The instruments used in this study were an HPLC system equipped with a UV-Vis detector and Empower software for data acquisition and processing. The HPLC system consisted of a Waters e2695 separation module and a Waters 2489 UV-Vis detector operated at 254 nm. Chromatographic separation was performed using a C18 column, L1 USP category, with dimensions of 4.6 mm × 250 mm and particle size of 5 µm. Other instruments included a dissolution tester (Hanson), pH meter (Mettler Toledo), analytical balance (Mettler Toledo), ultrasonic bath (Hanson), volumetric glassware (Pyrex), HPLC vials (Waters), volumetric pipettes (Pyrex), and 0.22 µm membrane filters (Merck).

The materials used were isoniazid working standard (Second Pharma Co. Ltd.), 2 FDC dispersible tablets containing isoniazid and rifampicin (PT. Chemia Farma), methanol gradient grade for HPLC (Merck), dipotassium phosphate analytical grade (Merck), monopotassium phosphate analytical grade (Merck), hydrochloric acid analytical grade (Merck), placebo, and distilled water/aquadest (PT. Kimia Farma).

The analytical method was developed based on the monograph for rifampicin and isoniazid capsules in the Indonesian Pharmacopoeia Ed. VI. The method was adapted for the determination of isoniazid in 2 FDC dispersible tablets. Method development focused on chromatographic conditions, sample preparation, and dilution procedure suitable for dispersible tablet matrices. The final method used a C18 column, L1 USP category, with dimensions of 4.6 mm × 250 mm and particle size of 5 µm. The prepared sample solution was diluted with phosphate buffer at a ratio of 1:2 before HPLC analysis.

The HPLC analysis was performed using isocratic elution. The mobile phase consisted of water, phosphate buffer, and methanol in a ratio of 850:100:50. The flow rate was set at 1.5 mL/min, and the injection volume was 50 µL. Detection was carried out using a UV-Vis detector at 254 nm. The solvents used during sample preparation were 0.1 N HCl, phosphate buffer, and distilled water.

Isoniazid working standard was accurately weighed and dissolved in 0.1 N HCl using an ultrasonic bath for 10 minutes. The solution was then diluted to volume with 0.1 N HCl and mixed until homogeneous. An aliquot of the standard solution was transferred into a volumetric flask, mixed with phosphate buffer, and diluted with distilled water to volume. The final solution was filtered through a 0.22 µm membrane filter before injection into the HPLC system.

The analytical method was developed using reversed-phase HPLC with a C18 stationary phase and a polar mobile phase. This system was selected because isoniazid is a polar compound that can be separated using reversed-phase chromatography (Hakkimane & Guru, 2017). The C18 column is widely used in HPLC analysis because it provides suitable retention and separation for compounds with various polarity levels (Mishra et al., 2018)(Prasanthi et al., 2015)(Rusli et al., 2022).

In this study, the analytical method was modified from the previous L1 column, C18 3.90 mm × 300 mm, 10 µm, to an L1 column, C18 4.60 mm × 250 mm, 5 µm. The chromatogram comparison is shown in Figure 1. The modified column produced a shorter retention time and a sharper isoniazid peak. The retention time decreased from 5.811 minutes using the previous column to 3.599 minutes using the modified column. This result indicates that the modified column improved the efficiency of the analysis. The smaller particle size of the modified column may increase chromatographic efficiency, resulting in a sharper peak and faster elution of isoniazid.

The HPLC analysis was performed using a mobile phase consisting of water, phosphate buffer, and methanol with UV detection at 254 nm. Phosphate buffer was used to maintain the pH of the analytical system because pH can affect the elution strength and chromatographic behavior of the analyte ((Alvarez-Segura et al., 2019); (Gandjar & Rohman, 2012)). The wavelength of 254 nm was selected because isoniazid provides adequate UV absorbance at this wavelength.

Method development was also carried out by comparing the ratio of sample solution to phosphate buffer. The previous preparation used a sample solution:phosphate buffer ratio of 1:2, whereas the modified preparation used a ratio of 1:1. As shown in Figure 2, the 1:1 ratio produced a cleaner chromatographic profile with lower interfering peaks than the 1:2 ratio. The isoniazid peak remained detectable at the expected retention time, indicating that reducing the amount of phosphate buffer did not interfere with isoniazid detection. Therefore, the 1:1 ratio was selected because it provided sufficient chromatographic performance, reduced buffer use, and minimized interfering peaks in the chromatogram.

Validation of analysis is a technique that uses laboratory tests to ensure that the procedure's performance characteristics fulfill the requirements for its intended usage (Health, 2020). The validation parameters evaluated in this study included linearity and range, LOD, LOQ, accuracy, precision, and selectivity.

Linearity refers to an analytical method's ability to deliver a proportional response to analyte concentration. Linearity is typically stated in terms of variation around the regression line's direction, which is derived using calculations based on data received from analyte test results in samples of varying concentrations ((Raposo & Ibelli-Bianco, 2020); (Riyanto, 2014)). Linearity is reached under typical conditions when the correlation coefficient (r) value is ≥ 0.98. The range is defined as the interval between the highest and lowest limits of verified analyte levels that can be determined with sufficient accuracy, precision, and linearity using recognized analytical processes. The International Council for Harmonization (ICH) recommends that linearity be established use at least five commonly used concentrations. Dissolution Test ± 20% of the specific range (for example, in controlled release preparations, after 1 hour, 20%, and after 24 hours, more than 90%, then the range is from 0% to 110% of the concentration stated on the label) (5,11). Data from the regression line can help to show an approximate degree of linearity by relating the response (y) to the concentration (x). Linearity can be described in the form of a slope (b), an intercept (a), and a regression coefficient (r).

Linearity was evaluated using five concentration levels of isoniazid, namely 60%, 70%, 80%, 100%, and 120%. The calibration curve showed a linear relationship between isoniazid concentration and peak area, as presented in Figure 3. The regression equation obtained was y = 54389523x – 4007.7 with a correlation coefficient r = 0.9995. The correlation coefficient close to 1 indicates that the HPLC response was proportional to isoniazid concentration within the tested range. Therefore, the method demonstrated acceptable linearity for isoniazid determination.

LOD and LOQ were determined based on the standard deviation of the response and the slope of the calibration curve ((Raposo & Ibelli-Bianco, 2020); (Riyanto, 2014)). Testing the LOD and LOQ of an analyte can be seen from the standard deviation value of the response (σ) of the analyte being analyzed. The standard deviation of response was 6382.381, and the slope obtained from the regression equation was 54389523. The calculated LOD and LOQ values were 0.00038724 mg/mL and 0.00117346 mg/mL, respectively. These results indicate that the developed method was sufficiently sensitive to detect and quantify isoniazid at low concentrations. LOD and LOQ are important parameters in analysis because they help to determine reliable analyte concentration limits in the analytical method used.

Accuracy is a value that indicates how near the analyst's results are to the real analyte levels. The percent recovery of the additional analyte is used to calculate accuracy (Visconti et al., 2023). Accuracy was determined using the recovery method by adding isoniazid standard to the placebo matrix at three concentration levels: 60%, 100%, and 120%.

The accuracy results are presented in Table 1. The recovery values ranged from 98.57% to 100.97%, with an average recovery of 99.51% and RSD of 0.68%. These results met the acceptance criterion of 98–102%, indicating that the method was accurate for determining isoniazid in the 2 FDC dispersible tablet matrix ((Gandjar & Rohman, 2012); (Indonesian Food and Drug Authority, 2014); (Health, 2020)).

Precision is a parameter that represents the level of consistency between individual measurements, as assessed by the deviation of individual results from the average when the operation is repeated on samples drawn from the same mixture. The standard deviation or relative standard deviation (coefficient of variance) is used to calculate precision.

Precision was evaluated at 100% concentration using six replicate preparations. The precision results are presented in Table 2. The recovery values ranged from 100.32% to 101.19%, with an average recovery of 100.79% and RSD of 0.32%. The RSD value was less than 2%, indicating that the method showed good precision and produced consistent results for repeated isoniazid determination ((Raposo & Ibelli-Bianco, 2020); (Riyanto, 2014)).

Selectivity is the degree to which an analytical method can reliably measure analytes in the presence of interference (error) under predetermined test situations for the sample to be examined. Selectivity describes an analytical method for measuring analytes specifically and precisely (Raposo & Ibelli-Bianco, 2020). Selectivity was evaluated by comparing chromatograms of the isoniazid standard solution, sample solution, placebo, mobile phase, phosphate buffer, dissolution medium, and distilled water. The method was considered selective when no interfering peak appeared at the retention time of isoniazid. The chromatogram showed that the isoniazid peak in the sample appeared at the same retention time as the isoniazid standard, while placebo, distilled water, phosphate buffer, and dissolution medium did not produce peaks at the isoniazid retention time. HCl produced a peak, but it did not appear at the same retention time as isoniazid; therefore, it did not interfere with the analysis. These results indicate that the method was selective for determining isoniazid in the 2 FDC dispersible tablet matrix (Marson et al., 2020).

After the method was confirmed to be linear, accurate, precise, sensitive, and selective, it was applied to determine isoniazid levels in dissolution test samples of 2 FDC dispersible tablets. The dissolution test is the process of dissolving an active substance or compound from a solid dosage form into a certain medium (Merwe et al., 2020). This test is used to determine compliance with the dissolution requirements stated in each monograph for dosage forms used orally. The dosage unit in question is 1 tablet, 1 capsule, or a specified amount (Health, 2020). The Q value for isoniazid was 80%, and the stage-one acceptance criterion required that each dosage unit should not be less than Q + 5%, equivalent to 85%. The dissolution results showed that the lowest isoniazid dissolution value was 94.63%, with an average value of 99.06%. The lowest value was higher than the stage-one acceptance criterion of 85%, indicating that all tested units met the dissolution requirement. The average dissolution value of approximately 99% indicates that isoniazid was effectively released from the 2 FDC dispersible tablet formulation. This result supports the quality expectation for dispersible tablets, which should disintegrate and release the active substance efficiently in the dissolution medium.