A sensitive analytical liquid chromatography-tandem mass spectrometry method for the estimation of Topiramate in bulk and pharmaceutical formulation

Chemicals

The working standard Topiramate was obtained from the Indian Pharmacopoeia Commission, New Delhi. Chemicals (ammonium acetate) and solvents (acetonitrile and formic acid) were of LC-MS grade procured from SD Fine Chemicals. Ultrapure water was obtained from Milli-Q RO system.

Instrument and chromatographic conditions

Shimadzu LC-MS/MS 8,030 system was equipped with electrospray ionization interface, SIL-20AC autosampler, CBM-20 alite controller, CTO-20AC column oven, LC-20AD pump, and SPD-M20 PDA detector and operated using Lab Solutions data station.

A stationary phase consisting of Zorbax RP-C₁₈ column (50 mm × 4.6 mm i.d., 5 μ) was selected, and ammonium acetate and acetonitrile were used as a mobile phase at the ratio of 15:85 v/v and pumped at a flow rate of 0.5 ml/min. 10 μl of injection volume was employed at an ambient column temperature.

Preparation of stock and working solution

About 10 mg of Topiramate was weighed and dissolved into 10 ml of ethanol (1 mg/ml). From the above stock solution, 1 μg/ml of working concentration was prepared. Further, from the working standard, calibration standard ranging from 1 to 1,000 ng/ml of Topiramate was prepared. Three different quality control (QC) levels were prepared, i.e., low (LQC; 3 ng/ml), medium (MQC; 100 ng/ml), and high (HQC; 900 ng/ml) in the mobile phase.

Optimization of mass range

The optimization of mass conditions was performed using standard Topiramate (1,000 ng/ml). The transition was obtained at m/z 338.05 (precursor ion) → 77.95 (product ion) and m/z 338.05 → 95.90 which were used to monitor Topiramate (Fig. 1).

Method validation

As per the ICH guidelines, analytical method was validated using various validation parameters such as specificity, linearity range, accuracy and precision, Detection limit (LOD) and quantification limit (LOQ), robustness, and system suitability (ICH, 1996).

Linearity

Six different concentrations of Topiramate ranging from 1 to 1,000 ng/ml were used to determine the linearity. The linearity curve was used to measure the correlation coefficient (R²), slope (m), and intercept values.

Accuracy and precision

As per the ICH guidelines, the accuracy and precision of the method were determined at three QC levels by analyzing six replications. Recovery of the method was used to determine the accuracy of the method. Intra- and interday precision was carried out to determine the method precision, and the results were recorded in terms of percent relative standard deviation (% RSD).

Figure 1. (a) Mass scan spectra and (b) product ion scan spectra for Topiramate in the negative mode. [Click here to view]

Detection limit and quantification limit

LOD and LOQ were calculated by signal-to-noise ratio. Signal-to-noise ratio of 3:1 was used to determine the LOD, and signal-to-noise ratio of 10:1 was used to determine the LOQ.

Robustness

For the determination of robustness, chromatographic parameters such as flow rate, column temperature, wavelength detection, injection volume, mobile phase, and pH were studied.

Specificity

At the retention time of Topiramate, there were no potential interference peaks found (Fig. 2a). Therefore, the method was found to be selective and highly sensitive.

Accuracy and precision

The method accuracy was determined by recovery studies for three QC samples. The recovery ranged between 93.3% and 99.7% with % RSD ranging from 0.10% to 1.85% for precision study (Table 1).

Linearity

The linearity curve was determined at six different concentration levels, and the regression equation was found to be 0.999 for the concentration ranging from 1 to 1,000 ng/ml. The standard deviations (SDs) of calibration curve results were found to be within the limits, with the regression equation of y = 1,055.x + 1,287 (Fig. 3).

Figure 2. Multiple reaction monitoring (MRM) chromatogram of Topiramate (a) standard and (b) sample formulation. [Click here to view]

Table 1. Accuracy and precision studies of Topiramate. [Click here to view]

Figure 3. Six-point calibration curve of Topiramate (1–1,000 ng/ml). [Click here to view]

LOD and LOQ

The LOD and LOQ were obtained at 0.5 and 1.0 ng/ml for the developed method, respectively, based on the minimum level of peak area and signal-to-noise ratio.

Robustness

The conditions such as flow rate (0.2, 0.5, and 0.8 ml/min), pH (5.5, 6.0, and 6.5), and acetonitrile percentage (75%, 85%, and 90%) were studied to determine the robustness of the method. The % RSD was calculated for flow rate (1.10%, 0.82%, and 0.25%), pH (1.20%, 0.91%, and 0.33%), and acetonitrile percentage (1.30%, 0.79%, and 0.22%) which were found to be within the limit and demonstrated that the developed method was found to be robust (Table 2).

Table 2. Robustness studies. [Click here to view]

System suitability

The system suitability study was carried out to determine parameters such as tailing factor (T), number of theoretical plates (N), and retention factor (R_t), as per the ICH guidelines. The results were found to be within the limits (Table 3).

Estimation of Topiramate dosage form

Twenty tablets of Topiramate tablet were accurately weighed and crushed into powder, and the weight equivalent to 10 mg was taken. The powder was dissolved using 5 ml of organic solvent by sonication for 15–20 minutes and filtered through 0.45-μm membrane, and the volume was made up to 10 ml using organic solvent (ethanol). From the resulting solution (1 mg/ml), the QC samples (3, 100, and 900 ng/ml) were prepared by appropriate dilution and analyzed (Table 4 and Fig. 2b).

Table 3. System suitability. [Click here to view]

Table 4. Recovery studies for the Topiramate formulation. [Click here to view]