INTRODUCTION:

Tocotrienol (Figure 1) are distinguished from tocopherols by the presence of unsaturation in the isoprenic side chain. They both have four naturally occurring forms (α, β, γ and δ) that differ in the number and position of methyl group attached to the chromanol head¹. It is official listed in Merck index. Chemically it is 2-methyl-2-[(3E,7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-yl]-3,4-dihydro-2H-1 benzopyran-6-ol². Several studies have reported that Tocotrienols may have more potent antioxidation³and anticancer effects^4,5than tocopherols. On literature survey, several analytical methods has been identified to be reported for the quantification of Tocotrienol employing techniques such as HPLC,^6-8 GC-MS and LC-MS,⁹ Mass Spectrometry¹⁰. The current technique was developed with the aim to use simple statistical methodology employing Multivariate Calibration Technique with the aid of UV Spectrophotometry.

Figure 1: Chemical structure of Tocotrienol

Spectrophotometric techniques due to their accurate, precise, reproducible of procedure and low cost are employed as the favourable method in most of the laboratories^11-14. The preferred procedure is based on the direct estimation of Tocotrienol with a high scope of accuracy and precision. The technique is simple, economical and can be applied for the evaluation of Tocotrienol. The proposed method characterizes the use of the Ultraviolet spectral multilinear regression technique employing simple mathematical contents for the evaluation of Tocotrienol in pharmaceutical formulation.

Multilinear regression represents the conversion of single common class determination from one dependent variable to “m” dependent variables, which can be all the while incorporated into the calibration model¹⁵.

Under optimized experimental conditions, the statistical technique gives appreciable resolving ability and sensitivity at low cost for routine quality control analysis¹⁶.

Confirmation of the applicability of the developed method validated according to the International Conference on Harmonization (ICH)¹⁷. The developed technique was proposed to be validated as per ICH Q2(R1) protocol for analytical method validation to check the validness of the developed method.

The present work was framed with the target to develop simple, accurate, precise and sensitive with rapid analytical method for the quantification of Tocotrienol amidst the already reported complex analytical methods. Based on the above statement, a simple analytical method employing UV spectrophotometry aided multivariate calibration technique was proposed to be developed^18,19.

EXPERIMENTAL:

Chemicals and solvents employed:

· Ethanol

· The reference standard of Tocotrienol (99.7% w/w) was obtained as gift sample from Softgel Healthcare Private Ltd., Chennai, India. The marketed capsule formulation was procured from the local market (Tocofil softgel capsules – 200mg of Tocotrienol, by Fourrts India Laboratories Pvt. Ltd.,)

Solubility:

· Very freely soluble in ethanol.

Instrumentation:

· UV-Vis double beam Spectrophotometer (Lab India UV- 3092)

· Electronic balance (Shimadzu BL-220H)

· Ultra Sonic Bath (ILE, ILTC)

METHOD DEVELOPMENT:

Selection of solvent:

Tocotrienol was found to be freely soluble in ethanol, which was used as the solvent to solubilise the drug throughout the analysis.

Preparation of standard stock solution:

10mg of Tocotrienol was weighed accurately and transferred into a 10mL volumetric flask. 5mL of the solvent was added and the mixture was sonicated for 15 min. The final volume was made to 10mL with the solvent and mixed well (1mg/mL). Further 5mL of the above solution was pipetted out and transferred into a 50 mL volumetric flask and the final volume was made up to the mark employing the solvent and mixed well. The resultant solution was further diluted to yield concentrations ranging from 30 - 90µg/mL employing the solvent.

Determination of λ_max:

The standard stock solution of Tocotrienol was diluted with the solvent to obtain a concentration of 60µg/mL. This solution was scanned under the UV range from 400-200nm. Figure 2 represents the UV spectra of Tocotrienol.

Preparation of sample solution:

20 capsules of Tocotrienol (Tocofil softgel capsules, Label claim - 200mg of Tocotrienol) were weighed and their average weight was determined. The capsules were cut open and the contents were collected in a mortar and mixed well. A weight equivalent to 10mg of Tocotrienol was weighed from the mixed content and dissolved in 5 mL of solvent with the aid of sonication for 15min and the volume was finally made up to 10mL with the solvent. The above solution was mixed well and filtered. The filtrate was suitably diluted for further analysis.

METHOD VALIDATION:

The developed technique was validated as per the ICH Q2(R1) protocol to examine the validation parameters such as linearity, sensitivity, precision and accuracy²⁰.

Linearity:

The standard stock solution of Tocotrienol was diluted with the solvent to obtain concentrations of 30, 45, 60, 75 and 90µg/mL. To build up linear correlation and to cut off the instrumental fluctuations,^21,22 the absorbance of the prepared concentrations were recorded at five different wavelengths selected around the λ_max (293nm) of the drug, i.e., 289, 291, 293, 295 and 297nm respectively (Table 1) and the overlay UV spectra showing linearity is represented in Figure 3. The correlation coefficient values were calculated for the constructed linear regression equations individually at the five selected wavelengths (Table 2). The calibration graphs constructed at five selected wavelengths is represented in Figure 3.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

Limit of detection (LOD) and Limit of quantification (LOQ) values were calculated employing the slope and intercept values of the linear regression line, thereby the sensitivity of the developed method was established. (Table 2).

Precision:

Intraday and interday precision studies were performed by measuring the absorbance of 100% concentration level (60µg/mL) of the linearity solution at all the five selected wavelengths. The selected concentration was scanned 6 times a day (intraday precision) and at three different days (interday precision). The absorbance values recorded at the selected wavelengths for intraday and interday precision studies were provided in Tables 3 – 6 and the spectral representation in Figure 4 represents the overlay UV spectra for inter and intraday precision studies. The Standard deviation (SD) and percentage relative standard deviation (%RSD) values were calculated.

Assay:

The absorbance of the extracted sample solution was recorded at 293nm. The amount of drug present in the formulation was calculated and the assay results are tabulated in Table 7.

Accuracy (Recovery studies):

Recovery studies were carried out by standard addition method to ascertain the accuracy of the developed technique at 50%, 100% and 150% concentration levels. From the prepared stock solutions of standard and sample, 0.2mL of sample solution was pipetted into three different 10mL volumetric flasks and 0.1, 0.4 and 0.7mL from the standard stock solution were added into the above volumetric flask respectively. The final volume was made up to the mark with ethanol. The % recovery values were calculated. The results of recovery studies were tabulated in Table 8 and its overlay UV spectra is represented in Figure 5.

RESULTS AND DISCUSSION:

The absorption maxima of Tocotrienol were observed at 293 nm.

Figure 2: UV Spectra of Tocotrienol

Linearity:

The developed method was reported to be linear within the specified concentration range of 30 – 90µg/mL. Linear regression equation was constructed for all the five selected wavelengths of 289, 291, 293, 295 and 297 nm. The correlation coefficient values obtained were found to be more than 0.998.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ values calculated were found to range between 0.34 – 0.53µg/mL and 1.05 – 1.6µg/mL respectively.

Precision:

Intraday and interday precision studies were carried out. % RSD values for intraday and interday precision were observed to be in the range of 1.59 -1.70 and 1.43-1.55 respectively, which was found well below the acceptance criteria of 2% as per ICH guidelines. The low estimated %RSD value demonstrates that the developed technique was precise.

Assay:

The absorbance of the sample solution was recorded at 293 nm and the quantity of Tocotrienol present in the capsule formulation was calculated. The assay percentage of the drug was found to be 99.58% w/w and the calculated %RSD was found to be less than 2%.

Recovery:

The percentage recovery of the drug was calculated and observed to be within the range of 99.35 - 102.9% w/w, which was found to be within the limit of 97 – 103% w/w according to ICH protocol. Hence the method can be reported as accurate.

CONCLUSION:

The developed simple and rapid UV spectrophotometric aided Multivariate calibration technique was found linear, sensitive, accurate and precise for the evaluation of Tocotreinol in capsule formulation. Multivariate calibration technique was reported to be superior over the other reported techniques as the absorbance of the drug is measured at five different selected wavelengths, which makes the method more reliable. The quantification of Tocotrienol by the current developed methodology was compared with the other reported methods and the present method was found more sensitive with low reported LOD and LOQ values. All the validation parameters assessed were reported to be within the limit specified in accordance to ICH guidelines. The developed method can be extended for the quantification of Tocotrienol in various other available dosage forms of the drug such as tablets, injectables and inhalation powders. Therefore, a simple and rapid method using mathematical contents was developed, which was found more predictable than the other spectrophotometric methods and is strongly recommended for the routine quality control analysis of Tocotrienol in Pharmaceutical formulations.

Figure 3: Calibration graph at five selected wavelengths

Figure 4: Overlay UV Spectra of Tocotrienol showing inter and intraday precision studies

Figure 5: Overlay UV Spectra of Terbutaline sulphate showing accuracy

Table 1: Multivariate UV calibration at five selected wavelengths

Concentration (µg/mL)	Absorbance (nm)
Concentration (µg/mL)	289	291	293	295	297
30	0.230	0.238	0.242	0.238	0.230
45	0.343	0.355	0.361	0.356	0.342
60	0.457	0.473	0.479	0.473	0.455
75	0.574	0.594	0.602	0.594	0.572
90	0.686	0.710	0.719	0.709	0.683

Table 2: Linearity data showing system suitability parameters at the selected wavelengths

Wavelength (nm)	Regression equation	r²	Average of Slope	SD of Intercept	LOD (µg/mL)	LOQ (µg/mL)
289	Y=0.0076x+0.0018	0.999	0.00768	0.00114	0.48	1.48
	Y=0.0077x+0.0006	0.998
	Y=0.0077x+0.002	0.998
	Y=0.0077x+0.0004	0.998
	Y=0.0077x+0.0032	0.998
291	Y=0.0079x+0.0006	0.999	0.00796	0.001163	0.48	1.46
	Y=0.008x+0.0008	0.998
	Y=0.008x+0.002	0.999
	Y=0.0079x+0.0032	0.998
	Y=0.008x+0.0028	0.998
293	Y=0.0079x+0.005	0.999	0.00806	0.001192	0.48	1.47
	Y=0.0081x+0.002	0.998
	Y=0.0081x+0.002	0.999
	Y=0.0081x+0.003	0.998
	Y=0.0081x+0.002	0.998
295	Y=0.0078x+0.0032	0.999	0.00796	0.000841	0.34	1.05
	Y=0.008x+0.0016	0.998
	Y=0.008x+0.002	0.999
	Y=0.008x+0.001	0.998
	Y=0.008x+0.0014	0.998
297	Y=0.0076x+0.002	0.999	0.00766	0.001238	0.53	1.61
	Y=0.0076x+0.002	0.998
	Y=0.0077x+0.0032	0.999
	Y=0.0077x+0.0006	0.999
	Y=0.0077x+0.0038	0.999

Table 3: Absorbance values for intraday precision studies

Concentration (µg/mL)	Number of repetitions	Absorbance
Concentration (µg/mL)	Number of repetitions	289 nm	291 nm	293 nm	295 nm	297 nm
60	1	0.462	0.478	0.485	0.478	0.46
	2	0.462	0.479	0.484	0.478	0.461
	3	0.482	0.498	0.504	0.498	0.48
	4	0.474	0.49	0.496	0.489	0.471
	5	0.467	0.482	0.489	0.483	0.471
	6	0.464	0.48	0.486	0.479	0.462

Table 4: Absorbance values for interday precision studies

Concentration (µg/mL)	Number of repetitions	Absorbance
Concentration (µg/mL)	Number of repetitions	289 nm	291 nm	293 nm	295 nm	297 nm
60	1	0.475	0.49	0.496	0.489	0.471
	2	0.465	0.481	0.487	0.48	0.463
	3	0.466	0.482	0.489	0.481	0.464
	4	0.483	0.499	0.505	0.498	0.48
	5	0.469	0.485	0.491	0.484	0.467
	6	0.479	0.495	0.501	0.494	0.476

Table 5: Intraday precision study

Concentration (µg/mL)	Description	289 nm	291 nm	293 nm	295 nm	297 nm
60	Mean	0.4685	0.4845	0.490667	0.484167	0.4675
	SD	0.007994	0.007893	0.00784	0.007985	0.007868
	%RSD	1.70	1.62	1.59	1.64	1.68

Table 6: Interday precision study

Concentration (µg/mL)	Description	289 nm	291 nm	293 nm	295 nm	297 nm
60	Mean	0.472833	0.488667	0.494833	0.487667	0.470167
	SD	0.007333	0.007285	0.007111	0.007285	0.006795
	% RSD	1.55	1.49	1.43	1.49	1.44

Table 7: Assay of Tocotrienol in marketed pharmaceutical formulation

Label claim (mg)	Amount estimated (mg)	%Assay
200.0	198.21	99.10
	199.44	99.72
	198.98	99.49
	199.52	99.76
	199.74	99.87
Average		99.58
SD		0.30
% RSD		0.30

Table 8: Recovery studies

Wavelength (nm)	Conc. levels (%)	Sample Conc. Present (µg/mL)	Standard Conc. (µg/mL)	Final Conc. (µg/mL)	Amount recovered (µg/mL)	% Recovery
289	50	20	10	30	29.84	99.46
	100	20	40	60	60.15	100.25
	150	20	70	90	89.65	99.61
291	50	20	10	30	29.97	99.90
	100	20	40	60	59.98	99.96
	150	20	70	90	89.76	99.73
293	50	20	10	30	30.89	102.9
	100	20	40	60	59.89	99.81
	150	20	70	90	90.91	101.01
295	50	20	10	30	29.97	99.9
	100	20	40	60	59.61	99.35
	150	20	70	90	90.97	101.07
297	50	20	10	30	29.82	99.40
	100	20	40	60	59.99	99.98
	150	20	70	90	89.96	99.95

ACKNOWLEDGEMENT:

The authors are thankful to The Management of SRM Institute of Science and Technology and SRM College of Pharmacy, Kattankulathur for providing various reprographic sources for carrying out this research work successfully.

CONFLICT OF INTEREST:

The authors report no conflicts of interest on the study.

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Received on 29.09.2020 Modified on 17.07.2021

Research J. Pharm. and Tech 2022; 15(10):4503-4508.

DOI: 10.52711/0974-360X.2022.00755