The acetate buffer solution was prepared by weighing 1.64 g of anhydrous sodium acetate CH₃COONa and dissolving it in 1L HPLC grade water, then adjusting its pH to a value of 4.25 using acetic acid.

The mobile phase was filtered by passing it through 0.45 μm pore size membrane filter (Agilent technologies) at a pumping speed of 30 L/min (Glassco diaphragm Vacuum pump) and degassed for 30 min using SONOMATIC^R LANGFORD ultrasonic sonicator.

A 0.1 N sodium hydroxide solution was prepared by weighing 4 g of NaOH and dissolving it in 1 L of HPLC grade water. To the above prepared solution an equal amount of methanol was added.

The solution was prepared by accurately transferring 2 mg of LT4 standard powder into a 10-mL volumetric flask. The powder was dissolved and diluted to volume with 50% methanolic NaOH.

The chromatographic analysis was performed at ambient temperature with isocratic elution using HiQ sil NH₂ column at a flow rate of 1.2 mL/min and at a wavelength of 252 nm.

The mobile phase used for the chromatographic separation was prepared by adding HPLC grade acetonitrile to the acetate buffer solution of pH 4.25 in the ratio of (55:45, %V/V), respectively.

Aliquots of LT4 standard solution were transferred into a series of 10 mL volumetric flasks and completed to volume with 50% methanolic NaOH to give the final concentration ranges stated in Table 2.

The above solutions were filtered using 0.2 µm disposable filters and 20 µL portion of these solutions were injected in triplicates and chromatographed under the conditions mentioned above. The standard calibration graph was prepared by plotting the peak area values against the corresponding concentrations. Later, the concentration of LT4 was computed from this calibration graph.

Ten tablets of Euthyrox (100 µg) and Eltroxin (100 µg) were accurately weighed and finely powdered. Powder amount equivalent to 250 µg was accurately weighed and transferred to a 25-mL volumetric flask, dissolved in around 10 mL 50% methanolic NaOH, shaken for 15 minutes, filtered using Whatmann No. 41 filter paper and completed to volume with the same solvent.

The above solutions were filtered using 0.2 µm disposable filters and 20 µL portion were injected in five replicates and chromatographed under the chromatographic conditions mentioned above. The peak area values were measured and the corresponding concentrations in the tablets were derived by referring to the calibration graph.

Various physiochemical properties like pKa value, log P, solubility and absorption maximum (λ_max) of the drug must be known, as such parameters are important for HPLC method development. Log P and solubility helps in the selection of the mobile phase and sample solvent while pKa value helps in the determination of the pH of the mobile phase (Bakshi et al. 2002).

The HPLC method was developed to provide simple rapid and reliable quality control analysis of LT4. So, the most important aspect is to achieve elution with acceptable retention time, peak symmetry, and high sensitivity.

To attain this goal, the preliminary investigations were directed towards studying the effect of different variables on the system suitability in order to maximize the sensitivity of the method. The parameters assessed and optimized included column type, detection wavelength, type and quantity of organic modifier, aqueous phase concentration and pH, mobile phase additives and flow rate.

The absorption spectrum of the standard solution LT4 in 50% methanolic NaOH was performed over the wavelength range of 200–400 nm as shown in Fig. 2. Different wavelength values were tested; 252 nm was found to be the most effective λ for LT4 detection as it provided better detector responses together with the best sensitivity. Hence, 252 nm was set as the UV-detector wavelength for the determination of LT4.

Figure 2. 

Absorption spectrum of 30 μg.mL^-1 of standard LT4 in methanolic NaOH.

Trials were performed on three different column types: two reversed-phase (C₈ and C₁₈) and the third one was an ion exchange column NH2. Upon using the RP columns, several problems were faced of which peak splitting and tailing, in addition to baseline noise. However, upon the use of anion exchange column (HiQ sil NH2), all these problems were resolved.

Mobile phase composition has a major effect on peak spacing, shape and retention time.

Methanol and acetonitrile have been tried as organic modifiers. Acetonitrile, due to its high eluting power, has been chosen, introduced into the mobile phase and allowed the elution of LT4 at reasonable retention time.

As LT4 contains acidic and basic functional groups, the retention time can vary with both, ionic strength and pH of the aqueous phase. Thus, the use of buffer- acetonitrile as mobile phase allows better elution of LT4. Phosphate buffer and acetate buffer were tried. The use of acetate buffer having the concentration of 0.02 M provided better ionization of LT4 and hence resulted in better chromatograms as an outcome.

The pH value of the acetate buffer was adjusted to 4.25 using acetic acid. At this pH value LT4 carboxylic group was completely ionized which provided optimum separation with the most symmetric, well-defined peaks, eluted after 3 min (Fig. 3).

Figure 3. 

HPLC chromatogram of a 20µL injection of 20 µg.mL^-1 of standard LT4 using the optimized chromatographic conditions.

The ratio of constituents of the mobile phase was also studied with respect to change in the ratio of acetonitrile: acetate buffer. Different ratios were tried including: 40:60, 45:55, 50:50, 55:45, 60:40, The optimal mobile phase composition was found to be acetonitrile: acetate buffer in the ratio of (55:45, %v/v). This mobile phase composition resulted in well-defined peaks with good shape and symmetry.

Various flow rate values in the range of 0.5–1.5 mL/min were tried. The best separation was achieved by adjusting the flow rate at 1.2 mL/min in an isocratic mode. Injection volume and run time were 20 μL and 10 minutes, respectively.

Under the optimized experimental conditions, the developed method was validated for system suitability, along with linearity, limits of detection (LOD) and quantification (LOQ), specificity, accuracy, precision (repeatability and intermediate precision) and robustness according to the procedures described in ICH guidelines (ICH 2005).

According to FDA guidance 1994, system suitability tests are an integral part of any liquid chromatographic method (FDA). Under the optimized chromatographic conditions, system suitability parameters including capacity factor (k’), column efficiency expressed by the number of theoretical plates (N), tailing factor (T), and repeatability (RSD of peak response and retention time) were performed and listed in Table 1. The calculated parameters were satisfactory and indicative of good efficiency of the method for the determination of LT4.

Under the optimized experimental conditions, the standard calibration curve was constructed. The linear regression equation was obtained by the method of least squares by plotting the peak area against the concentrations of LT4. Linearity of the method was studied by injecting five concentrations prepared in methanolic NaOH keeping the injection volume constant.

Linearity parameters including correlation coefficient, intercept, slope, and the standard deviation of the residuals for the calibration data and concentration range are summarized in Table 2.

The correlation coefficient (r) obtained was higher than 0.999 with high values of F (low significant F) which confirmed the linearity of the calibration curves. An important statistical parameter for indicating the random error in the estimated values of y is the standard deviation of the residuals S_y/x. Also, the importance of S_y/x originates from being used to calculate S_a and S_b, the standard deviation of the intercept (a) and the slope (b). These values showed the good linearity of the calibration graphs and the compliance to Beer’s law. The variance test for the regression lines revealed that, for equal degrees of freedom, the increase in the variance ratio (F-values) means an increase in the mean squares due to regression and a decrease in the mean squares due to residuals, (i.e., the less is the scatter of experimental points around the regression line). Consequently, regression lines with high F-values (low significance F) are much better than those with lower ones. In conclusion, good regression lines show high values for both r and F statistical parameters (Mileer 2005).

The LOD and LOQ were derived by calculating the signal-to-noise ratio. LOD and LOQ represent the concentration of the analyte that would yield signal-to-noise ratios of 3 for LOD and 10 for LOQ. To determine the LOD and LOQ, serial dilutions of LT4 were prepared from the standard stock solution. The samples were injected in LC system and the measured signal from the samples were compared with those of blank samples. LOD and LOQ were calculated according to the ICH guidelines (ICH 2005). The sensitivity of the proposed method can be confirmed by the low LOD and LOQ values obtained in Table 2.

The accuracy of the proposed method was determined through a recovery study. Five different LT4 samples of known concentrations ranging from 10–100 μg/mL were prepared and chromatographed in triplicates under the optimized conditions. The amount of LT4 was estimated by measuring the peak area and fitting these values to the calibration curve equation. Good accuracy expressed as % recovery was obtained. The results, summarized in Table 3, show that the % recovery values do not exceed the accepted limits (98–102%), which demonstrate the accuracy of the developed method.

In order to evaluate the precision of the proposed method, repeatability (intra-day precision) and intermediate precision studies (inter-day precision) were performed.

Repeatability studies were performed by triplicate injections of standard LT4 solutions performed for three different concentrations within the calibration range (intra-day precision). The intermediate precision of the method was checked by repeating studies on three different days (inter-day analysis).

High precision, expressed as percentage RSD, were obtained proving the high precision of the method The results are summarized in Table 4.

The specificity of the method was determined by comparing test results obtained from the analysis of sample solutions containing excipients with that of the test results obtained from standard LT4 solutions. The peaks were well separated under the described chromatographic conditions where no interference from the excipients of the pharmaceutical tablets were shown.

Robustness of the method was determined by small deliberate changes in flow rate, mobile phase pH and mobile phase ratio. The content of the drug was not adversely affected by these changes as evident from the low value of %RSD indicating that the method was robust. This study demonstrated that slight intended variations in the previously mentioned parameters have no significant effect in the determination of LT4 (Table 5).

The applicability of the proposed method was tested in two different pharmaceutical formulations; Euthyrox tablet and Eltroxin tablet. LT4 peak was eluted at its specific retention time. The results for LT4 were comparable to the labelled amount expressed by high percentage recoveries with low % RSD which indicate high accuracy and precision in the determination of LT4. The absence of additional peaks indicates no interference of the excipients used in the tablets. The results obtained are summarized in Table 6. The proposed method, by the use of NH2 column, can be considered superior to the compendial method described in the European pharmacopeia. It was found to be more cost time effective due to a shorter run time of analysis in comparison to Ph. Eur. Method (CEEP 2019), where officially it is stated to be around 11 min. Based on literature review, statistical comparison wasn’t possible as the two pharmaceutically available tested drugs were not analyzed previously using the Eur. Ph. method, pointing to the innovative work. Further investigations should be targeted to investigate bioequivalence and interchangeability between the two LT4 brands.