Systematic development and validation of RP-HPLC method for simultaneous estimation of tamoxifen and sulphoraphane with specific application for nanolipidic formulations

2.2.1 HPLC instrumentation and chromatographic conditions

The HPLC system consisted of a model Shimadzu-10ATVP binary pump connected by photodiode array detector (SPD-M20A) with EMPOWER software. Both analyte was separated on reverse phase (RP) C₁₈ column (250 mm × 4.6 mm × 5 μm) at their respective wavelength i.e. 210 nm for SFN and 275 nm for TAM (Mangla et al., 2020b). Absorption maxima of both drugs are shown in Fig. S1. For this study, Methanol: water (pH 3.5) with ratio 70:30% v/v was used as mobile phase that degassed daily and filtered by 0.22-µm nylon membrane filter with before use. The column temperature was maintained at 30 °C. Total run time analysis was less than 25 min, with flow rate of 0.8 mL/min.

2.2.2 Preparation of standard and working solutions

The standard solution of both analytes has been separately prepared with methanol in 10 mL volumetric flask to obtained 1000 µg/mL concentration and considered as standard solution A₁ for TAM and A₂ for SFN. From this solution, took 1 mL and diluted with methanol to obtained 100 µg/mL of concentration (stock B₁ and B₂). Stock B₁ and B₂ was further appropriately diluted with methanol to get mixed working stock solution with concentrations 6, 9, 12, 15, 18, 21, and 24 μg/mL, respectively (Nakagawa et al., 2006). Further, the quality control (QC) samples of both analyte were similarly prepared to obtain 10 μg/mL i.e. lower quality control (LQC), 15 μg/mL i.e., middle quality control (MQC) and 20 μg/mL i.e. higher quality control (HQC). All solutions were filtered through a 0.22-µm filter (Millifilter, Milford, MA, USA) before analysis.

2.2.3 Optimization of RP-HPLC method

The proposed method was optimized by Box Behnken design (BBD) using three independent variables and six dependent variables. BBD produces higher-order responses of independent variables with less possible runs than a standard factorial approach. In this design, % methanol concentration (A), pH of aqueous mobile phase (B), flow rate (C) were considered as independent variables and retention time (RT) of TAM (Y1); SFN (Y4), tailing factor of TAM (Y2); SFN (Y5), No. of theoretical plates of TAM (Y3); SFN (Y6) were taken as dependent variables. With the help of design Expert® (STATEASE Inc., USA) software, 17 test runs were conducted (Table S1) and analyzed the response of dependent variables (Y) via polynomial equations.

2.3.1 System suitability test

This test is an integral part of the liquid chromatographic techniques that check the method’s reproducibility and sure that the whole testing system is suitable for the intended application. The system suitability test was assessed at MQC level (15 µg/mL) by injecting six replicate and outcomes have been studied by RT, peak area, theoretical plates of the column and tailing factor of both analytes. US-FDA guidelines considered the approval criterion for this test that the relative standard deviation (%RSD) for RT and peak area should be less than 2%. For tailing factor, the range should not exceed by two and for theoretical plates of the column, it should more than 2000 (N > 2000).

2.3.2 Specificity

Specificity is the essential characteristic of HPLC and refers to the capability of the analytical system to separate the analyte from the complex mixture. Specificity has been evaluated by comparing the chromatogram of individual solutions, their mixture and blank solution independently at MQC level. The blank solution was prepared as the sample solution, but without adding both the drugs (TAM and SFN).

2.3.3 Linearity

The linearity was determined by preparing seven different concentrations of a mixed standard solution in the range of 6–24 µg/ml of TAM and SFN, which cover 40%, 60%, 80%, 100%, 120%, 140% and 160% of the target concentration, respectively. The individual calibration curves of TAM and SFN were plotted between peak area on the Y-axis versus their respective concentration X-axis and regression equations were calculated. Also, calculate the response ratio (response factor) by dividing the peak area with concentration.

2.3.4 Robustness

This experiment has been conducted to assess the suitability of the method by changing the small variations in the experimental parameters such as a change in detection wavelength of TAM (273 to 277 nm); SFN (207 to 211 nm), column oven temperature (25 to 35 °C) and injection volume (15 to 25 μL). Further, the effect caused due to these variations in method conditions was investigated and analyze the results. At each variation, 100% of target concentration (MQC, 15 µg/mL) was taken and three replicate (n = 3) of each sample injected into the chromatographic system. The method robustness was evaluated from the %RSD of the mean peak area and mean % recovery of TAM and SFN. Their limit should not be exceed 2% (Ganorkar et al., 2017). Further measure the tailing factor, theoretical plates and RT of both analyte.

2.3.5 Accuracy

The accuracy of an analytical method means the nearness between the expected value and the value found. The accuracy of the proposed method was determined by % recovery of both analytes at three QC levels (50%, 100% and 150%) and from each concentration, three sample replicate (n = 3) were injected. The mean % recovery of TAM and SFN with their %RSD and standard error (SE) were analyzed using the following formula. The mean % recovery should be within 90–110% and %RSD as well SE should NMT 2%, to be accepted for the accuracy.

2.3.6 Precision

The precision is the extent to which the technique is repeatedly used to analyze several replicates in different instances. The precision of the method for intra-day and inter-day analysis was determined by studying with the QC samples for both analytes. For intra-day, three replicates of samples for each drug (n = 3) at each QC level were analyzed on the same day, whereas for inter day, samples were assessed every day during three consecutive days. Peak area and % recovery of both analytes were noted. Then calculate %RSD with the acceptance criteria of not more than 2%. Moreover, calculate the RT, theoretical plates and tailing factors of both analytes.

2.3.7 Ruggedness

The ruggedness is defined as the ability to reproduce the testing result under various conditions, like different instruments and different analysts (n = 3). Ruggedness was determined by analyzing three different samples (LQC, MQC and HQC) by two analysts in the same and different laboratory having identical HPLC equipment with the same general specification and analytical column. The % recovery of both analytes was calculated, and the results obtained from two different HPLC and different analysts were compared with each other to examine the ruggedness of the method (Dejaegher and Heyden, 2007).

2.3.8 Sensitivity

The sensitivity of the analytical technique was estimated in terms of LOD and LOQ. LOD is the concentration which provides a signal-to-noise ratio of approx.3:1, while LOQ is the concentration which provides a signal-to-noise ratio of approx. 10:1 having %RSD (n = 3) of less than 10%. LOD and LOQ of both analytes were determined using the following formula.

A = kσ/S where A is LOD or LOQ value, σ is the standard deviation (SD) of the response (peak area), and S represents the slope obtained from the calibration curve. k for LOD = 3.3; for LOQ = 10.

2.3.9 Stability studies

Stability studies were conducted to assess the stability of mixture solution at all QC levels under different stability conditions i.e., freeze and thaw, short term and long-term stability. Three aliquots were processed for freeze and thaw stability for the 24 h at −20 °C and thawed at room temperature unassisted. QC samples were placed at room temperature for short-term stability and analyzed for 4 and 12 h. Long term stability studies were carried out for 2 weeks at −20 °C. Furthermore, the stability of standard solutions of both analyte was determined for 6 h at room temperature and 2 weeks at −20 °C. Experiments were done in triplicate and then analyzed by % recovery ± SD. This should be within the acceptable range.

2.4.1 Preparation of TAM-SFN-SLNs

TAM-SFN-SLNs was developed by the method of melt emulsification ultrasonication where Compritol® ATO 888 was used as a solid lipid. For the preparation of TAM-SFN-SLNs, precisely weighed solid lipid was melted at a temperature 5–10 °C beyond its melting point. After that, accurately weighed quantity of TAM and SFN were added in the melted lipid. Subsequently, surfactant solution was prepared by adding the surfactants Poloxamer188, Tween 80 and Tween 20 in the ratio of 2:1:1 in milli-Q water and heated at the same temperature. Under constant stirring at 12,000 rpm for 30 min, the melted lipid was added to the heated aqueous surfactant solution to acquire a primary emulsion.

Further, the primary emulsion thus obtained was nano sized through probe sonicator using ultrasonication technique with an amplitude of 70%. The acquired dispersion was immediately cooled down at room temperature to obtain SLNs. After that, the resultant TAM-SFN-SLNs were subjected to ultracentrifugation for 20 min at 6000 g at 4 °C to remove unentrapped drug from the SLNs. The supernatant was discarded, whereas the pellet obtained after centrifugation was washed using milli-Q water and then evaluated for the percentage entrapment efficiency (Harivardhan Reddy et al., 2006).

2.4.2 Preparation of TAM-SFN-LIPO

Phospholipon 90G and cholesterol (70:30 M ratio) were used for the preparation of TAM-SFN-LIPO by a thin-film hydration method. Appropriately weighed the amount of lipid, cholesterol, TAM and SFN were dissolved in 5 mL of chloroform: methanol (2:1). Thereafter, the solvent was evaporated using a rotary evaporator (Buchi, Switzerland) in a round bottom flask (RBF) under decreased pressure to form a thin lipid film. Further, the vacuum was released and the RBF was kept in a vacuum desiccator for 12 h to remove the residual traces of organic solvent. Using 5 mL of milli-Q water the thin film of lipid was hydrated by rotating the RBF at 100 rpm for 45 min in water maintained above the transition temperature (T_m) of the lipid. The flask was rotated until the thin film was utterly hydrated and the liposome dispersion was obtained. The resulting liposome dispersion was ultrasonicated with a probe sonicator for 5–10 min and stored at 4 °C until further use. Then the liposomes were centrifuged at 20000g for 15 min 4 °C; the supernatant was separated and the pellet obtained was washed thrice using milli-Q water and evaluated it for drug encapsulation efficiency (Zhang, 2017).

2.4.3 Preparation of TAM-SFN-NLCs

TAM-SFN-NLCs were formulated by melt emulsification ultrasonication technique utilizing Precirol® ATO 5 and Transcutol HP as solid and liquid lipids respectively. Accurately, weighed solid lipid was heated 5–10 °C beyond its melting point and thereafter, liquid lipid was added to it to form an oily phase, to which the prerequisite amount of TAM and SFN was added. At the same time, an aqueous surfactant phase was developed by dispersing Tween 80 and Poloxamer 188 (1:1) in milli-Q water and heated at the same temperature. After that, this hot aqueous surfactant solution was poured dropwise to the above prepared oily phase with constant stirring for 30 min at 12,000 rpm using a magnetic stirrer. The primary emulsion thus obtained was ultrasonicated with a probe sonicator for 60 sec to obtain the dispersion, which was kept to cool down wherein the lipid recrystallizes to develop NLCs. TAM-SFN-NLCs thus obtained were ultracentrifuged at 6000 g for 20 min at 4 °C to remove the unentrapped drug. The supernatant was disposed of, whereas the pellet was rinsed using milli-Q water and evaluated the for percentage entrapment efficiency (Singh et al., 2019; Soni et al., 2020).

3.2.1 System suitability test

The %RSD of peak area, as well as RT of analytes, was calculated and found to be within 2%, indicating the suitability of the system as shown in Table 1A and 1B. The %RSD of tailing factor and number of theoretical plates of the column for the six replicate injections were found to be 1.52 ± 0.50% and 5716.50 ± 1.32% for TAM; 1.77 ± 0.43% and 6551.17 ± 1.08% for SFN, respectively. The number of theoretical plates was greater than 2000 and considered to be acceptable for the system suitability test. The %RSD of tailing factor was within the specified limits as per the guidelines. These results ensure that the proposed HPLC method was capable of providing data of acceptable quality.

3.2.2 Specificity

Specificity was determined by comparing the chromatograms of the same target concentration of a blank solution, a standard solution of TAM and SFN and their mixture solution. For this purpose, 20 µL of the samples were separately injected in HPLC system and RT of TAM and SFN was recorded at 4.13 ± 0.034 min and 7.6 ± 0.073 min, respectively as shown in Figs. 3 and 4. The findings revealed that RT of analytes in the mixture solution did not change to their individual solution. Moreover, no co-eluting peaks of interference were found. This indicates the specificity of the HPLC method developed.

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Fig. 3

Chromatogram of blank solution (A), TAM (B) and SFN (C).

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Fig. 4

Chromatogram of TAM and SFN at their respective wavelength.

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3.2.3 Linearity

Linearity was described in terms of calibration curve. This curve was obtained by plotting the mean peak area of TAM and SFN against their corresponding concentrations. The results (Fig. S2) provide a linear relationship over the concentration range of 6–24 µg/mL for both analytes. From the regression data, a linear equation and r² of TAM and SFN were found to be y = 44275x −32825; 0.9972 and y = 58306x−37166; 0.998. These results indicated a linear relationship between the mean peak area and concentration of the analyte. The response factor of the analytes are shown in Fig. S3.

3.2.4 Robustness

The robustness of the analytical process was evaluated by assessing the effect of small variations in HPLC conditions i.e., change in column oven temperature, detection wavelength of both analysts, and injection volume (15–25 µL). The results are summarized in Table S4 (A) & (B) and found that a minor change in method condition did not significantly affect the tailing factor, theoretical plates and RT of analyte. Thus the proposed method is reliable and robust.

3.2.5 Accuracy

The accuracy expresses in terms of % recovery means the proximity to the actual value. The % recovery at all three QC levels ranged from 97.07–103.0% and 99.65–101.0% for TAM and SFN, respectively, as summarized in Table 2. These results were found to be within the accepted limits, which demonstrated the applicability of the developed method for routine drug analysis. The excellent recovery values for accuracy study ascertain that method is accurate.

3.2.6 Precision

The %RSD of peak area for inter-day precision and intra-day precision at all QC levels was found to be in the range of 0.33–1.05 and 0.19–0.65 for TAM, whereas 0.22–0.34 and 0.03–0.72 for SFN, respectively. The %RSD of % recovery for inter-day precision and intra-day precision at all QC levels was found to be in the range of 0.65–1.33 and 1.28–1.80 for TAM, whereas 0.64–1.72 and 0.45–1.33 for SFN respectively. The developed method indicates good precision as well as reproducible with %RSD found to be less than 2% as listed in Table S5 (A, B, C & D). This signifies the repeatability and reliability of the method.

3.2.7 Ruggedness

Ruggedness studies carried out in two different HPLC instruments with two different analysts and exhibited a negligible change in the mean % recovery values for the LQC, MQC and HQC of both analytes. The results are shown in Table S6 (A and B), indicating a high degree of the ruggedness of proposed method.

3.2.8 Sensitivity

The signal-to-noise ratio of 3:1 and 10:1 was obtained for the LOD and LOQ, respectively. The LOD and LOQ were found to be 0.5 μg/mL and 0.9 μg/mL for TAM and 0.44 μg/mL and 1.1 μg/mL for SFN respectively. The results have shown that the method is highly sensitive to the methods previously reported.

3.2.9 Stability studies

Stability study of the analyte was evaluated under different storage conditions at QC levels (10 μg/mL, 15 μg/mL and 20 μg/mL). The accuracy of the predicted concentration obtained after three freeze–thaw cycles, short-term storage at room temperature and long-term storage at −20 °C for 2 weeks was in the range 90–101.4% for TAM and 87.4–100.2% for SFN, as shown in Table 3. These results indicate that both drugs did not degrade during the different storage conditions except the SFN that was less stable at 12 h at room temperature as compared to TAM. This implies that QC samples were only stable up to 4 h. Also, TAM and SFN standard solutions were observed to be stable for at least 6 h at room temperature and 2 weeks at −20 °C.