INTRODUCTION:

Raloxifene hydrochloride (RAL), a non-steroidal benzothiophene acts as a selective estrogen receptor modulator and antiosteoporotic approved by the FDA for prevention and treatment of invasive breast cancer and osteoporosis in postmenopausal women and women at high risk of developing breast cancer.^1,2

Naringin, (NAR), (4′,5,7-Trihydroxyflavanone 7-rhamnoglucoside) is a flavanone glycoside, a well-known natural flavonoid in citrus fruit that exhibited a wide variety of biological activities.^3-5

Flavonoids are interested in dietary bioactive compounds with a wide promising beneficial effect on health especially as cancer chemopreventive with decreased toxicity and synergistic effect when co-taken with synthetic chemopreventive.⁶

Moreover, flavonoids have been shown to have inhibitory effects on various cancer cells by a particular inhibition mechanism suggesting that they may serve as possible adjunctive chemopreventive agents for the treatment and prevention of various cancers(62).^7,8 Therefore, to increase patient compliance for long-term medication and to ensure the healthcare system's sustainability, we were interested in developing a new drug delivery system that might contain both the drugs. But a significant challenge in terms of simple and rapid simultaneous determination of drug concentrations in their combinations or therapeutic delivery systems. Numerous estimation techniques have been developed recently to enable mixture resolution, including Vierordt's method.^9,10 Studies have been published that demonstrate the utility of the simultaneous equation or Vierordt's method for estimating the concentrations of two or more drugs in a combined mixture of pharmaceutical formulations.^11,12

According to the literature survey, it was found that some analytical methods for estimation of RAL and NAR separately or in combination with other drugs have been reported. for raloxifene estimation; spectrophotometric methods,¹³ chromatographic HPLC methods¹⁴, LC/MS/MS method¹⁵ have been reported and for naringin, a number of chromatographic HPLC methods,¹⁶ LC-MS-MS methods¹⁷ have also been reported.

The current study is part of a larger effort to develop and validate a quick, specific, accurate, economical, and precise UV spectrophotometric method for the simultaneous estimation of two drugs in a nanoformulation dosage form. Since the combination of these two drugs is not recognized by any pharmacopeia, there is no recognized procedure for simultaneously estimating raloxifene and naringin in their combined nanoformulation. Based on the above-mentioned fact, it was decided to develop and validate a simple, new, precise, accurate method for quantification of raloxifene and naringin in a bulk binary mixture and utilizing it to estimate active substances in the pharmaceutical formulations.

MATERIALS AND METHODS:

Raloxifene hydrochloride was gifted by Aarti Drugs Ltd., India, while naringin was purchased from Tokyo Chemical Industry CO., LTD. (Tokyo, Japan). Analytical grade methanol was purchased from SRL (Mumbai, India). Glyceryl monostearate and Tween 80 were obtained from Gattefosse India Pvt. Ltd. (Mumbai, India).

Standard and working solutions:

Both RAL and NAR are insoluble in water. Methanol was chosen as the solvent to ensure that both drugs were completely soluble. Stock solutions of RAL and NAR were prepared by dissolving 10mg of each compound in methanol in a volumetric flask with sonication for 10 min. The final volumes of both solutions were adjusted with methanol to obtain stock solutions of RAL and NAR at a concentration of 100µg/ml. Working standard solutions of RAL and NAR were prepared by diluting the standard stock solution of 100μg/mlin a 10ml volumetric flask. The final volumes of both the solutions were adjusted with methanol to get working standard solutions with a concentration of 2, 4, 6, 8, 10, 15, and 20μg/mlof RAL and NAR, separately.

Preparation of sample solutions of binary mixture:

In a ratio of 1:1, a methanolic sample solution of RAL and NAR binary mixture was prepared. 5ml of each drug's standard stock solution (100µg/ml) was transferred to a 10ml volumetric flask to obtain a sample solution at a concentration of 50µg/ml for each drug, from which the different dilutions were made.

Selection of wavelengths and method selection:

Working standard solutions of both the drugs were scanned in the UV range 200-400nm. The UV overlain spectrum of RAL and NAR is shown in Fig.1. From the overlain spectra, wavelengths 289nm (λ_max of RAL) and 284nm (λ_max of NAR) were observed. Due to a small difference in the absorbance maxima that can lead to absorbance interference with each other and no isosbestic point exist, so both drugs can be simultaneously estimated by Vierordt's method at their respective maximum wavelengths.

Validation of the concentration range:

The absorbance was measured for RAL and NAR in the concentration range of 2, 4, 6, 8, 10, 15, and 20μg/mland at 289nm and 284nm for both the RAL and NAR respectively. Calibration graphs plotted for RAL at 289 nm and NAR at 284nm at a concentration range of 2-20 μg/ml for both drugs are shown in Fig.2.

Vierordt’s method:

As the λ_maxof RAL and NAR are closer and not even exhibiting a difference of ±10nm, henceforth no isoabsorption point (λ _iso) was exhibited as seen in Fig. 2, hence estimation of both RAL and NAR was not possible using the absorptive ratio method. Henceforth simultaneous estimation or Vierordt’s method was selected for the determination of RAL and NAR in the binary mixture and combined nanoformulation dosage forms. From overlain spectra, wavelengths 289 nm (𝜆_max of RAL) and 284 nm(𝜆_max of NAR) were selected for analysis of both drugs using the simultaneous equation method. Consequently, it may be possible to determine both drugs following Vierordt’s method, the concept of absorptivity was employed to develop a method for simultaneous estimation of both these drugs.^18,19 The absorptivity (a) value represents the extinction coefficient and is calculated using the following equation:

a= A/C (1)

Where, a = Absorptivity, A = Absorbance (nm), and C = Concentration (gm/100ml).

Using equation (1), absorptivities of raloxifene and naringin were calculated at both λ1 (289nm) and λ2 (284 nm), for which, the absorbance of any three dilutions (n=3) of raloxifene was determined at λ1 (289nm). The absorbance values were then divided by the corresponding concentrations to determine the absorptivity of raloxifene at λ1 and λ2. An average of absorptivity values was taken and denoted as ar1 and ar2, where ‘a’ denotes absorptivity, ‘r’ denotes raloxifene, ‘1’ denotes λ1, and ‘2’ denotes λ2 i.e., 289 nm and 284nm, meaning, the absorptivity of raloxifene at λ1 and λ2 respectively. The absorptivity values of RAL at λ1 and λ2 were calculated using the formula (1) and the results are presented in Tables 1. Similarly, the process was repeated for naringin, that is, the absorbance of three dilutions of naringin was determined at λ1 (289nm) and λ2 (284nm) separately and an1 and an2 were calculated, where ‘a’ denotes absorptivity, ‘n’ denotes naringin, ‘1’ denotes λ1 (289nm) and ‘2’ denotes λ2 (284nm). The absorptivity values of NAR at λ1 and λ2 were calculated using the formula (1) and the results are presented in Tables 2.

To determine the levels of both drugs in unknown samples the subsequent two simultaneous equations derived from equation (1) are solved.

A1 = ar1Cr + an1 Cn (2)

A2 + ar2 Cr + an2 Cn (3)

Where, A1 = Absorbance of test sample at λ1, A2 = Absorbance of test sample at λ2, ar1 = Absorptivity of raloxifene at λ1 (289 nm), ar2 = Absorptivity of raloxifene at λ2 (284 nm), an1 = Absorptivity of naringin at λ1 (289 nm), an2 = Absorptivity of naringin at λ2 (284 nm), Cr = Concentration of raloxifene (to be determined), Cn = Concentration of naringin (to be determined).

The concentration of drugs r (RAL) and n (NAR) in sample solutions were determined by the simultaneous equation method using the following formula:

For estimation of raloxifene (Cr) = an1 A2 – an2 A1/ an1ar2 – an2 ar1 (4)

For estimation of naringin (Cn) = ar2 A1 – ar1 A2 / an1 ar2 – an2 ar1 (5)

Method validation:

For the validation of the developed method, the absorbance was measured for RAL and NAR in the concentration range of 2, 4, 6, 8, 10, 15, and 20μg/mlat 289nm and 284nm for both the RAL and NAR respectively. Calibration graphs were plotted for RAL at 289nm and NAR at 284nm at a concentration range of 2-20 μg/ml for both drugs are shown in Fig. 2.

Linearity and range:

To verify the linearity and range of both drugs, the diluted stock solutions of each drug was been used. The dilutions of raloxifene in the range of 2-20µg/ml were prepared and a calibration curve was plotted between the concentration and absorbance at 289 and 284nm, respectively. Similarly, dilutions of naringin stock solution in the range of 2-20µg/ml were prepared, and the calibration curves at 289 and 284nm were plotted separately.

Accuracy:

Three dilutions of the stock binary mixture solution were prepared to determine the developed method's accuracy. The developed method was then used to analyze these solutions with known concentrations. To verify the developed method's accuracy further, the pre-analyzed sample was spiked with an additional 50%, 100%, and 150% of the drug concentrations, and the binary mixtures were analyzed using the developed method.

Precision:

To verify the developed method's precision, three different dilutions of the stock mixture binary solution were made, and the method's precision (intra- and inter-day precision) was determined by determining the concentration levels of both drugs in the binary mixture. The method's precision and repeatability were determined by determining the concentrations of raloxifene and naringin in three different dilutions of stock mixture solution at three different times across the day. While the method's intermediate precision was assessed by determining the concentrations of raloxifene and naringin in three different dilutions of the binary mixture solution over three days.

Limit of detection and limit of quantification:

The limit of detection (LOD) is defined as the smallest amount of analyte identified but not necessarily computed in a sample. While the limit of quantification (LOQ) is the smallest amount of analyte which can be counted with sufficient precision and accuracy in a sample. LOD and LOQ are calculated by the following equation: LOD = 3.3 N/B, LOQ = 10 N/B. Where 'N' is the standard deviation of the drugs' absorbance (n = 3), which is used to quantify noise, and 'B' is the slope of the accompanying calibration curve.

Robustness:

The robustness of an analytical process refers to its ability to remain unaffected by minor intentional changes to system parameters. It serves as a measure of the procedure's reliability for simultaneous estimation. The proposed method's robustness was calculated by altering the wavelengths by ±2 nm.

RESULTS AND DISCUSSION:

Method development:

Both RAL and NAR are highly hydrophobic and soluble in methanol. Hence methanol was used as a solvent for standard preparation. The use of Vierordt’s method allowed these drugs to be determined simultaneously. The absorbance values were taken at 289 nm for RAL and 284 nm for NAR, respectively, and then the calibration curves were plotted at their respective wavelength for both the drugs, and their absorptivity values were calculated for each concentration as shown in Tables 1 and 2.

Fig 1. Overlain spectra of raloxifene and naringin.

Fig 2. Calibration curves of RAL at 289 nm and NAR at 284 nm.

Table 1. Calibration data and absorptivity of RAL at 289 and 284nm in methanol

Conc. μg/ml	Absorbance nm at (289) ±SD	Conc. Found μg/ml	Absorptivity of RAL at 289 nm	Absorbance nm at (284) ±SD	Conc. Found μg/ml	Absorbavity of RAL at 284 nm
2	0.134±0.001	1.89	670.00	0.133±0.0030	1.89	665.00
4	0.288±0.003	4.15	720.00	0.285±0.0020	4.14	712.50
6	0.406 ±0.001	5.89	677.77	0.401±0.0010	5.86	668.33
8	0.549±0.004	7.97	686.25	0.545±0.0035	7.98	680.83
10	0.704 ±0.001	10.23	703.66	0.698±0.0021	10.24	697.66
15	1.015±0.005	14.79	676.66	1.011±0.0040	16.19	674.22
20	1.374±0.034	20.05	687.00	1.358±0.0056	19.99	679.16
Average			689.50			683.13

Table 2. Calibration data and absorptivity of NAR at 289 and 284 nm in methanol

Conc. μg/ml	Absorbance nm at (289) ±SD	Conc. Found μg/ml	Absorptivity of NAR at 289 nm	Absorbance nm at (284) ±SD	Conc. Found μg/ml	Absorbivity of NAR at 284 nm
2	0.05467±0.006	2.10	273.33	0.0630±0.005	2.09	316.66
4	0.11067±0.005	4.00	276.66	0.1250±0.006	4.01	313.33
6	0.16133±0.015	5.72	268.88	0.1810±0.015	5.74	302.22
8	0.21500±0.001	7.55	268.75	0.2396±0.015	7.54	299.58
10	0.29133±0.015	10.14	291.33	0.3236±0.015	10.14	323.67
15	0.46300±0.008	15.74	308.66	0.5060±0.056	15.78	337.33
20	0.56130±0.003	19.31	280.66	0.6250±0.002	19.47	312.50
Average			282.45			316.75

Linearity:

The statistical results of the linear regression for RAL and NAR are shown in Table 3. The coefficient of determination indicated good linearity: 0.9994 and 0.9951 for RAL and NAR, respectively. For the concentration of 2-20 μg /mlfor RAL and NAR, the absorbance values for these concentrations ranged from (0.1 - 1.36) for RAL and (0.06 -0.62 ) for NAR.

Table 3: Statistical and Validation Parameter

Statistical parameters	RAL	NAR
λ_max	289 nm	284 nm
Linearity range (μg /ml)	2-20	2-20
Regression equation (y = mx+c) Slope (m) Intercept (c)	Y=0.0971x+0.0011 0.0971 0.0011	Y=0.1002x- 0.0049 0.1002 0.0049
Coefficient of correlation (R²)	0.9998	0.9999
LOD (μg/ ml)	0.57	4.25
LOQ (μg/ ml)	0.613	4.55

Accuracy:

The average recovery percentages were 102.26 % and 96.45 % for RAL and NAR, respectively (Table 4). These results indicate the accuracy of the method.

Table 4. Accuracy study of simultaneous UV method in methanol (N = 3)

Drug	Recovery level (%)	Sample conc. (μg/ml)	Conc. added (μg/ml)	Total conc. (μg/ml)	Conc. recovered ±SD (μg/ml)	%Recovery
RAL	50	10	5	7.5	7.36±1.43	98.13
	100	10	10	10	10.62±0.90	106.2
	200	10	20	15	15.37±1.10	102.46
Average						102.26
NAR	50	10	5	7.5	7.25±0.89	96.66
	100	10	10	10	9.490.1.32	94.90
	200	10	20	15	14.67±0.97	97.80
Average						96.45

Precision:

The precision parameters (% recovery) expressed as repeatability (intraday) and as intermediate precision (inter-day) are presented in Table 5. For RAL and NAR, all % recovery values were within the acceptable limits for repeatability in analyses performed during 3 consecutive days, and also for intermediate precision. Thus, the proposed method has good precision in the simultaneous determination of RAL and NAR.

Limit of detection and limit of quantification:

LOD and LOQ for RAL and NAR were found to be 0.57 µg/ml and 0.613 µg/ml for RAL and 4.25 µg/ml and 4.55 µg/ml for NAR. respectively. These values show that the proposed method has good sensitivity and results are presented in Table 3.

Robustness:

The responses of RAL and NAR did not change significantly when the analytical conditions were modified (Table 6). The % RSD value calculated from the robustness study was found to be less than 2% for RAL and NAR, indicating that the method is robust. These observations confirm the robustness of the method for the determination of RAL and NAR in the pure binary mixture and dosage form.

Table 5. Precision of simultaneous UV method in methanol

Repeatability (Intraday precision) (n = 3)					Intermediate (Interday precision) (n=3)
Drug	Conc	Found Conc ±SD (μg/ml)	Recovery %	Mean % Recovery	Found Conc ± SD (μg/ml)	% Recovery	Mean % Recovery
RAL	5	5.07±1.12	101.4	102.78	5.22±0.29	104.4	102.58
	10	10.28±0.65	102.8		10.103±0.15	101.03
	20	20.62±1.5	104.15		20.463±0.75	102.316
NAR	5	4.87±1.89	97.4	96.13	4.86±0.25	97.2	96.02
	10	9.46±1.04	94.6		9.43±0.12	94.3
	20	19.28± 3.18	96.4		19.311±1.5	96.55

Table 6: Robustness studies (by changing of wavelength)

Analyte	Wavelength (±nm)	Amount Present (20µg/ml)	Amount Present %	% RSD
RAL	289	20.49	102.45	1.12
RAL	287	19.83	99.14	1.71
NAR	284	19.01	95.05	1.09
NAR	286	19.27	96.35	1.23

Apply the developed and validated method to the prepared nanoformulations:

20 ml of SLN loaded with RAL and NAR, equivalent to 20 mg of each drug, was transferred to a 100 ml volumetric flask and dissolved in methanol. The solutions were then subjected to a 20-minute sonication treatment. Following that, the final volume was adjusted with methanol and vacuum filtered. For a total of 20 minutes, the filtrate was centrifuged at 6,000 RPM. Then clear supernatant solutions were transferred to a separate flask for drug content analysis. Then absorbance was measured at 289 nm and 284 nm a minimum of three times. The amount of RAL and NAR in each sample was calculated and results are presented in Table 7. These results showed a good agreement between the results found and the quantities labeled by the formulator.

Table 7. Validation assay for the content of RAL and NAR in their prepared SLNs dosage form, by applying Vierordt’s method

RAL		NAR
Amount present (mg)	Amount present (%) label claim	Amount present (mg)	Amount present (%) label claim
0.2032	101.6	0.1913	95.65
0.1981	99.05	0.1925	96.25
0.2015	100.75	0.1952	97.6
Average	100.47	96.5	96.5
SD	1.3		0.9987
%RSD	1.29		1.035

CONCLUSION:

The proposed UV spectrophotometric method for simultaneous estimation of RAL and NAR in their binary mixture has been successfully established by Vierordt’s method. The precision, accuracy, specificity, linearity and range, the limit of detection (LOD) and limit of quantitation (LOQ), and robustness were all validated for the proposed process. The proposed technique was validated in compliance with the guidelines established by the International Conference on Harmonization. The assay of RAL and NAR in lab-prepared SLN was found to be 100.47% and 96.5 % respectively. Thus, this method provides a precise, accurate, and cost-effective method for determining the presence of these drugs simultaneously in a binary mixture or a composition.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

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Received on 28.10.2021 Modified on 08.04.2022

Research J. Pharm. and Tech 2023; 16(3):1199-1204.

DOI: 10.52711/0974-360X.2023.00199