Develop And Validate RP-HPLC Method for The Determination of Process Related Impurity in Tolbutamide Drug

(a)                                                                           (b)

Figure 1. Structures of (a) Tolbutamide and (b) p-Toluenesulfonamide

METHOD

UV spectroscopic method

Preparation of Diluent.

Mix thoroughly and remove gas after measuring and transferring a mixture of 10% v/v water and 90% v/v methanol. To dissolve active pharmaceutical ingredient.

Selection of wavelength

Tolbutamide (10PPM): After precisely measuring 20 milligrams of tolbutamide, the standard was dissolved by adding 70 milliliters of diluent to a 100 milliliter volumetric flask (VF). The mixture was then sonicated until it reached the desired concentration of 200 parts per million (PPM).  Using a diluent, 5 mL was further diluted to 100 mL.

P-Toluenesulfonamide (4 PPM): To prepare the standard, we precisely measured 2 milligrams of p-toluenesulfonamide and transferred it to a 50 milliliter VF. We then added 30 milliliters of diluent and sonicated it to dissolve it fully, and diluted it up to mark by 40 parts per million of diluent.  Diluted 5 mL with diluent till it reaches 50 mL.

Determination of λ_max

A range of 400 nm to 200 nm was used to scan the standard solutions independently. Display a strong absorption from the spectrum [6].

IR spectroscopy:

Each of the two components, tolbutamide (20 mg API) and potassium bromide (Kbr), was thoroughly combined and triturated using a mortar and pestle. Finally, the combination was placed on a plate and analyzed using the Diffused Attachment reflectance infrared spectrophotometer.

RP-HPLC method development and optimization

Mobile phase

Get a 75:25 v/v combination of methanol and 0.1% orthophosphoric acid (OPA) ready. Combined thoroughly.

Standard stock solution for Chromatographic development:

Transfer approximately 50 milligrams of the Tolbutamide standard into a 100 milliliter VF after carefully weighing it. To dissolve, sonicate approximately 70 mL of diluent, and then add enough diluent to reach the mark. Blended thoroughly. Fill the 25 mL VF with diluent until the mark is reached after further diluting 5 mL of Tolbutamide stock solution. (100 PPM) [7].

Method development and optimization

Chromatographic conditions

Tolbutamide and p-Toluenesulfonamide were separated by chromatography with the following parameters: a Phenomenex column (5 µm, 250 mm × 4.6 mm) running at 1.2 mL/min with an injection volume of 20 µL, detection at 230 nm, a column oven temperature of 40 °C, and a total run time of 7 minutes. Tolbutamide eluted at around 3.29 minutes, while p-Toluenesulfonamide eluted at 4.62 minutes. The seal wash and needle wash were composed of water and acetonitrile in a 90:10 v/v and 10:90 v/v, correspondingly.

Preparation of Tolbutamide Standard stock solution: Transfer around 50 milligrams of the Tolbutamide standard, measured precisely, into a 100 milliliter VF. Shake to dissolve, then add 70 mL of diluent and fill to mark by diluent. Completely combined. To further dilute Tolbutamide stock solution, add 1 milliliter of diluent to a 10-milliliter VF and fill to mark. (50 ug Tolbutamide).

Preparation of Sample solution:

Weigh 10 tablets were and then crumpled to powder using a mortar and pestle. Combine the ingredients thoroughly. Measured 651 milligrams of crush powder, which is comparable to 417 milligrams of Tolbutamide. Filling a VF with 500 milliliters of liquid. Add approximately 350 mL of diluent, sonicate for roughly 30 minutes along by intermittent shaking to dissolve complete. Allow it to cool and make up to volume with diluent. Filter through 0.45µ Nylon membrane syringe filter discard 3 ml of Filtrate. For a more accurate volumetric measurement, add 3 milliliters of sample stock solution to 50 milliliters of diluent and mix thoroughly [8].

Evaluation of System Suitability: Tolbutamide peak areas should not deviate from the mean by more than 2.0% when measured with five independent injections. Tolbutamide peak USP theoretical plate factors should be more than or equal to 2000[9].

% Assay of Tolbutamide as:

%Assay=AT×WS×5×500×50×P×100AS×100×50×Sample wt.×3×100×LC×Avg. Wt.

Where

AS: Average peak area of Tolbutamide in chromatogram of Standard solution.

LC: Label claim of Tolbutamide in mg

AT: Peak area of Tolbutamide in chromatogram of sample solution.

Spl. Wt.: Weight of tablet taken in mg

WS: Weight of Tolbutamide working standard taken in mg

P: % Potency of Tolbutamide working standard on as is basis.

Avg. Wt.: Average weight of Tolbutamide tablet.

Optimized Chromatographic Conditions

Chromatographic condition mentioned above was an optimized chromatographic condition and subjected for validation kept constant throughout the method.

Method validation

System suitability: To ensure the analytical system is functioning correctly and producing accurate and exact findings, a system suitability test was conducted. Chromatograms were recorded after six injections of the standard solution.

Specificity: The capacity to determine with absolute certainty the presence or absence of an analyte in the presence of components that are likely to be present is known as specificity. Typical examples of such substances are matrix, degradants, etc. For an assay to be considered specific, it must be possible to prove that the inclusion of excipients had no effect on the technique.

Interference from blank, samples and Impurity

Preparation of Sample solution:

Weigh 10 tablets were and then crumpled to powder using a mortar and pestle.  Efficiently combined the ingredients. Calculated that 417 milligrams of tolbutamide (651 milligrams of crush powder) was the weight of the crushed material. Fill a VF with 500 mL of the mixture. Shake occasionally while sonicating for 30 minutes to dissolve completely, then add 350 mL of diluent. Wait for it to cool before adding diluent to get it up to volume. The filtrate should be 3 milliliters after passing through a 0.45µ nylon membrane syringe filter. Step 3: Fill a 50 mL VF with diluent until the mark is reached. Add 3 mL of the sample stock solution. Combine by stirring well.

Preparation of p-Toluenesulfonamide

Fill a 100 mL VF by measured 2.5 mg of in p-Toluenesulfonamide standard. After adding approximately 70 mL of diluent, sonicate until dissolved. Then, add enough diluent to reach volume, mix well. To further dilute the impurity stock solution, add 1 milliliter to 50 milliliters of VF and stir thoroughly. (Concentration of Imp: 0.5 PPM)

Preparation of spike solution

Combine 3 ml of the sample stock solution with 1 ml of the impurity stock solution. Add diluent to bring volume up to specified level (50 ml), and stir thoroughly. (Concentration of sample solution: 50 PPM, Concentration of Impurity: 0.5 PPM) [10].

Linearity

In order to prepare standard solutions with varying concentrations of analytes, certain amounts of an intermediate stock solution were diluted with the diluent. The regression coefficient 'r²', y-intercept, and slope were determined by plotting concentration (ppm) against area. Within the 80–100% of the working concentration level range, determine linearity.

Linearity stock solution:

Transfer around 50 milligrams of the Tolbutamide standard, measured precisely, into a 100 milliliter VF. Make up to the mark with diluent, then add approximately 70 mL of dissolved sonicated diluent. Mixed properly (Table 1) [11].

Table 1. Preparation of linearity sample

Accuracy (Recovery)

Evaluate accuracy at 3 levels 80, 100 and 120% of working concentration level for Tolbutamide. As working concentration of Tolbutamide. Make three copies of each level. According to the information in table 1, transfer the appropriate amounts of placebo and Tolbutamide API/standard into a 500 mL VF. To dissolve completely, add 350 mL of diluent and sonicate for 30 minutes while shaky intermittently. After it has cooled, add enough diluent to bring it to volume. Pour 3 milliliters of filtrate by a 0.45µ nylon membrane syringe filter and discard liquid. In a separate VF, combine 3 milliliters of sample stock solution with 50 milliliters of diluent, then fill to the mark. Mix thoroughly. Tolbutamide standard or API should be prepared to the following recovery levels according to tables 2 [12].

Table 2. Preparation of accuracy sample

Calculate % recovery at every level as

% Recovery=Recovered AmountAmount Initially Present×100

Precision:

Method Precision (Repeatability)

The consistency of test results across several replicates of a homogenous sample was defined as the precision of the analytical method. It was reported as the coefficient of variation (CV) and gave an indication of results that were influenced by random error. For purpose of determining if approach consistently produced same findings for each batch, this was carried out. Six repetitions of injecting the same amount of standard and sample solutions into the assay were used to ensure method precision. Through comparing sample solution's response to that of reference solution, % assay of sample to label claim was computed. % relative standard deviation of the test findings was determined.

Intermediate Precision (Ruggedness)

On separate days, we compared standard solution to sample solution in order to gauge the intermediate precision. % of assay and RSD were determined. Six separate samples were prepared and put into the HPLC system on separate days by separate analysts.

Robustness:

Testing an analytical technique's robustness ensures that it maintains its accuracy even when subjected to intentionally slight changes to its parameters and gives a sense of how reliable the method is when used regularly. Chromatograms were acquired after six injections of the standard solution into each of the tested flow, column temperature, and wavelength combinations. The study of this parameter involved making minor, intentional adjustments to the chromatographic settings and assay parameters. Then, the system appropriateness and the findings obtained from injecting the standard and sample solutions were examined according to these changes. 

Change in Flow rate (±0.1mL/min)

Original flow rate 1.2 mL/min. When flow rate was greater than before, it became 1.3 mL/min, and when flow rate was decreased, it was reduced to 1.1 mL/min.

Change in wavelength (±2nm)

Original wavelength was set at 230 nm. When wavelength was increased by 2 nm, it changed to 232 nm, while a decrease of 2 nm resulted in a wavelength of 228 nm.

RESULTS AND DISCUSSION

For the purpose of estimating tolbutamide using QbD technique, a direct, accurate, and profitable RP-HPLC technique was formed and verified. Linearity, precision, specificity, accuracy, and robustness were between validations criteria used to ensure the approach complied with ICH standards.

Wavelength selection

Figure. 2. A) UV Spectra of Blank, B) Tolbutamide, C) p-Toluenesulfonamide, D) Overlay of Tolbutamide and p-Toluenesulfonamide

The UV–visible spectral analysis of Tolbutamide and p-Toluenesulfonamide was carried out to determine their respective absorption maxima (λmax). Tolbutamide exhibited prominent absorption peaks at 228 nm and 203 nm, whereas p-Toluenesulfonamide showed absorption maxima at 263, 225 and 203 nm. Overlaying the spectra of both compounds revealed a common intersection point (Q-point) at 230 nm, where both drugs demonstrated appreciable absorbance. Therefore, 230 nm was certain as the optimum wavelength for further chromatographic technique development, as it allows simultaneous and effective detection of both analytes.

 Identification by IR Spectroscopy:

Figure 3. FTIR spectrum of A) p-Toluenesulfonamide and B) Tolbutamide Standard Solution

The FT-IR spectra of tolbutamide and p-toluenesulfonamide exhibit distinct and diagnostic absorption bands that enable clear differentiation between the active pharmaceutical ingredient and the related impurity. The spectrum of tolbutamide shows a characteristic broad band at 3300–3200 cm⁻¹, corresponding to N–H stretching of the sulfonylurea group, along with prominent aliphatic C–H stretching bands in the region of 2960–2870 cm⁻¹, which are absent in the impurity spectrum. A strong absorption band observed at 1715–1690 cm⁻¹ is attributed to the urea carbonyl (C=O) stretching vibration, as a main diagnostic marker for tolbutamide and clearly distinguishing it from p-toluenesulfonamide, which lacks this functional group. In contrast, p-toluenesulfonamide exhibits a broader N–H stretching band in the region of 3400–3300 cm⁻¹, characteristic of the sulfonamide moiety, along with a distinct aromatic C=C stretching band around 1600–1580 cm⁻¹. Although both compounds display intense sulfonyl (S=O) stretching vibrations in the regions of 1335–1310 cm⁻¹ (asymmetric) and 1165–1140 cm⁻¹ (symmetric), the absence of the carbonyl band and the presence of para-substituted aromatic C–H out-of-plane bending near 820–780 cm⁻¹ in p-toluenesulfonamide provide clear spectral evidence for impurity identification. These distinctive IR features confirm the suitability of FT-IR spectroscopy as a rapid and reliable tool for qualitative impurity profiling and structural discrimination of tolbutamide and its sulfonamide impurity.

RP-HPLC Method Development different trials

Table 3. Chromatographic Settings

Figure 4. Typical chromatogram for (1-6) Tolbutamide Trial, (7) Tolbutamide and p-Toluenesulfonamide

The chromatographic technique for estimation of Tolbutamide was optimized through a series of systematic trials. In Trial 1, Tolbutamide eluted at 3.42 minutes; however, the peak was unacceptable due to pronounced tailing, indicating the need for method optimization. In Trial 2, the drug eluted at 3.2 minutes with a broad and poorly defined peak, which again failed to meet system suitability requirements. In Trial 3, Tolbutamide eluted at 3.0 minutes, but peak fronting was observed, suggesting inadequate chromatographic conditions and necessitating further optimization. In Trial 4, Tolbutamide eluted at 4.7 minutes with a sharp and symmetrical peak that satisfied all system suitability parameters; however, to achieve a more cost-effective and time-efficient method, a reduction in retention time was desired. In Trial 5, Tolbutamide again eluted at 4.7 minutes with a symmetrical peak and successfully met all system suitability criteria, making these conditions suitable for further analytical evaluation. In Trial 6, Tolbutamide eluted at 5.7 minutes with a well-defined and symmetrical peak that also complied with system suitability requirements and was considered acceptable for further analysis. For the simultaneous estimation of Tolbutamide and p-Toluenesulfonamide, Tolbutamide eluted at 3.9 minutes while p-Toluenesulfonamide eluted at 5.6 minutes, both exhibiting sharp, symmetrical, and well-resolved peaks, confirming specificity and suitability of developed technique for combined examination.

Method validation

System suitability: As part of the pharmacopoeial process, a system suitability test is conducted to confirm that chromatographic system's resolution also reproducibility meet necessary standards for analysis.

Table 4. System suitability parameter of Tolbutamide

Statistics was collected by a chromatograph from 5 replicate injections of a standard solution and one injection of a blank (diluent). Table 4 summarizes the data that has been obtained. Based on the facts, it may be concluded that the system meets the acceptance criteria for appropriateness.

Specificity:

Data found is concise in Table 5.

Table 5. Specificity statistics

Figure 5. Chromatogram of Blank, Standard solution, Test sample, Sample spiked with impurity at 1% level, Impurity 100PPm and Placebo

Linearity

Working concentrations of Tolbutamide ranging from 80% to 120% were used to assess linearity. Tolbutamide in solution has a working concentration of 50 µg/mL.  This information is condensed within Table 6.

Table 6. Tolbutamide Linearity

Figure 6. Linearity of Tolbutamide

Accuracy (Recovery):

Assessed accuracy as of 50-150% of Tolbutamide tablet, working concentration level. Every level prepared in 3 times.

Table 7. % Recovery for Tolbutamide

According to the numbers, the average recovery rate for 80% to 120% falls between 98.0% and 102.0 percent, while the recovery rate for individuals falls between 95.0 and 105.0 percent.  Precision:

Method and Intermediate Precision:

To check the accuracy of the procedure, we injected the system with one blank (diluent) injection, five standard solution replication injections, and six sample solution injections. Six separate sample preparations, performed by separate analyzers on separate days, were injected into the HPLC apparatus to evaluate intermediate precision.

Table 8. Method precision

Furthermore, the system suitability criteria were fulfilled under intermediate precision conditions, and the %RSD of the % assay for six sample preparations did not exceed 2.0. The % assay values were in specified acceptance criteria, thereby confirming ruggedness of the method.

Robustness

Table 9. Tolbutamide Robustness