MATERIAL AND METHODS:

Instruments:

Spectrophotometer: Shimadzu UV-1800 PC, dual beam UV–visible spectrophotometer with two matched 1-cm quartz cells, connected to an IBM compatible personal computer. Bundled UV-PC personal spectroscopy software version (2.43) was used to process the absorption and the derivative spectra. The spectral band width was 0.2 nm with wavelength scanning speed of 2800 nm/min and 0.1 nm interval. Balance: Sartorius CPA224S, Sonicator: Elma S 30 H Elmasonic.

Samples:

Pure form:

THC, BEN and CNCo were kindly supplied by Eva pharma for pharmaceuticals and medical appliances, Kafr El Gabal, Giza, Egypt. Their purity was found to be 99.69% ± 0.65, 100.01% ± 0.32 and 100.37% ± 0.78 for the three drugs, in order, according to the Manufacturer method.

Pharmaceutical formulation:

Thiotacid compound^® capsules batch No. 607659. Each capsule is labeled to contain 300 mg of THC, 40 mg of BEN and 0.25 mg of CNCo and manufactured by Eva Pharma for Pharmaceuticals and Medical Appliances, Kafr El Gabal, Giza, Egypt.

Solvents:

Ethyl alcohol; spectroscopic grade (El-NASR Pharmaceutical Chemicals Co., Abu-Zaabal, Cairo, Egypt), Acetonitrile, HPLC grade (Sigma-Aldrich, St. Louis, USA) and Deionized Water.

Procedure:

Standard solutions:

Thioctic acid standard solution (2.00 mg/mL), was prepared by dissolving 0.2 gm of THC in the least amount of solvent mixture (acetonitrile: water, 50:50 v/v) in 100-mL volumetric flask and then the volume was completed with ethanol.

Benfotiamine standard solution (0.10 mg/mL), was prepared by dissolving 0.01 gm of BEN in the least amount of solvent mixture (acetonitrile: water, 50:50 v/v) in 100-mL volumetric flask and then volume was completed with ethanol.

Cyanocobalamin stock standard solution (2.00 mg/mL), was prepared by dissolving 0.2 gm of CNCo in least amount of water in 100-mL volumetric flask then the volume was completed with ethanol.

Cyanocobalamin working standard solution (0.20 mg/mL) in ethanol.

The prepared solutions were used within 4 weeks when stored at 4 ^◦C and covered by aluminium foil.

Spectral characteristic of THC, BEN and CNCo:

The absorption spectrum of 5000µg/mL of THC, 500.00µg/mL of BEN and 20.00µg/mL of CNCo is scanned against blank over the range of 200–650 nm.

Construction of calibration curves:

Aliquots equivalent to 2.00 –18.00mg of THC and 0.02–0.30 mg BEN were accurately transferred from their respective standard solutions into two separate series of 10-mL volumetric flasks then completed to volume with ethanol. Aliquots equivalent to 0.10–2.00 mg CNCo were accurately transferred from its working standard solution into series of 10-mL volumetric flasks then completed to volume with ethanol. The absorption spectra of the prepared solutions were scanned in the range 200.0-450.0 nm for THC and BEN and 200.0- 650.0nm for CNCo and stored in the computer.

For first derivative spectrophotometric method (¹D):

This method was applied for determination of CNCo. The previously scanned spectra of CNCo were subjected to derivative calculation using Δλ = 4 and scaling factor = 100. The values of the ¹D amplitudes were recorded at 546.0 nm for CNCo.

For ratio difference spectrophotometric method (RDSM):

This method was applied for determination of THC and BEN. The stored spectra of THC were divided by the spectrum of 14.00 µg/mL BEN, while BEN spectra were divided by the spectrum of 1000.00 µg/mL THC.

Calibration curves for THC and BEN were constructed by plotting the difference between the amplitudes of ratio spectra at 237.2 and 306.6 nm for THC and 236.0 and 276.0 nm for BEN, versus the corresponding concentrations and the regression equations are computed.

For first derivative of the ratio spectra method (¹DD): This method was applied for determination of THC and BEN. The stored spectra of THC are divided by the spectrum of 14.00 µg/mL BEN, then the first derivative of the ratio spectra (¹DD) was calculated using Δλ = 4 nm and scaling factor =1. The amplitude of the ¹DD is recorded at 330.8 nm. A calibration graph relating the peak amplitude at 330.8 nm to the corresponding concentrations of THC was constructed. Similarly, for determination of BEN, its stored spectra were divided by spectrum of 1000.00 µg/mL THC, then the first derivative of the ratio spectra (¹DD) with Δλ = 4 nm and scaling factor = 10 was obtained. The amplitude of the ¹DD was recorded at 286.8 nm. A calibration graph relating the peak amplitude at 286.8 nm to the corresponding concentrations of BEN was constructed.

For ratio subtraction method (RSM):

This method was applied for determination of BEN. A calibration curve was constructed relating the absorbance of BEN at 241.5 nm (λ_max) to the corresponding concentrations and the regression equation is computed.

Application of the proposed methods for determination of THC, BEN and CNCo in their laboratory prepared mixtures:

Different aliquots of THC were transferred from its standard solution (2.00mg/mL) into a series of 10-mL measuring flasks to which different aliquots of BEN using its standard solution (0.10mg/mL) were added and the volume was completed with ethanol. The absorption spectra of the prepared solutions were recorded from 200.0 to 450.0 nm and stored in the computer. Aliquots equivalent to 2.10 mg of THC and 0.28 mg of BEN were accurately transferred from their standard solutions (2.00 mg/mL) and (0.10 mg/mL) while aliquots equivalent to 0.00175 mg of CNCo were transferred from its working standard solution (0.20 mg/mL), were added into 10-mL measuring flask, completed to volume with ethanol. The spectrum of the prepared solution was recorded from 200.0 to 650.0 nm. For the determination of BEN through ratio subtraction method, the spectra of the laboratory-prepared mixtures were divided by the spectrum of 1000.00 µg/mL THC. The absorbance in the plateau region was subtracted (the constant). The obtained curves were multiplied by the spectrum of 1000.00 µg/mL THC. The obtained curves were used for the determination of BEN from the corresponding regression equation. For the rest of the proposed methods followed as under calibration of each one. The concentration of each drug was calculated using the specified regression equation.

Application of the proposed methods to the analysis of THC, BEN and CNCo in Thiotacid compound^® capsules:

Two solutions have been prepared, one for determination THC and BEN and the other for determination of CNCo.

For determination of THC and BEN, the content of 10 capsules were accurately weighed, an amount of the powder equivalent to one capsule was accurately weighed and transferred to100-mL volumetric flask, extracted with the least amount of acetonitrile: water mixture (50:50, v/v) and sonicated for 20 min. The volume was completed with ethanol re-sonicated for 20 min, filtered into a 100-mL volumetric flask. Aliquot of 0.7 mL was taken from the filtrate solution to a 10-mL volumetric flask and completed to volume with ethanol to obtain solution with a final concentration 210.00 µg/mL THC and 28.00 µg/mL BEN. The spectra of the prepared solutions were recorded from 200.0 to 450.0 nm. Proceed as under laboratory prepared mixture. The concentration of each drug was calculated using the specified regression equation.

For determination of CNCo, an amount of the powder equivalent to one capsule was accurately weighed and transferred to a 25-mL volumetric flask, extracted in least amount of water and sonicated for 15 min then completed to volume with ethanol and sonicated again for 15 min then the solutions were filtered into a 25-mL volumetric flask to obtain solution with a final concentration 10.00 µg/mL CNCo. The spectra of the prepared solution were recorded from 200.0 to 650.0 nm. The procedure was followed as under laboratory prepared mixture.

Results and discussion:

Thioctic acid is co-formulated with BEN and CNCo, the three compounds were only determined by HPLC ⁴¹, however spectrophotometric methods are favorable during the rush hours in the quality control laboratories. Spectrophotometric methods are considered non-selective; hence a lot of mathematical manipulations were developed to deal with the uv-signals in order to resolve the overlapped spectral bands. In this work four methods were developed and validated for determination of THC, BEN and CNCo in bulk powders, laboratory prepared mixture and capsules.

As shown in Fig. 2, the absorption spectra of the three drugs were found to be overlapped in the region 200.0 – 400.0 nm, which hinders their simultaneous determination by direct methods. CNCo is a colored compound and hence its absorption spectrum is extended in the visible region as shown in Fig.2 while THC and BEN have no absorption.

Fig. 2. Absorption spectra of 5000.00 µg/mL Thioctic acid (—), 500.00 µg/mL Benfotiamine (- - -) and 20.00 µg/mL Cyanocobalamin (....) in ethanol.

On the other hand, CNCo concentration in pharmaceutical formulations is very small relative to the other two components where THC and BEN could be considered and determined as binary mixture due to the lack of interference of CNCo in the ratio of dosage form (210.00 :28.00: 0.175 mg) THC: BEN: CNCo, respectively (Fig.3).

Fig. 3. Absorption spectra of 210.00 µg/mL Thioctic acid (—), 28.00 µg/mL Benfotiamine (- - -) and 0.175.00 µg/mL Cyanocobalamin (....) in ethanol (in dosage form ratio).

First derivative spectrophotometric method (¹D):

This method is a convenient and accurate way for simultaneous analysis of multiple drugs in its pharmaceutical dosage form^42-45 . It was applied for determination of CNCo. The values of the ¹D amplitudes were measured at 546.0 nm for the determination of CNCo where THC and BEN have zero contribution (Fig. 4).

Linear relationships between derivative amplitude and drug concentration were obtained over the concentration range of 10.00-200.00 µg/mL CNCo. Regression parameters are shown in Table 1.

Fig. 4. First-derivative spectra of 5000.00 µg/mL Thioctic acid (—), 500.00 µg/mL Benfotiamine (- - -) and 20.00 µg/mL Cyanocobalamin (....) in ethanol.

Ratio difference spectrophotometric method (RDSM):

This method was applied for determination of THC and BEN. This technique having the ability of solving overlapped spectra without prior separation. The utility of ratio difference method is to calculate the unknown concentration of a component of interest without interference of other component in the mixture. The only requirement is the contribution of the two components at the two selected wavelengths λ₁ and λ₂ where the ratio spectrum of the interfering component gives the same amplitudes (constant amplitude) and the component of interest shows significant difference in its two amplitudes at the two selected wavelengths. Similarly, another two wavelengths were selected for the estimation of the second component.

For determination of THC, absorption spectra of the laboratory-prepared mixtures (THC and BEN) were divided by the standard spectrum of BEN' (14.00 µg/mL) producing ratio spectra which represent THC/BEN' + constant as shown in (Fig. 5), the difference in amplitude at 237.2 nm and 306.6 nm was calculated, hence the constant values for BEN was cancelled. The concentration of THC was calculated using the corresponding regression equation (obtained by plotting the difference in the amplitudes at 237.2 and 306.6 nm of the ratio spectra of THC/BEN' against the corresponding concentrations). Similarly, the two selected wavelengths for the determination of BEN using standard THC' (1000.00 µg/mL) as a divisor were 236.0 and 276.0 nm as shown in (Fig. 6).

Fig. 5. Ratio spectra of laboratory prepared mixtures of Thioctic acid and Benfotiamine (—) and ternary mixture of Thioctic acid, Benfotiamine and Cyanocobalamin (-..-..-) and 14.00 µg/mL Benfotiamine (- - -) using 14.00 µg/mL of Benfotiamine as a divisor and ethanol as blank.

Fig. 6. Ratio spectra of laboratory prepared mixtures of Thioctic acid and Benfotiamine (—) and ternary mixture of Thioctic acid, Benfotiamine and Cyanocobalamin (-..-..-) and 1000.00 µg/mL Thioctic acid (−.−.−) using 1000.00 µg/mL of Thioctic acid as a divisor and ethanol as blank.

First derivative of the ratio spectra method (¹DD):

This method was applied for determination of THC and BEN. The effect of variables such as divisor concentration and Δλ were studied to optimize the ¹DD method⁴⁶^,⁴⁷. Different divisors were tried, a standard spectrum of 14.00 µg/mL of BEN as divisor was considered as suitable for the THC determination and a standard spectrum of 1000.00 µg/mL of THC as divisor was considered as suitable for the BEN determination. For determination of THC Δλ = 4 nm was selected with a scaling factor = 1 while Δλ = 4 nm was selected with a scaling factor = 10 for determination of BEN. Thioctic acid was divided on BEN and BEN was divided on THC and the corresponding first derivative of the ratio-spectra was shown in (Figs. 7 and 8). For calibration graph, the wavelengths 330.8 nm for THC and 286.8 nm for BEN were selected which exhibited the best linear response to the analyte concentration. The proposed method is applicable over the ranges 200.00 - 1800.00 µg/mL for THC and 2.00 - 30.00 µg/mL for BEN. Regression parameters are shown in Table 1.

Fig. 7. First derivative of ratio spectra of laboratory prepared mixtures of Thioctic acid and Benfotiamine (—) and ternary mixture of Thioctic acid, Benfotiamine and Cyanocobalamin (-..-..-) using 14.00 µg/mL of Benfotiamine as a divisor and ethanol as blank.

Fig. 8. First derivative of ratio spectra of laboratory prepared mixtures of Thioctic acid and Benfotiamine (—) and ternary mixture of Thioctic acid, Benfotiamine and Cyanocobalamin (-..-..-) using 1000.00 µg/mL of Thioctic acid as a divisor and ethanol as blank.

Ratio subtraction method (RSM):

This method was applied for determination of BEN. This technique⁴⁸ depends on that, when a mixture of BEN and THC where the spectrum of THC is more extended (Fig. 2), the determination of BEN could be done by scanning the zero order absorption spectra of the laboratory prepared mixtures of BEN and THC, dividing them by a chosen concentration (1000.00 µg/ml) of standard THC (THC' = divisor) producing a new ratio spectra that represent (BEN/ THC') + constant as shown in (Fig. 9), then subtraction of the absorbance values of these constants (THC/ THC') in plateau as shown in (Fig. 10), followed by multiplication of the obtained spectra by (THC') the divisor as shown in (Fig. 11). At the end, the original spectra of (BEN) could be obtained which are used for direct determination of BEN at 241.5 nm and calculation of the concentration from the corresponding regression equation.

Fig. 9. Ratio spectra of laboratory prepared mixtures of Thioctic acid and Benfotiamine (—) and ternary mixture of Thioctic acid, Benfotiamine and Cyanocobalamin (-..-..-) using 1000.00 µg/mL of Thioctic acid as a divisor and ethanol as a blank.

Fig. 10. Ratio spectra of laboratory prepared mixtures of Thioctic acid and Benfotiamine (—) and ternary mixture of Thioctic acid, Benfotiamine and Cyanocobalamin (-..-..-) using 1000.00 µg/mL of Thioctic acid as a divisor and ethanol as a blank after subtraction of the constant.

Fig. 11. The estimated absorption spectra of Benfotiamine obtained by the proposed ratio subtraction method for the analysis of laboratory prepared mixtures after multiplication by the divisor.

Method validation:

Validation was done according to ICH recommendations⁴⁹.

Linearity and Range:

The linearity of the methods was evaluated by analyzing six concentrations of THC, eight concentrations of BEN and eleven concentrations of CNCo ranging among 200.00 –1800.00 µg/mL, 2.00 –30.00 µg/mL and 10.00 –200.00 µg/mL, respectively. Each concentration was repeated three times. The assay was performed according to the experimental conditions previously mentioned.

Accuracy:

The accuracy of the results was checked by applying the proposed methods for determination of different samples of THC, BEN and CNCo. The concentrations were obtained from the corresponding regression equations and percentage recoveries were calculated. The obtained results suggested good accuracy of the proposed methods, Table 1.

Precision:

Three concentrations of THC (200.00, 600.00, 1200.00 µg/mL), BEN (4.00, 16.00, 28.00 µg/mL) and CNCo (60.00, 160.00, 200.00 µg/mL) were analyzed three times intra-daily (Repeatability) and inter-daily (Intermediate precision) using the proposed methods. The relative standard deviations were calculated (Table 1).

Selectivity:

Selectivity of the methods was achieved by the analysis of different laboratory prepared mixtures of THC and BEN within their linearity ranges. Satisfactory results were shown in Table 2.

Application of the method in analysis of capsules:

The proposed UV methods were applied for the determination of THC, BEN and CNCo in their combined pharmaceutical formulation Thiotacid compound capsules. The high percentage recoveries values confirm the suitability of the proposed methods for the routine determination of these compounds in combined formulation. The accuracy of the proposed procedures is further assessed by applying the standard addition technique. The results obtained are shown in Table 3.

Statistical analysis:

Results obtained by the proposed methods for determination of pure form of THC, BEN and CNCo were statistically compared to those obtained by applying the manufacturer’s method. The results showed no significant differences (Table 4).

Table 1: Regression and validation parameters of the proposed methods for determination of thioctic acid, benfotiamine and cyanocobalamin.

Parameter	Thioctic acid		Benfotiamine			Cyanocobalamin
Parameter	¹DD	RDSM	RSM	¹DD	RDSM	¹D
Range	200.00 – 1800.00 µg/mL		2.00 – 30.00 µg/mL			10.00 – 200.00 µg/mL
Slope	0.0052	0.0076	0.0451	0.1064	0.1564	0.0194
Intercept	-0.0095	-0.0670	-0.0042	-0.0195	-0.0408	0.00072
Correl. Coef. (r)	1	0.9999	0.9999	0.9998	0.9998	0.9999
Accuracy (mean ± SD)	99.34 ± 0.37	99.36 ± 0.54	99.83 ± 0.36	99.64 ± 0.71	99.70 ± 0.79	100.81 ± 1.22
%RSD^a	0.43	1.67	0.38	0.47	1.30	0.45
%RSD^b	0.93	1.91	1.06	1.49	1.46	0.70
LOD*	9.876	17.789	0.443	0.467	0.538	1.842
LOQ*	29.929	53.908	1.341	1.415	1.632	5.582

^a and ^b are the intra-day, inter-day precision, respectively (n = 9) relative standard deviation of 3 concentrations repeated three times on the same day and 3 successive days calculated for 200.00, 600.00, 1200.00 µg/mL Thioctic acid, 4.00, 16.00, 28.00 µg/mL of Benfotiamine and 60.00, 140.00, 200.00 µg/mL of Cyanocobalamin.

*LOD (Limit of Detection) = 3.3 × SD/slope & LOQ (Limit Of Quantification) = 10 × SD/slope.

Table 2: Determination of thiocitc acid and benfotiaminein their laboratory prepared mixtures by the proposed methods.

Ratio in µg/mL	Thioctic acid (recovery%^b)		Benfotiamine (recovery%^b)
	¹DD at 330.8 nm	RDSM at 237.2 nm - 306.6 nm	RSM at 241.5 nm	¹DD at 286.8 nm	RDSM at 236.0 nm – 276.0 nm
210:28^a	101.21	98.71	100.48	101.55	102.66
225:30	101.25	99.52	98.31	101.89	100.94
400:20	101.01	98.54	98.35	98.61	101.95
500:20	101.37	99.33	98.19	98.84	102.71
700:10	100.68	101.80	100.59	101.17	99.99
700:15	100.39	100.28	100.42	96.61	102.76
Mean	100.98	99.69	99.39	99.78	101.83
SD	0.38	1.20	1.21	2.08	1.14
%RSD	0.37	1.21	1.22	2.09	1.12

^a the ratio in Thiotacid compound capsules (including 0.175 µg/mL CNCo).

^baverage of three determinations.

Table 3: Determination of thiocitc acid, benfotiamine and cyanocobalamin in Thiotacid compound^® capsules by the proposed spectrophotometric methods and application of standard addition technique.

Product		¹D	¹DD	RDSM	RSM
Product		Found% ± SD	Found% ± SD	Found% ± SD	Found% ± SD
Thiotacid compound capsules 300 mg B.N. (608016) *Labelled to contain 300 mg thioctic acid, 40 mg benfotiamine and 250 µg cyanocobalamin.**	Thioctic acid	-	99.32 ± 0.73	95.73 ± 1.79	-
	Standard addition	Recovery %	Recovery %	Recovery %	Recovery %
	Mean ± SD	-	99.03 ± 1.71	100.69 ± 1.35	-
		Found% ± SD	Found% ± SD	Found% ± SD	Found% ± SD
	Benfotiamine	-	115.20 ± 0.17	117.04 ± 0.23	114.84 ± 0.13
	Standard addition	Recovery %	Recovery %	Recovery %	Recovery %
	Mean ± SD	-	101.31 ± 0.62	100.11 ± 1.20	101.34 ± 0.64
		Found% ± SD	Found% ± SD	Found% ± SD	Found% ± SD
	Cyanocobalamin	113.92 ± 0.80	-	-	-
	Standard addition	Recovery %	Recovery %	Recovery %	Recovery %
	Mean ± SD	101.88 ± 0.70	-	-	-

*Acceptance limit for thioctic acid is 90% - 110%, benfotiamine is 90% - 150% and cyanocobalamin 90% - 150% according to dosage form manufacturer.

Table 4: Statistical comparison between the results obtained by the proposed methods and the Manufacturer method for the determination of thiocitc acid, benfotiamine and cyanocobalamin in pure powdered form.

Parameter	Thioctic acid			Benfotiamine				Cyanocobalamin
Parameter	Manufacturer method*	¹DD	RDSM	Manufacturer method*	RSM	¹DD	RDSM	Manufacturer method*	¹D
Mean	99.69	99.34	99.36	100.01	99.83	99.64	99.70	100.37	100.81
SD	0.65	0.37	0.54	0.32	0.36	0.71	0.79	0.78	1.22
Variance	0.425	0.138	0.298	0.108	0.131	0.509	0.631	0.611	1.511
n	5	5	5	5	5	5	5	4	5
Student's t-test	_	1.015 (2.306)**	0.856 (2.306)**	_	0.785 (2.306)**	1.047 (2.306)**	0.785 (2.306)**	_	0.625 (2.365)**
F	−	3.06 (6.39)**	1.42 (6.39)**	−	1.21 (6.39)**	4.69 (6.39)**	5.82 (6.39)**	−	2.47 (9.12)**

*Manufacturer method of thiocitc acid and benfotiamine is HPLC using C8 column (5 µm, 25 cm x 4.6 mm i.d.), 0.025 M phosphoric acid: acetonitrile (50:50, v/v) as a mobile phase, at a flow rate of 2 mL/min and UV detection at 220.0 nm and the same for cyanocobalamin but with UV detection at 550.0 nm.

** Theoretical values of t and F at p=0.05.

Conclusion:

From the previous discussion, it could be concluded that the proposed methods are simple, do not require sophisticated techniques or instruments. They are also sensitive and selective and could be used for routine analysis of THC, BEN and CNCo in their combined dosage form without prior separation. The methods are also suitable to be an alternative to HPLC assays in quality control laboratories.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

References:

1. The British Pharmacopoeia. 2015: The Stationary Office on behalf of the Medicines and Healthcare products Regulatory Agency (MEHRA)-© Crown Copyright.

2. Martindale, The Complete Drug Reference, . 37 ed. Vol. A. 2011: Pharmaceutical Press.

3. Walash M, Metwally M-S, El-Brashy A, and Abdelal A. Kinetic Spectrophotometric Determination of Some Sulfur Containing Compounds in Pharmaceutical Preparations and Human Serum. Il Farmaco. 2003; 58(12): 1325-1332.

4. Walash M, El-Brashy A, Metwally M, and Abdelal A. Spectrophotometric and Kinetic Determination of Some Sulphur Containing Drugs in Bulk and Drug Formulations. B. Kor. Chem. Soc. 2004; 25(4): 517-524.

5. El-Enany N, Belal F, and Rizk M. Spectrophotometric Determination of Thioctic Acid in its Dosage Forms Through Complex Formation with Pd (II). J. Chin. Chem. Soc-Taip. 2007; 54(4): 941-948.

6. Korićanac Z, Čakar M, Tanasković S, and Jovanović T. Spectrophotometric Determination of Thioctic (α-Lipoic) Acid in Water and Pharmaceutical Preparations. J. Serb. Chem. Soc. 2007; 72(1): 29-35.

7. Taha EA, Hassan NY, Aal FA, and Fattah LE-SA. Fluorimetric Determination of Some Sulfur Containing Compounds Through Complex Formation with Terbium (Tb^{+ 3}) and Uranium (U^{+ 3}). J. Fluoresc. 2007; 17(3): 293-300.

8. Ibrahim F, Ali F, Ahmed S, and Tolba M. Kinetic Spectrofluorometric Determination of Thioctic Acid in Bulk and Pharmaceutical Preparations Via its Oxidation with Cerium (IV). J. Chin. Chem. Soc-Taip. 2007; 54(4): 925-932.

9. Satoh S, Toyo’oka T, Fukushima T, and Inagaki S. Simultaneous Determination of α-Lipoic Acid and its Reduced Form by High-Performance Liquid Chromatography with Fluorescence Detection. J. Chromatogr. B. 2007; 854(1): 109-115.

10. Durrani AI, Schwartz H, Nagl M, and Sontag G. Determination of Free α-Lipoic Acid in Foodstuffs by HPLC Coupled with CEAD and ESI-MS. Food Chem. 2010; 120(4): 1143-1148.

11. Khan A, Khan MI, Iqbal Z, Ahmad L, Shah Y, and Watson DG. Determination of Lipoic Acid in Human Plasma by HPLC-ECD Using Liquid–Liquid and Solid-Phase Extraction: Method Development, Validation and Optimization of Experimental Parameters. J. Chromatogr. B. 2010; 878(28): 2782-2788.

12. Siangproh W, Rattanarat P, and Chailapakul O. Reverse-Phase Liquid Chromatographic Determination of α-Lipoic Acid in Dietary Supplements Using a Boron-Doped Diamond Electrode. J. Chromatogr. A. 2010; 1217(49): 7699-7705.

13. Poongothai S, Ilavarasan R, and Karrunakaran C. Simultaneous and Accurate Determination of Vitamins B₁, B₆, B₁₂ and Alpha-Lipoic Acid in Multivitamin Capsule by Reverse-Phase High Performance Liquid Chromatographic Method. Int. J. Pharm. Pharm. Sci. 2010; 2(S4): 133-9.

14. Khan A, Iqbal Z, Watson DG, Khan A, Khan I, Muhammad N, Muhammad S, Nasib HA, Iqbal N, and Kashif M. Simultaneous Determination of Lipoic Acid (LA) and Dihydrolipoic Acid (DHLA) in Human Plasma Using High-Performance Liquid Chromatography Coupled with Electrochemical Detection. J. Chromatogr. B. 2011; 879(20): 1725-1731.

15. Kumar SA, Debnath M, Rao JS, and Sankar DG. A new, simple, sensitive, accurate and rapid analytical method development and validation for simultaneous estimation of Pregabalin, Mecobalamin and Alpha Lipoic Acid in bulk as well as in pharmaceutical dosage form by using RP-HPLC. Asian J. Res. Chem. 2014; 7(4): 426-433.

16. Kumar SA, Debnath M, Rao JS, and Sankar DG. Stability Indicating Analytical Method Development and Validation for Simultaneous Estimation of Pregabalin, Mecobalamin and Alpha Lipoic Acid in Bulk as well as in Pharmaceutical Dosage Form by using RP-HPLC. Research J. Pharm. and Tech. 2014; 7(9): 7.

17. Vani R, Kumar BV, and Mohan GK. Analytical Method Development and Validation for the Determination of Allopurinol and Alphalipoic Acid Using Reverse Phase HPLC Method in Bulk and Tablet Dosage Form. Research J. Pharm. and Tech. 2015; 8(2): 207.

18. Corduneanu O, Garnett M, and Brett AMO. Anodic Oxidation of α-Lipoic Acid at a Glassy Carbon Electrode and Its Determination in Dietary Supplements. Anal. Lett. 2007; 40(9): 1763-1778.

19. Abbas M and Radwan A. Novel Lipoate-Selective Membrane Sensor for the Flow Injection Determination of α-Lipoic Acid in Pharmaceutical Preparations and Urine. Talanta. 2008; 74(5): 1113-1121.

20. Miranda M, Del Rio R, Del Valle M, Faundez M, and Armijo F. Use of Fluorine-Doped Tin Oxide Electrodes for Lipoic Acid Determination in Dietary Supplements. J. Electroanal. Chem. 2012; 668(2012): 1-6.

21. Tan X, Li Q, Zhang X, Shen Y, and Yang J. A Novel and Sensitive Turn-On Fluorescent Biosensor for the Determination of Thioctic Acid Based on Cu ²⁺-Modulated N-Acetyl-l-Cysteine Capped CdTe Quantum Dots. RSC Adv. 2015; 5(55): 44173-44182.

22. Hegazy MA, Abdelwahab NS, and Fayed AS. A Novel Spectral Resolution and Simultaneous Determination of Multicomponent Mixture of Vitamins B₁, B₆, B₁₂, Benfotiamine and Diclofenac in Tablets and Capsules by Derivative and MCR–ALS. Spectrochim. Acta A. 2015; 140: 524-533.

23. Agrawal O and Telang N. Development and Validation of UV Spectrophotometric Method for Estimation of Benfotiamine in Bulk and Dosage Form. Asian J. Pharm. Anal. 2016; 6(3): 133-137.

24. Adithya BP and Mahesh MVJ. Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Benfotiamine and Metformin hydrochloride in Tablet Dosage Form. RGUHS J. Pharm. Sci. 2012; 2(4): 87-91.

25. Adithya BP and Vijayalakshmi M. Development and Validation of RP-HPLC Method for the Estimation of Benfotiamine in Bulk and Dosage Form. Int. J. Pharm., Chem. Biol. Sci. 2012; 2(3): 354-360.

26. Nanaware DA, Bhusari VK, and Dhaneshwar SR. Validated HPLC Method for Simultaneous Quantitation of Benfotiamine and Metformin Hydrochloride in Bulk Drug and Formulation. Int. J. Pharm. Pharm. Sci. 2013; 5(2): 138-42.

27. Hadad G, Salam RA, and Hameed EA. Application of LC and HPTLC-Densitometry for the Simultaneous Determination of Two Multicomponent Mixtures Containing Pyridoxine Hydrochloride and Cyanocobalamine. Acta Chromatogr. 2013; 25(3): 431-450.

28. Kim J, Hopper CP, Connell KH, Darkhal P, Zastre JA, and Bartlett MG. Development of A Novel Method for the Bioanalysis of Benfotiamine and Sulbutiamine in Cancer Cells. Anal. Methods-UK. 2016; 8(28): 5596-5603.

29. Fayed AS, Hegazy MA-M, and Wahab NSA. Chromatographic Analysis of a Multicomponent Mixture of B₁, B₆, B₁₂, Benfotiamine, and Diclofenac; Part I: HPLC and UPLC Methods for the Simultaneous Quantification of These Five Components in Tablets and Capsules. J. AOAC Int. 2016; 99(6): 1513-1521.

30. Fayed AS and Hegazy MA-M. Chromatographic Analysis of Multicomponent Mixture of Vitamins B₁, B₆, B₁₂, Benfotiamine and Diclofenac; Part II: LC-Tandem MS/MS Method for Simultaneous Quantification of Five Components Mixture in Pharmaceutical Formulations and Human Plasma. RSC Adv. 2016; 6(45): 39409-39423.

31. Sakhare RS, Pekamwar SS, and Mohkare DP. Development and Validation of Stability Indicating HPTLC Method for the Determination of Metformin Hydrochloride and Benfotiamine in Bulk and Combined Dosage Form. Indian J. Pharm. Educ. 2017; 51(2): S8-S16.

32. Karşilayan H. Quantitation of Vitamin B₁₂ by First-Derivative Absorption Spectroscopy. Spectrochim. Acta A. 1996; 52(9): 1163-1168.

33. Elzanfaly ES, Nebsen M, and Ramadan NK. Development and Validation of PCR, PLS, and TLC Densitometric Methods for the Simultanous Determination of Vitamins B₁, B₆ and B₁₂ in Pharmaceutical Formulations. Pak. J. Pharm. Sci. 2010; 23(4): 409-415.

34. Li H-B and Chen F. Determination of Vitamin B₁₂ in Pharmaceutical Preparations by A Highly Sensitive Fluorimetric Method. Fresen. J. Anal. Chem. 2000; 368(8): 836-838.

35. Song Z and Hou S. Sub-Picogram Determination of Vitamin B₁₂ in Pharmaceuticals and Human Serum Using Flow Injection with Chemiluminescence Detection. Anal. Chim. Acta. 2003; 488(1): 71-79.

36. Kucukkolbasi S, Bilber O, Ayyildiz HF, and Kara H. Simultaneous and Accurate Determination of Water-and Fat-Soluble Vitamins in Multivitamin Tablets by Using An RP-HPLC Method. Quím. Nova. 2013; 36(7): 1044-1051.

37. Abano E and Dadzie RG. Simultaneous Detection of Water-Soluble Vitamins Using the High Performance Liquid Chromatography (HPLC)-a review. Croat. J. Food Sci. Technol. 2014; 6(2): 116-123.

38. Antakli S, Sarkees N, and Sarraf T. Determination of Water-Soluble Vitamins B₁, B₂, B₃, B₆, B₉, B₁₂ and C on ODS Column 5 µm by High Performance Liquid Chromatography with UV-DAD Detection. Asian J. Chem. 2014; 26(20): 7035-7039.

39. Tomčik P, Banks CE, Davies TJ, and Compton RG. A Self-Catalytic Carbon Paste Electrode for The Detection of Vitamin B₁₂. Anal. Chem. 2004; 76(1): 161-165.

40. Chen J-H and Jiang S-J. Determination of Cobalamin in Nutritive Supplements and Chlorella Foods by Capillary Electrophoresis− Inductively Coupled Plasma Mass Spectrometry. J. Agr. Food Chem. 2008; 56(4): 1210-1215.

41. Salem H. LC Simultaneous Determination of Thioctic Acid, Benfotiamine and Cyanocobalamin in Thiotacid Compound Capsules. Chromatographia. 2010; 72(3-4): 327-330.

42. Bhatia NM, Deshmane SJ, More HN, and Choudhari PB. Simultaneous Spectrophotometric Estimation of the Amlodipine Besylate and Hydrochlorothiazide in Pharmaceutical Preparations and Biological Samples. Asian J. Res. Chem. 2009; 2(4): 394-397.

43. Vijayalakshmi R, Archana S, and Dhanaraju M. A Simultaneous Estimation of Perindopril and Losartan in Solid Dosage Forms by UV Spectrophotometry. Asian J. Res. Chem. 2010; 3(3): 571-573.

44. Seervi C, Pawar K, Dhabale P, Gonjari I, Raut C, and Gharge D. Development and Validation of UV Spectrophotometric Method of Ambroxol Hydrochloride in Bulk and Pharmaceutical Formulation. Asian J. Res. Chem. 2009; 2(4): 547-549.

45. Sharma M, Sharma A, Jain A, and Banerjee P. Spectrophotometric Estimation of Valsartan in Tablet Dosage Form. Asian J. Res. Chem. 2009; 2(4): 464-466.

46. Bhadoriya A, Padekar S, Patil V, Shinde S, Choudhari V, and Kuchekar B. Development and Validation of Ratio Spectra Derivative Spectrophotometric Method for Determination of Ternary Mixture of Aspirin, Atenolol and Amlodipine Besylate in Formulation. Asian J. Res. Chem. 2010; 3: 562-565.

47. Chabukswar A, Shinde S, Kuchekar B, Jagdale S, Lokhande P, Choudhari V, and Ingale K. Development and Validation of Novel UV Methods for Irbesartan and Hydrochlorothiazide Combination. Asian J. Res. Chem. 2010; 3(3): 728-731.

48. El-Bardicy MG, Lotfy HM, El-Sayed MA, and El-Tarras MF. Smart Stability-Indicating Spectrophotometric Methods for Determination of Binary Mixtures Without Prior Separation. J. AOAC Int. 2008; 91(2): 299-310.

49. International Conference on Harmonization (ICH), Q2B: Validation of Analytical Procedures: Methodology, US FDA 1997: Federal Register.

Received on 14.02.2019 Modified on 18.03.2019

Research J. Pharm. and Tech. 2019; 12(6): 2648-2656.

DOI: 10.5958/0974-360X.2019.00443.8