Characterization of forced degradants of Tegafur, Gimeracil, and Oteracil potassium by Liquid Chromatographic-Electrospray Ionization-Mass Spectrometry and simultaneous estimation of triple combination in drug substance and finished pharmaceutical Dosage Form

1. Introduction

The cytochrome P-450 enzyme in the liver progressively transforms the prodrug tegafur into the antibiotic fluorouracil. Fluorouracil's catabolic enzyme, dihydropyrimidine dehydrogenase (DPYD), is competitively inhibited by uracil, increasing the serum levels of the drug (1). Gimeracil (5-chloro-2,4-dihydroxypyridine) is an inhibitor of DPYD that prevents the blood from degrading pyrimidines such as 5-fluorouracil. The oral fluoropyrimidine derivative S-1 is first combined with gimeracil to produce sustained 5-fluorouracil (5-FU) in the body, specifically in serum and tumor tissues (2). Oteracil potassium is a chemoprotective agent that modulates the action of 5-FU and inhibits the enzyme orotate phosphoribosyl-transferase (OPRT). The gastrointestinal (GI) tract is where oteracil potassium is primarily localized. By inhibiting OPRT, it slows down the conversion of 5-FU into its active metabolite, 5-fluorouridine-5'-monophosphate. This lessens the GI toxicity brought on by the activated 5-FU (3). For the chemical structure, refer to figure 1. Tegafur, gimeracil, and oteracil potassium are widely used pharmaceuticals to treat lung cancers of the GI tract, such as those of the oral cavity, esophagus, colon and rectum, and pancreas, as well as non-small cell lung cancers. They also received approval as a therapy for head and neck tumors (progressive or recurrent) in 2001 (4,5). The literature survey for tegafur showed that tegafur drug substance was analyzed by high-pressure liquid chromatography (HPLC) (6,7), gas-liquid chromatography (8), and liquid chromatography-mass spectroscopy (LC-MS) (9). Gimeracil was estimated by HPLC (10) and LC-MS (11-13) mostly in human plasma and blood samples withdrawn from subjects. Oteracil potassium was always used in combination with tegafur and gimeracil for treating cancer. Tegafur, gimeracil, and oteracil potassium combinations were estimated by LC-MS (14-19). Tegafur, gimeracil, and oteracil potassium were estimated simultaneously; nevertheless, the stability and forced degradation tests have not been satisfactorily carried out yet. None of the previous studies reported the characterization of the degradation products formed during the shelf life of the formulation. Moreover, no forced degradation studies were reported on the finished pharmaceutical dosage form. The information presented above can help understand the chemical stability of tegafur, gimeracil, and oteracil potassium, create a suitable formulation, and check for optimal storage conditions. The literature review revealed that no study has yet offered a completely stability-demonstrating, validated liquid chromatography-mass spectrometric approach for the concurrent estimation of tegafur, gimeracil, and oteracil potassium, along with all known degradation products. The simultaneous detection of tegafur, gimeracil, and oteracil potassium and their forced degradation product characterization necessitated the invention of a simpler, faster, and less expensive method. The goal of the study was to follow the ICH method validation standards to develop and validate a fast, easy, and rugged liquid LC-MS technique for the concurrent estimation of tegafur, gimeracil, and oteracil potassium in the drug substance and the finished dosage.

2. Materials and Methods

2.1. Chemicals and Drugs

Merck supplied all of the solvents, which were HPLC-grade. All of the solvents and solutions used were filtered using 0.45 µm PVDF filters. Shree Icon Labs sent tegafur, gimeracil, and oteracil potassium medicinal substance samples as a gift. Tegafur, gimeracil, and oteracil potassium drug products (Tegonat 20) were manufactured by Natco Pharma LTD (Hyderabad, Iran) and bought from the market.

2.2. Instruments

HPLC (Make: Waters, Model: Alliance HPLC e-2695) combined with the mass spectrometer of the SCIEX QTRAP 5500 was used and endowed with an interface capable of electrospray ionization. The SCIEX software was used for the interpretation of the data from the chromatogram.

2.3. Method

The separation of tegafur, gimeracil, and oteracil potassium was successfully achieved by an Inertsil ODS column with dimensions of 150 mm×4.6 mm, 3.5µm, a mobile phase composition of formic acid (0.1%) and acetonitrile in the proportion of 40:60 in isocratic mode, with a flow rate of the mobile phase of 1.0 ml/min, and an auto-injector injection volume of 10 μL. The autosampler and column maintained a temperature of 25°C (ambient or room temperature). In the forced degradation study, the liquid chromatography system was linked to a mass spectrometer. On the positive electrospray ionization mode, the usual operational basic settings for mass spectrometer scans of tegafur, gimeracil, and oteracil potassium were improved. The collision energy was set at 15 V, the ion spray voltage was set at 5500 V, and the de-clustering potential was set at 40 V. The collision gas was ultrapure nitrogen gas. The source temperature was 550°C, the drying gas temperature was 120-250°C, and the drying gas flow rate was 5 l/min. The entrance potential was 10 V while the exit potential was 7 V. The dwell time was set at 1 sec.

2.4. Diluent Preparation

In this stage, 0.1% formic acid and acetonitrile were mixed in a ratio of 40:60.

2.5. Preparation of the Standard Solution

2.5.1. Preparation of the Standard Stock Solution

Tegafur 200 mg, gimeracil 58 mg, and oteracil 158 mg working standards were carefully weighed and put into a 100-cleaned dried volumetric flask. A total of 70 ml of diluent was included and sonicated for over 30 min. The remaining volume was then made up using a diluent and used as the default stock solution.

2.5.2. Standard Solution Preparation

A total of 5 ml of the above-prepared stock was transferred using a pipette into a 50 ml volumetric flask, which was then filled with the diluent to the final volume, which was considered the standard solution.

2.6. Sample Solution Preparation

A total of 327 mg of the sample was weighed and put in a 10 ml cleaned and dried volumetric flask. It was then filled with 7 ml of diluent, sonicated over 30 minutes, and diluted up to the final volume using the diluent. After that, 1 ml of the aforesaid stock solution was pipetted into a 10 ml volumetric flask, and the remaining volume was filled up with the diluent.

2.7. Method Validation Activity

2.7.1. System Suitability Parameter

Six replicate injections of tegafur, gimeracil, and oteracil standard solutions were introduced to the system to validate system suitability, and parameters such as plate count-USP (N), resolution, tailing factor, and analyte peak asymmetry were studied.

2.7.2. Linearity

Six distinct concentrations of tegafur, gimeracil, and oteracil were synthesized in various amounts of 50-300 µg/ml, 14.5-87 µg/ml, and 39.5-237 µg/ml, respectively. The peaks of each solution were recorded when they were injected into the instrument. After that, the regression coefficient was computed by drawing a line from the average peak area to the concentration.

2.7.3. Accuracy

Recovery experiments were carried out to confirm the procedure's accuracy at three levels of 50%, 100%, and 150%. Tegafur, gimeracil, and oteracil were discretely introduced into the pre-analyzed samples in the specified amounts. After each spike level was delivered into the liquid chromatographic system, the recovery percentage of each level was determined.

2.7.3. Method Precision

Tegafur, gimeracil, and oteracil samples were spiked at 100% of the concentration of the sample specification limit in six replicate preparations to demonstrate method precision. Six identical replicates were injected. The relative standard deviation was then calculated using the amounts of tegafur, gimeracil, and oteracil.

2.7.4. Intermediate Precision

In six preparations, the intermediate precision was obtained by sample spiking with tegafur, gimeracil, and oteracil at 100% of the prescribed limit in terms of sample concentration. The intermediate precision study was carried out over several days by numerous experts.

2.7.5. Sensitivity of the Method

The signal-to-noise ratios of 3:1 for the limit of detection (LOD) and 10:1 for the limit of quantification (LOQ) were used for evaluation.

2.7.6. Robustness

To verify the method's durability, small changes were made in chromatographic parameters, such as organic phase (+) (65B:35A) (-) (55B:45A) and mobile phase flow (+) (1.1 ml/min) (-) (0.9 ml/min).

2.7.7. Specificity

Two distinct samples were administered for comparison to placebo controls. Any interference peaks found in LC chromatograms for the therapeutic matrix (combination of medication and placebos) were investigated.

2.8. Forced Degradation Studies

2.8.1. Preparation of Buffer

In this stage, formic acid (1 ml) was dissolved in 1 liter of water.

2.8.2. Mobile Phase Preparation

The mobile phase was made by combining acetonitrile and buffer at a 60:40 ratio. The mobile phase was further filtered through 0.45 µm membrane filter paper.          

2.8.3. Preparation of Standard Stock Solution for Forced Degradation

A sample of 327 mg was measured and then added to a 10 ml volumetric flask. Afterward, 7 ml of the diluent was added. Later, this solution was further sonicated for 15 min to dissolve the contents before they were diluted to a final volume of 10 ml with the diluent and combined.

2.8.4. Acid Degradation Procedure

In this stage, 1 ml of the sample stock solution was taken into a 10 ml volumetric flask, and 1 ml of 1 N HCl was added. It was placed at room temperature (RT) for 30 min. After that, 1 ml of 1 N NaOH was added slowly and diluted to volume with the diluent.

2.8.5. Base Degradation Procedure

A 10 ml volumetric flask was taken. The sample stock solution quantity of 1 ml was transferred, and 1 ml of 1 N NaOH was added. It was then placed at RT for 30 min. Further, 1 ml of 1 N HCl was added and diluted to volume with the diluent.

2.8.6. Peroxide Degradation Procedure

A 10 ml volumetric flask was taken. The sample stock solution quantity of 1 ml was transferred, and 1 ml of 10% H₂O₂ was added. It was then placed at RT for 30 min and diluted to volume with the diluent.

2.8.7. Reduction Degradation Procedure

A 10 ml volumetric flask was taken. The sample stock solution quantity of 1 ml was transferred, and 1 ml of 10% NaHSO4 was added. It was then placed at RT for 30 min and diluted to volume with the diluent.

2.8.8. Thermal Degradation (105°C/6 h) Procedure

A total of 500 mg of the sample was exposed for 6 h at 105°C, and the exposed standard was examined. Afterward, 327 mg of the exposed sample was placed in a 10 ml flask and diluted with the diluent according to volume. Finally, 1 ml of the above stock was diluted to 10 ml with the diluent.

2.8.9. Photodegradation Procedure

A total of 500 mg of the sample was exposed to sunlight for 6 h, and the exposed sample was analyzed. A total of 327 mg of the exposed sample was placed in a 10 ml flask and diluted with the diluent according to volume. After that, 1 ml of the above stock was diluted to 10 ml with the diluent.

2.8.10. Hydrolysis Degradation Procedure

A 10 ml volumetric flask was taken. A sample stock solution quantity of 1 ml was transferred, and 3 ml of HPLC grade water was added. It was then placed at room temperature (RT) for 3 min. Finally, 1 ml of the above stock was diluted to 10 ml with diluent. The forced degradation studies are summarized in table 1.

3. Results

3.1. Method Development and Optimization

The analytical method development activity was initiated by selecting a mobile phase with varied combinations of acid and organic phases. Trials were conducted on 0.1% orthophosphoric acid (OPA) with acetonitrile in varied concentration ratios. It was observed that in most of the trials with 0.1% orthophosphoric acid with acetonitrile, the baseline was not stable and had high noise. Finally, trials were conducted on 0.1% formic acid with acetonitrile in varied concentration ratios, and the peak response was found to be better than the OPA and acetonitrile combination. Various C₁₈ columns were also scanned for better separation. The final optimized LC-MS method was developed on an Inertsil ODS column with dimensions of 150 mm×4.6 mm, 3.5 µm, and a mobile phase composition of formic acid (0.1%) and acetonitrile in the proportion of 40:60 injected in isocratic mode. The mobile phase flow rate was 1.0 ml/min, and the auto-injector volume was 10 µl. The column oven temperature was maintained at 25°C (ambient or room temperature). The wavelength maxima of tegafur, gimeracil, and oteracil were 224, 219, and 212 nm, respectively. The mobile phase was used as the diluent. The isosbestic point was found to be 220 nm (refer to figure 2), and this wavelength was selected for estimating the combined response of tegafur, gimeracil, and oteracil. In the optimized chromatographic conditions, tegafur, gimeracil, and oteracil peaks elute at 2.3, 3.7, and 5.3 min, respectively. Therefore, each injection was run for only 7 min. The resolution between tegafur and gimeracil and the resolution between gimeracil and oteracil were greater than 2.0. The USP plate count (N) and the resolution for tegafur, gimeracil, and oteracil were more than 5000 each, and the peak asymmetry of tegafur, gimeracil, and oteracil ranged from 0.9 to 1.1, which was optimum. The optimized chromatogram is shown in figure 3.

3.2. Method Validation

ICH Q2 standards were used to validate the optimized analytical technique. The estimated approach was validated well using ICH principles, and the results were within acceptable limits. Therefore, this validated analytical technique can be used for the estimation of tegafur, gimeracil, and oteracil.

3.2.1. System Suitability Test

Analytical techniques include workability testing of the system as the primary step of any analysis. To find the appropriate settings, the system suitability indicators were examined and used. For this objective, the retention duration, the number of theoretical plates, and the tailing factor were investigated. The theoretical plate number count exceeded 2000, which was judged adequate. According to the standards, the tailing factor was within the specified limitations. These results demonstrate that the suggested approach may deliver findings of satisfactory quality. All of the system's proper parameters were agreed upon and found to be within the parameters. The findings are summarized in table 2.

3.2.2. Linearity

Linearity parameters displayed a direct proportionate connection between the concentration and test outcome. Tegafur, gimeracil, and oteracil linearity were examined in the ranges of 50-300 µg/ml, 14.5-87 µg/ml, and 39.5-237 µg/ml, respectively. The regression coefficient (R²) values were calculated using the calibration curve. The calibration curve was made by calculating the acquired peak area and concentrations. Tegafur, gimeracil, and oteracil calibration curve R² values were 0.99956, 0.99986, and 0.99979, respectively, which were within the acceptable limits (NLT 0.99). As a result, the data demonstrated a high association between the peak area and analyte concentration. The outcomes are depicted in figure 4 and table 3.

3.2.3. Accuracy

The average recovery of tegafur, gimeracil, and oteracil from varied amounts of spiked sample solutions was 99.9%, 99.9%, and 99.4%, respectively. The percentage of recovery was estimated to be between 98 and 102%. This means that the suggested method was very accurate, and the findings were within the permissible limits of the ICH recommendations. The results are tabulated in tables 4 to 6.

3.2.4. Precision and Intermediate Precision

For tegafur, gimeracil, and oteracil method precision results, the percent relative standard deviation values were 0.52%, 0.26%, and 0.72%, respectively. Tegafur, gimeracil, and oteracil intermediate precision findings were 0.93%, 0.36%, and 1.01%, respectively. For tegafur, gimeracil, and oteracil method precision results, the total percent relative standard deviation values were 0.75%, 0.30%, and 0.84%, respectively.  The results were substantially below the commonly accepted 2% limit. Consequently, the precision of the new procedure has been proven. Tables 7 and 8 display the results.

3.2.5. Limit of Detection and Limit of Quantitation

The LOD results of tegafur, gimeracil, and oteracil were estimated at 0.6, 0.174, and 0.474 µg/ml, respectively. Tegafur, gimeracil, and oteracil exhibited LOQs of 2.0, 0.58, and 1.58 mg/ml, respectively, suggesting that the procedure was sensitive. The LOD and LOQ values for tegafur, gimeracil, and oteracil are shown in table 9.

3.2.6. Robustness

To examine the robustness of the experimental process, the influence of minor adjustments in chromatographic parameters was considered. In all of the purposely modified chromatographic conditions, the RSD percentage of tegafur, gimeracil, and oteracil was less than 2.0. The system suitability requirements were not changed significantly and were all passed. The results are shown in table 10.

3.2.7. Specificity

The technique's specificity to one analyte is assessed in each analysis by looking for interference peaks in blank matrix samples. The approach's specificity was assessed with regard to interference resulting from the existence of extra placebo peaks. In LC-MS, the blank and placebo chromatograms exhibited essentially no intrusive peaks throughout the retention time ranges. As a consequence, the LC-MS technique presented in this article was particular and discriminating. The chromatograms of the placebo and blank solutions are shown in figures 5 and 6, respectively.

3.3. Forced Degradation Studies

According to the ICH guidelines, many forms of stressful circumstances have been investigated. During the investigation, a few degradation products (DP) were discovered. System suitability parameters, such as plate count, tailing factor, percentage relative standard deviation, and percentage deteriorated, were all within the parameters for forced degradation investigations. This shows that the method was accurate and stability-demonstrating. The findings are summarized in table 11, and chromatograms are shown in figures 7-13.  No significant degradation was found in gimeracil.

3.3.1. Forced Degradation Studies of Tegafur

DP1: DP1 disintegration process is presented in figure 14a. For the strongest [M+H] ion, the m/z was -236.03. This peak was observed after degeneration by acid stress, as indicated by the ESI spectrum. Several degradative product (consequential) ions were indicated at m/z of -165.99 (loss of C₈H₈O) and -117.02 (loss of NH₃Cl) by the MS spectra of DP1.

DP2: Under alkali degeneration conditions, the ESI fields confirmed the greatest [M+H] ion of m/z -186.08. The fragmentation process of DP2 is illustrated in figure 15a. Numerous product (consequential) ions were identified at m/z of -113.03 (expulsion of C₄H₈O) and -61.03 (expulsion of C₂H₅NO) in the DP2 MS spectra.

DP3: The process of disintegration of DP3 is depicted in figure 16a. The most powerful [M+H] ion of m/z -216.05

was detected under peroxide deterioration conditions indicated by the ESI field. Many product ions were indicated at m/z of -146.01 (removal of C₄H₆O) and -61.01 (removal of C₃H₄FNO) in the DP3 MS spectra.

DP4: The ion with an m/z of -204.09 was the strongest [M+H] ion that was observed in the ESI spectrum under thermal degeneration conditions. Figure 17 illustrates the mechanism for DP4 fragmentation. The DP4 MS spectra at m/z of -134.04 (removal of C₄H₈O) and -61.03 (removal of C₂H₇NO₂) indicated many product ions.

3.3.2. Forced Degradation Studies of Oteracil

DP5: Figure 14.5 depicts the DP5 breakup mechanism. The strongest ion of [M+H] with m/z -230.94 was observed after acid degeneration, as indicated by the ESI spectrum. The MS spectra of DP5 indicated multiple product (consequential) ions at m/z -148.99 (CHKO₂ removal) and m/z-100.02 (NH₃Cl removal).

DP6: A prominent [M+H] ion was discovered when subjected to alkali degeneration studies. The ESI range was m/z -180.98 for the prominent alkali degradant, and the process of DP6 fragmentation is depicted in figure 15b. Several product ions were found in the DP6 aMS ranges at m/z -99.04 (elimination of CHKO₂) and m/z -44.03 (elimination of C₂H₅NO).

DP7: The most prominent [M+H] ion found in peroxide

deterioration was at m/z -210.96 in the ESI spectra. Figure 16b depicts the DP7 fragmentation process. The existence of multiple product (consequential) ions at m/z -129.01 (elimination of CHKO₂) and m/z -72.03 (elimination of CH₃NO₂) was indicated by the MS spectra of DP7.

4. Discussion

An accurate and sensitive LC-MS technique was developed and validated for the concurrent quantification of tegafur, gimeracil, and oteracil potassium in the drug material and the medicinal dosage form. The proposed method was extremely simple, exact, and robust. The present method is superior to the existing method as it is a stability-indicating chromatographic method where characterization of the degradants was performed. During the stability investigations, just a handful of degradation products were found. The system suitability parameters, such as plate count, tailing factor, percentage relative standard deviation, and percentage deteriorated, were all within the parameters for forced degradation investigations. This illustrates how exact and reliable the technique was. Furthermore, no gimeracil degradation products were found. As a result, this technique can be utilized in the quality control departments to detect tegafur, gimeracil, and oteracil potassium.