Estimation of loxoprofen sodium dihydrate in tablets by reverse phase high performance liquid chromatography

R. Nanthakumar; V. Prabakaran; K. Chitra; C. Uma Maheswara Reddy

doi:10.1016/j.arabjc.2011.07.010

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ORIGINAL ARTICLE

9 (

1_suppl

); S632-S636

doi:

10.1016/j.arabjc.2011.07.010

Estimation of loxoprofen sodium dihydrate in tablets by reverse phase high performance liquid chromatography

R. Nanthakumar^{^a,⁎}, V. Prabakaran^{^b}, K. Chitra^{^a}, C. Uma Maheswara Reddy^{^a}

a Faculty of Pharmacy, Sri Ramachandra University, Porur, Chennai 600 116, India

b Research Scholar, Jawaharlal Nehru Technological University, Hyderabad, India

⁎Corresponding author. Tel.: +91 9790710015. pharma_chemistry1980@rediffmail.com (R. Nanthakumar)

Received: 2011-4-27, Accepted: 2011-7-11, Published: 2011-09-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

A simple and rapid reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the estimation of loxoprofen sodium dihydrate (LSD) in tablets. An isocratic HPLC analysis was performed on Phenomenex, Luna C₁₈(2) (250 × 4.6 mm, 5μ) column. The compound was separated with the mixture of 0.4% orthophosphoric acid and acetonitrile in the ratio of 45:55 (v/v) as a mobile phase at a flow rate of 1.0 mL/min. UV detection was performed at 210 nm. Run time per sample was 8 min with the retention time of 4.9 min. The system suitability parameters, such as theoretical plate count, tailing and% RSD between six standard injections were within the limits. The method was validated according to ICH guidelines. The method was validated for specificity, precision, linearity, stability of sample solution, intermediate precision, robustness and accuracy. The stability of the sample solution was checked for 24 h at one hour intervals. The results show that the sample solution is stable for at least 24 h. Calibration plots were linear over the concentration range of 10–90 μg/mL as indicated by the correlation coefficient of 0.9992. The% RSD was 0.115 and 1.288 for system precision and method precision, respectively. The % RSD values for intermediate precision studies were less than 2%. The robustness of the method was evaluated by deliberately altering the chromatographic conditions and the results were adhered with the limits. The high recovery and low relative standard deviation confirm the suitability of the method for the estimation of loxoprofen sodium dihydrate in tablets.

Keywords

RP-HPLC

Loxoprofen sodium dihydrate

Validation

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1 Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) are used for the management of pain and inflammation associated with musculo-skeletal, joint disorders and operative procedures. The uses of these drugs are prevalent in India due to the high rates of occurrence of rheumatoid disorders. Many NSAIDs are being marketed here such as ibuprofen, diclofenac sodium, and piroxicam etc. The new one among is loxoprofen sodium dihydrate (LSD), a derivative of propionic acid and is chemically sodium-2-[4-(2-oxocyclopentyl-1-methyl) phenyl] propionate dihydrate (Fig. 1) (Martindale, 2005). It is a non-selective COX inhibitor and a NSAID. The drug is marketed in Brazil, Mexico and Japan as its sodium salt with the trade name of Loxonia. In India it is marketed under the trade name of Loxomac. It is official in the Japanese Pharmacopoeia and they have used internal standard for the estimation of LSD (Japanese Pharmacopoeia, 2001).

Chemical structure of loxoprofen sodium dihydrate.

Several studies for the estimation of the drug using various techniques have been carried out, some of them being: The identification of degradation products in loxoprofen sodium adhesive tapes by liquid chromatography-mass spectrometry and dynamic pressurized liquid extraction–solid-phase extraction coupled to liquid chromatography–nuclear magnetic resonance spectroscopy (Murakami et al., 2008), direct and simultaneous analysis of loxoprofen and its diastereometric alcohol metabolites in human serum by on-line column switching liquid chromatography and its application to a pharmacokinetic study (Cho et al., 2006), high-performance liquid chromatographic determination of loxoprofen and its diastereomeric alcohol metabolites in biological fluids by fluorescence labelling with 4-bromomethyl-6,7-methylene dioxycoumarin (Naganumah and Kawahara, 1990), column liquid chromatography for the simultaneous determination of the enantiomers of loxoprofen sodium and its metabolites in human urine (Nagashima et al., 1985), simultaneous determination of loxoprofen and its diasteriomeric alcohol metabolites in human plasma and urine by a simple HPLC-UV detection method (Choo et al., 2001), stereo specific analysis of loxoprofen in plasma by chiral column liquid chromatography with a circular dichroism based detector (Kanazawa et al., 2002), liquid chromatography–tandem mass spectrometry method of loxoprofen in human plasma (Lee et al., 2009). In the present study, a very simple and rapid isocratic RP-HPLC method was developed and validated for the determination of LSD in its tablet dosage form.

2 Experimental procedure

2.1

2.1 Apparatus

Shimadzu LC-2010 AHT HPLC system (Class-VP software), Sartorius electronic analytical balance was used for weighing, Crest sonicator is used for degassing solvents, Phenomenex, Luna C₁₈(2) (250 × 4.6 mm, 5μ) column was used.

2.2

2.2 Reagents and materials

Loxoprofen sodium dihydrate (LSD) was supplied by Macleod’s Pharmaceuticals Limited, Mumbai and Loxomac tablets 60 mg/tablet Macleod’s pharmaceuticals Ltd were procured from the local pharmacy. All the solvents and chemicals used were of HPLC grade (Merck). 0.4% of ortho phosphoric acid (OPA) was prepared by adding 4 mL of OPA mixed with water and the volume made up to 1000 mL with water. It was filtered through 0.45 μ nylon membrane filter. The mobile phase was prepared by mixing 0.4% v/v OPA and acetonitrile in the ratio of 45:55 v/v and 0.4% v/v OPA and acetonitrile (27:73 v/v) was used as a diluent for the preparation of the sample and standard.

3 Method

3.1

3.1 Chromatographic conditions

Chromatographic separation was achieved at 30 °C and the detection was carried at 210 nm at a flow rate of 1 mL/min. Run time was kept at 8 min. Prior to the injection of drug solution, column was equilibrated for 60 min with mobile phase flowing through the system. The injection volume was 10 μL for assay level. Blank was injected to check the solvent interference (Fig. 2).

3.2

3.2 Standard preparation

Working standard solution containing 50 μg/mL of LSD was prepared by weighing accurately 100 mg of LSD into a 100 mL volumetric flask. 30 ml of diluent was added and sonicated to dissolve and diluted upto the mark with the diluent. A representative chromatogram of the standard is shown in (Fig. 3).

Chromatogram of loxoprofen sodium dihydrate standard.

3.3

3.3 Sample solution

Twenty tablets containing LSD were weighed and finely ground into a fine powder. A quantity of powder equivalent to 100 mg of LSD was accurately weighed into a 100 ml volumetric flask. 30 ml of diluent was added and sonicated for 15 min with intermittent shaking, made up to the volume with diluent and mix. Filter the solution through 0.45 μ nylon membrane filter. Dilute 5 ml of the above solution to 100 ml with diluent. A representative chromatogram of the sample is shown in (Fig. 4).

Chromatogram of loxoprofen sodium dihydrate sample (Loxomac).

3.4

3.4 Evaluation of system suitability

Ten microlitre of standard solution was injected in duplicate before and after the analysis and the chromatograms were recorded. System suitability parameters like column efficiency, plate count and tailing were also recorded. % RSD of six injections of standard were also taken. The column efficiency as determined from LSD peak is not less than 2000 USP plate count and USP tailing for the same peak is not more than 2.0 and % RSD of six injections of the standard solution is not more than 2.0%.

4 Analytical method validation (ICH, 2005)

4.1

4.1 Specificity

Prepare a placebo solution by following the procedure for the test solution using equivalent weight of the placebo in a portion of the test preparation. Placebo solution was injected into the HPLC system following test conditions, the chromatogram was recorded and measures the responses of the peaks if any. Chromatogram of the placebo has not shown any interference at the retention time of LSD.

4.2

4.2 Precision

Precision was measured in terms of repeatability of application and measurement. Repeatability of standard application (System precision) was carried out using six replicates of the same injection (50 μg/mL). Repeatability of sample measurement (Method precision) was carried out in six different sample preparations from the same homogeneous blend of the marketed sample (50 μg/mL). The % RSD for repeatability of standard preparation is 0.115% whereas the % RSD for repeatability of sample preparation is 1.288%. This shows that the precision of the method is satisfactory as % RSD is not more than 2%. Table 1 represents the precision data obtained for the method.

Table 1 System precision and method precision of loxoprofen sodium dihydrate

System precision		Method precision
Injection	Area	Sample	Assay (mg/tablet)
1	910747	1	60.70
2	911219	2	59.12
3	910899	3	60.89
4	912002	4	59.19
5	913596	5	59.55
6	911599	6	59.52
Mean	911677	Mean	59.83
SD	1046.348	SD	0.771
% RSD	0.115	% RSD	1.288

4.3

4.3 Linearity

The linearity of response for LSD assay method was determined by preparing and injecting solutions with concentrations of about 10–90 μg/mL of LSD. The linear regression data for the calibration curve indicates that the response is linear over the concentration range studied for LSD with a correlation coefficient of 0.9992 (Fig. 5).

Linearity graph of loxoprofen sodium dihydrate.

4.4

4.4 Stability of sample solution

The sample solution was prepared as per test method, analysed initially and at different time intervals by keeping the solutions at room temperature. The % difference of response between the initial and different time intervals shows that the sample solution is stable for at least 24 h at room temperature.

4.5

4.5 Intermediate precision

Intermediate precision of the method was determined by analysing solutions by two different analysts, using different instruments, using multiple lots of column, in two different labs and on different days. The % RSD obtained under different conditions were below 2. As the values of % RSD for different conditions were below 2% intermediate precision of the method is established. Table 2 represents the intermediate precision of LSD.

Table 2 Intermediate precision of loxoprofen sodium dihydrate.

Parameter	Normal conditions	Changed conditions
Column make	Phenomenex, Luna C₁₈(2)	Waters symmetry C18,
	(250 × 4.6 mm), 5μ	(250 × 4.6 mm), 5μ
Analyst	R. Nanthakumar	Analyst-II
% Assay	101.18	100.71
Reagent made	Merck	Rankem

4.6

4.6 Robustness

Robustness of the method was determined by analysing standard solutions at normal operating conditions and by changing some operating analytical conditions such as flow rate, column oven temperature, detection wavelength and mobile phase acetonitrile content. The conditions with the variation and the results were included in Table 3. The tailing factor is around unity indicative of peak symmetry and theoretical plate counts were also above 2000. Hence robustness of the method is established to the extent of variations applied to analytical conditions.

Table 3 Robustness parameters.

Mobile phase–acetonitrile content (%)				Flow rate (mL/min)				Column temperature (°C)				Wavelength (nm)
53%		57%		0.9		1.1		25		35		205		215
Plate count	Tailing	Plate count	Tailing	Plate count	Tailing	Plate count	Tailing	Plate count	Tailing	Plate count	Tailing	Plate count	Tailing	Plate count	Tailing
8021	1.38	9043	1.31	10,275	1.32	9270	1.36	8381	1.33	9985	1.36	9684	1.35	9709	1.33
9571	1.32	8627	1.31	10,528	1.36	9258	1.36	8702	1.42	8766	1.34	9786	1.37	9716	1.30
9118	1.32	8308	1.41	10,470	1.35	9160	1.35	8945	1.34	9555	1.37	9685	1.33	9981	1.38
9571	1.32	8650	1.32	10,359	1.31	9323	1.25	8783	1.40	9321	1.36	9709	1.31	10,004	1.39
10,050	1.29	8571	1.30	10,024	1.41	9114	1.32	9399	1.42	8837	1.40	9923	1.40	9568	1.30
9602	1.32	9784	1.34	9781	1.38	9410	1.39	8789	1.32	9321	1.34	9815	1.36	9666	1.34

4.7

4.7 Accuracy

The recovery experiments were performed by adding a known quantity of pure standard drug in to the pre-analysed sample .The tablet powder equivalent to 100 mg of LSD was accurately weighed in to a 100 ml volumetric flask. The sample was then spiked with standard at levels 80%, 100% and 120% of test concentration. The resulting spiked sample solutions were assayed in triplicate and the results were compared with the expected results and expressed as percentage. The mean % recoveries of LSD were found to range between 100.78 and 101.58 which are within the acceptance limit.

5 Results and discussion

A different combination of mobile phases and chromatographic conditions were tried and a degassed mixture of 0.4% OPA and acetonitrile (45:55v/v) mobile phase, Phenomenex, Luna C₁₈(2) (250 × 4.6 mm, 5μ) column, 1 mL/min flow rate, 10 μl injection volume, 30 °C column oven temperature, 210 nm wavelength and 8 min run time was found to be suitable for all combinations. These chromatographic conditions gave a retention time of 4.9 min. Specificity of the method was checked by injecting the placebo solution, no peaks were found at the retention time of LSD. The stability of the sample solution was evaluated by preparing a sample solution as per the proposed method and analysed initially and at 1 h time intervals up to 24 h by keeping the sample solution at room temperature. The results of the stability studies showed that the solution of the drug was found to be stable for 24 h at room temperature. System precision and method precision results showed the % RSD of 0.115 and 1.288, respectively. A good linear relationship as indicated by correlation coefficient value 0.9992 was observed between the concentrations of 10 μg/mL and 90 μg/mL of LSD. Intermediate precision was done by changing the analyst, column, chemical make with the same chromatographic conditions and the obtained results were within the limits. The robustness of the method was evaluated by deliberately varying the chromatographic conditions of the method such as mobile phase-acetonitrile content, flow rate, column temperature and wavelength. The parameters like tailing factor and retention times showed adherence to the limits. The accuracy of the method was determined by recovery studies and the percentage recovery was calculated. The data indicates an average of 101.18% recovery of the standard sample.

6 Conclusion

The method developed for loxoprofen sodium dihydrate was found to be a simple process and the procedure does not involve any critical experimental conditions. The reagents used in this method are inexpensive and readily available. The validation results show that the method is a specific, accurate, linear, precise, rugged and robust. The run time is relatively short (8 min) which enables rapid quantification of many samples in routine and quality control analysis of tablets.

Acknowledgement

The author extends his sincere gratitude to the Management, Faculty of Pharmacy, Sri Ramachandra University, Porur, Chennai for providing all the facilities needed to conduct the experiment. He also thanks Macleods Pharmaceuticals limited for giving the authentic sample of loxoprofen sodium dihydrate required for the study.

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