1758 Mohamed et al.: Journal of AOAC International Vol. 94, No.

6, 2011
DIETARY SUPPLEMENTS

Spectrofluorimetric Determination of Some Water-Soluble Vitamins

Abdel-Maaboud I. Mohamed, Horria A. Mohamed, Niveen M. Abdel-Latif,1 and Marwa R. Mohamed Assiut University, Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, Assiut, Egypt Two simple and sensitive spectrofluorimetric methods were developed for determination of three water-soluble vitamins (B1, B2, and B6) in mixtures in the presence of cyanocobalamin. The first one was for thiamine determination, which depends on the oxidation of thiamine HCl to thiochrome by iodine in an alkaline medium. The method was applied accurately to determine thiamine in binary, ternary, and quaternary mixtures with pyridoxine HCl, riboflavin, and cyanocobalamin without interference. In the second method, riboflavin and pyridoxine HCl were determined fluorimetrically in acetate buffer, pH 6. The three water-soluble vitamins (B1, B2, and B6) were determined spectrofluorimetrically in binary, ternary, and quaternary mixtures in the presence of cyanocobalamin. All variables were studied in order to optimize the reaction conditions. Linear relationship was obeyed for all studied vitamins by the proposed methods at their corresponding exc or em. The linear calibration curves were obtained from 10 to 500 ng/mL; the correlation ranged from 0.9991 to 0.9999. The suggested procedures were applied to the analysis of the investigated vitamins in their laboratoryprepared mixtures and pharmaceutical dosage forms from different manufacturers. The RSD range was 0.461.02%, which indicates good precision. No interference was observed from common pharmaceutical additives. Good recoveries (97.6 0.7101.2 0.8%) were obtained. Statistical comparison of the results with reported methods shows excellent agreement and indicates no significant difference in accuracy and precision. fat-soluble and water-soluble. This research dealt with some water-soluble vitamin mixtures of thiamine, riboflavin, pyridoxine, and cyanocobalamin. Their molecular structures are shown in Figure 1. Deficiency of these vitamins can cause beriberi, corneal inflammation photophobia, and pernicious anemia, which results from malabsorption of the vitamin and decreases growth rate in children (1). There are many reported methods for the analysis of the vitamins singly or in combination with drugs. The methods are volumetric, electrochemical, spectroscopic, separation, flow injection, and immunoassay. The British Pharmacopoeia describes a spectrophotometric method for pyridoxine analysis in tablets (2). For determination of multicomponent mixtures of vitamins, spectrophotometric methods were used. Binary mixtures of pyridoxine with riboflavin (3) or thiamine (4) with drugs like isoniazid (5) and metronidazole (6) were determined. Quaternary mixtures of thiamine, pyridoxine, riboflavin, and nicotinamide were determined using multivariate analysis (7). Derivative spectroscopy (614) and derivative ratio spectra (15) methods were used for analysis of mixtures of riboflavin, carminic acid, curcumin, and erythrosine. A chemometric-assisted spectroscopic method was used to determine binary mixtures of thiamine and pyridoxine (1618) and for vitamin mixtures with drugs (19); it was also used to determine quaternary mixtures of thiamine, pyridoxine, riboflavin, and folic acid (20). The Kalmanfiltering UV-spectrophotometric method was used for simultaneous determination of thiamine, riboflavin, pyridoxine, and nicotinamide (21). The simultaneous spectrofluorimetric determination of mixtures of thiamine, pyridoxine, and riboflavin in pharmaceutical multivitamin preparations was reported (22, 23). A synchronous spectrofluorimetric method was reported for determination of a mixture of riboflavin and pyridoxine (24). HPLC was used to determine binary mixtures of vitamin B (2528), ternary (29, 30), ternary vitamin B with vitamin C (31), or niacin (32). HPLC was also used to determine mixtures of vitamins B1, B2, B3, B6, B12, and folic acid (33), and eight water-soluble vitamins (34). Many of the reported and official spectrophotometric methods were used for determination of the studied compounds as sole medications or in combination with

itamins are organic compounds required for normal health, growth, and maintenance of human life. There are two types of vitamins:

Received June 22, 2010. Accepted by AP October 9, 2010. 1 Corresponding authors e-mail: niveenam5606@yahoo.com DOI: 10.5740/jaoacint.9-493

Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1759 purchased from a local market and subjected to analysis by the proposed procedures. Solvents, Reagents, and Solutions Solvents.Analytical grade acetonitrile, dioxan, dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, methanol, and n-propanol were used throughout the investigation. Freshly boiled and cooled distilled water were used according to the indicated requirements. Buffer solutions.Phosphate buffer, acetate buffer, pH 38, and borate buffer solution, pH 410 (37) were prepared in freshly boiled and cooled distilled water. The solutions were refrigerated in light-protected flasks and used only for 1 week. Sodium hydroxide solution.Sodium hydroxide solution (1.0 M) was prepared by dissolving 4 g pure sodium hydroxide pellets (El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt) in 100 mL carbon dioxidefree distilled water. Iodine solution.An aqueous iodine solution (0.03 M) was prepared fresh daily by dissolving 0.42 g pure iodine (El-Nasr Pharmaceutical Chemicals Co.) and 1.08 g potassium iodide (El-Gomhouria Co., Cairo, Egypt) in 100mL distilled water. Preparation of Standard Solutions An accurately weighed amount of the studied drug (25.0 mg) was transferred into a 50 mL volumetric flask, dissolved in about 30 mL distilled water, then diluted to the mark with the same solvent to obtain the stock standard solution of the studied drug containing 0.5 mg/mL. The working standard solutions were prepared by further dilution of a suitable volume of the stock solution with the same solvent to obtain concentration ranges of 2005000 ng/mL for thiamine HCl and 1001200 ng/mL for pyridoxine HCl and riboflavin. The stock and working standard solutions were kept refrigerated in lightprotected flasks. Preparation of Sample Solutions Tablets.Twenty tablets were weighed, finely powdered, and mixed thoroughly. An accurately weighed quantity of the powdered tablets equivalent to 25 mg of the studied drug was transferred into a 50 mL volumetric flask containing 30 mL distilled water. The contents of the flask were shaken well for at least 10 min, then completed to the mark with distilled water. The resulting solution was filtered and the first portion of the filtrate was rejected. A suitable aliquot of the obtained solution was diluted quantitatively with the same solvent to obtain a concentration within the linearity range of each vitamin and suitable for the determinations. The general assay procedure was applied according to the vitamin determined.

Figure 1. Molecular structures of (I) thiamine, (II) pyridoxine, (III) riboflavin, and (IV) cyanobalamin.

other drugs, but few were used to determine vitamin mixtures and multivitamin formulations. The present work describes simple, rapid, and sensitive spectrofluorimetric methods for determination of some water-soluble vitamins, namely, thiamine HCl, riboflavin, and pyridoxine HCl in pure form and in laboratoryprepared mixtures, as well as pharmaceutical multivitamin preparations, in the absence or presence of cyanocobalamin. Experimental Apparatus Spectrofluorimeter SFM 23/B (Kontron, Gunzgen, Switzerland); UV-1601 PC, UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan); and pH meter, Model 3305 (Jenway, London, UK). Materials Pharmaceuticals Thiamine HCl, % purity 99.9 1.2; pyridoxine HCl, % purity 100.8 1.7; and riboflavin, % purity 100.6 0.3 (CID; Chemical Industries Company, Giza, Egypt) and cyanocobalamin, % purity 97.6 1.8 (The Nile Co. for Pharmaceuticals and Chemical Industries, Cairo, Egypt) were used as working standards without further purification. They were analyzed according to the official methods (35, 36) to determine their purity and compliance with the pharmacopeial requirements. Commercial Multivitamin Preparations The pharmaceutical preparations listed in Table 1 were

1760 Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011

Table 1. Studied commercial multivitamin pharmaceutical preparations

Pharmaceutical preparations Tri-B ampules Ingredients Vitamin B1 Vitamin B6 Vitamin B12 Trivitacid ampules Vitamin B1 Vitamin B6 Vitamin B12 Trivarol ampules Vitamin B1 Vitamin B6 Vitamin B12 Neurobion ampules Vitamin B1 Vitamin B6 Vitamin B12 Neurobion coated tablets Vitamin B1 Vitamin B6 Vitamin B12 Neurorubine tablets Vitamin B1 Vitamin B6 Vitamin B12 Neurovit ampules Vitamin B1 Vitamin B6 Vitamin B12 Tetraviton ampules Vitamin B1 Vitamin B6 Vitamin B12 Neurorubine ampules Vitamin B1 Vitamin B6 Vitamin B12 Lidocaine Neuroton ampules Vitamin B1 Vitamin B2 Vitamin B6 Vitamin B12 Lidocaine Nominal content, mg 100/amp. A 40/ amp. A 1/amp. B 100/amp. A 25/ amp. A 1/amp. B 100/amp. A 25/ amp. A 1/amp. B 100 100 1 100 200 0.2 200 50 1 150 100 1 200/amp. A 100/amp. A 3/amp. A 1/amp. B 200 50 1 10 200 4 100 1 10 Merck KGaA, Darmstadt, Germany Merck KGaA, Darmstadt, Germany Mepha Ltd, Basel, Switzerland Amriya Pharm., Ind. Alexandria, Egypt Kahira, Cairo, Egypt 023195A Manufacturer The Nile Co., Cairo, Egypt CID, Giza, Egypt Memphis Co., Cairo, Egypt Batch No. 92174

771002

301049

021609A

011034

866513

0380022

Mepha Ltd, Basel, Switzerland

030652

Amoun, Cairo, Egypt

2436

Ampules.The contents of 10 ampules were mixed well, and an accurately measured volume equivalent to 25mg of the studied drug was transferred into a 50 mL volumetric flask. The solution was diluted to volume with distilled water. The procedure was completed as described above for a suitable aliquot. General Assay Procedures Method (1) for thiamine HCl.A 1 mL volume of standard or sample solution was transferred into a 10mL volumetric flask, followed by addition of 1 mL iodine solution (0.030 M) and 1 mL sodium hydroxide solution (0.053 M). The solution was shaken well, then diluted with ethanol to the mark. The relative fluorescence

intensity (RFI) of the solution was measured after 20 min at exc 360 nm and em 430nm against a blank solution treated similarly. Method (2) for riboflavin or pyridoxine HCl.A 1 mL volume of standard or sample solution was transferred into a 10 mL volumetric flask, followed by 3 mL acetate buffer, pH 6. The flask contents were shaken and diluted to the mark with the same buffer solution. The RFI was measured at exc 325 nm and em 415 nm for pyridoxine hydrochloride, or at exc 457 nm and em 527nm for riboflavin. Results and Discussion The first proposed method depends on oxidation of thiamine to the fluorescent thiochrome using iodine/NaOH

Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1761 without interference from vitamins B2, B6, and B12. The second method depends on using acetate buffer of pH 6 for simultaneous determination of riboflavin and pyridoxine HCl utilizing their native fluorescence without interference from thiamine HCl. Different variables that affect the fluorescence intensity related to the two methods were studied. Method 1 A number of oxidants were tried for thiamine oxidation, but all oxidized to thiochrome with interference from other vitamins. However, iodine in alkaline medium produced stable and highly fluorescent product without interference from other vitamins in their mixtures. This is may be due to the highly quenching activity of the I2/I- system used, in addition to the alkaline medium, which is not suitable for determination of both drugs spectrofluorimetrically, especially pyridoxine HCl. Thiamine HCl was oxidized with iodine in alkaline medium to produce the highly fluorescent thiochrome. The RFI of the formed product was measured at em430nm (exc 360 nm) against a reagent blank treated similarly. Figure 2 shows excitation and emission spectra of the produced product. Optimization of Variables The parameters affecting the oxidation of thiamine HCl include iodine concentration, sodium hydroxide concentration, order of addition, reaction time, solvents, and stability time. Effect of iodine concentration.The effect of variation of iodine concentration was investigated by using 1.0mL of different concentrations ranging from 0.01 to 0.05M of iodine solution (Figure 3). It was found that the RFI increased as the concentration of iodine was increased to about 0.025 M, then remained unchanged. Thus, maximum fluorescence intensity and most reproducible results were obtained by using iodine concentration in the range of 0.0250.035 M. Effect of sodium hydroxide concentration.For this study 1.0 mL of different concentrations ranging from 0.03 to 0.08 M of sodium hydroxide solutions were used. Figure 4 shows that the RFI increased as the concentration of sodium hydroxide was increased to about 0.05 M, then became constant at higher concentration levels (up to 0.055M). Therefore, 1.0 mL of 0.053 M was used in all subsequent work. Effect of order of addition.The possible sequences of addition of reactants were tested. The most proper orders of addition were drug + iodine, then sodium hydroxide, or iodine + sodium hydroxide, then the drug. Effect of reaction time.Thiochrome was produced at ambient temperature (25 2C) without heating. It was

Figure 2. Fluorescence spectra of thiochrome formed by oxidation of thiamine HCl (100 ng/mL) with iodine/sodium hydroxide. (1) Excitation spectrum, (2) emission spectrum, and (3) spectra of blank.

Figure 3. Effect of iodine concentration on the fluorescence intensity induced due to oxidation of thiamine HCl, 100 ng/mL.

Figure 4. Effect of sodium hydroxide concentration on the fluorescence intensity induced due to oxidation of thiamine HCl, 100 ng/mL.

1762 Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011

Figure 5. Effect of reaction time on the fluorescence intensity induced due to oxidation of thiamine HCl, 100 ng/mL.

Figure 6. Effect of time on the stability of the induced fluorescence intensity due to oxidation of thiamine HCl, 100 ng/mL.

noted that the RFI increased with time, and maximum intensity was reached after about 15 min, then remained approximately constant up to 60 min. Therefore, measurements were performed in a 1550 min period (Figure 5). Reaction time between 20 and 25 min was selected for all subsequent work. Effect of diluting solvents.Effect of various solvents on the position and intensity of the produced fluorescence of the studied drug was investigated. Table 2 shows the obtained results for the effect of different diluting solvents on the RFI. It was found that the type of diluting solvent used slightly affects the wavelength of maximum excitation or emission, and does not significantly affect the fluorescence intensity for the same concentration of the drug (100 ng/mL thiamine HCl). The maximum fluorescence intensity was obtained by using ethanol as diluting solvent. DMF and DMSO solvents cause turbidity of solutions that cannot be measured spectrofluorimetrically. Effect of time on the stability of the oxidation product of thiamine HCl.The stability of the fluorescent product was investigated by following the fluorescence intensity at 5min intervals for at least 60 min after dilution with ethanol. The results are shown in Figure 6. It was found that the induced fluorescence remained stable for more than 60 min.
Table 2. Effect of different diluting solvents on the fluorescence intensity of the oxidation product of thiamine HCl (100 ng/mL)
No. 1 2 3 4 5 6
a

Figure 7. Fluorescence spectra of (1) pyridoxine HCl (50 ng/mL; ----); and (2) riboflavin (50 ng/mL; ) in acetate buffer, pH 6.

Method 2 Both pyridoxine and riboflavin exhibit intrinsic native fluorescence with excitation and emission maxima at 325, 457 and 415, 527 nm for pyridoxine HCl and riboflavin, respectively. The spectra of both compounds are shown in Figure 7. The intensity, as well as the stability, of the fluorescence for both drugs was affected by the pH of the solution, as well as the type and concentration of the buffer solution used, in addition to the diluting solvent. Therefore, such variables were investigated in order to obtain the most suitable conditions to maximize the fluorescence intensity of the studied drugs. Optimization of Variables Effect of pH variation.The effect of pH and the type of buffer solutions on the fluorescence intensity was studied using three types of buffer systems: phosphate, acetate, and borate buffer solutions. The results obtained are listed in Table 3. It was found that the fluorescence of

Solvent Water Ethanol Methanol n-Propanol Acetonitrile Dioxan

exc, nm 360 360 352 365 358 350

em, nm 430 430 415 424 448 420

RFIa SD 50.6 1.9 62.2 2.2 14.9 0.7 25.1 2.1 22.8 2.1 43.6 2.2

Average of five readings SD.

Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1763

Table 3. Effect of pH variation and buffer type on the RFI of riboflavin and pyridoxine HCl
RFIa Riboflavinb pH 3 4 5 6 7 8 9 10
a b

Pyridoxine HClb Borate bufferc 33.6 41.5 53.8 60.5 57.3 55.6 53.4 Phosphate bufferc 27.5 30.1 38.4 47.7 46.2 50.9 42.9 Acetate bufferc 10.4 27.5 51.8 55.4 54.6 43.7 Borate bufferc 29.5 32.9 37.4 40.5 42.1 45.0 44.2

Phosphate bufferc 38.7 40.5 46.8 54.3 50.5 48.2 43.2

Acetate bufferc 52.4 57.8 72.6 75.1 70.0 59.7

Average of five determinations Concentration is 50 ng/mL for each vitamin. c Ionic strength for all buffers is 0.01 M.

both pyridoxine HCl and riboflavin is dependent not only on the pH of the solution, but also on the type and ionic strength of the buffer solution used. The fluorescence intensity increased by raising the pH of the solution, and reached its maximum reading at pH around 6 for all buffer solutions used, but acetate buffer, which is the simplest with respect to composition, gave the maximum fluorescent intensity. In addition, when various ionic strengths of acetate buffer, pH 6, ranging from 0.005 to 1.0 M were tested, slightly significant differences were observed. However, more diluted solutions are preferable and gave the highest fluorescence intensity at the specified exc and em for each drug. In the present investigation, 0.01 M concentration of acetate buffer components was used for all subsequent work. Effect of diluting solvents.Various solvents, such as water, ethanol, methanol, n-propanol, DMF, DMSO, dioxin, and acetonitrile, were tested in order to select the most suitable for the determinations. The RFI was

measured after addition of acetate buffer, pH 6, and then diluting the solution to the mark with either of the previously mentioned solvents or with the acetate buffer. The results in Table 4 show that the fluorescence intensity was maximized by using the same buffer solution as diluting solvent. DMSO, dioxin, and acetonitrile solvents gave turbid solutions that cannot be measured spectrofluorimetrically. Effect of time on the stability of the measured fluorescence.The stability of the native fluorescence of both pyridoxine HCl and riboflavin in acetate buffer was studied by measuring the RFI after dilution with acetate buffer at 5 min intervals. Results are shown in Figure 8. The fluorescence of pyridoxine HCl remained stable for more than 60 min, but that of riboflavin was stable until 30 min, then begin to decrease.

Table 4. Effect of solvent type on the RFI of riboflavin and pyridoxine HCl
Riboflavina Solvent Water Ethanol Methanol n-Propanol DMF Acetate buffer, pH 6b
a b

Pyridoxine HCla RFI 72.5 66.1 65.7 68.3 57.7 75.4 exc, nm 325 325 325 322 320 330 em, nm 380 384 384 379 378 390 RFI 50.3 47.8 44.7 46.4 40.1 55.4

exc, nm 457 454 455 455 450 457

em, nm 527 513 520 524 525 527

Concentration is 50 ng/mL. Ref. 37.

1764 Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011

The regression analysis of the results was done according to the following equation: RFI = a + b C where RFI = relative fluorescence intensity measured in 1 cm cell, a = intercept, b = slope (regression coefficient), and C = concentration of the analyzed drug in ng/mL. The relation of RFI and concentration plots were linear with intercepts (2.24.3), and slopes (0.51.5), which reflects the sensitivity of the proposed methods, and good correlation coefficients (0.99910.9999) in the concentration range of 10500 ng/mL. The detection limit is calculated as 3/b, where b is the slope and is the SD of the intercept. The quantitative limit is calculated as 10 /b. The LOD and LOQ values were 2.004.20 and 6.6714.0 ng/mL, respectively, as shown in Table 5. The proposed method was found to be more sensitive than most of the reported methods. The LOD in spectrofluorimetric method was 0.43.5 g/mL (22), first-derivative spectrophotometric method was 0.010.25 g/mL (14), and zero-crossing technique was 840 g/mL. Precision The precision of the proposed method was checked by replicate analysis of 10 separate solutions of the working standard of each drug at three concentration levels. Results obtained (Table 6) show that the RSD (CV) for all concentration levels is <2%, indicating good repeatability of the proposed methods. Selectivity and Interferences In order to access the possible analytical application of the proposed method, the effect of some common excipients used in pharmaceutical preparations was studied by analyzing sample solutions containing a fixed amount of the drug with various amounts of each excipient. The studied excipients are sucrose, glucose, lactose, citric acid, gum acacia, talc, starch, and propylene glycol. Good recoveries were obtained, which indicated no interferences from the classical additives tested at a tolerance ratio of 1:100 (drug:excipient). The proposed methods are able to access the analyte in the presence of common excipients.

Figure 8. Effect of time on the stability of the fluorescence intensity of (1) riboflavin (50 ng/mL) and (2) pyridoxine HCl (50 ng/mL) in acetate buffer, pH 6.

Figure 9 Suggested reaction mechanism for thiamine oxidation.

Reaction Mechanism Thiamine oxidation has been previously carried out with different oxidizing agents (3847), but the major drawback was the high instability of thiochrome obtained in presence of the excess oxidant used. In the present investigation, iodine in alkaline medium was used for the oxidation of thiamine to thiochrome. The oxidation product is stable and has strong blue fluorescence that can be used for accurate determination of thiamine (Figure 9). Validation of the Proposed Methods Linearity, detection, and quantitation limits.Under the previously mentioned experimental conditions, calibration curves for all the studied drugs were constructed by plotting RFI versus the drug concentration.

Table 5. Quantitative parameters and statistical data for spectrofluorimetric determination of the studied vitamins
Linearity range, ng/mL 20500 10100 10120 Correlation coefficient (r) 0.9999 0.9991 0.9995 Determination coefficient (r2) 0.9998 0.9983 0.9991 LOD, ng/mL 4.20 2.75 2.00 LOQ, ng/mL 14.00 9.17 6.67

Drug Thiamine HCl Pyridoxine HCl Riboflavin

Equation Y = 2.2(0.7) + 0.5(0.002) X Y = 4.3(1.1) + 1.2(0.02) X Y = 3.2(1.0) + 1.5(0.01) X

Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1765

Table 6. Results of 10 replicate samples of the studied vitamins at three different concentration levels
Found Thiamine HCla Sample No. 1 2 3 4 5 6 7 8 9 10 Mean % Recovery SD RSD
a

Pyridoxine HCla 400 402.84 399.48 398.16 399.72 398.12 397.12 399.28 402.48 398.40 399.04 399.46 99.9 1.853 0.464 40 40.34 39.82 40.16 39.73 40.07 39.56 39.90 40.25 39.47 39.65 39.89 99.7 0.300 0.752 60 59.73 59.56 59.12 59.81 58.78 59.05 58.87 59.99 59.47 59.56 59.39 99.0 0.415 0.699 80 80.34 80.42 81.37 80.59 79.98 79.82 80.76 79.73 79.22 79.65 80.19 100.2 0.630 0.786 40 39.26 39.54 39.68 39.88 39.20 39.34 40.29 39.47 40.09 39.61 39.64 99.1 0.358 0.903

Riboflavina 60 60.16 60.49 61.18 59.75 60.49 60.29 59.61 60.09 59.41 59.06 60.05 100.1 0.615 1.024 79.33 79.47 79.41 79.67 79.26 79.88 79.62 79.82 79.54 79.33 79.53 99.4 0.212 0.266

100 100.60 99.71 100.37 101.04 100.60 100.37 101.48 102.15 99.49 99.27 100.51 100.5 0.893 0.888

200 202.82 200.60 198.36 199.84 201.48 202.36 199.72 199.94 197.26 197.94 200.03 100.0 1.844 0.922

Drug concentration expressed in ng/mL.

Table 7. Robustness of the proposed spectrofluorimetric methoda

% Recoveryb SD Variation No variation
c

Table 8. Determination of thiamine HCl and pyridoxine HCl in their laboratory-prepared binary mixtures
Recovery, %a SD Mixture 1 2 Actual concn, ng/mLb 500:10 400:20 300:30 200:40 200:50 100:40 100:50 Thiamine HCl 98.7 0.5 98.8 0.6 100.1 1.2 98.5 0.7 98.5 0.7 98.7 0.7 98.6 0.7 Pyridoxine HCl 100.3 1.0 99.1 0.7 99.2 0.9 98.4 0.9 97.5 0.6 97.8 0.5 99.5 0.8

Thiamine HCl 99.7 1.0

Pyridoxine HCl 100.6 0.2

Riboflavin 99.8 0.7

Iodine concn For thiamine HCl 2.5 102 M 3.5 10

2

100.4 1.0 99.8 0.6

3 4 5 6

NaOH conc. For thiamine HCl 5.2 10 5.4 10

2 2

M M

99.1 1.2 98.9 0.7

7
a b

Average of five determinations SD.  The declared mixtures for thiamine HCl and pyridoxine HCl, respectively.

pH of buffer For pyridoxine and riboflavin pH 5.5 pH 6.5 Reaction time (min) For thiamine HCl 14 min 16 min
a b c

98.9 0.9 99.5 0.4

98.5 0.7 99.8 0.6

99.9 0.9 98.7 0.6

Drug concentration is 50 ng/mL. Average of three determinations.

 No variation in the assay conditions of the proposed methods.

1766 Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011 Table 9. Determination of thiamine HCl, pyridoxine HCl, and riboflavin in their laboratory-prepared ternary mixtures
Recovery, % a SD Mixture 1 2 3 4 5
a b

Actual concn, ng/mLb 200:100:10 300:90:20 350:80:30 400:70:40 500:60:50

Thiamine HCl 98.6 0.6 98.7 0.6 99.4 0.7 97.6 1.1 100.2 0.6

Pyridoxine HCl 98.7 0.8 98.4 0.9 99.5 0.8 98.6 0.4 98.2 0.3

Riboflavin 98.8 0.5 99.8 0.6 98.9 0.5 99.9 0.6 98.7 0.6

To assess the selectivity of the iodine method for analysis of thiamine HCl, pyridoxine HCl, riboflavin, cyanocobalamin, and other related B-complex vitamins were each tested under the developed reaction conditions. No fluorescence readings at the specified exc and em for thiamine HCl were given by any of these compounds, indicating the high selectivity of the method to thiamine HCl. Robustness Robustness was examined by evaluating the influence of small variations of method variables, including concentration of iodine, sodium hydroxide, and reaction time on the method suitability and sensitivity. The results in Table 7 show that none of these variables significantly affect the method, meaning that it is robust and reliable. Applications

Average of five determinations SD.  The declared mixtures for thiamine HCl, pyridoxine HCl, and riboflavin, respectively.

Table 10. Determination of thiamine HCl and pyridoxine HCl in the presence of cyanocobalamin in their laboratory-prepared ternary mixtures
Recovery, %a SD Mixture 1 2 3 4 5 6
a b

Actual concn, ng/mLb 300:40:100 200:50:80 150:100:60 100:40:40 100:50:20 50:100:10

Thiamine HCl 99.0 0.7 98.5 0.8 99.5 0.5 98.8 1.0 99.6 1.1 99.9 0.6

Pyridoxine HCl 99.5 0.3 100.2 1.2 99.9 0.7 100.4 0.5 98.8 0.4 99.3 1.1

Average of five determinations SD.  The declared mixtures for thiamine HCl, pyridoxine HCl, and cyanocobalamin, respectively.

Different laboratory-prepared mixtures (Tables 811) and pharmaceutical dosage forms, including tablets and ampules (Tables 1214), were successfully analyzed by the proposed and some reported methods. The obtained results indicated the selectivity of the proposed method for analysis of the investigated drugs. The t- and F-values were calculated at 95% confidence limit (48), and no significant differences were found between results obtained by the proposed and reported methods with respect to accuracy and precision. In addition, recovery experiments were carried out for the studied drugs in their respective pharmaceutical formulations by the standard addition method. The results in Table 15 indicate that there is no interference from the frequently encountered excipients and additives. Generally, in addition to their marked sensitivity, the proposed methods are accurate, precise, and suitable for the analysis of the studied drugs in pure form and in their dosage forms. Conclusions The present study developed simple, sensitive, inexpensive, and accurate spectrofluorimetric methods for the analysis of the studied vitamins. One of these methods was based on oxidation of thiamine HCl using iodine and sodium hydroxide, with fluorescence measured at em 430 nm (exc 360 nm). This method is valuable for separate determination of thiamine HCl in mixtures with riboflavin and pyridoxine HCl. The native fluorescence of both pyridoxine and riboflavin was utilized for simultaneous efficient determination of the two vitamins in acetate buffer, pH 6, at excitation and emission maxima at 325, 457 and 415, 527 nm for pyridoxine HCl and riboflavin, respectively. The developed methods were successfully applied to the determination of the studied vitamins in their

Table 11. Determination of thiamine HCl, pyridoxine HCl, and riboflavin in the presence of cyanocobalamin in their laboratory-prepared quaternary mixtures
Recovery, %a SD Mixture 1 2 3 4 5
a b

Actual concn, ng/mLb 100:50:30:10 200:40:40:20 300:30:50:30 400:20:60:40 500:10:70:50

Thiamine HCl 99.5 0.5 98.4 0.8 98.5 0.8 98.2 0.7

Pyridoxine HCl 98.8 0.7 99.0 0.4 97.5 1.0 98.4 0.9

Riboflavin 99.1 0.9 99.2 1.0 99.4 0.8 99.7 0.8

98.5 1.3 100.6 0.9 98.2 0.2

Average of five determinations SD.  The declared mixtures for thiamine HCl, pyridoxine HCl, riboflavin, and cyanocobalamin, respectively.

Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1767

Table 12. Determination of thiamine HCl, pyridoxine HCl, and riboflavin in pharmaceutical multivitamin preparations using the proposed and reported methods
Recovery, % SD Drug content (mg) Thiamine HCl (100) Pyridoxine HCl (40) Thiamine HCl (100) Pyridoxine HCl (25) Thiamine HCl (100) Pyridoxine HCl (25) Thiamine HCl (200) Pyridoxine HCl (100) Riboflavin (3) Proposed method 100.6 1.2 98.7 1.2 97.8 0.7 99.4 1.3 98.9 1.4 99.6 1.2 99.0 1.3 98.5 1.1 98.8 0.7 Reported method 101.1 0.5 97.7 1.0 98.5 0.9 98.3 1.0 99.3 1.0 98.4 0.5 97.8 0.7 97.9 0.5 98.2 0.8 F-value 5.760 1.174 1.653 1.690 1.960 5.760 3.448 4.840 1.306 t-Value 0.932 1.863 2.236 1.892 0.639 2.236 2.064 1.219 1.917

Table 13. Determination of thiamine HCl and pyridoxine HCl in the presence of cyanocobalamin in multivitamin preparations using the proposed and reported methods
Recovery, %a SD Product Neurorubine (tablets) Drug content (mg) Thiamine HCl (200) Pyridoxine HCl (50) Cyanocobalamin (1) Neurobion (ampules) Thiamine HCl (100) Pyridoxine HCl (100) Cyanocobalamin (1) Neurovit (ampules) Thiamine HCl (150) Pyridoxine HCl (100) Cyanocobalamin (1) Neurovit (tablets) Thiamine HCl (250) Pyridoxine HCl (100) Cyanocobalamin (0.25) Tri-B (ampules) Pyridoxine HCl (40) Thiamine HCl (100) Cyanocobalamin (1) Trivarol (ampules) Thiamine HCl (100) Pyridoxine HCl (25) Cyanocobalamin (1) Trivitacid (ampules) Thiamine HCl (100) Pyridoxine HCl (25) Cyanocobalamin (1) Neurorubine (ampules) Thiamine HCl (200) Pyridoxine HCl (50) Cyanocobalamin (1) Lidocaine (10)
a b c

Proposed method 98.2 1.1 100.6 1.3 98.3 0.9 98.7 1.2 99.0 1.3 98.6 0.9 98.3 1.1 98.6 1.0 99.2 1.0 99.1 0.8 98.6 0.7 98.1 1.0 98.4 1.1 98.6 0.9 98.2 1.2 98.2 1.2

Reported methodb 99.0 0.6 101.2 0.8 98.1 1.2 97.6 1.1 100.0 1.2 97.9 1.1 98.0 0.8 98.4 1.0 98.2 1.3 98.8 0.8 98.0 1.0 97.6 1.0 98.1 1.0 99.5 0.7 97.2 0.9 98.3 0.7

F-value 3.361 2.641 1.778 1.190 1.174 1.494 1.891 1.000 1.690 1.000 2.041 1.000 1.210 1.653 1.778 2.939

t-valuec 1.626 1.032 0.497 1.677 1.720 1.739 0.609 0.447 2.236 0.838 1.917 1.118 0.609 2.236 1.863 0.186

Average of five determinations SD. Ref. 22.

Theoretical values at 95% confidence limit; t = 2.306, F = 6.388.

1768 Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011 Table 14. Determination of thiamine HCl, pyridoxine HCl, and riboflavin in the presence of cyanocobalamin in multivitamin preparations by the proposed and reported methods
Recovery, %a SD Product Tetraviton (ampules) Drug content (mg) Thiamine HCl (200) Pyridoxine HCl (100) Riboflavin (3) Cyanocobalamin (1) Neuroton (tablets) Thiamine HCl (250) Pyridoxine HCl (200) Riboflavin (15) Cyanocobalamin (0.25) Neuroton (ampules) Thiamine HCl (200) Pyridoxine HCl (100) Riboflavin (4) Cyanocobalamin (1) Lidocaine (10)
a b c

Proposed method 99.6 1.2 100.5 0.8 97.5 1.2 98.1 1.1 97.2 0.9 98.0 0.5 97.5 0.9 99.0 1.2 97.9 1.1

Reported methodb 99.0 1.0 100.0 1.0 98.7 0.9 97.2 0.5 96.6 1.1 97.6 0.6 97.2 0.7 98.4 1.1 97.7 0.8

F-value 1.440 1.562 1.778 4.840 1.494 1.440 1.653 1.190 1.891

t-Valuec 1.118 1.398 2.236 1.829 1.491 1.789 0.646 1.234 0.502

Average of five determinations SD. Ref. 22.

Theoretical values at 95% confidence limit; t = 2.306, F = 6.388 (Ref. 48).

Table 15. Recovery of standard drugs added to their commercial dosage forms by the proposed methods
Dosage form Tri-B (ampules) Tetraviton (ampules) Neurorubine (ampules) Trivarol (ampules) Tetraviton (ampules) Neurobion (ampules) Tetraviton (ampules) Neuroton (ampules)
a

Drug Thiamine HCl

Declared amount, mg 100 200 200

Added, mg 100 150 200 300 200 300 100 150 100 150 100 150 3 6 4 8

% Recoverya SD 99.1 0.7 98.5 0.2 98.6 0.6 99.2 0.5 98.5 1.1 99.2 0.4 98.8 0.6 99.5 0.3 98.8 0.8 98.6 0.6 98.8 0.8 98.7 0.4 98.8 0.7 99.1 0.5 98.7 0.3 98.7 0.7

Pyridoxine HCl

100 100 100

Riboflavin

3 4

Average of five determinations.

Mohamed et al.: Journal of AOAC International Vol. 94, No. 6, 2011 1769
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