The reagents used were analytical grade. They consisted of: Ibuprofen BP purchased from Palghar, India, with a purity of 98%; acetonitrile (≥99.8%); triethylamine (95.5%); orthophosphoric acid (85%); 1N sodium hydroxide; 1N hydrochloric acid; 10% hydrogen peroxide and ultrapure water.

The analyses focused on a specialty containing ibuprofen tablets dosed at 400 mg marketed in Ivory Coast.

The apparatus consisted of a HPLC (Waters) system and a precision balance (Mettler Toledo), a pH meter (seven compact), an oven (prolabo), a UV lamp, an ultrasonic bath (cleaner-TH), millipore filters (Millex-HN nylon 0.45 µm) and classic laboratory glassware.

The reversed-phase liquid chromatography method was performed on a stationary phase consisting of a Nucleodur column octadecylsylanized (125 mm × 4.6 mm, 5 μm). The mobile phase consisted of an acetonitrile/buffer solution mixture at pH 7.05 (60:40, v/v). The buffer solution was composed of HPLC-grade water, triethylamine, and orthophosphoric acid (1000:1:0.5 v/v/v).

The flow rate was set at 1.5 ml/min for an injection volume of 10 μl, the column temperature was maintained at 25˚C and the detection of ibuprofen was carried out in UV at 220 nm.

The analyses were carried out at the National Public Health Laboratory (LNSP).

2.3.1. Mobile Phase

The mobile phase consisted of a mixture of acetonitrile and triethylamine buffer solutions at pH 7.05. The buffer solution triethylamine was prepared in a 1000 ml volumetric flask by adding 1 ml of triethylamine to 400 ml of milliliter water, followed by 0.5 ml of orthophosphoric acid. After manual stirring for 30 seconds, the solution was filled to the mark with milliliter of water and then homogenized using ultrasound for 15 minutes. The pH of the solution was then measured using a pH meter and adjusted to 7.05 with orthophosphoric acid. The mobile phase was prepared in a 1000 ml volumetric flask by mixing 600 ml of acetonitrile with 400 ml of pH 7.05 buffer solution (60:40 v/v).

2.3.2. Reference Solution

A quantity of 40.01 mg of ibuprofen was weighed and introduced into a 100 ml volumetric flask to which 70 ml of diluent-1, consisting of a buffer/acetonitrile mixture (20:80 v/v), was added. The mixture was dissolved ultrasonically for 15 min and made up to volume with diluent-1. After homogenization, 5 ml of this preparation (stock solution) was transferred into a 25 ml volumetric flask and made up to the mark with diluent-2, consisting of a buffer/acetonitrile mixture (40:60 v/v).

2.3.3. Preparation of the Solution to Analyze

Twenty tablets were weighed and pulverized using a mortar and pestle. The mass of powder equivalent to 400 mg of ibuprofen was dissolved in a 250 ml volumetric flask with 175 ml of diluent-1 and placed in an ultrasonic bath for 30 minutes with intermittent stirring. The mixture was then made up to volume with diluent-1 and homogenized. Approximately 25 ml of the mixture was centrifuged at 3000 rpm for 5 minutes. Five ml of the supernatant was transferred to a 25 ml volumetric flask, made up to 25 ml with diluent-2, homogenized, filtered, and injected for HPLC analysis.

The process was validated by determining specificity, linearity, precision, accuracy, limits of detection (LOD) and limits of quantification (LOQ) according to the guidelines of the International Conference on Harmonisation (ICH) [15] and the United States Pharmacopeia (USP) guidelines [16].

The linearity study (n = 5) was performed with reference solutions of concentrations 5.33 µg/ml, 6.857 µg/ml, 9.6 µg/ml, 12 µg/ml, and 16 µg/ml. Two statistical tests were subsequently carried out. These were, on the one hand, the Student’s t-test comparing the y-intercept with the null hypothesis (H0: the y-intercept is equal to zero at the risk α = 5%) and, on the other hand, the test for the existence of a significant slope with the null hypothesis (H0 = the slope is significantly different from zero at the risk α = 5%). Repeatability (n = 6) was performed on solutions of concentrations 8 µg/ml and 12 µg/ml. The accuracy of the method was performed on (n = 3) increasing quantities of ibuprofen powder: 800 µg, 1000 µg, and 1200 µg/ml. µg ) were added to the initial quantity of 1000 µg.

The limit of detection (LOD) and the limit of quantification (LOQ) were determined from the analytical curve as defined by ICH [15][17]. Its formula is as follows:

LD = 3.3σ/S and LQ = 10σ/S

σ = the standard deviation of the response;

S = the slope of the calibration curve.

Forced degradation studies were undertaken to deliberately degrade the active drug. These studies served to evaluate the analytical method’s ability to measure the active ingredient and its degradation products without interference. These degradation studies were performed under acidic, basic, and oxidative conditions.

The standard mixture was exposed to acid (HCl, 1 N), a base (NaOH, 1 N) and an oxidizing agent (H₂O₂, 10%) for 24 to 48 hours [18][19]. Samples were taken for each type of degradation at 24 hours and 48 hours of exposure.

Ibuprofen was identified (Figure 2), using a Nucleodur C18 stationary phase (125 mm × 4.6 mm, 5 μm), with a retention time of 1.28 min; this time is shorter than that obtained with a column Hypersil BDS (150 × 4.6 mm, 5 μm), which was approximately 3.29 minutes [20]. This time difference is due to the column length, which is longer than that used in our study. This time saving will reduce the amount of mobile phase and consequently, the cost of the analysis. The method was specific because no degradation peak interfered with that of ibuprofen, as shown in the chromatogram of the standard ibuprofen solution (Figure 2).

Furthermore, a linearity domain was identified for ibuprofen concentrations between 5.33 μg/ml and 16 μg/ml, with a straight-line equation.

Y = 82320X + 23142, and a coefficient of determination R² = 0.9995 (Figure 3). The test for the y-intercept showed that it is not significantly different from 0 at the probability level α = 5% (Table 1). Similarly, the test for the existence of a significant slope showed that the slope b is significantly different from zero (Table 2). The coefficients of variation reflecting repeatability, obtained for the solutions concentrated at 8 µg/ml and 12 µg/ml were less than 2% with respective values of 0.23 and 0.30%. The repeatability of the method is in accordance with ICH guidelines [15].

The limits of detection and quantification were 0.447 μg/ml and 1.356 μg/ml, respectively. The mean percentage recovery, reflecting accuracy, was 100.45% (Table 3). The validation criteria all met ICH requirements and demonstrated satisfactory linearity, precision, and sensitivity for the assay of ibuprofen in tablet dosage forms [17].

Figure 2. Chromatogram of standard ibuprofen (9.6 µg/ml)/sample ibuprofen.

Figure 3. Ibuprofen regression curve.

Table 1. Intercept test.

t tabulated (0.975; 13) < calculated < tabulated (0.025; 13).

Table 2.Test for the existence of a significant slope.

t b calculated < tabulated t (0.975; 13).

Table 3.Accuracy parameters.

The validated method was used for the assay of the drug ibuprofen ubi tablets 400 mg. The ibuprofen content was 103.51% (for n = 3). This value is within the USP specifications of 90% to 110% [16].

The results of the degradation studies showed that ibuprofen degraded under strong basic conditions (NaOH; 1N). Based on the relative retention time, the ibuprofen-related compound C, described in the United States Pharmacopeia 47—NF 42 [16], was identified at a retention time of 1.7 min after 48 h in the presence of NaOH. Similarly, an unidentified peak was observed at a retention time of 1 min under basic conditions. The degradation results in a basic medium are comparable to those obtained under basic conditions (NaOH; 6N) by Raja et al. [18].

Ibuprofen remained relatively stable under acidic (HCl; 1N) and oxidative (H₂O₂; 10%) conditions between 24 and 48 hours of exposure. Rahman et al. obtained similar results after simultaneous forced degradation studies of paracetamol and ibuprofen [19]. Furthermore, harsher stress conditions with (H₂O₂; 30 %) for a longer exposure time of 9 days, conducted by Sherri et al., led to the identification of the presence of 4-Isobutylacetophenone (4-IBAP) [21].

When applying the stability indicator method to the analysis of ibuprofen 400 mg tablets under normal conditions, no degradation products were observed.