Development and Validation of UV Spectrophotometric Method for the Estimation of Naproxen in Bulk and Formulation

Pharmaceutical analysis serves as the backbone of ensuring drug safety, efficacy, and consistency. It encompasses the evaluation of chemical substances, whether isolated compounds or complex mixtures, across various dosage forms. With components derived from both natural and synthetic sources, pharmaceutical analysis plays a fundamental role in modern medicine and its applications.

Analytical chemistry, particularly spectrophotometric methods, provides powerful tools for assessing pharmaceutical formulations. These techniques are categorized into two primary approaches: qualitative analysis, which identifies chemical constituents, and quantitative analysis, which measures their concentration. Both methods are indispensable in pharmaceutical development and quality control.

Figure 1: Parts of UV- Visible Spectrophotometer

Spectroscopic Characteristics of Naproxen:

Solubility is one of the most critical pre formulation properties which play a significant impact on performance of a molecule. Solubility and permeability are the two important properties of Biopharmaceutical Classification System (BCS). Biopharmaceutical Classification System (BCS) provides the scientific framework for designing of drug delivery systems and many regulatory decisions. Solubility determination is one of the first most important and extensively studies of pre formulation. Aqueous solubility is a solubility which effects on the bioavailability of the drug.

A spectrophotometer is an essential tool for analysing the absorption characteristics of various substances, including pharmaceuticals. One notable example is naproxen, a widely used nonsteroidal anti-inflammatory drug (NSAID) that helps manage pain, inflammation, and fever. The analysis of naproxen’s absorption spectrum is crucial in ensuring its purity, concentration, and efficacy during formulation. Spectrophotometry offers an efficient and reliable method to study naproxen in pharmaceutical research and quality control, making it a vital technique for drug selection and analysis.

Naproxen is a nonsteroidal anti-inflammatory drug (NSAID). It works by reducing hormones that cause inflammation and pain in the body. Naproxen is used to treat pain or inflammation caused by conditions such as arthritis, ankylosing spondylitis, tendinitis, bursitis, gout, or menstrual cramps. Naproxen is also available in combination with other medications under the following brand names: Aleve PM, Aleve-D Sinus and Cold, Treximet, and Vimovo.

Table 1. Drug Profile

MATERIALS AND METHODS

1. Drug Sample:

Pharmaceutically pure samples of Naproxen were obtained from Arti Pharma in Mumbai. The 10gram API Powder were purchased from Arti Pharma Bulk Manufacturing factory. 

2. Chemicals & Solvents Used:

All of the analytical-grade chemicals and solvents that were used were purchased     from Loba Chem. Ltd. in India. The study's solvent system was made up of:  

• Methanol
• Ethanol
• DMF
• Acetone

3. Instrument Used:

Double beam UV-Visible Spectrophotometer Shimadzu 1900 using UV Probe Software. The spectra were recorded over range 200 - 400 nm against solvent in 1 cm Quarts cells.

Analytical balance (Sartorius) and Ultra-sonic cleaner (Fisher scientific FB15061) were used. Micropipette of Variable volume 10-1000 μL (Capp Eco pipette single channel) and Digital balance (Mettler Toledo XP 105). UV Probe Software used.

4. Marketed Formulation:

Marketed drug formulation: Naprosyn; Two types of formulation of Naproxen tablet were used.

One is Naproxen IP 750mg Tablets and Other is Naproxen Sodium USP 550mg Tablets. 10 Tablets of each were purchased from retail Medical Shop.

Figure 2: Marketed Drug Formulation

• Method:

1. Selection of solvent system:

The solubility of drugs was determined in a variety of polar to non-polar solvents as per Indian Pharmacopeial standards. I performed Trial and Error method to select the best possible solvent which can dissolve both drugs and tablet formulation. The Solubility study and solvent selection for the Naproxen API Powder is carried out by using differential solvents namely Ethanol, Methanol, Acetone, and Water. Along with individual solvent and Binary Solvent Mixture were prepared and solubility studies were done.

Figure 3: Selection of Solvent System (trail & error basis)

The solvents and Binary Mixture used were enlisted in following chart along with Solubility Result.

Table 2. Drug Solubility

The drug is found to be highly soluble in methanol an DMF. Further Binary mixtures these solvents were prepared and solubility is checked. Following table shows the ratio and solubility.

Table 3. Drug Solubility in Binary Mixture

The best solvent system was found to be 85% Methanol and 15% DMF (N, N, Dimethyl Formamide), chosen on account of its ready availability, cost factor and solubility for the analysis of Naproxen for the proposed method. For the preparation of standard stock solution 500ml solvent were prepared using selected solvent i.e. Methanol and DMF (85:15).

2. Preparation of Standard Stok Solution:

Accurately weighed 10 mg of Naproxen API powder were transferred into a clean & dried 250 ml Conical flask and then volume was made up to 100ml by addition of prepared solvent mixture (85% Methanol and 15% DMF) to get a standard concentration of 100 µg/ml for drugs. This standard stock solution (100 µg/ml) was further diluted with solvent system to obtain a series of dilution - 10, 20, 30, 40, 50 and 60 µg/ml for Naproxen.

Figure 4: Standard stock solution of drugs & series of dilution

3. Determination of λ max:

The samples were scanned in UV spectrophotometer from a range of 200 – 400 nm against prepared solvent mixture (85% Methanol and 15% DMF) as blank and the wavelength corresponding to maximum absorbance in it was determined. The λ Max was found to be 272nm

Figure 5: Spectrum Graph of Naproxen

4. Preparation of Calibration Curve:

The Naproxen drugs appropriate aliquots were pipetted out from the standard stock solution into a series of 10 ml volumetric flasks and the volume was made up to the mark with the prepared solvent system - 85% Methanol and 15% DMF to get series of concentrations - 1, 2, 3, 4, 5 and 6 µg/ml of Naproxen API. (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mg/ml) Solutions prepared by this serial dilution method for each drug were analysed at their respective λ wavelengths and absorbances were recorded.

5. Validation of Method:

Validation can be defined as (ICH) establish documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics. The method was validated for several parameters like linearity, accuracy, precision, ruggedness, robustness, repeatability, Limit of detection (LOD), Limit of quantification (LOQ) according to ICH guidelines.

1. Linearity

The linearity of the analytical method was its ability to elicit test results which are directly proportional to analyte concentration in samples within a given range. The drug showed linearity in the range of 10-50µg/mL with correlation coefficient of 0.9705 as maximum absorbance in methanol 272 nm was obtained.

2. Accuracy

Accuracy of the proposed method was determined using recovery studies. The recovery studies were carried out by adding different amounts (50%, 100% and 150%) of the pure drug to the pre-analysed formulation. The solutions were prepared in triplicates and the % recovery was calculated.

3. Ruggedness

Ruggedness assesses how reliably a method performs under slightly different conditions. In this case, two analysts independently measured absorbance values using the same method. The consistency of their results was evaluated by calculating the % Relative Standard Deviation (% RSD). A low % RSD indicates the method produces stable and reproducible results, showcasing its ruggedness.

4. Robustness

Analysis was carried out at 3 different wavelengths at room temperature to determine the robustness of the method and the respective absorbance was measured.

5. Precision

Precision studies were carried out to ascertain the reproducibility of the proposed method. Repeatability was determined by preparing three replicates of same concentration of the sample and the absorbance was measured. Intraday precision study was carried out by preparing drug solution of same concentration and analyzing it at three different times in a day.

6. Repeatability

Repeatability is the consistency of analytical measurements when the UV-spectrophotometric method is applied under identical conditions. It ensures that the same concentration of Naproxen produces reproducible absorbance values, confirming the method’s reliability for routine pharmaceutical analysis.

7. LOD & LOQ

Limit of detection (LOD) is the lowest amount of analyte in the sample that can be detected. Limit of quantification (LOQ) is the lowest amount of analyte in the sample that can be quantitatively determined by suitable precision and accuracy. LOQ and LOD were determined using the following equation LOQ-10s/m, LOD-3.3s/m where s is the standard deviation of the response and m is the slope of the related calibration curve. The values of LOQ and LOD were found to be      and     µg/mL respectively.

Standard Deviation: Square root of the variance: 0.3323

LOD & LOQ Calculated using the standard sensitivity formulas:

• LOD = {3.3×SD /Slope} = 3.3×0.0021/0.022 = 0.31 µg/ml
• LOQ ={10×SD/Slope} = 10×0.0021/0.022 = 0.95 µg/ml

6. Preparation of Tablet Solution for Assay:

Twenty Naproxen and Naproxen sodium tablets (750 mg Naproxen and 550 mg Naproxen Sodium) were weighed and powdered. A portion equivalent to 355.64 mg of Naproxen and 449.13 mg of Naproxen sodium was weighed and added into 10 ml Standard Solvent separately and volume was made up to 100ml with selected solvent system. The contents of the solution were mixed well and filtered through Whatman filter paper No. 41. Hence the 100ml standard stalk solution were prepared for both Tablets respectively.

Figure 4: Standard

stock solution of Tablet & series of dilution

Then the dilutions of both Naproxen and Naproxen sodium tablet are prepared in series of concentrations - 1, 2, 3, 4, 5 and 6 µg/ml of Naproxen API. (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mg/ml). The absorbance of dilutions was recorded at respective maximum wavelengths.

RESULT AND DISCUSSION:

Table 4. Linearity Range

Figure 5: Linearity Graph of Naproxen API

Tabel 5. Accuracy Results of Naproxen

Table No. 6.  Ruggedness results of Naproxen

Table No. 7. Robustness results of Naproxen

Table No. 8. Intraday precision

Table No. 9. Inter day precision

Table No. 10. Repeatability Results

The present study introduces a method for the analysis of Naproxen through UV Spectrophotometry, offering a straightforward, stable, rapid, accurate, precise, and reproducible approach. Additionally, this technique is resource-efficient, minimizing reagent consumption and operational complexity while ensuring robust analytical performance. The results obtained, summarized in Table No. 11, validate the effectiveness of this method in the quantitative determination of Naproxen, demonstrating its suitability for pharmaceutical quality control and analytical applications.

Table No. 11. Summary Of Developed Method

• Results of Calibration Curve of Naproxen Drug:

Figure 6: Calibration Curve of Naproxen

Figure 6: Graph of Naproxen Tablet

Figure 6: Graph of Naproxen Sodium Tablet

• Results of Assay: