Rapid determination of thiamine, riboflavin

Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 151–157

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Rapid determination of thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, folic acid and ascorbic acid in Vitamins with Minerals Tablets by high-performance liquid chromatography with diode array detector Pengfei Jin ∗ , Lufeng Xia, Zheng Li, Ning Che, Ding Zou, Xin Hu Department of Pharmaceutical Science, Beijing Hospital of the Ministry of Health, No. 1 Dahua Road, Dongcheng District, Beijing 100730, China

a r t i c l e

i n f o

Article history: Received 4 May 2012 Received in revised form 13 June 2012 Accepted 15 June 2012 Available online 23 June 2012 Keywords: HPLC Vitamins with Minerals Tablets Water-soluble vitamins Ascorbic acid Pharmaceutical preparation

a b s t r a c t A simple, isocratic, and stability-indicating high-performance liquid chromatography (HPLC) method has been developed for the rapid determination of thiamine (VB1 ), niacinamide (VB3 ), pyridoxine (VB6 ), ascorbic acid (VC), pantothenic acid (VB5 ), riboflavin (VB2 ) and folic acid (VB9 ) in Vitamins with Minerals Tablets (VMT). An Alltima C18 column (250 mm × 4.6 mm i.d., 5 ␮m) was used for the separation at ambient temperature, with 50 mM ammonium dihydrogen phosphate (adjusting with phosphoric acid to pH 3.0) and acetonitrile as the mobile phase at the flow rate of 0.5 ml min−1 . VB1 , VB3 , VB6 , VC and VB5 were extracted with a solution containing 0.05% phosphoric acid (v/v) and 0.3% sodium thiosulfate (w/v), and were then simultaneously analyzed by using the mobile phase of phosphate buffer–acetonitrile (95:5, v/v), while VB2 and VB9 were extracted with a solution containing 0.5% ammonium hydroxide solution (v/v), and were then simultaneously analyzed by using the mobile phase of phosphate buffer–acetonitrile (85:15, v/v). The detection wavelengths were 275 nm for VB1 , VB3 , VB6 , VC, 210 nm for VB5 , and 282 nm for VB2 and VB9 . The method showed good system suitability, sensitivity, linearity, specificity, precision, stability and accuracy. All the seven water-soluble vitamins were well separated from other ingredients and degradation products. Method comparison indicated good concordance between the developed method and the USP method. The developed method was reliable and convenient for the rapid determination of VB1 , VB3 , VB6 , VC, VB5 , VB2 and VB9 in VMT. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Vitamins, natural constituents of food can be classified into 2 main groups: oil- and water-soluble vitamins. A well-balanced diet supplies all of the required vitamins. However, appropriate vitamin supplementation is advisable in case of poor vitamins intake or in pathological conditions with nutritional requirements increasing. Vitamins with Minerals Tablets (VMT) containing 5 oil-soluble vitamins, 9 water-soluble vitamins and 17 mineral elements, is considered as a relatively complete formula, and is among the pharmaceutical preparations most widely used for the supplementation of vitamins and minerals. The determination of water-soluble vitamins in VMT is rather difficult due to their instability and high polarity as well as the complexity of the matrices. Although numerous methods including spectrophotometry, titration, high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), high-performance thin layer chromatography (HPTLC) and microbiological assays

∗ Corresponding author. Tel.: +86 10 85133621; fax: +86 10 65283696 5. E-mail address: [email protected] (P. Jin). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.06.020

have been reported for the determination of water-soluble vitamins in various matrices, only a limited number of publications are available for the quantitative analysis of water-soluble vitamins in pharmaceutical and dietary supplement multivitamin preparations with or without minerals. These methods include spectrophotometry [1–5] and HPLC with ultraviolet (diode array) [4–17], electrochemical [17], fluorescence [18], mass spectrometric [19], ultraviolet/mass spectrometric [20], and diode array/fluorescence/mass spectrometric [21] detectors. Among these HPLC methods, only one method equipped with a 4 ◦ C thermostated autosampler [21] was found in our preliminary study to be suitable for the simultaneous determination of ascorbic acid together with B-complex vitamins in VMT in the presence of minerals, especially copper and iron, which could significantly accelerate the oxidation of ascorbic acid [8]. However, the use of mass spectrometry could be hurtling for the popularity of the method. Moreover, except for [14], these studies did not investigate the potential interferences from the degradation products, therefore were not rigorously validated as stability-indicating assays. In respect of the official methods, the United States Pharmacopeia [22] uses HPLC methods with C8 and C18 columns for the simultaneous determination of niacin or niacinamide, thiamine,

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riboflavin and pyridoxine in the monograph of “Oil- and WaterSoluble Vitamins with Minerals Tablets”. Other water-soluble vitamins are analyzed separately using different methods including titration, HPLC or microbiological assays. This paper reports a simple, isocratic and stability-indicating HPLC method for the rapid determination of thiamine (VB1 ), niacinamide (VB3 ), pyridoxine (VB6 ), ascorbic acid (VC), pantothenic acid (VB5 ), riboflavin (VB2 ) and folic acid (VB9 ) in VMT at ambient temperature. VB1 , VB3 , VB6 , VC and VB5 were simultaneously analyzed in a single chromatographic run, while VB2 and VB9 were simultaneously analyzed in another chromatographic run. The developed method only required very simple sample pretreatment, and a whole analysis for all seven water-soluble vitamins was achieved within 4 h. Method comparison showed good concordance between the developed method and the USP methods.

was prepared by dissolving accurately weighed amounts of reference standards in diluting solution I containing 0.05% phosphoric acid (v/v) and 0.3% sodium thiosulfate (w/v), while stock solution II containing 288.00 mg l−1 of VB2 and 73.80 mg l−1 of VB9 was prepared by dissolving accurately weighed amounts of reference standards in diluting solution II containing 0.5% ammonium hydroxide solution (1 ml of ammonium hydroxide diluted into 200 ml with water). Standard solution I/II were made by transferring 2 ml, 3 ml, 4 ml, 5 ml or 6 ml of stock solution I/II into 50 ml amber-colored volumetric flasks and making up to volume with diluting solution I/II. The standard solutions of mid-point concentrations were used as working standard solutions for system suitability and stability studies.

2. Experimental

Weigh and finely powder not less than 20 tablets. Transfer an accurately weighed quantity of the powder (equivalent to about 40 mg of VC) into a 50 ml amber-colored volumetric flask, the analytical sample solution I (for determination of VB1 , VB3 , VB6 , VC and VB5 ) was prepared by adding 30 ml of diluting solution I into the volumetric flask, shake vigorously for 5 min, and make up to volume with diluting solution I, while the analytical sample solution II (for determination of VB2 and VB9 ) was prepared by adding 30 ml of diluting solution II into the volumetric flask, ultrasonicate (320 W, 50 Hz) for 25 min at 25 ◦ C, and make up to volume with diluting solution II. The resulting solutions were then analyzed by HPLC after filtration through 0.22-␮m PTFE membrane syringe filter (Anachem, Cheshire, UK).

2.1. Reagents and chemicals Ascorbic acid reference standard (99.5%) was purchased from Fluka (Steinheim, Germany). d-Pantothenic acid hemicalcium salt reference standards were purchased from Sigma (St. Louis, USA). Thiamine hydrochloride, pyridoxine hydrochloride, niacinamide, folic acid and riboflavin reference standards were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Analytical grade ammonium dihydrogen phosphate, sodium thiosulfate, phosphoric acid (85%, w/w), ammonia solution (25%, w/w), hydrochloric acid, sodium hydroxide and hydrogen peroxide (30%, w/w) were obtained from Beijing Reagent Company (Beijing, China). HPLC grade acetonitrile was purchased from Fisher (Fair Lawn, NJ, USA). Purified water was prepared by Millipore Milli-Q system (Bedford, MA, USA). All solvents were filtered through 0.22-␮m PTFE filters (HPLC Technology, Cheshire, UK) before use. VMT (a complete formula from A to Zinc of Centrum® , lot: 1,011,365, 1,012,232 and 1,101,344) were manufactured by Wyeth company in Suzhou city, Jiangsu province, China and were obtained commercially. The labeled active ingredients included 5 oil-soluble vitamins, 9 water-soluble vitamins and 17 mineral elements. The excipients (details kindly provided by the manufacturer) included microcrystalline cellulose, lactose, magnesium stearate, crospovidone, stearic acid, silicon dioxide, triethyl citrate (coating material), tween 80 (coating material) and persicine pigment (coating material). 2.2. Instruments and chromatographic conditions The study was performed on a Waters 2695 quaternary pump system. A Waters 2996 photodiode array detector and an Empower professional® software were used for data acquisition and processing. The chromatographic separations were carried out on an ˚ Alltech Alltima C18 column (250 mm × 4.6 mm i.d., 5 ␮m, 100 A, Associates Inc., Deerfield, IL, USA). The mobile phase composed of 50 mM ammonium dihydrogen phosphate (adjusting with phosphoric acid to pH 3.0) and acetonitrile was delivered at a flow rate of 0.5 ml min−1 . A buffer–acetonitrile ratio of 95:5 (v/v) was set for VB1 , VB3 , VB6 , VC and VB5 , and 85:15 (v/v) for VB2 and VB9 . The detection wavelengths were 275 nm for VB1 , VB3 , VB6 and VC, 210 nm for VB5 , and 282 nm for VB2 and VB9 . The injection volume was 20 ␮l. Analysis was conducted at ambient temperature. 2.3. Preparation of standard solutions Stock solution I containing 244.8 mg l−1 of VB1 , 3205 mg l−1 of VB3 , 2714 mg l−1 of VB5 , 397.6 mg l−1 of VB6 and 10.26 g l−1 of VC

2.4. Preparation of analytical samples

2.5. System suitability The key system suitability parameters, including theoretical plates, asymmetry factors and resolutions between two adjacent peaks were performed by analyzing five consecutive injections of the working standard solutions, and were calculated as FDA CDER guidance for validation of chromatographic methods [23] described. 2.6. Validation procedure The developed method was validated for specificity, linearity, precision, accuracy, stability, limit of detection (LOD) and limit of quantitation (LOQ) following ICH recommendations [24,25]. 2.6.1. Linearity Linearity of the method was evaluated at five concentration levels, covering about 50%, 75%, 100%, 125% and 150% of the targeted assay concentration (n = 5). The resulting peak areas were processed and calibration curves were generated by Microsoft Excel. 2.6.2. LOD and LOQ The LOD and LOQ were determined by injecting progressively low concentration of standard solutions which generated S/N ratios of about 3 and 10 (n = 9). 2.6.3. Specificity To assess the method specificity, reconstituted tablet placebo I (without VB1 , VB3 , VB5 , VB6 and VC) and placebo II (without VB2 and VB9 ) were prepared. The placebo I solution and placebo II solution were prepared following Section 2.4, and were then subjected to HPLC analysis to evaluate the potential interferences from other active ingredients and excipients. Forced degradation studies of tablet powders under different stress conditions (heat, light, oxidation, acid and base) were conducted to evaluate the potential interferences of degradation


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Fig. 1. Chromatograms for placebo I solution (A), standard solution I (B), analytical solution I (C), placebo II solution (D), standard solution II (E) and analytical solution II (F). The mobile phase of phosphate buffer–acetonitrile (95:5, v/v) was used for A, B and C, while phosphate buffer–acetonitrile (85:15, v/v) was used for D, E and F.

products. To prepare the heat degradation products, the powders (equivalent to about 40 mg of VC) were heated at 150 ◦ C for 4 h in dark, cooled down to room temperature and transferred into a 50 ml amber-colored volumetric flask, and prepared solutions following Section 2.4. To investigate the light induced degradation products, the powders were exposed to light (10,000 lx) for 4 h, and then prepared solutions as described above. For preparing the oxidation induced degradation products, 2 ml of 3% hydrogen peroxide (v/v) solution was added to the powders in a 50 ml amber-colored volumetric flask, stored in dark for 24 h, and then the degraded samples were prepared solutions as described above. To study the acid and base induced degradation products, 2 ml of 1 M hydrochloric acid solution or 2 ml of 1 M sodium hydroxide solution was added to the powders in 50 ml amber-colored volumetric flasks, stored in dark for 24 h, and then the degraded samples were neutralized with 1 M sodium hydroxide solution or 1 M hydrochloric acid solution and prepared solutions as described above. All those resulting solutions were immediately analyzed by HPLC after filtration through 0.22-␮m PTFE membrane syringe filter (Anachem, Cheshire, UK).

investigated by analyzing the working standard solutions and analytical solutions at 0, 2, 4, 8 and 12 h, while the stability of analytical solutions at ambient temperature was investigated by analyzing analytical solutions at 0, 0.5, 1, 1.5 and 2 h. The %R.S.D. values (n = 5) of peak areas were used for evaluation.

2.6.4. Precision The instrumental precision was investigated by analyzing six consecutive injections of an analytical sample solution and the %R.S.D. values (n = 6) of peak areas were used to evaluate. For the evaluation of method precision (repeatability and intermediate precision), six independent sample solutions were prepared and analyzed everyday in six consecutive days. The intra-day %R.S.D. values (n = 6) of the assay results in the first day were used to examine the repeatability of the method, and the inter-day %R.S.D. values (n = 6) of six average assay results were used to evaluate the intermediate precision.

2.6.7. Method comparison To compare the developed method with the official method, seven vitamins were also analyzed using the methods described in the monograph of “Oil- and Water-Soluble Vitamins with Minerals Tablets” in USP [22]. VB1 , VB2 , VB3 and VB6 were simultaneously analyzed following the assay method 3 and using an ˚ Alltech Alltima C8 column (250 mm × 4.6 mm i.d., 5 ␮m, 100 A, Associates Inc., Deerfield, IL, USA). VB5 was analyzed following the assay method 1 and using an Alltima C18 column ˚ Alltech Associates Inc., Deer(150 mm × 4.6 mm i.d., 5 ␮m, 100 A, field, IL, USA). VB9 was analyzed following the assay method 2 and using the same C8 column with VB1 , VB2 , VB3 and VB6 . VC was analyzed following the assay method 1 using standard dichlorophenol–indophenol solution as volumetric solution.

2.6.5. Stability The stability of standard solutions at 4 ◦ C and at ambient temperature and the stability of analytical solutions at 4 ◦ C were

2.6.6. Accuracy To assess accuracy, freshly prepared placebo I/II were spiked with various amounts of stock solution I/II, and diluted with the diluting solution I/II to concentrations at about 75%, 100%, and 125% of the targeted concentrations. The spiked solutions of each concentration level were prepared in triplicate and their peak areas were used to calculated mean and standard deviation values, and compared with those obtained with the same concentration of standard solutions.

154


Fig. 2. Chromatograms for forcedly degraded samples: (A and F) light treatment; (B and G) heat treatment; (C and H) acidic condition; (D and I) basic condition; (E and J) oxidation treatment. The mobile phase of phosphate buffer–acetonitrile (95:5, v/v) was used for A–E, while phosphate buffer–acetonitrile (85:15, v/v) was used for F–J.

3. Results and discussion 3.1. Method development The aim of this study was to develop simple, isocratic, and stability-indicating HPLC method for the determination of VB1 , VB3 , VB6 , VC, VB5 , VB2 and VB9 in VMT. It was our intention to avoid the use of ion-pairing reagents in the mobile phase. VC is extremely unstable in basic and neutral solutions, but relatively stable in acidic solutions, therefore a solution containing phosphoric acid was used as a diluting solution for VB1 , VB3 , VB6 , VC and VB5 . The test of concentrations of phosphoric acid (0.02%, 0.05%, 0.08% and 0.10%) indicated that VC was more stable at higher concentrations, however a shoulder peak was observed in the peak of VB1 when 0.08% or 0.10% phosphoric acid was used, thus, 0.05% phosphoric acid was selected. In addition, it was also found that 0.05% phosphoric acid solution could only guarantee the stability of VC in standard

solutions, but not in analytical solutions, therefore, an antioxidant, sodium thiosulfate was introduced into the diluting solution. The concentrations of sodium thiosulfate (0.1%, 0.3%, 0.5% and 1.0%) were then investigated and the results showed that concentrations over 0.3% did not significantly improve the stability of VC, therefore 0.3% sodium thiosulfate was performed in the final experimental condition. As for the extraction of VB2 and VB9 , both vitamins were found slightly soluble in water and acidic aqueous solutions, but soluble in basic aqueous solutions, therefore the acidic diluting solution for VB1 , VB3 , VB5 , VB6 and VC was not suitable for VB2 and VB9 , and a basic diluting solution of ammonium hydroxide solution was selected. The test of concentrations of ammonium hydroxide (0.2%, 0.5%, 1.0% and 2.0%) showed that VB9 was not fully extracted at lower concentration (0.2%), while VB2 was not stable enough for 8 h (%R.S.D. > 2.0%) at higher concentration (1.0% and 2.0%), thus the diluting solution of 0.5% ammonium hydroxide solution was selected. Further, the investigation of ultrasonication time (10 min,

282 1.69 × 105 ± 1.93 × 102 1.142 ± 0.006 – 11.52–34.56 Y = 96,872X − 180,790 94,353–99,391 −238,828–122,752 0.9999 0.026 0.077

VB9 VB2 VB5

210 1.79 × 104 ± 2.18 × 102 1.134 ± 0.011 – 108.6–325.7 Y = 10,534X − 18,511 10,303–10,765 −64,839–27,817 1.0000 0.181 0.543 275 4.37 × 104 ± 3.94 × 102 1.078 ± 0.031 4.275 ± 0.015 0.4102–1.2307 Y = 15,676,236X + 258,699 15,346,236–16,006,236 69,102–586,500 1.0000 0.137 0.410

VC VB6

275 1.89 × 105 ± 5.27 × 102 1.221 ± 0.009 6.928 ± 0.202 15.90–47.71 Y = 48,340X − 32,501 46,985–49,695 −71,554–6552 0.9999 0.042 0.127

15 min, 20 min, 25 min and 30 min) for VB2 and VB9 extraction indicated that higher extraction rates and better precisions for both compounds were achieved when the time was 25 min or longer, therefore, the ultrasonication time of 25 min was selected. In the course of the optimization of mobile phase, phosphoric acid, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate solutions mixed with methanol and acetonitrile were tested. Relatively bigger retention factors and better resolutions for all seven compounds were obtained when performed with a mobile phase consisting of ammonium dihydrogen phosphate buffer and acetonitrile. To optimize the separation, effects of the buffer concentrations (10, 25, 50 and 100 mM), the buffer pH values (2.5, 3.0, 3.5, 4.0), the buffer–acetonitrile ratios (97:3, 95:5, 93:7, 90:10, 85:15, 80:20), the flow rates (0.5, 1.0 and 1.5 ml min−1 ) and the column temperatures (ambient, 30 ◦ C, 40 ◦ C) were systematically investigated. The results showed that neither flow rate nor column temperature had significant effect on separations, while the buffer concentrations, the buffer pH values and the buffer–acetonitrile ratios were critical to separations. Higher buffer concentrations, but not higher than 50 mM, could improve the peak shapes and resolutions for all seven vitamins, therefore 50 mM was selected; shoulder peaks for VB1 and VB3 were observed in a buffer of pH 2.5 while instability of VC was indicated in a buffer of pH 3.5 or 4.0, therefore a buffer of pH 3.0 was used. Higher acetonitrile ratio in the mobile phase could shorten the analysis time, however, the forced degradation products could interfere when the ratio was higher than 5% for VB1 , VB3 , VB6 , VC and VB5 , or higher than 15% for VB2 and VB9 , therefore, the buffer–acetonitrile ratios of 95:5 and 85:15 were selected. Furthermore, although the maximum UV absorbance of VB1 , VB3 , VB6 and VC in the mobile phase were 246, 261, 291 and 244 nm, respectively, when the window of 275 nm was detected, all these compounds showed suitable peak heights, thus 275 nm was selected as the detection wavelength for VB1 , VB3 , VB6 and VC. VB5 only showed end absorption at 194 nm, the detection wavelength was set at 210 nm as reported in the literature [14]. The detection wavelength for VB2 and VB9 was set at 282 nm for it was the maximum UV absorbance for VB9 (0.4 mg/tablets) and also showed adequate UV absorption intensity for VB2 (1.7 mg/tablets).

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282 1.75 × 105 ± 4.44 × 102 1.357 ± 0.006 20.89 ± 0.10 2.952–8.856 Y = 113,905X − 11,187 111,741–116,069 −12,776–1589 1.0000 0.010 0.030


c

a

b

Expressed as mean ± SD (n = 5). The unit for VC was g l−1 . 95% confidence interval.

275 1.32 × 105 ± 2.36 × 102 0.778 ± 0.001 3.551 ± 0.251 128.2–3205 Y = 15,427X − 6758 15,047–15,807 −96,311–82,795 1.0000 0.143 0.428

VB3 VB1

3.3.2. Specificity The representative chromatograms (Fig. 1) for the placebos, vitamin standards, and analytical samples showed that other active ingredients and excipients were well separated from the seven vitamins. The chromatograms for forcedly degraded samples (Fig. 2) indicated that VC and VB1 significantly degraded under all stress conditions, while VB3 , VB5 , VB6 , VB2 and VB9 degraded under some

275 1.18 × 105 ± 2.61 × 102 1.432 ± 0.021 2.827 ± 0.254 9.792–29.376 Y = 43,387X + 119,049 42,085–44,689 89,076–149,022 0.9999 0.041 0.123

3.3.1. Linearity, limit of detection and limit of quantitation The results of linearity, LOD and LOQ are listed in Table 1. All those parameters indicated the method with satisfactory linear correlations and sensitivities for all seven compounds.

Wavelength (nm) Theoretical platea Asymmetry factora Resolutiona Linear range (mg l−1 )b Linear equation CIc for slope CIc for intercept Correlation coefficient (r) LOD (mg l−1 ) LOQ (mg l−1 )

3.3. Method validation

Parameters

The results of system suitability test are listed in Table 1. All those parameters met the acceptable criteria of the FDA CDER guidance for validation of chromatographic methods (>2000 for theoretical plates, ≤2.0 for asymmetry factors, and > 2.0 for resolutions between two adjacent peaks) [23], and the recommended criteria for the proposed method was > 4000 for theoretical plates, 0.7–1.5 for asymmetry factors, and > 2.0 for resolutions between two adjacent peaks.

Table 1 System suitability, linearity, limits of detection and limit of quantitation.

3.2. System suitability

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Table 2 Results of precision and stability. Analyte

VB1 VB3 VB6 VC VB5 VB2 VB9

Precision (n = 6, %R.S.D.)

Stability of standard solutions (n = 5, %R.S.D.)

Stability of analytical solutions (n = 5, %R.S.D.)

Instrumental

Repeatability

Intermediate

Ambient temp. (within 12 h)

4 ◦ C (within 12 h)

Ambient temp. (within 2 h)

4 ◦ C (within 12 h)

0.88 0.27 1.01 1.97 0.34 0.53 0.91

1.41 0.92 1.27 1.75 0.99 1.42 1.37

1.66 1.05 0.94 1.92 1.11 1.87 1.75

0.45 0.35 1.06 0.85 0.18 1.72 0.91

0.42 0.36 0.86 0.60 0.27 1.55 0.77

1.20 0.18 1.05 1.41 0.86 1.84 0.96

1.44 0.83 0.88 1.90 1.43 1.55 1.82

Table 3 Results of accuracy: % recoveries expressed as mean ± SD (n = 3). % of target conc.a

VB1

VB3

VB6

VC

VB5

VB2

VB9

75 100 125 Average

99.78 ± 0.44 100.2 ± 0.3 99.67 ± 0.23 99.88

99.75 ± 0.04 100.1 ± 0.4 99.72 ± 0.17 99.86

100.4 ± 1.0 99.45 ± 0.74 99.82 ± 0.35 99.89

100.5 ± 0.7 100.5 ± 0.8 100.2 ± 0.7 100.4

99.77 ± 0.15 100.3 ± 0.1 99.77 ± 0.24 99.95

102.3 ± 0.4 100.1 ± 0.3 100.1 ± 0.7 100.8

97.95 ± 0.71 98.94 ± 1.30 99.51 ± 1.75 98.80

a

100% of target concentration is the concentration of working standard solutions.

Table 4 Results of sample assay: % of label claima expressed as mean ± SD (n = 3). Analyte

The developed method Lot 1,011,365

VB1 VB3 VB6 VC VB5 VB2 VB9

100.5 100.0 116.6 101.7 123.9 93.82 109.2

± ± ± ± ± ± ±

1.6 0.9 1.3 1.8 0.9 1.31 1.9

USP methods Lot 1,012,232 98.73 99.40 119.7 101.3 122.5 96.68 109.3

± ± ± ± ± ± ±

1.47 0.87 1.5 1.7 1.0 1.58 1.6

Lot 1,101,344 100.5 102.3 124.5 96.58 126.1 101.3 119.1

± ± ± ± ± ± ±

1.8 1.1 1.6 2.16 1.3 1.8 2.1

Lot 1,011,365 101.8 101.4 115.4 103.8 123.4 94.74 107.9

± ± ± ± ± ± ±

1.9 1.2 1.4 0.4 1.1 1.67 2.3

Lot 1,012,232 99.88 98.95 119.0 103.5 122.9 95.11 111.0

± ± ± ± ± ± ±

1.61 0.99 1.4 0.5 1.3 1.80 1.5

Lot 1,101,344 102.4 102.7 123.3 98.82 125.5 103.1 117.9

± ± ± ± ± ± ±

1.7 1.3 1.5 0.3 1.4 2.2 2.4

a 100% of label claim equal to 1.5 mg/tablet of VB1 , 20 mg/tablet of VB3 , 2 mg/tablet of VB6 , 60 mg/tablet of VC, 10 mg/tablet of VB5 , 1.7 mg/tablet of VB2 and 0.4 mg/tablet of VB9 in a tablet of about 1.5 g.

stress conditions, however, the degradation products did not interfere with the components of interest. 3.3.3. Precision and stability Results of precision and stability tests are shown in Table 2. In all cases, the %R.S.D. values were less than 2%. VC was stable within 12 h in standard solutions at ambient temperature and in analytical solutions at 4 ◦ C, but only stable within 2 h in analytical solutions at ambient temperature. 3.3.4. Accuracy Results obtained are shown in Table 3. The values demonstrated that the method was accurate within the desired ranges. 3.3.5. Analysis of VMT and method comparison The validated method was successfully applied to the determination of VB1 , VB3 , VB6 , VC, VB5 , VB2 and VB9 in three batches of VMT (Centrum® ). The representative chromatograms are shown in Fig. 1(C and F). The assay results (see Table 4) indicated that the developed method was coincident with the USP methods in the determination of all seven vitamins. 4. Conclusions Determination of water-soluble vitamins in VMT by HPLC was challenging due to their high polarity and diverse physico-chemical properties, especially when extremely unstable VC was included. In this paper, we developed a simple, isocratic, and stabilityindicating HPLC method for the rapid determination of VB1 , VB3 , VB6 , VC, VB5 , VB2 and VB9 in VMT with good system suitability,

sensitivity, linearity, specificity, precision, stability and accuracy. All seven water-soluble vitamins were well separated from other ingredients and degradation products. Method comparison showed good concordance between the developed method and the USP methods.

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