INTRODUCTION:

Raloxifene (RLX) is a benzothiophene-class selective oestrogen receptor modulator (SERM) with antiestrogenic actions on the endometrium and breast¹. RLX is a drug that helps postmenopausal women to minimize their risk of breast cancer more than Tamoxifen². It was authorized by the US Food and Drug Administration (FDA) in 2007 again as an anticancer drug³. This drug is classified as class II in the Biopharmaceutical Classification System, which is characterised by high metabolism, low solubility, and high permeability⁴.

As a result, a combinatorial approach of a synthetic drug (RLX) with a bioactive compound (CUR) that would lessen the underlying difficulties linked to toxic effects and augment anti-cancer efficacy would be a novel way to sidestep the problems outlined above. The chemical structure of RLX is shown in Fig.1

Figure 1: Chemical structure of Raloxifene

The second active constituent, Curcumin (CUR) is hydrophobic in nature and a rich source of polyphenols, extracted from Curcuma longa rhizomes and is 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione structurally⁵ and the chemical structure of CUR is shown in Fig.2. CUR is a phenolic compound that was used as a spice or natural food colouring ingredient. In recent years, it has sparked attention due to its antioxidants, anti-inflammatory, and cancer chemo-preventive properties⁶. Curcumin has low water solubility, inadequate gastrointestinal intake, rapid metabolic rate, limited bioavailability, and poor stability. Their little clinical application is due to their inability to function in neutral pH settings⁷. It has been reported that these compounds are bioactive but inactive in the neutral and alkaline aqueous phases⁸.

Figure 2: Chemical structure of Curcumin

The quantitative estimation of RLX and CUR individually in various pharmaceutical formulations and biological fluids via high-performance liquid chromatography (HPLC) methods has been^9-11. It helps in the determination of exact amount of the substance, which is very useful for many applications like food, chemical, diary, paint and pharmaceutical^12.Such quantitative estimations are helpful to check the progress of a reaction in the reaction mixture, to check the purity and identity of the compound, to check the stability of that chemical compound, to check how much amount of the drug is released in the body and many more¹³. According to International Organization for Standardization (ISO), validation is the affirmation via inspection and provision of verifiable data that the specified requirements for a specific intended application are satisfied¹⁴. Calibration is the process of assessing accuracy, which validates the qualification and validation procedures and aids in quantifying and managing errors within various measurements¹⁵. There are several methods reported to measure curcumin. CUR detection has also been reported using capillary electrophoresis techniques¹⁶. CUR has also been analyzed using several high-performance liquid chromatography identification (HPLC) techniques, with fluorescence detectors to assess biological fluids¹⁷. In another study, CUR was measured quantitatively in natural materials using HPLC-UV techniques¹⁸. Various analytical approaches for quantifying total and separated CUM in different samples, especially spectrophotometric methods for determining total curcuminoids, have also been documented¹⁹. Single curcuminoids, however, cannot be quantified using this method²⁰. The most popular technique among all for determining curcuminoids and CUR in turmeric materials, clinical materials, or dosing schedules is HPLC.Analytical techniques reported for RLX, is isocratic RP HPLC method for determination of RLX hydrochloride in tablets¹⁴. While HPLC techniques are known for their short run times, to the best of our knowledge, no researcher has published a validated test for RLX-CUR that uses simple extraction processes. Our goal was to develop an HPLC approach for determining RLX-CUR retention time that was rapid, robust, specific, sensitive, and accurate.

However, according to the literature, there is currently no simultaneous HPLC method for identifying and quantifying RLX and CUR in pharmaceutical samples and we are able to develop the method of quantitative estimation, the next step is validation of the developed method. The validation process is an essential aspect of research and development, and its goal is to assess whether the instrument is performing its intended duties consistently and adequately^21,22. For simultaneous estimations, several treatments can be done which are inexpensive, but they required complex methodology like sample preparation in laboratories and making them difficult to execute in small laboratories²³. As a result, developing result-oriented, simple, precise, and cost-effective methods that can overcome the needs of patients²⁴. The present study intends to verify the U.V. spectroscopic strategies, which implies several parameters, such as predictability, versatility, device appropriateness, the quantification limited detection limit, accuracy, precision, selectivity, and reliability, as well as forced degradation tests, as per ICH counselling. The precision of an analytical technique explains how closely a set of measurements acquired coincide from a serial sampling of the same homogenous sample. In contrast, the accuracy of an analytical approach explains how near the test findings developed by the technique are to the analyte's expected value. We validated a new RP-HPLC method for measuring RLX and CUR in bulk that was easy, affordable, effective, and accurate. System appropriateness, uniformity, precision (LOD) and (LOQ), correctness (recoveries), durability, robustness, stabilization, and forced degradation tests were have been used to verify the proposed approach (specificity). Thus, the present work is an attempt to develop a simple and precise method for simultaneously estimating RLX and CUR using the HPLC technique, followed by validation. This method was used for the quantification of RLX and CUR in various lipid-based nanoformulations such as nanostructured lipid carriers (NLCs). Furthermore, simultaneous estimate of RLX and CUR in these nanoformulations could have clinical implications and potentially benefit pharmacokinetic studies. This is the first time this work has been done, to the best of our knowledge.

MATERIALS AND METHOD:

Material:

Cadila Pharmaceuticals Limited in Gujarat, India provided gift samples of Raloxifene and Curcumin, and other analytical grade chemicals and reagents were procured and were used as received.

Method development of RLX and CUR:

Mobile phase selection:

The mobile phase is the liquid that passes through the sheet, bringing various compounds with it and help in separation of component based on partition coefficient. The stationary phase remains stationary on the page and does not migrate. First of all, several mobile phase solvents were prepared by a fixed ratio of RLX and CUR. Based on several trials, the most suitable mobile phase screened for the experiment was 10mM KH2PO4: Acetonitrile (pH 3.0 with ortho-phosphoric acid) in the proportion of 20:80v/v. For this purpose, device suitability parameters were noticed, such as R.T., Tailing factor, numerous theoretical plates, and HETP. The mobile phase was strained through 0.45m filter paper and then sonicated to eliminate particulate particles. Hence, the flow rate of 1.0ml/min was used for the analysis.

Chromatographic conditions:

Shimadzu HPLC was employed with LC-20AT prominence liquid chromatography for the current study with an SPD-20A prominence U.V./Visible sensor (Spinchrom CFR version 2.4.01.093 database management system).

The mobile phase preparation:

The mobile phase found to be most suitable for analysis was 20mM KH₂PO₄: acetonitrile (pH 3.0 adjusted with OPA) in the ratio of 20:80v/v. and filtered through 0.45 filter paper. The sharp peak was observed for both 20mM KH₂PO₄and Acetonitrile along with various separation variables mentioned in (Table 1).

Selection of diluents:

Acetonitrile was utilized as a diluent after several trials. The diluent used to prepare the sample was found to be compatible with the mobile phase and had no effect on the analyte's retention or resolution. HPLC peak patterns may be affected by the solvent (diluent) used to make HPLC samples²⁵.

Table 1. Separation Variables for both RLX and CUR for HPLC analysis using C18 column

Variable		Condition
Column Dimension		250mm x 4.60mm
Particle Size		5m
Bonded Phase		Octadecylsilane (C₁₈)
Mobile Phase Ratio	20mM KH₂PO₄	20
	Acetonitrile	80
Diluent		Acetonitrile
Flow rate		1.0 ml/min
Temperature		Ambient
Sample Size		20 ml
Wavelength	RLX	292nm
	CUR	428nm
Retention Time	RLX	5.116 ± 0.3min
	CUR	3.225 ± 0.3min

Standard Stock Solution Processing:

For a practical case, a stock solution is a concentrated solution that will be diluted to a lower percentage. Stock solutions are utilized to save time, resources, and storage space while also increasing the accuracy with which functioning lower concentration solutions are created. To prepare standard stock solution, first of all, 10mg of RLX and CUR each were precisely weighed and placed in 10ml volumetric flasks, where they were mixed in 5 ml acetonitrile, sonicated for 10 minutes, and then diluted up to 10ml with acetonitrile. The concentrations of RLX and CUR in acetonitrile were 1000µg/ml and named to solution as (Standard Stock - A).

Sub Stock Solution Preparation:

At this part, stock solution will be used which was prepared earlier. To prepare sub stock solution, first of all 1ml of RLX and CUR solution was taken from standard stock-A, and placed in a 10ml volumetric flask separately, after that, diluted with acetonitrile up to 10 ml to prepare concentration of 100µg/ml which was named as (Stock-B).

RLX and CUR Solution Preparation:

Five volumetric flasks of 10ml were taken and filled with 0.1ml, 0.2ml, 0.3ml, 0.4ml, and 0.5ml of stock-B in each flask and filled up to 10ml with acetonitrile in each flask. This produces RLX solutions with concentrations of 1-5µg/ml. In same manner of CUR also prepared.

Laboratory Sample Analysis:

For quantitative analysis, various standard solutions were created, yielding good findings. The mixed standard was produced in the same way as the stock solution, with additional dilutions to achieve the necessary concentration of 1-5mg/ml for RLX and CUR.

Validation of the Developed Techniques:

Linearity:

An analyzing procedure's capacity to provide tests that are proportional to the concentration of analyte in a sample (within a certain range) is known as Linearity. Five different concentrations (range from 1-5mg/ml) were tested, the calibration plot was condensed, and areas for each concentration were recorded in triplicates and the mean area was determined.

Specificity:

The specificity of the approach was examined to see whether the analyte contained constituents that may be predicted to be present, such as pollutants, degradation products, and cellular elements. The capacity of a procedure to distinguish the analyte from all possibly interfering substances is referred to as specificity.

Accuracy:

The degree to which the experimental result matches the real quantity of the chemical in the matrix is known as accuracy. To test the accuracy of the devised technique on a pre-analysed sample solution, a particular concentration of standard medication (80, 100 and 120 %) was added, and the recovery was measured.

Precision:

Accuracy and precision are the two most significant characteristics of a chromatographic test procedure. Individual measurements are compared to see how close they are to each other. Precision was measured using intra-day and inter-day variance experiments. Pharmaceutical solutions ranging from 1-5mg/ml were prepared for the inter-assay, and uptake was measured three times a week. Uptake was measured three times on the same day for intraday assay. Relative standard deviation (RSD) percentage was used to demonstrate the scattering of data around the mean value.

Robustness:

Robustness is an assessment of an analytical method in which the values obtained are reliable even when conducted under slightly different conditions. It refers to a method's ability to remain unaffected when minor changes are made and small but controllable variations in the mobile phase concentration were performed. ICH guidelines were followed to validate the test's ability to stay unaltered.

Factors of System Suitability:

The separation factors were determined and permitted the column to saturate with the mobile phase at a rate of 1.00 ml/min. Following full saturation of the column, triplicates of the RLX and CUR working standards were injected independently. A peak report and a column performance report were included in every chromatogram.

Sensitivity:

The specified procedure was used to estimate the quantification limit (LOQ) and detection limit (LOD) of RLX and CUR values. The suggested approach's LOD and LOQ were estimated using the standard deviation of the output and the slopes of the linearity curve. The detection limit, also known as the LOD, is the smallest quantity of analyte that can be identified but not necessarily measured in a sample. The limit of quantification, or LOQ, refers to the smallest amount of analyte in the sample that can be quantified with reasonable precision and accuracy.

RESULTS AND DISCUSSION:

The proposed technique was validated using ICH criteria. The process was adjusted before validation by changing the pH of the column, solvent, and mobile phase. After multiple trials, the proposed strategy was finalized. The validation parameters studied were linearity, LOD, LOQ, accuracy, precision, and robustness.

Linearity:

Linearity of analytical procedure is its ability (within a given range) to obtain test, which are directly proportional to area of analyte in the sample. The calibration plot was contracted after analysis of five different concentrations (from 1 to 5µg/ml) and areas for each concentration were recorded five times, and mean area was calculated. The regression equation and correlation coefficient of curve are given, and the standard calibration curve of the drug. From the mean of AUC observed and respective concentration value, the response ratio (response factor) was found by dividing the AUC with respective concentration mentioned in Table 2.

Table 3. Recovery Study (accuracy) of RXL and CUR

Sample % Level	Sample Conc. (mg/ml)	Amt. added (mg/ml)	Recovered conc. (mg/ml)			% conc. found (mg/ml)			Mean % conc.	% Recovery	SD	% RSD
Sample % Level	Sample Conc. (mg/ml)	Amt. added (mg/ml)	Rep -1	Rep-2	Rep-3	Rep-1	Rep-2	Rep-3	Mean % conc.	% Recovery	SD	% RSD
RLX (80%)	1	0.8	0.79	0.78	0.79	98.75	97.50	98.75	98.84	98.84	0.561	0.567
	2	1.6	1.59	1.58	1.57	99.38	98.75	98.13	98.33
	3	2.4	2.39	2.38	2.39	99.58	99.17	99.58	98.75
RLX (100%)	1	1	0.95	0.99	0.98	95.00	99.00	98.00	97.33	98.26	0.804	0.818
	2	2	1.98	1.99	1.95	99.00	99.50	97.50	98.67
	3	3	2.95	2.96	2.98	98.33	98.67	99.33	98.78
RLX (120%)	1	1.2	1.18	1.18	1.19	98.33	98.33	99.17	98.61	99.29	0.617	0.621
	2	2.4	2.38	2.39	2.39	99.17	99.58	99.58	99.44
	3	3.6	3.59	3.58	3.61	99.72	99.44	100.28	99.81
CUR (80%)	1	0.8	0.78	0.81	0.79	97.50	101.25	98.75	99.17	99.03	0.241	0.243
	2	1.6	1.59	1.58	1.57	99.38	98.75	98.13	98.75
	3	2.4	2.38	2.37	2.39	99.17	98.75	99.58	99.17
CUR (100%)	1	1	0.98	0.99	0.98	98.00	99.00	98.00	98.33	98.70	0.390	0.495
	2	2	1.95	1.99	1.98	97.50	99.50	99.00	98.67
	3	3	2.95	2.98	2.99	98.33	99.33	99.67	99.11
CUR (120%)	1	1.2	1.18	1.19	1.21	98.33	99.17	100.83	99.44	99.49	0.080	0.081
	2	2.4	2.39	2.38	2.4	99.58	99.17	100.00	99.58
	3	3.6	3.58	3.59	3.57	99.44	99.72	99.17	99.44

Precision:

The precision of the method was determined by intra- day and inter-day precision. Assessing intra-day and inter-day coefficients of variance were used to measure the accuracy (% RSD) of the obtained data set and examined Intra-day and inter-day variability scales from 1 to 5µg/ ml for all three RLX and CUR values in five repetitions (n = 5) to determine the accuracy of an analytical methodology (Tables 4 and 5).

Table 4. Intra-day variability of RLX and CUR

Conc. Rep.	Raloxifene Concentration found (mg/ml)					MEAN
Conc. Rep.	1	2	3	4	5
1	0.98	1.99	2.98	3.99	4.98
2	0.99	1.98	2.99	3.98	4.99
3	0.99	1.95	2.95	3.95	5.01
4	0.98	1.98	2.99	3.96	4.96
5	0.98	2.01	3.01	3.95	4.98
Mean	0.984	1.982	2.984	3.966	4.984
% Mean	98.40	99.10	99.47	99.15	99.68	99.159
SD	0.005	0.022	0.022	0.018	0.018	0.017
% RSD	0.006	0.022	0.022	0.018	0.018	0.017
Conc. Rep.	Curcumin Concentration found (mg/ml)					Mean
Conc. Rep.	1	2	3	4	5
1	0.99	1.98	2.95	3.98	4.95
2	1.02	2.01	2.98	4.01	4.98
3	0.99	1.99	2.99	3.95	4.99
4	0.98	1.98	3.01	3.98	4.98
5	0.99	1.97	2.98	3.99	4.95
Mean	0.994	1.986	2.982	3.982	4.97
%Mean	99.4	99.3	99.4	99.55	99.4	99.41
SD	0.015	0.015	0.022	0.022	0.019	0.0185
% RSD	0.015	0.015	0.022	0.022	0.019	0.0186

Table 5. Inter day variability of RLX and CUR

Conc. Rep.	Raloxifene Concentration found (mg/ml)					Mean
Conc. Rep.	1	2	3	4	5
1	0.98	1.99	2.98	3.99	4.95
2	0.97	1.98	2.96	3.98	4.98
3	0.98	1.97	2.99	3.97	4.99
4	0.99	1.98	2.99	3.99	4.96
5	0.98	1.99	2.98	4.01	4.98
Mean	0.98	1.982	2.98	3.988	4.972
% Mean	98.00	99.10	99.33	99.70	99.44	99.115
SD	0.007	0.008	0.012	0.015	0.016	0.012
% RSD	1.054	0.989	0.410	0.489	0.260	0.012
Conc. Rep.	Curcumin Concentration found (mg/ml)					Mean
Conc. Rep.	1	2	3	4	5
1	0.99	1.98	2.95	3.98	4.95
2	0.98	1.97	2.98	3.99	4.98
3	0.97	1.99	2.99	3.96	4.99
4	0.98	1.99	2.98	3.98	4.96
5	0.99	1.98	2.99	3.99	4.98
Mean	0.98	1.982	2.978	3.98	4.972
% Mean	98.20	99.10	99.27	99.50	99.44	99.101
SD	0.008	0.008	0.016	0.012	0.016	0.012
% RSD	0.009	0.008	0.017	0.012	0.017	0.012

Intra-day variability assessments evaluate accuracy over a short period inside a day while operating under the corresponding circumstance. The precision analysis over three days is referred to as inter-day variability. Some other factors were also determined as relative standard deviation percentage (RSD%) and was found to be less than 2% in both situations. The RSD is a measurement that determines the accuracy of the average value of the observed values^27,28. It may be expressed as a percentage or a simple number, and it may be added to or removed from your primary measurement. The RSD values were all within the tolerable level, indicating that the proposed analysis is correct.

Specificity:

Specificity of the method was carried out to assess unequivocally the analyte presence of the components that might be expected to be present, such as impurities, degradation products and matrix components. Specificity was determined by comparing the chromatograms of the same target concentration of a blank solution, a standard solution of RLX and CUR and their mixture solution. For this purpose, 20ml of the samples were separately injected in HPLC system and RT of RLX and CUR was recorded at 5.116±0.3min and 3.225±0.3min, respectively as shown in Figs. 5-7. The results showed that the RT of the analytes in the combination solution did not change after they were separated into their individual solutions. Furthermore, no interference co-eluting peaks were discovered. This demonstrates the specificity of the established HPLC technique²⁹.

Figure 5: Chromatogram of Curcumin

Figure 6: Chromatogram of RLX

Figure 7: Representative chromatogram of Curcumin and Raloxifene

Robustness:

Robustness of the method was determined by making slight changes in the chromatographic conditions. The robustness of the formulated analytical method was determined by lowering injection volume to 10L from 20L, flowing capacity to 0.6 and 0.8ml/min from 1 ml/min, the temperature of chromatographic column to 35°C, and mobile phase ratio to (20:80% v/v). The analysis of the robustness of chromatographic procedures typically is difficult and tedious, taking into account the high number of analytical factors that need to be addressed to carry out the test^29,30. For each metric, injections were provided in five different ways. To differentiate any variations in retention time attributable to improvements in these parameters, the %RSD was determined. As indicated in (Tables 6), the RSD percentage for RLX and CUR was stated to be in the range of 0.020 percent and 0.015 percent, respectively. It was observed that there were no marked changes in the chromatograms, which demonstrated that the RP-HPLC method developed is robust.

Table 6. Robustness of RLXand CUR

Conc. Rep.	Raloxifene Concentration found (mg/ml)					Mean
Conc. Rep.	1	2	3	4	5
1	0.98	1.99	2.98	3.99	4.95
2	0.99	1.98	2.95	3.98	4.98
3	1.01	1.95	2.96	4.01	4.96
4	0.97	1.96	2.99	3.99	4.97
5	0.96	1.99	3.01	3.95	4.99
Mean	0.982	1.974	2.978	3.984	4.97
% Mean	98.20	98.70	99.27	99.60	99.40	99.033
Sd	0.019	0.018	0.024	0.022	0.016	0.020
% Rsd	0.020	0.018	0.024	0.022	0.016	0.020
Conc. Rep.	Curcumin Concentration found (mg/ml)					Mean
Conc. Rep.	1	2	3	4	5
1	0.99	1.98	2.99	3.98	4.98
2	0.98	1.98	2.97	3.96	4.95
3	0.99	1.98	2.98	3.95	4.99
4	1.01	1.99	3.01	3.96	4.96
5	1.02	1.98	2.98	4.01	4.98
Mean	0.998	1.982	2.986	3.972	4.972
% Mean	99.80	99.10	99.53	99.30	99.44	99.435
Sd	0.016	0.004	0.015	0.024	0.016	0.015
% Rsd	0.016	0.005	0.015	0.024	0.017	0.015

Sensitivity:

The specified procedure was used to estimate the quantification limit (LOQ) and detection limit (LOD) of RLX and CUR values. The LOD and LOQ of developed method were calculated based on the standard deviation of response and slope of the linearity curve and results stated that LOD and LOQ of 0.15mg/ml and 0.45mg/ml of RXL and that's of CUR was 0.24mg/ml and 0.75 mg/ml respectively as shown in Table 7.

System suitability:

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 7. The %RSD of tailing factor and number of theoretical plates of the column for the six replicate injections were found to be 1.19±0.49% and 3461.16±0.98% for RLX; 1.20± 0.38% and 3529.667±0.91% 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.

CONCLUSION:

For measuring RLX and CUR, the RP-HPLC approach was found to be rapid, responsive, specific, and consistent and hence the elution is via isocratic technique. To our knowledge, no attempts have been made to use the stability-indicating analytical approach to estimate this multidrug combination. All the active components were effectively resolved and quantified with good resolution. As a result, the recommended validated stability-indicating approach was effectively used to determine the concentrations of RLX and CUR in bulk. The present study followed the ICH guidelines for method development and validation, and it satisfied the fundamental approval criteria. The technique was considered accurate, specific, linear, consistent, and robust within the boundaries. The development and validation of the RLX and CUR processes given here may be utilized for the intended purpose. It will be expanded to include a review of its estimation in plasma and other biological fluids, as well as the possibility of using it for quality control sample estimation and cleaning method analysis during cleaning validation and method may also be used to measure the amount of RLX and CUR drugs in nanostructured lipid carriers that we will manufactured. As a result, it can be easily used in future in vitro and in vivo research to assess therapeutic potential.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Amity University Uttar Pradesh,Noida for their kind support for research work.

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Received on 28.10.2021 Modified on 27.11.2021

Research J. Pharm. and Tech 2022; 15(9):4218-4225.

DOI: 10.52711/0974-360X.2022.00709

Replicates	Concentration (mg/ml)	Mean AUC		Response Ratio
Replicates	Concentration (mg/ml)	RLX	CUR	RLX	CUR
1	1	95.920	116.912	95.92	116.912
2	2	184.624	239.185	92.312	119.5925
3	3	271.890	348.7213	90.630	116.2404
4	4	362.357	481.4765	90.589	120.3691
5	5	470.238	611.4037	94.047	122.2807
			Mean SD % RSD	56.425	119.119
				1.002	3.770
				1.762	3.362

Figure 1: Chemical structure of Raloxifene

Figure 2: Chemical structure of Curcumin

MATERIALS AND METHOD:

Material:

1

Figure 3: Calibration Curve of RLX

Accuracy: