INTRODUCTION:

Ubidecarenone (UBD) chemically it is 2, 5-cyclohexadiene-1,4-dione,2- [ (2E, 6E, 10E, 14E, 18E, 22E, 26E, 30E, 34E)- 3, 7, 11, 15, 19, 23, 27, 31, 35, 39-decamethyl - 2, 6, 10, 14, 18, 22, 26, 30, 34, 38-tetracontadecaenyl]-5, 6-dimethoxy-3- methyl(Figure 1). It is official in USP¹, BP2, Ph.Eur³ and JP⁴.

Figure 1: Molecular structure of Ubidecarenone.

UBD is naturally occurring coenzyme structurally similar to vitamin K. It is involved in electron transport and energy production in the mitochondria. It is also found in dietary supplements and has been tried in conditions associated with coenzyme deficiency and also as an adjunct in cardiovascular disorders including mild and moderate heart failure^5,6. UBD is primarily indicated in conditions like cardiomyopathy^7,8. Literature survey revealed that there is a RP-HPLC method was reported for the estimation of UBD in combination with other drugs^9-15. HPLC method for the analysis of coenzyme Q10 in human plasma was developed to investigate its clinical application¹⁶. HPLC and Mass spectrometry method was reported for the determination of Ubidecarenone Q10 and its synthesis related impurities¹⁷. 1H NMR Spectroscopic method was reported for the determination of Coenzyme Q10 in food supplements¹⁸. No methods were reported for the estimation of UBD by UV Spectroscopy. Hence the present study aims to develop a simple, sensitive, precise, accurate, reproducible and rapid UV Spectroscopic methodfor the estimation of UBD in bulk and in formulation.

MATERIALS AND METHODS:

Apparatus:

A Perkin Elmer Double Beam UV-Visible Spectrophotometer with spectral width of 1nm or less, wavelength accuracy of ± 0.1nm and a pair of 10mm matched quartz cell was used to measure absorbance of all the solutions. A Shimadzu AUX-220 Digital analytical balance was used in the study.

Reagents and Materials:

UBD was obtained as a gift sample from Sai Mirra Inno pharm Private Limited, Chennai. Ubi Q 300 Capsule containing UBD 300mg (Fourrts Laboratories Pvt. Limited, Chennai) was procured from the local market. Ethanol (AR grade) was purchased from Loba Chemie India Limited, Mumbai.

Experimental conditions:

According to the solubility characteristics, ethanol was selected as a solvent for the analysis of UBD.

Preparation of standard stock solution:

10mg of UBD raw material was weighed accurately and transferred in to 10ml volumetric flask, dissolved in ethanol and made up to the volume with ethanol. This solution contains 1mg/ml concentration.

Preparation of working standard stock solution:

1ml of the above standard stock solution was transferred in to 10ml volumetric flask and made up to the volume with ethanol. This solution contains 100µg/ml concentration.

Selection of wavelengths for estimation and stability studies:

The above working standard stock solutions was further diluted with ethanol to get the concentration of 10µg/ml and the solution was scanned between 200 to 400nm using ethanol as blank. From the spectra, λmax was found to be 275nm and was selected as an analytical wavelength. The stability was performed by measuring the solution at different time intervals.It was observed that UBD in ethanol was stable up to 24 hours in ethanol at 275nm.

Preparation of calibration graph:

The working standard stock solution of UBD (1-8ml) was transferred into a series of 10ml volumetric flasks and made up to volume with ethanol. The absorbance of different concentration solution was measured at 275 nm. The calibration curve was constructed by plotting concentration Vs absorbance. UBD was found to be linear with the concentration range of 10- 80µg/ml.

Quantification of raw material:

3.0ml of working standard stock solution was taken into a series of six 10ml standard flasks and the volume was made up to mark with ethanol. The absorbance of these solutions was measured at 275nm. The amount of UBD present in the raw material was determined by using slope and intercept values from the calibration graph.

Quantification of tablet formulation:

Ten capsule of formulation (Ubi Q 300 equivalent to UBD 300mg) were weighed accurately and the average weight of each capsule was found. The capsule content equivalent to 10mg of UBD was weighed and transferred in to 100ml volumetric flask. Pipette out 3 ml and volume made up to 10ml of ethanol to obtain 30 µg/ml. The absorbance of six replicates was measured and the amount was calculated by using regression equation. This procedure was repeated for six times.

Recovery studies:

Preparation of UBD raw material stock solution:

40mg of UBD was accurately weighed and transferred into 10ml volumetric flask and sufficient ethanol was added to dissolve the substance and made up to the mark with the same. This contains 4mg/ ml concentration.

Procedure:

The recovery experiment was done by adding known concentrations of raw material stock solution of UBD to the pre-analyzed formulation. The capsule content equivalent to 10mg of UBD was taken into a series of six 100ml volumetric flask. To that 2ml, 2.5ml and 3ml of above raw material stock solutions (30µg/ml) were added into a series of each six 100ml volumetric flask and dissolved with ethanol and sonicated for 15 minutes. The supernatant liquid was filtered through a Whatmann filter paper No.41. From each series, 3ml of the clear solution was transferred into 10ml volumetric flask and made up to 10ml with ethanol. The absorbance of three replicates was measured at their selected wavelength. The amount of drug recovered from formulation was calculated. The procedure was repeated for three times of each solution.

VALIDATION:

The method was validated as per ICH guidelines^22,23. The methods were validated with respects to linearity, LOD (Limit of detection), LOQ (Limit of quantitation), precision, accuracy and ruggedness. The validation parameters are explained as given below.

Linearity:

A calibration curve was plotted between concentration and absorbance. UBD was linear in the concentration range of 10–80µg/ml at 275nm. The optical characteristics such as Beer’s law limits, correlation Coefficient, slope, intercept, Sandell’s sensitivity and molar absorptivity were calculated.

Sensitivity:

The limit of detection (LOD) and limit of quantitation (LOQ) parameters were calculated using the following equations; LOD = 3.3σ/ S and LOQ = 10σ/ S, where σ is standard deviation of y intercept of calibration curve (n = 6) and s is slope of regression equation.

Precision:

The precision of the method was confirmed by repeatability and intermediate precision. The repeatability was performed by the analysis of formulation was repeated for six times with the same concentration. The amount of each drug present in the tablet formulation was calculated. The % RSD was calculated. The intermediate precision of the method was confirmed by intraday and inter day analysis i.e. the analysis of formulation was repeated three times in the same day and on three successive days. The amount of drugs was determined and % RSD also calculated.

Accuracy:

Accuracy of the method was confirmed by recovery studies. To check the accuracy of the developed method and to study the interference of formulation excipients, analytical recovery experiments were carried out by using standard addition method in three different concentrations viz 80%, 100% and 120% of the formulation concentration. From the total amount of drug found, the percentage recovery was calculated. This procedure was repeated for three times for each concentration. The % RSD was calculated.

Ruggedness:

The ruggedness test of analytical assay method is defined as the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions such as different labs, different analysis, different lots of reagents etc. Ruggedness is a measure of reproducibility of test results under normal expected operational conditions from laboratory to laboratory and from analyst to analyst.

RESULTS AND DISCUSSION:

A simple, precise, accurate and rapid UV Spectrophotometric method was developed for the estimation of UBD in bulk and in capsule formulation. From the solubility data, ethanol was used as a solvent for the analysis. The spectrum of UBD was recorded in the wavelength range of 200-400nm. From the spectrum, the wavelength selected for the analysis was 275nm (Figure 2). The stability of UBD was studied by measuring the absorbance at different time intervals. It was observed that the drug was stable up to 24 hours in ethanol.

Figure 2: Uv Spectrum Of Ubidecarenone In Ethanol (10 Μg/ Ml)

Validation data of the proposed methods:

Beer’s law obeyed in the concentration range of 10-80 μg/ml at 275nm. The correlation coefficient values were found to be 0.9996; this indicates that the absorbance was linear with the concentration range of 10-80μg/ml. The data for the optical characteristics are shown in (Table 1).

Table 1: Spectral and Linearity Characteristics Data

Parameters	values
lmax (nm)	275
Beer’s law limit (mg/ ml)	10 - 80
Sandell’s sensitivity (mg/ cm²/ 0.001 A.U)	0.0458
Molar absorptivity (L mol^–1 cm^–1)	1.7405×10⁴
Correlation coefficient (r)	0.9996
Regression equations (y = mx + c)	Y = 0.0128x + 0.0166
Slope (m)	0.0128
Intercept (c)	0.0166
LOD (mg/ ml)	0.0371
LOQ (mg/ ml)	0.1125
Standard error	0.0155

Mean of six observations

The LOD and LOQ were found to be 0.0371 and 0.1125, respectively. The low values indicated that the sensitivity of the method.

To confirm the developed method, quantification of raw material was done and the amount was calculated. The percentage amount of UBD was found to be 99.02% ± 0.7232. The amount found by this method was close to 100%. Hence this method can be applied for the analysis of formulation. The results are shown in (Table 2).

The percentage label claim present in tablet formulation was found to be 98.78 ± 1.3682. Precision of the method was confirmed by the repeated analysis of formulation for six times. The % RSD values were found to be 1.3850. The low % RSD values indicated that the method has good precision were shown in (Table 3).

Table 3: Results of Analysis of Capsule Formulation

Sample No.	Labelled Amount (mg/Tab)	Amount Found (mg/Tab)*	Percentage Obtained	Mean (%)	SD	% RSD	SE
1		297.6	99.22
2		296.5	98.85
3		298.6	99.54
4	300 mg	292.7	97.58	98.78	1.3682	1.3850	0.5584
5		290.6	96.88
6		301.9	100.66

*Mean of six observations

Table5: Recovery Analysis Of Formulation By UV Method

Sample

Amount Present

(µg/ml)

Amount Added*

(µg/ml)

Amount Found*

(µg/ml)

Amount recovered*

(µg/ml)

% Recovered*

RSD

29.9495

23.9096

23.7108

23.7060

53.4939

53.5903

53.6060

23.5444

23.6408

23.6565

98.47

99.70

99.79

0.5166

0.5190

29.9495

29.6807

29.5843

29.7409

59.6385

59.5602

59.6626

29.6890

29.6107

29.7131

100.02

100.08

99.90

29.9495

35.8674

35.8075

35.9325

65.5361

65.5542

65.5848

35.5866

35.6047

35.6353

99.22

99.43

99.17

Mean

99.53

Mean of three observations

CONCLUSION:

A simple, sensitive, precise, accurate and rapid method was developed and validated for the analysis of UBD in Bulk and in formulation. The low % RSD values of all the validation parameter indicated that the developed method can be effectively applied for the routine quality control analysis of UBD in bulk and in capsule dosage form.

ACKNOWLEDGEMENT:

The authors are very much thankful to Dr. G. Murugananthan, Principal, Swamy Vivekanandha College of Pharmacy, Tiruchengode, for his support and also thankful to the management for providing the necessary facilities to carry out the research work successfully.

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Received on 13.08.2019 Modified on 28.09.2019

Research J. Pharm. and Tech 2020; 13(5):2233-2237.

DOI: 10.5958/0974-360X.2020.00401.1

Sample No.	Amount Found (µg/ ml)*	*Percentage Obtained (%)**	Mean (%)	SD	% RSD	SE
1	29.3614	97.86
2	29.5421	98.46
3	29.8132	99.36	99.02	0.7232	0.7303	0.2952
4	29.8253	99.40
5	29.7771	99.22
6	29.9518	99.83