INTRODUCTION:

Naproxen or (2S)-2-(6-methoxynaphthalen-2-yl)propanoic acid (Fig. 1) is a nonsteroidal anti-inflammatory drug (NSAID) of the propionic acid class that uses to relieve pain, fever, swelling and stiffness^1,2. Mechanism of action of the drug involves a reversible inhibition of both COX-1 and COX-2 enzymes, resulting in the inhibition of prostaglandin synthesis. Therefore, it exhibits an anti-inflammatory activity and is used to treat those symtoms³. Due to poor solubility (15.9 mg/l) of naproxen, naproxen sodium is prepared to enhance the dissolution properties.Naproxen sodium has molecular weight of 252.24 g/mol.

It is soluble in water with one pKa value at 4.15⁴. The orally effective dose is 500 - 1,000 mg/day and 10-20 mg/kg/day in adult and children, respectively⁵.

Fig. 1:Chemical Structure of Naproxen Drug.

However, the drug is mostly available in the tablet form of immediate release or extended release formulation. Extemporaneous preparation in an oral liquid form is suitable for children who are unable to swallow tablets. A number of high performance liquid chromatography (HPLC) methods were developed for analysis of naproxen in raw material and tablet preparation^6-8. A previous study developed a fast HPLC method to determine naproxen in tablet dosage form⁹. They performed an isocratic chromatography with C18 column (4.6x150mm; 5µm) and mobile phase of phosphate buffer pH 6.1 and acetonitrile (40:60). The flow rate was 1.50 ml/min and the drug was monitored at 302 nm. It showed that naproxen was eluted at 1.75 min. Linearity was found in a range of 9.38 - 300 µg/ml and limit of detection (LOD) and limit of quantification (LOQ) were 0.12 and 0.40 µg/ml, respectively. The method was precise and accurate that complied with ICH guideline. Slightly changes in mobile phase composition, flow rate and buffer pH had negligible effects on the chromatographic parameters. In addition, the method could separate the degradation products produced during forced degradation studies (acidic, basic, oxidative and thermal stresses)⁹. Therefore, it could be used for regular determination of naproxen and stability studies. Another study developed a HPLC method for estimation of naproxen simultaneously with other three NSAID drugs in bulk and tablet formulations¹⁰. It was carried out on a C18 column with mobile phase, consisting of 50% acetonitrile in the deionized water acidified with 1% acetic acid (solvent A) and 100%acetonitrile (solvent B). The flow rate was maintained in a gradient mode at 1.50 ml/min and the detection was set at 264 nm. The result revealed that the retention time of the drug was 4.78 min. The method was linear in the concentration of 1 - 200 µg/ml with LOD and LOQ of 0.13 and 0.40 µg/ml, respectively. However, the method performance in precision and accuracy did not present in the study¹⁰. A C18 column with mobile phase of ammonium acetate buffer pH 4.0 and methanol (40:60) was also used for analysis of naproxen in tablet. The flow rate was 1.00 ml/min and UV wavelength was set at 210 nm. The method was specific to the drug as judged by good shape and resolution of the peak. The retention time of naproxen was observed at 3.06 min and quantitative linearity was in the concentration ranges of 20- 80 μg/ml. The method was quite precise (% RSD less than 2) and accurate (98.89 - 101.50%) with LOD and LOQ of 0.13 and 0.40µg/ml, respectively¹¹. However, stress testing did not include in those studies. The majority of the methods were developed and validated for analysis of naproxen in raw material, tablet, cream and biological fluids. There were a limited number of methods applied for other dosage forms such as suspension.

The present study was to develop and validate a HPLC method to determine naproxen in an extemporaneous suspension, according to ASEAN guideline for validation of the analytical procedure. It included specificity, range, linearity, accuracy, precision, LOD, LOQ and robustness. Forced degradation studies such as hydrolysis, oxidation and thermal degradation were also carried out to present the stability-indicating capability of the method.

MATERIAL AND METHODS:

Chemicals and Reagents:

Naproxen sodium tablets (275 mg) and naproxen working standard were generous gift from Siam Bheasach Co., Ltd, Thailand. Sodium phosphate monobasic and sodium phosphate dibasic were analytical grade from LOBA Chemie, India. Hydroxy propyl methyl cellulose (HPMC)-4000, methylparaben, propylparaben, saccharine and sucrose were purchased from P.C. drug center Co. Ltd, Thailand. Methanol and acetronitrile were HPLC grade from RCI Labscan, Thailand.

Preparation of ExtemporaneousSuspension:

Naproxen sodium tablets (equivalent to naproxen base 1,250 mg; ANNOXEN^®-S) were reduced to a fine powder. A small aliquot of vehicle, containing blue-lemon syrup, 0.5% HPMC-4000, 1% paraben concentrate, was added to the fine powder and mixed into a uniform paste. The remaining vehicle was mixed and adjusted using a graduated cylinder to obtain an extemporaneous suspension with the nominal strength of 25 mg/ml. The preparation was then filled in the amber glass bottles.

Instrumentation and Chromatographic Condition:

V-630 double beam spectrophotometer (JascoCo., Ltd, Japan) was used to determine the l_max of naproxen^12-16. Naproxen standard solution (250 µg/ml) was dissolved in methanol and a matched pair of 1 cm quartz cells was used in the study. UV spectrum was measured from 200- 400nm and analyzed by Spectra Manager II software. Ultimate 3000 HPLC system (Dionex Corporation, USA) was utilized for the drug analysis and Chromeleon 7 software was used to perform data collection and interpretation. Separation was achieved on an Inertsil^® ODS-3C18 column (4.6 x 250 mm, GL Sciences)¹⁷. The elution was isocratic with mobile phase of 50 mM sodium phosphate buffer pH 7.8 and acetronitrile (70:30) at the flow rate of 0.7 ml/min. The column temperature was maintained at 25°C. The injection volume was 20 μl with UV detection at 230 nm.

Preparation of Standard and Sample Solutions:

Naproxen working standard was accurately weighted into a 100 ml volumetric flask. It was dissolved with methanol to the concentration of 250 µg/ml.The standard stock solution was diluted with methanol to achieve standard solutions of 2.5, 12.5, 25.0, 40.0 and 50.0 µg/ml. Sample solution was prepared by shaking and transferring 5 ml of naproxen extemporaneous suspension (25 mg/ml) to a 50 ml volumetric flask. Methanol was used as a diluent and then the sample mixture was sonicated for 10 min. Sample solution (25 mg/ml) was obtained by 100-fold dilution with methanol. The standard and sample solutions were filtered through 0.45 mm nylon membrane before injection.

Method Validation:

The method was validated according to ASEAN guideline for validation of the analytical procedure. The following validation characteristics were evaluated: specificity, range, linearity, precision, accuracy, LOD, LOQ and robustness.

System Suitability Test:

System suitability test was performed by injections of five replicates of the standard solution before sample analysis. General acceptance criteria were the relative standard deviation (RSD) of the peak area less than 2.0%, theoretical plate of the column greater than 2000 and tailing factor of the peak less than 2.0.

Specificity:

Specificity is referred to the ability of the method to distinguish the analyte from other chemicals in the sample. It was evaluated by comparing the chromatograms of standard solution, sample solution, vehicle solution and standard-spiked sample solution.

Linearity, Limit of detection (LOD) and limit of quantification (LOQ):

Standard calibration curves were prepared by using11 different concentrations ranging from 2.56 ng/ml to50 µg/ml. It was performed by accurately weighing naproxen standard in triplicate to prepare stocksolutions. Linear least-squareregression analysis was then used to correlate peak area and drug concentrations. LOD and LOQwere calculated by calibration curve method. They wereobtained by multiplying factors of 3.3 and 10 to a ratio of the standard deviation of the response and the slope of thecurve, respectively.

Accuracy:

Accuracy of the method was determined by addition of naproxen standard in the sample solution in triplicate at three levels of 80%, 100% and 120%of test concentration (20, 25 and 30 µg/ml, respectively). Methanol as diluent was used to adjust the volume. The standard-added sample solutions were filteredthrough a 0.45 µm membrane filter. Percent recoveryand its standard deviation (SD) were calculated to determine the accuracy.

Precision:

The method precision was determined by injection of six sample solution in the same day for intraday precision (repeatability) and on another day for interday precision (intermediate precision). Precision was expressed by the percent relative standard deviation (%RSD) of the peak area.

Robustness:

The robustness refers to the ability of the method to remain unaffected by small change of the chromatographic condition. It indicates the method reliability during normal usage by analyzing the standard and sample solutions after varying the flow rate (±0.3ml/min), mobile phase composition (±10%), detection wavelength (±5 nm) and injection volume (±50%). The % RSD of robustness testing under these conditions was calculated in all cases.

Forced Degradation Studies:

The method was used to determine the degradation of naproxen in the extemporaneous suspension. Various stress conditions such as hydrolysis, thermal degradation and oxidative stress were carried out in triplicate.

Hydrolytic Studies:

Acid and basic hydrolysis was carried out by transferring 5 ml of stock sample solution (250 mg/ml) into a 50ml volumetric flask. Methanol was added to the samples and then the mixture was sonicated for 10 min. Samples were refluxed at 80°C for 1 h with 5 ml of 1.0 N HCl as acid hydrolysis, 1.0 N NaOH as basic hydrolysis or distilled water as neutral hydrolysis. Subsequently, 5 ml of 1.0 N NaOH or 1.0 N HCl was used to neutralize the acid and basic samples, respectively. Samples were filtered through a 0.45 µm membrane filter before analysis.

Thermal Studies:

Thermal degradation studies were evaluated by keeping extemporaneous suspension in a hot air oven at 80°C for 1week. Samples were shaken well and 5 ml of the drug product was transferred to a 50 mL flask. They were mixed with methanol and sonicated for 10 min. The samples were diluted 100-fold with methanol and filtered before analysis.

Oxidation Studies:

Oxidative degradation studies were performed by pipetting 5 ml of stock sample solution (250 mg/ml) to a 50 ml flask. Samples were diluted with methanol and sonicated for 10 min. An aliquot of 30% H₂O₂ solutionwas added to the samples, making a final concentration of 0.1% or 1.0% H₂O₂. They were incubated at room temperature for 1 h in the darkand filtered in the vials prior to injection.

RESULT AND DISCUSSION:

UV Spectrum of Naproxen:

UV spectrum of naproxen standard solution (250 µg/ml) was measured between 200-400 nm by using methanol as a blank. The result showed that the drug had the l_maxat 230 nm (Fig. 2). The aromatic naphthalene ring absorbed the UV light and displayed this common characteristic.

Fig. 2:Absorption Spectrum of Naproxen Standard Solution against Methanol Blank.

System Suitability Test:

The test is used to assure that the chromatographic system isreliable and reproducible for drug analysis. The result revealed that the drug was eluted as a narrow and symmetrical peak at 8.18 min with tailing factor of 1.24 ± 0.02. The relative standard deviation (% RSD) of the peak area was 1.04 and the theoretical plate number was 9854.80±46.57. Therefore, the method satisfies the acceptance criteria of the system suitability parameters.

Specificity Study:

HPLC chromatogram of naproxen standard solution was compared with those of sample and vehicle solutions (Fig. 3). It showed that the mixture of the inactive ingredients in the suspensionhad no peaks to interfere withthe drug response. Chromatogram of the standard-added sample solution also presented a large peak at the same retention time. Therefore, the used method was found to be specific for determination of naproxen in the extemporaneous suspension.

Fig.3: Chromatograms of (A) naproxen standard (B) extemporaneous naproxen sample (c) standard-added extemporaneous sample and (D) vehicle solution. * Indicated the Naproxen Drug Peak.

Linearity, LOD and LOQ Studies:

Regression analysis of the calibration curves of naproxen standard solution was summarized in Table 1. It was linear over the concentration range of 2.56 ng/ml - 50 µg/ml with the correlation coefficient (r²) of 0.9999. LOD and LOQ values of the method were calculated based on the standard deviation of the response and the slope of calibration curves. They were found to be 0.19 and 0.59 μg/ml, respectively. It indicated that the method was more sensitive and suitable for druganalysis in the extemporaneous suspension.

Table 1.Regression analysis of calibration curves for determining linearity, LOD and LOQ values (n=3).

Parameters	Results
Linearity range	2.56 ng/ml - 50 µg/ml
Slope	8.6715
y-intercept	3.6620
Correlation coefficient	0.9999
LOD	0.19 µg/ml
LOQ	0.59 µg/ml

Accuracy Study:

Accuracy of the method was determined by standard addition procedure. Three replicate injections, containing known amount of naproxen standard at 80%, 100% and 120% of test concentration, were added to the sample solutions. Percent recovery was found in the range of 96.91±1.84 to 98.14±3.53 while the mean percent recovery was 97.45±0.63 (Table 2). They were in the acceptable criteria in the range of 95-105%¹⁸. It indicated that the method was accurate forroutine application.

Table 2. Accuracy of the proposed method for determination of naproxen in drug product (mean ± SD, n = 3).

Level	Concentration added (µg/ml)	Concentration measured (µg/ml)	%Recovery
80%	18.38	18.04 ± 0.65	98.14 ± 3.53
100%	22.97	22.35 ± 0.16	97.29 ± 0.72
120%	27.57	26.72 ± 0.51	96.91 ± 1.84
Overall			97.45 ± 0.63

Precision Study:

Six replicate injections of the sample solutions were used to determine the method precision. Repeatability was expressed as the relative standard deviation (%RSD) of the response and they were 2.33 and 1.33 in the first and second day, respectively (Table 3). In addition, intermediate precision was presented by % RSD of all sample injections and it was found to be 2.28. They complied with the general acceptable criteria (less than 3.7)¹⁸. Hence, the method displayed a good precision.

Robustness Studies:

It is important to demonstrate whether minor changes in the chromatographic conditions affect the experimental result. The effects of flow rate, mobile phase composition, wavelength and injection volume were evaluated (Table 4). However, these changes had an influence on the method parameters. Shorter retention time of the drug was observed when using high flow rate of the mobile phase. Reduction of polarity of the mobile phase by increasing ratio of organic solvent (acetonitrile) made the drug eluted faster. Slight change on UV detection near the l_max gave lower peak signal. Increase on injection volume caused the broader peak. However, the quantitative result was still reliable as the %RSD of all parameters was less than 2%. The method was robust to analyze the content of naproxen in the extemporaneous suspension

Table 4. Robustness of the proposed method for determination of naproxen extemporaneous suspension (mean±%RSD; n=3).

Parameters	Conditions	%Recovery	Retention time (min)	Theoretical plate number	Tailing factor
Flow rate (ml/min)	0.4	100.16 ± 0.94	13.93 ± 0.07	17525.00 ± 0.20	1.20 ± 1.78
	0.7	99.09 ± 0.10	8.10 ± 0.06	13701.50 ± 0.54	1.14 ± 1.87
	1.0	100.00 ± 1.94	5.64 ± 0.00	11209.50 ± 0.11	1.12 ±0.63
Mobile phase composition (Buffer:ACN)	60:40	99.35 ± 1.73	5.39 ± 0.00	2524.50 ± 1.20	0.94 ± 0.76
	70:30	99.90 ± 0.10	8.62 ± 0.25	9704.50 ± 0.30	1.14 ± 1.24
	80:20	100.99 ± 0.21	33.61 ± 0.06	9144.50 ± 0.69	1.05 ± 1.35
Wavelength (nm)	225	97.71 ± 0.09	8.51 ± 0.11	9571.50 ± 0.44	1.19 ± 0.60
	230	100.93 ± 1.51	8.61 ± 0.22	9454.50 ± 0.44	1.17 ± 0.61
	235	98.28 ± 0.76	8.61 ± 0.11	9155.50 ± 0.50	1.18 ± 0.60
Injection volume (µl)	10	96.39 ± 0.20	8.39 ± 0.00	9627.50 ± 0.27	1.18 ± 1.81
	20	98.46 ± 1.51	8.62 ± 0.14	9554.50 ± 0.44	1.16 ± 0.61
	30	95.28 ± 0.12	8.48 ± 0.12	9206.00 ± 0.34	1.17 ± 0.61

Forced Degradation Studies:

A number of common reactions (hydrolysis, oxidation and thermal stresses) were used to determine the capability of the proposed method to analyze chemical stability of the drug samples during storage (Table 5). The decomposition of naproxen sample was found in the acid-catalyzed hydrolysis (0.1 N HCl) by which the percent drug content was15.52 ± 1.06. The hydrolytic degradation products were eluted at less than 6.00 min, suggesting they became more polarity (Fig.4). On the other hand, the drug remained intact in the basic (0.1 N NaOH) and neutral (H₂O) hydrolysis. The drug content was 97.76 ± 0.51% and 101.67 ± 0.79%, respectively. It implied that naproxen in the extemporaneous suspension was sensitive to acid hydrolysis whereas the drug product was stable under basic and neutral condition¹⁹. Furthermore, the drug content was more than 97.30 ± 1.99% under the chemical oxidation (0.1% and 1.0% H₂O₂) and dry heat (80°C), showing that the drug preparation was stable towards those conditions. These indicated that the method was selective and could distinguish naproxen from other inactive compounds and degradation products. It could serve as a stability-indicating method that uses for determination of naproxen in the preparation during product development and stability studies.

Table 5.Forced degradation studies of extemporaneous naproxen suspension (mean±SD, n= 3).

Conditions	% Drug content
Acid hydrolysis/ 0.1 N HCl, 80°C/ 1 h	15.52 ± 1.06
Basic hydrolysis/ 0.1 N NaOH, 80°C/ 1 h	97.76 ± 0.51
Neutral hydrolysis/ Water, 80°C/ 1 h	101.67 ± 0.79
Dry heat/ 80°C/ 1 week	99.21 ± 1.53
Oxidation/ 0.1% H₂O₂/ 1 h	97.30 ± 1.99
Oxidation/ 1.0% H₂O₂/ 1 h	101.35 ± 1.13

Fig. 4: Forced Degradation Studies of Extemporaneous Naproxen Extemporaneous Suspension. (A) Acid Hydrolysis, (B) Basic Hydrolysis, (C) Neutral Hydrolysis, (D) Thermal Degradation, (E) 0.1% and (F) 1.0% H₂O₂Oxidative Degradation. *Indicated the Naproxen DrugPeak.

Application of the Method for Determination of Naproxen in the Extemporaneous Suspension:

The method was used to analyze naproxen in the extemporaneous preparation. It was found that the average drug content was 101.57±1.25% from 6 different samples.The value was in the general acceptant criteria of the labeled amount of the drug (90 – 110%). The proposed method was sensitive, precise and accurate to determine the drug in the extemporaneous suspension.

CONCLUSION:

This HPLC method was validated, according toASEAN guideline for the validation of analytical procedure, to determine naproxen in an extemporaneous suspension. It was useful for analysis of the drug at low concentration. The method was simple, precise and accurate. It was demonstrated to be robust for intentional minor changes of flow rate, mobile phase composition, wavelength and injection volume. In addition, it could separate any degradation products from the naproxen drug peak, indicating that it was served asa stability-indicating method. Hence, it can be used for routine determination of naproxen in the suspension and during stability study.

ACKNOWLEDGEMENT:

This work was supported by a grant from Walailak University. In addition, we would like to thank Centre of

Scientific and Technological Equipments, Walailak University for research facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 25.07.2018 Modified on 11.08.2018

Research J. Pharm. and Tech 2018; 11(10): 4332-4338.

DOI: 10.5958/0974-360X.2018.00793.X

Sample	Day 1		Day 2
Sample	Concentration measured (µg/ml)	%LA	Concentration measured (µg/ml)	% LA
1	25.99	103.96	25.85	103.42
2	25.43	101.71	26.35	105.38
3	24.99	99.97	25.68	102.7
4	26.08	104.31	25.36	101.45
5	24.81	99.24	25.54	102.15
6	26.21	104.86	25.60	102.41
Average	25.59	102.34	25.60	102.41
Repeatability (%RSD)	2.33		1.33
Overall average	25.25	101.01
Intermediate precision (%RSD)	2.28