 60 F 254 (20 cm × 10 cm) with 250 µm thickness, E.Merck, Darmstadt, Germany, supplied by Anchrom Technologists, (Mumbai) using a Camag Linomat IV (Switzerland). The plates were prewashed by methanol and activated at 110⁰C for 5 min prior to chromatography. A constant application rate of 0.1 µl/s was employed and space between two bands was 5 mm. The slit dimension was kept at 5 mm × 0.45 mm and 10 mm/s scanning speed was employed. The mobile phase consisted of ethylacetate-methanol-chloroform-triethylamine (9:0.5:0.5:0.2 v/v/v/v) and 15 ml of mobile phase was used per chromatography. Linear ascending development was carried out with the mobile phase. The optimized chamber saturation time for mobile phase was 30 min at room temperature. The length of chromatogram run was 9 cm approximately 30 min. Subsequent to the development, TLC plates were dried and the densitometric scanning was performed on Camag TLC scanner III in the reflectance – absorbance mode at 254 nm for all measurements and operated by CATS software (V 3.15, Camag). The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm. Concentrations of the compound chromatographed were determined from the intensity of diffusely reflected light. Evaluation was via peak areas with linear regression.

 

Standard Solutions and Calibration graphs:

Stock standard solution was prepared by dissolving 0.10 g of LOR in 100 ml methanol. A mixed standard solution was prepared by placing 0.50 g of PAR and 8 ml of stock solution of LOR in 100 ml volumetric flask. The volume was made up to the mark with methanol. One to six µl of their solution was spotted on the TLC plate to obtain the concentration of 5-30 µg/spot of PAR and 0.8-4.8 µg/spot of LOR. Each concentration was spotted six times on the TLC plate. The plate was developed on previously described mobile phase. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs.

 

Sample Preparation:

To determine the content of PAR and LOR simultaneously in conventional tablets (label claim: 500 mg Paracetamol and 8 mg Lornoxicam per tablet, combination tablet containing both analytes), the twenty tablets were weighed, their mean weight was determined and they were finely powdered and powder equivalent to 8 mg LOR and 500 mg PAR was weighed. The equivalent weight of the drug was transferred into a 100 ml volumetric flask containing 50 ml methanol, sonicated for 30 min and diluted to 100 ml with methanol. Supernatant containing 80 µg/ml of LOR and 5mg/ml of PAR was taken and filtered using 0.45µm filter (Millipore, Milford, MA). Different microlitres (1, 2 and 3) of sample solution were applied six times to the HPTLC plate to give concentrations of 5, 10 and 15 µg/spot and 0.8, 1.6 and 2.4 µg/spot for PAR and LOR respectively. The plate was developed in the previously described chromatographic conditions. The peak area of the spots were measured at 254 nm for PAR and LOR respectively and their concentrations in the samples were determined using multilevel calibration developed on the same plate under the same conditions using linear regression equation.

METHOD VALIDATION:

The method was validated in compliance with ICH guidelines. The following parameters were validated.

 

Precision:

Precision of the method was determined by weighing amount of the product powder equivalent to 100% of the label claim of PAR and LOR and assayed. System suitability was determined by six replicate applications and six times measurement of a sample solution at the analytical concentration. The repeatability of sample application and measurement of peak area for active compound were expressed in terms of relative standard deviation (%RSD) and standard error (SE). Method repeatability was obtained from RSD value by repeating the assay six times in same day for intra-day precision. Intermediate precision was assessed by the assay of two; six sample sets on different days (inter-day precision). The intra-day and inter-day variation for determination of PAR and LOR was carried out at three different concentration levels 5,15, 25 µg/spot and 0.8, 2.4, 4.0 µg/spot respectively (Table 2).

 

Limit of Detection and Limit of Quantitation:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and / or degradation products. In order to estimate limit of detection (LOD) and limit of quantitation (LOQ), blank methanol was spotted six times following the same method as explained above. The signal to noise ratio (S/N) of 3 and 10 was determined for six replicate determinations.

 

Specificity:

The specificity of the method was ascertained by analyzing standard drug and sample. The spot for PAR and LOR in sample was confirmed by comparing the Rf and spectra of the spot with that of standard. The peak purity of PAR and LOR was assessed by comparing the spectra at three different levels, i.e., peak start (S), peak apex (M) and peak end (E) positions of the spot.

 

Table 1: Linear Data Regression for Calibration Curves (n=6)

^a 95% Confidence limit

 

Table 2: Intra and Inter day precision of Paracetamol and Lornoxicam

 

Table 3: Standard addition technique for determination of Paracetamol and Lornoxicam by TLC densitometry (n=6)

Excess drug added to the analyte (%)	Theoretical Content (µg)	Recovery (%)	% RSD	SE
Paracetamol
0	10	98.34	1.21	1.02
80	18	99.24	1.32	0.97
100	20	99.65	1.06	1.23
120	22	101.23	1.03	1.32
Lornoxicam
0	4.0	99.45	1.63	1.67
80	7.2	100.34	1.45	1.32
100	8.0	100.66	1.32	1.08
120	8.8	101.45	1.33	0.82

 

Recovery Studies:

For both methods recovery studies was carried out by applying the method to drug sample to which known amount of PAR and LOR corresponding to 80, 100 and 120 % of label claim had been added (standard addition method). At each level of the amount six determinations were performed and the results obtained were compared with expected results.

 

RESULTS AND DISCUSSION:

Optimization of Procedures:

Initially ethylacetate and methanol in the ratio of 5:5 (v/v) was tried for both drugs simultaneously. The spots were not separated. Then ethylacetate and methanol in the ratio of 7:3 (v/v) was tried. Spots were developed and were near to solvent front. Then 1ml of chloroform was added to ethylacetate and methanol in the ratio 8:2 v/v. The spots developed were dense, compact and typical peak nature for both PAR and LOR was observed but resolution between them was less. To improve the resolution, the volume of ethylacetate was increased by 1ml and that of methanol was reduced by 1 ml and 0.2 ml of triethylamine was added. Ultimately mobile phase consisting of ethylacetate – methanol – chloroform -triethylamine (9:0.5:0.5:0.2 v/vv/v) gave good resolution. Both the peaks were symmetrical in nature and no tailing was observed when plates were scanned at 254nm [Figure 3].

 

Linearity:

PAR showed good correlation coefficient in concentration range of 5-30 µg/spot (r = 0.9998 ± 0.08) where as LOR in the concentration range of 0.8-4.8 µg/spot (r = 0.9998 ± 0.06) respectively. Linearity was evaluated by determining six standard working solutions containing 5-30 µg/spot, 0.8-0.48 µg/spot of PAR and LOR twice in triplicate (Table 1).

 

Precision:

The repeatability of sample application and measurement of peak area were expressed in terms of % RSD and were found to be 0.198, 0.342 and 0.214, 0.431 for PAR and LOR respectively. The % RSD values depicted in Table 3 shows that proposed method provides acceptable intra-day and inter-day variation of PAR and LOR.

 

Figure 3: Densitogram of Paracetamol (Rf 0.52) and Lornoxicam (Rf 0.05) showing Resolution

 

Precision:

The repeatability of sample application and measurement of peak area were expressed in terms of % RSD and were found to be 0.198, 0.342 and 0.214, 0.431 for PAR and LOR respectively. The % RSD values depicted in Table 3 shows that proposed method provides acceptable intra-day and inter-day variation of PAR and LOR.

 

Limit of Detection and Limit of Quantitation:

The signal / noise ratios 3:1 and 10:1 were considered as LOD and LOQ respectively. The LOD and LOQ were found to be 1, 2.5 µg/spot and 0.2, 0.6 µg/spot respectively for PAR and LOR respectively.

 

Specificity:

The peak purity of PAR and LOR was assessed by comparing their respective spectra at peak start, peak apex and peak end positions of the spot, ie, r (S,M) = 0.9993, 0.9996 and r (M,E) = 0.9994, 0.9996. Good correlation (r = 0.9994 and r = 0.9995) was also obtained between standard and sample spectra of PAR and LOR respectively.

 

Table 4: Stability of Paracetamol and Lornoxicam in sample solutions (n=6)

^a Average of three concentrations

 

Table 5: Applicability of the proposed methods for the determination of PAR and LOR in commercial tablets (n=6)

 

Recovery Studies:

The proposed method when used for extraction and subsequent estimation of PAR and LOR from pharmaceutical dosage form after spiking with additional drug afforded recovery of 98-102% and mean recovery for PAR and LOR from the marketed formulation are listed in Table 5 a and b. The data of summary of validation parameters are listed in Table 3.

 

Stability in Sample Solution:

Solutions of two different concentrations (10 and 25 µg/spot for PAR and 1.6 and 4.0 µg/spot for LOR) were prepared from sample solution and stored at room temperature for 0.5, 1.0, 2.0, 4.0 and 24 h respectively. They were then applied on the same TLC plate, after development the densitogram was evaluated as listed in Table 4 for additional spots if any. There was no indication of compound instability in the sample solution.

 

Analysis of Marketed Formulation:

The spots at Rf 0.52 (for PAR) and 0.05 (for LOR) were observed in the densitogram of the drug samples extracted from tablets. There was no interference from the excipients commonly present in the tablets. The drug content was found to be 99.50% ± 1.05 (%RSD of 0.63) and 98.75 % ± 1.12 (%RSD of 0.72) for PAR and LOR respectively. It may therefore be inferred that degradation of PAR and LOR had not occurred in the marketed formulations that were analyzed by this method as shown in Table 5. The low % RSD value indicated the suitability of this method for routine analysis.

 

CONCLUSION:

The proposed HPTLC method provides simple, accurate and reproducible quantitative analysis for simultaneous determination of PAR and LOR in tablets. The method was validated as per ICH guidelines. Six real samples of tablets were determined simultaneously by HPTLC method and the results were correlated. Statistical tests indicate that the proposed method appear to be equally suitable for routine determination of PAR and LOR simultaneously in pharmaceutical formulation.

 

REFERENCES:

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2.        The British Pharmacopoeia, 2007 edition, Vol II, 1575.

3.        USP-NF, Asian Edition 2007, Vol II, 1269.

4.        Merck Index – An encyclopedia of chemicals and drugs and biological, 13^th edition, 5612.

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13.     Nemutlu E, Demircal S and Kir S. Zero order and first order derivative UV spectrophotometric method for determination of Lornoxicam in pharmaceutical preparation. De Pharmazie. 2005; 60(6): 421-425.

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Received on 11.05.2012        Modified on 29.05.2012

Accepted on 09.06.2012        © AJRC All right reserved