2.1 Instruments and software

On an HPLC system (Agilent 1260, USA) outfitted with a UV–Vis detector, quaternary pump, vacuum degasser, column oven, and Chemstation software, chromatographic studies were carried out. Using 1.0 cm quartz cuvettes and UV-Probe software, spectrophotometric measurements were carried out on a dual beam path spectrophotometer (Shimadzu UV-1800, Japan). An Agilent Extend C₁₈ (250 × 4.6 mm, 5 μm) column was used for chromatographic separation. pH measurements were made with a pH meter (Mettler-Toledo, Switzerland) equipped with a glass electrode. Ultrapure water was produced with a water purification system (Millipore Milli-Q, USA).

2.2 Materials and reagents

All solvents were gradient purity for liquid chromatography. Lenalidomide (LDM) (≥98.0%), acetonitrile (≥99.9%), methanol (≥99.0%), ethanol (≥99%), and analytical grade potassium dihydrogen phosphate (≥99.0%) were purchased from Sigma-Aldrich Chemie GmbH (Istanbul, Turkey). LDM capsules (Revlimid, 10 mg) used in this study were purchased from a local pharmacy (Afyonkarahisar, Turkey). Each capsule contains 10 mg of lenalidomide. In addition, each capsule contains lactose anhydrous, microcrystalline cellulose, croscarmellose sodium, magnesium stearate, gelatin, shellac, propylene glycol, potassium hydroxide, titanium dioxide (E171), black iron oxide (E172) as excipients. Ultrapure water (0.075 μS cm⁻¹) was used for the preparation of all solutions and the mobile phase. Before analysis, the mobile phase was filtered through a 0.45 m membrane filter using a vacuum pump and sonicated.

2.3 Solutions

Stock standard solution (500 μg mL⁻¹): 25 mg of the reference LDM standard was accurately weighed, transferred to a 50 mL volumetric flask, topped up with 20 mL of ultrapure water, sonicated for 10 min to ensure dissolution, and the volume was topped up to 50 mL with ultrapure water.

Standard solutions (5, 10, 15, 20, 25, and 30 μg mL⁻¹): Different volumes of the stock solution were subjected to serial dilutions with ultra-pure water to obtain six standard solutions in the concentration range of 5–30 μg mL⁻¹.

Sample solution (20 μg mL⁻¹): The contents of 10 capsules (Revlimid, 10 mg) were emptied and weighed accurately, and the mass of an average capsule was recorded, crushed in a dry and clean mortar, ground to a fine powder, and mixed. The capsule powder equivalent to 50 mg of LDM was precisely weighed and transferred to a 100 mL volumetric flask. Approximately 40 mL of ultrapure water was added and shaken on a rotary shaker for 30 min to ensure complete dissolution. The volume was topped up with ultrapure water. The mixture was sonicated for 10 min and then filtered through a 0.45 mm membrane filter. This solution was called the stock sample solution. A sample solution was prepared at a concentration of 20 μg mL⁻¹ by diluting the stock sample solution with ultrapure water.

2.4 Determination of λ_max

To determine the λ_max value, standard solutions in the concentration range of (5–30 μg mL⁻¹, n = 6) were scanned against ultrapure water in the wavelength range of 200–800 nm in a spectrophotometer device.

2.5 Development of methods

The chromatographic conditions were optimized to obtain good peak parameters such as a good peak shape, the lowest tailing factor, a short retention time, and a high theoretical plate number. Initially, mobile phases consisting of various buffer systems were investigated, but the required system compatibility characteristics could not be achieved. Different types (X-Terra C₁₈ (250 × 4.60 mm × 5 μm), Extend C₁₈ (250 mm × 4.6 mm, 5 µm), Synergy Hydro C₁₈ (250 mm × 4.6 mm × 4 μm) and Luna C₁₈ (250 mm × 4.6 mm × 5 μm)) and different lengths of columns (X-Terra C₁₈ (150 × 4.60 mm × 5 μm), Extend C₁₈ (150 mm × 4.6 mm, 5 µm), Synergy Hydro C₁₈ (150 mm × 4.6 mm × 4 μm) and Luna C₁₈ (150 mm × 4.6 mm × 5 μm)) were tested but showed poor system compatibility parameters. Good peak parameters were obtained using an Extend C₁₈ (250 mm × 4.6 mm, 5 µm) column. Different ratios of water/methanol, water/acetonitrile, and water/ethanol mixtures were tested as mobile phases. Initially, acetonitrile and ultrapure water (20/80, V/V) were used as mobile phase, which resulted in a very long analysis time. To shorten the analysis time, the water component of the mobile phase was acidified with formic acid (pH:2.0). Under these conditions, the sample solution was injected to detect both impurities that may interfere with the analyte peak and the presence of drug matrix components that may remain longer on the column under the specified conditions. Furthermore, the sample solutions were injected sequentially into the system with an analysis time of 10 min and it was observed that no impurities were carried over from one analysis to the next. Therefore, the analysis time was set to 10 min. Furthermore, the column temperature was chosen as 30 °C due to its many advantages such as high column efficiency, low column pressure favorable peak shape, and cost-effectiveness. The spectral pattern of LDM was comprehensively investigated using different solvents (ultrapure water, ethanol, methanol, and isopropyl alcohol) for spectrophotometric analysis. Ultra-pure water was used as the solvent for spectrophotometric analysis since the best spectra of LDM were obtained with ultrapure water. Since the standard solutions of LDM have maximum absorbance at 304 nm wavelength, the absorbance values of the standard and sample solutions were measured at this wavelength.

2.6 Validation of analytical methods

Analytical methods developed for the quantification of LDM have been validated for “selectivity, system suitability, linearity, precision, sensitivity, and robustness” according to the ICH Q2 (R1) guidelines [35, 36].

Standard, sample, and mobile phase solutions were injected into the chromatographic system to evaluate the selectivity of the chromatographic method. The retention time (Rt) of LDM in commercial formulations and sample solution chromatograms was evaluated by comparing them with the chromatograms of the standard solution. To evaluate the selectivity of the spectrophotometric method, spectra of the standard, sample, and solvent (ultrapure water) were taken in the wavelength range of 200–400 nm in a spectrophotometer. The spectra obtained were compared and the presence of interfering bands was analyzed.

To evaluate the system suitability of the chromatographic method, LDM standard solution (25 μg mL⁻¹) was injected into the chromatographic system six times at short regular time intervals. Peak area, retention time, tailing factor, and the number of theoretical plates were recorded from the chromatograms. Relative standard deviation values were calculated for peak area and retention time. To evaluate the system suitability of the spectrophotometric method, absorbance values of LDM standard solution (20 μg mL⁻¹, n = 6) were measured. The relative standard deviation of the absorbance values was calculated.

The linearity of the chromatographic method was determined by injecting six standard solutions in the concentration range of 5–30 μg mL⁻¹ into the HPLC system for three replicates on three different days. The chromatographic responses (peak areas) obtained for each concentration were recorded. Calibration curves were plotted with concentrations on the x-axis and chromatographic responses (peak areas) on the y-axis. The linearity of the spectrophotometric method was determined by measuring the absorbance values of six standard solutions in the concentration range of 5–30 μg mL⁻¹ in the spectrophotometer for three replicates on three different days. The spectrophotometric responses (absorbance values) obtained for each concentration were recorded. Calibration curves were plotted with concentrations on the x-axis and spectrophotometric responses (absorbance values) on the y-axis. The data from both analytical methods were used for regression analysis, which was done using the least squares method. The linearity of the method was measured by the absolute mean recovery, RSD, and R2 of the calibration curve.

The sensitivity of the methods was evaluated by calculating the limits of detection (LOD) and limits of quantification (LOQ). The detection and quantification limits were determined from the standard deviation of the intercept and slope of the calibration curve using the equations ($\text{LOD}=3.3\hspace{0.17em}\mathrm{x}\hspace{0.17em}\sigma /\mathrm{S}$) and $(\text{LOQ}=10\hspace{0.17em}\mathrm{x}\hspace{0.17em}\sigma /\mathrm{S}$). In these equations, σ is the standard deviation of the point where the calibration curve crosses the y-axis, while S is the slope of the calibration curve.

The accuracy of the analytical methods was assessed by determining the % recovery values of the added analyte using the “standard addition method”. To a pre-analyzed LDM solution (10 μg mL⁻¹), an additional 75%,100%, and 125% LDM standard was added to obtain concentrations of 20 μg mL⁻¹. The resulting solutions were re-quantitatively analyzed using the developed methods. The amounts of LDM recovered were determined and % accuracy values were calculated (n = 6).

The analytical methods' accuracy was assessed using their intra-day and inter-day precision. For both procedures (n = 3), intra-day precision was assessed using quantitative measurement of a standard solution at a concentration of 20 g mL⁻¹ on the same day. Inter-day precision was determined by quantitative analysis of a standard solution at a concentration of 20 μg mL⁻¹ on three different days for both methods (n = 9). In the chromatographic method, peak areas and retention times were recorded and relative standard deviation values were calculated. In the spectrophotometric method, absorbance values were measured and relative standard deviation values were calculated.

The robustness of the chromatographic method was assessed by making small deliberate changes to the method conditions. Small modifications were made to the flow rate of the mobile phase (±0.1 mL min⁻¹), the organic modifier content in the mobile phase (±2%), the detection wavelength (±2 nm), and the effect of these modifications on the system suitability parameters were observed. To establish the system suitability parameters following each alteration, a standard solution (20 g mL⁻¹) was injected into the chromatographic system. The results were then compared to the results obtained under the original chromatographic circumstances. These effects were studied by three replicate analyses of the standard solution. Little changes were performed to the organic solvent (ethanol and isopropyl alcohol) and the detection wavelength (302 and 306 nm) in order to evaluate the spectrophotometric method's robustness. The results were compared with the results under the original spectrophotometric conditions. These effects were studied by three replicate analyses of the standard solution.

2.7 Application of analytical methods to commercial formulations

The contents of 10 capsules (Revlimid, 10 mg) were emptied and weighed accurately, and the mass of an average capsule was recorded, crushed in a dry and clean mortar, ground to a fine powder, and mixed. For quantitative analysis of commercial formulations of LDM, tablet powder containing 25 mg LDM was accurately weighed, dispersed in 50 mL of ultrapure water, and sonicated for 10 min. It was filtered to remove undissolved substances. This solution was referred to as the sample stock solution. The sample solution was prepared by taking 400 µL of the stock solution and diluting it to 10 mL with ultrapure water. The solutions were quantitatively analyzed by chromatographic and spectrophotometric methods.

2.8 Greennes profiling of analytical techniques

A metric system (AGREE) was used to assess the greenness of analytical methods (available at: https://git.pg.edu.pl/p174235/AGREEhttps://git.pg.edu.pl/p174235/AGREE). AGREE includes 12 basic principles of greenness assessment and allows weight assignment. It has user-friendly software, is easy to implement, and provides easy-to-interpret color pictogram output showing strengths and weaknesses. The AGREE score is a weighted average of the benchmark scores. It is shown in the center of the graph and its value ranges from 0.0 (lowest score) to 1.0 (perfect score). The graph is a visual representation of the score itself, the benchmark scores, and the benchmark weights [37, 38].

3.1 Determination of the wavelength

Standard solutions prepared using ultrapure water were scanned in a spectrophotometer device in the 200–800 nm wavelength range. The maximum absorption wavelength of LDM was determined as 304 nm (Fig. 1).

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The overlaid spectrum of LDM standard solutions in the 5–30 μg mL⁻¹ concentration range

Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01185

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3.2 Development of methods

The conditions of the developed analytical methods are given below.

Extend C18 column (250 × 4.6 mm, 5 μm) at a constant temperature of 25 °C was used for separation in the chromatographic method. The mobile phase contained 10 mM potassium dihydrogen phosphate solution (pH: 2.0 with orthophosphoric acid) and ethanol (50:50, V/V).

At a flow rate of 1.0 mL min⁻¹, isocratic elution was carried out and 304 nm was chosen for detection.

Spectrophotometric method; the spectral pattern of LDM was extensively analyzed. The best LDM spectra were obtained with ultrapure water and this solvent was used for spectrophotometric analysis. To determine the wavelength at which LDM standard solutions maximally absorbed UV light, the standard solutions were scanned in the 200–400 nm wavelength range. A wavelength of 304 nm was selected for detection.

3.3 Validation of analytical methods

The analytical methods developed for the quantification of LDM in oral formulations were validated for “selectivity, system suitability, linearity, precision, sensitivity, robustness, and specificity” according to ICH Q2 (R1) guidelines.

Standard, sample, and mobile phase solutions were injected into the chromatographic system to evaluate the selectivity of the chromatographic method. The interference peak(s) around the analyte peak were identified by comparing the three chromatograms. No peak interfering with the LDM retention time was observed in all chromatograms. The overlapping chromatogram of the standard, sample, and mobile phase solution is presented in Fig. 2.

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Chromatogram of standard (30 μg mL⁻¹), sample (20 μg mL⁻¹), and mobile phase solution

Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01185

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To evaluate the selectivity of the spectrophotometric method, spectra of standard solutions (5, 10, 15, 20, 25, 30 μg mL⁻¹), sample solution, and ultrapure water were scanned in the wavelength range 200–800 nm. The spectra were compared and the spectrum of the sample solution was analyzed for spectral band(s) interfering with the analyte spectrum. No bands interfering with the LDM bands were observed in the spectrum of the sample solution. The overlapping spectra of the standard solutions produced by the spectrophotometric method and the spectrum of the sample solution are presented in Figs 1 and 3, respectively.

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Spectrum of sample solution (20 μg mL⁻¹)

Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01185

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A standard solution (n = 6) containing 20 g mL⁻¹ was injected into the system, and the primary parameters were determined, to assess the system suitability of the chromatographic method.

LDM has excellent peak symmetry and the peak areas and retention times showed consistently low variability. To determine the spectrophotometric method's system suitability, absorbance values of the standard solution (n = 6) at a concentration of 20 μg mL⁻¹ were used to measure the results in a spectrophotometer. LDM absorbance values consistently showed low variability. The calibration curve's correlation coefficient in this research was 0.9999 for the chromatographic method and 0.9998 for the spectrophotometric method. This indicates that the methods are suitable for samples with highly complex matrices. The determined values are listed in Table 2. Therefore, the developed methods are highly suitable for LDM quantification in pharmaceuticals.

By diluted the stock solution of the standard (500 μg mL⁻¹) with ultrapure water, standard solutions (5, 10, 15, 20, 25, and 30 μg mL⁻¹) were organized in triplicate. For the chromatographic method, the standard solutions were injected into the system. The peak areas and retention times of the analyte were recorded. Average peak areas were calculated for each concentration level. A calibration graph was constructed with the peak area values versus the concentration of the standard solution. For the spectrophotometric method, the absorbance values of the standard solutions were measured against the blank solution. The average absorbance value for each concentration level was calculated. A calibration graph was plotted from absorbance data against standard solution concentration. Regression analysis was used to assess the linearity of the analytical data. The regression equation, slope, and intercept were calculated using linear regression analysis based on the least squares method. The results of the linearity studies are presented in Table 3. In the concentration range of 5–30 μg mL⁻¹, the calibration curve demonstrated a good linear relationship. In this study, the correlation coefficient of the calibration curve was 0.9999 for the chromatographic method and 0.9998 for the spectrophotometric method.

The sensitivity of the methods was evaluated by calculating the limits of detection (LOD) and limits of quantification (LOQ). Detection and quantification limits were determined as 0.10 and 0.30 for the HPLC method and 0.70 and 2.10 for the spectrophotometric method, respectively. The chromatogram of the LDM standard solution at LOQ concentration is presented in Fig. 4 and its spectrum is presented in Fig. 5.

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Chromatogram of standard solution (LOQ concentration)

Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01185

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Spectrum of sample solution (LOQ concentration)

Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01185

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Intraday precision was determined by recording the areas and retention times of LDM peaks obtained from three injections of the reference solution with a 20 g mL⁻¹ concentration on the same day in the chromatographic method (n = 3). In the spectrophotometric method, the absorbance values of the standard solution with a concentration of 20 μg mL⁻¹ on the same day were recorded (n = 3). Inter-day precision was measured by recording the areas and retention times of LDM peaks obtained from three injections of 20 μg mL⁻¹ standard solution on three days in a row (n = 9) in the chromatographic technique. In the spectrophotometric method, absorbance values of 20 μg mL⁻¹ standard solution were recorded for three consecutive days (n = 9). Peak areas, retention times, and the absorbance values' relative standard deviation were computed. Intra-day precision and inter-day precision results are given in Table 4. It was observed that the relative standard deviation values of peak areas, retention times, and absorbances of the analytical methods were below 1.00%. Our data show that the methods fulfill the validation requirements.

Three different concentrations of the LDM standard were added to the sample solution to test the accuracy of the analytical techniques. The standard was added to the sample solution (20 μg mL⁻¹) at the proportion of 75, 100, and 125% of the LDM content. The resulting solutions were analyzed by analytical methods. The % recovery values of the amount of standard added were calculated. Each concentration was tested three times. The recovery percentages ranged between 99.63% and 100.00% for the chromatographic method and between 99.45 and 99.85% for the spectrophotometric method. The maximum relative standard deviation values were 0.296 for the chromatographic method and 0.490 for the spectrophotometric method. Table 5 displays the findings of the recovery investigations.

To assess the robustness of the methods, small deviations from the optimal values of both analytical methods were made and these changes were observed to have an impact on the system suitability parameters. To determine the system suitability parameters in the chromatographic procedure, 20 g mL-1 of a standard solution was injected into the chromatographic system after each changes. The results were then compared to the results obtained under the original chromatographic circumstances. A reference solution (20 μg mL⁻¹) was injected into the HPLC system for the chromatographic method. In the spectrophotometric method, the effect of different solvents and detection wavelengths was studied and compared with the results under the original spectrophotometric conditions. These effects were studied by three replicate analyses of the standard solution. Small deviations from the optimum values for the method parameters did not have a significant effect on the results. According to the results obtained, the largest relative standard deviation value was calculated to be 0.44 (Table 6).

3.4 Application of analytical methods to pharmaceutical formulations

6 capsules (Revlimid, 10 mg) were quantitatively analyzed by the developed analytical methods. Table 7 shows the findings of both analytical methods, relative standard deviation, standard deviation and the mean, derived over six replicates. The Student (t) test and the Fischer (F) test were used to compare the outcomes of the two methods in terms of means and standard deviations, respectively. When the results in the table are analyzed, in terms of accuracy and precision, it can be shown that there is no noticeable distinction between the two analytical approaches. The t and F values obtained as a result of 6 trials were below the values indicated in the relevant tables.

3.5 Greennes profiling of analytical techniques

The pictograms of the greenness assessment of the analytical techniques are given in Fig. 5. The greenness score of the chromatographic technique is 0.75, while the spectrophotometric technique has a greenness score of 0.76. The scores relating to the green analytical chemistry standards 1, 7, and 8 in the AGREE pictogram of the chromatography technique were rather low, whereas the scores relating to the principles 4, 6, 9, 10, and 11 were extremely good (Fig. 6A). In the AGREE pictogram of the spectrophotometric technique, the scores for GAC principles 1 and 5 are quite low, while the performance for principles 3, 4, 6, 10, 11, and 12 is excellent (Fig. 6C). It can be said that both analytical methods are green, however, the chromatographic technique is more environmentally conscious than the spectrophotometric approach. The corresponding color scale for the reference is presented in Fig. 6B.

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A: AGREE pictogram of the chromatographic method, B: The color scale for reference, C: AGREE pictogram of the spectrophotometric method

Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01185

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In references 23, 24, 25, 26, 27, 28, 31, and 32, toxic methanol was used as the solvent. In references 26, 30, 33, and 34, toxic acetonitrile was used as the solvent. In reference 29, both toxic acetonitrile and toxic methanol were used as solvents. In the methods we developed, ultrapure water and ethanol were used from the sample preparation stage to the final detection stage, and no toxic solvents were used.