Stability-indicating HPLC method for determination of

vitamin B 2, 5 mL of vitamin B 3 and 4 mL of vitamin B6 were transferred to a 100 mL volumetric flask and the volume was made up with diluent and mixe...

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Indian Journal of Chemical Technology Vol. 15, November 2008, pp. 598-603

Stability-indicating HPLC method for determination of vitamins B1, B2, B3 and B6 in pharmaceutical liquid dosage form Shino Thomasa, Rakesh Kumarb, Ashutosh Sharmab, Roshan Issarania & Badri Prakash Nagoria* a

Pharmacy Wing, Lachoo Memorial College of Science and Technology, Jodhpur 324 003, India b Department of Analytical Research, Ranbaxy Research Lab., Gurgaon 122 015, India Email: [email protected] Received 1 November 2007; revised 21 July 2008

A simple, selective, precise and stability-indicating high-performance liquid chromatographic (HPLC) method for determination of vitamins B1, B2, B3 and B6 in pharmaceutical liquid dosage form was developed and validated. The chromatographic conditions comprised a reversed-phase C18 column (250 × 4.6 mm), 5 µ with a mobile phase consisting of a mixture of solution (0.015 M 1-hexane sulphonic acid sodium salt, pH 3.0 ± 0.05) and methanol in gradient elution. The flow rate was kept at 1.5 mL/min. and the detection was carried out at 280 nm. The retention times of vitamins B3, B2, B6 and B1 were 6.3, 15.1, 19.9 and 42.7 min. respectively. The linear regression analysis data for the calibration plots showed good linear relationship with coefficient of correlation values, r = 0.999 for vitamins B1, B2, B3 and B6 in the concentration ranges of 23.08-42.85, 9.61-17.84, 116.36-216.09 and 7.01-13.02 µg/mL respectively. The method was validated for precision, recovery and robustness. The vitamins undergo degradation under acidic, basic, peroxide, photochemical and thermal conditions. Statistical analysis proves that the method is reproducible and selective for the estimation of vitamins under study. As the method could effectively estimate the vitamins in presence of their degradation products, it can be employed as a stability-indicating method. Keywords: Vitamin B1, B2, B3 and B6, HPLC, Stability indicating, Validation

Vitamin B1 chemically, 3-[(4-amino-2-methyl-5pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium hydrochloride1; vitamin B2, 3,10-dihydro7,8-dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzopteridine-2,4-dione2; vitamin B3, pyridine-3carboxylic acid3 and vitamin B6, 5-hydroxy-6methylpyridine-3,4-dimethanol hydrochloride4 are official in I.P.5 and are mainly used as dietary supplements. Literature survey reveals that HPLC methods using colorimetric and ultraviolet detection in pharmaceutical formulations6,7 and in biological samples8,9; a reversed-phase highperformance liquid chromatographic procedure in multi-vitamin pharmaceutical formulations10; a reversed-phase ion-pair high performance liquid chromatographic method11-15; an electrokinetic chromatographic utilizing tetradecylammonium ions as the pseudostationary phase16; a capillary electrophoresis method17; and an in-capillary enzyme reaction method18 have been reported for the simultaneous determination of these vitamins. But there is no stability indicating highperformance liquid chromatography (HPLC) method for their determination in pharmaceutical liquid dosage form.

The ICH guideline requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substances19. It suggests that the degradation products that are formed under a variety of conditions should be identified and degradation pathways be established. It is stated that testing should include the effect of temperature, humidity (where appropriate); oxidation, photolysis and susceptibility to hydrolysis across a wide range of pH values. An ideal stability-indicating method is one that quantifies the drug per se and also resolves its degradation products. The aim of the present work is to develop an accurate, specific, reproducible and stability indicating method for the determination of vitamins B1, B2, B3 and B6 in the presence of its degradation products and related impurities as per ICH guideline20. Experimental Procedure Reagents and Materials

Vitamins B1, B2, B3 and B6 were supplied by Ranbaxy Research Labs., Gurgaon and syrup samples (Label claim: Vit. B1 5 mg/15mL, Vit. B2

THOMAS et al.: STABILITY-INDICATING HPLC METHOD FOR DETERMINATION OF VITAMINS B1, B2, B3 & B6

3 mg/15mL, Vit. B3 25 mg/15mL and Vit. B6 1.5 mg/15mL; product names: Polybion syrup, ViSyneral Z syrup and Riconia syrup; and manufacturers: Merck, Aviat Healthcare Pvt. Ltd., Rexin Pharmaceuticals Pvt. Ltd. respectively) were procured from the market. Methanol, acetonitrile and water (HPLC grade), 1-hexane sulphonic acid sodium salt, triethylamine and glacial acetic acid (all AR grade) were used. Apparatus

The apparatus used were Agilent 1100 series and Water-Alliance instruments equipped with an inbuilt solvent degasser, quarternary pump, photodiode array detector with variable injector and auto sampler. The columns used were ACE C18 (250 × 4.6 mm), 5 µm; Zorbax SB C18 (250 × 4.6 mm) 5 µm and Inertsil ODS 3 (250 × 4.6 mm) 5 µm. Methods Chromatographic conditions

Chromatographic separation was achieved at 30˚C on a reversed phase column using a mobile-phase consisting of solution (A) (6.2 g of 1-hexane sulphonic acid sodium salt dissolved in 2 L of water and 2 mL of triethylamine was added to it and pH was adjusted to 3.0±0.05 with glacial acetic acid) and methanol (B). Gradient elution was performed slowly at 99 : 1 of A : B (v/v) composition. After 7 min, isocratic elution, the composition was changed to 80 : 20 (v/v) of A : B and elution was continued isocratically for next 28 min. Thereafter the composition was changed back to 99:1 of A : B (v/v) and elution was continued isocratically for next 30 min. The flow rate was kept at 1.5 mL/min and detection was performed at 280 nm. Standard and sample preparations containing 30, 20, 168 and 10 µg/mL of vitamins B1, B2, B3 and B6 respectively were injected. The injection volume was 20 µL in all HPLC runs. Diluent preparation

Water: acetonitrile: glacial acetic acid in the ratio of 94 : 5 : 1 was used as the diluent. Water was used since the vitamins under question are soluble in water, while acetonitrile was used to increase the solubility of riboflavin and glacial acetic acid was used to adjust the pH approximately near to that of the mobile phase.

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Preparation of vitamins B1, B2, B3 and B6 stock solutions

Accurately weighed amounts, 30 mg of thiamine (vitamin B1), 20 mg of riboflavin (vitamin B2), 336 mg of niacinamide (vitamin B3) and 25 mg of pyridoxine hydrochloride (vitamin B6), were taken into 100 mL volumetric flasks separately and 50 mL of diluent was added and sonicated to dissolve. The volume was made up to the mark with diluent. The working standard solutions of vitamins contained 300 µg/mL of vitamin B1, 200 µg/mL of vitamin B2, 3360 µg/mL of vitamin B3 and 250 µg/mL of vitamin B6. Standard preparation

Stock solutions, 10 mL of vitamin B1, 10 mL of vitamin B2, 5 mL of vitamin B3 and 4 mL of vitamin B6 were transferred to a 100 mL volumetric flask and the volume was made up with diluent and mixed well. The solution was then filtered through 0.2 µm glass nylon filter. This final solution contains 30, 20, 168 and 10 µg/mL of vitamin B1, vitamin B2, vitamin B3, and vitamin B6 respectively. The filtered solution was injected into the HPLC system Sample preparation

The weight per mL of the syrup sample was determined and sample (5 mL of syrup) equivalent to about 0.5 mg of pyridoxine hydrochloride (vitamin B6) was accurately weighed into a 50 mL dry volumetric flask. To it, 35 mL of diluent was added and sonicated for 20-25 min with intermittent shaking. The volume was made up with diluent and mixed well. The solution was filtered through 0.2 µm glass nylon filter. The filtered solution was injected into the HPLC system Induced degradation of vitamins (i) Acid and base-induced degradation

Syrup (5 mL) equivalent to 0.5 mg of vitamin B6 was transferred to 50 mL volumetric flask. To it, 35 mL of diluent was added and sonicated for 20-25 min with intermittent shaking. To it, 5 mL of 1 N HCl was added and 5 mL of 1 N NaOH was added separately. The sample was diluted to volume with diluent and mixed well. The solution was immediately injected into the HPLC system.

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INDIAN J. CHEM. TECHNOL., NOVEMBER 2008

(ii) Hydrogen peroxide-induced degradation

The method described above (i) was followed except that 5 mL of 3% H2O2 was added in place of HCl/NaOH. (iii) Thermal degradation

The sample solution was heated on a boiling water bath for one hour, cooled to room temperature and injected into HPLC system. (iv) Photolytic degradation

The sample solution was kept in a photolytic chamber at 2600 lux for 24 h, followed by analysis as per proposed method.

Fig. 1 — Chromatogram of standard solution Table 1 — System suitability parameters

Results and Discussion Method development

The chromatographic conditions were optimized with a view to develop a stability-indicating assay method. Three different columns, namely, Zorbax SB C18 (250 × 4.6 mm) 5 µm, Inertsil ODS 3 (250 × 4.6 mm) 5µm and ACE C18 (250 × 4.6 mm) 5 µm were tried as under chromatographic conditions. The column ACE C18, gave good peak shape with response at affordable retention time with peak purity of the four vitamins on higher side. Also sodium salts of hexane, heptane and octane sulphonic acid were tried at concentration levels of 0.01 M and 0.015 M. The gradient profile, methanol in mobile phase varied from 1-20% (v/v), was also altered to give the best separation of the peaks. Using 0.015 M hexanesulphonic acid solution pH 3.0±0.05 and in gradient elution, all the four peaks for the mixture of water soluble vitamins were found resolved (resolution factor above 2.5). The final chromatographic system comprising a reversed-phase C18 column (250 × 4.6 mm), 5 µm with a mobile phase consisting of a mixture of solution (0.015 M hexane sulphonic acid sodium salt, pH 3.0 ± 0.05 adjusted with glacial acetic acid) and methanol in a gradient elution at a flow rate of 1.5 mL/min. was found optimum. Detection was performed at 280 nm. As per USP XXII21, system suitability tests were carried out on freshly prepared standard solution of the vitamins and parameters obtained with 20 µL injection volume are summarized in Table 1. A representative chromatogram is shown in Fig. 1.

Parameters

Vitamin B3 Vitamin B2 Vitamin B6 Vitamin B1

Retention time (min)

6.34

15.12

19.93

42.66

Tailing factor

1.16

1.07

1.18

1.17

Theoretical plates

1314

5715

11,294

35,946

Resolution factor between Vit B3 & B2

11.7







Vit B2 & B6



6.2





Vit B6 & B1

-

-

27.55

-

Calibration curves

The linearity responses for the four vitamins were determined by injecting solutions with concentrations of 23.1-42.9 µg/mL of vitamin B1, 9.6-17.9 µg/mL of vitamin B2, 116.9-217.1 µg/mL of vitamin B3 and 7-13 µg/mL of vitamin B6 prepared using working standards of each of the four vitamins. The linear regression data for the calibration curves indicate that the response is linear over the concentration range studied with coefficient of correlation, r2 value as 0.999 for all the four vitamins. The value of correlation coefficient, slope and intercept were 0.9992, 10928 and 3055, respectively for vitamin B1; .0.9993, 26521 and -2274, respectively for vitamin B2; 0.9992, 1969 and -1484, respectively for vitamin B3; and 0.9993, 22393 and -1113, respectively for vitamin B6. Validation of the method Precision

Precision was measured in terms of repeatability of application and measurement data. Repeatability of a standard sample was carried out using six replicates of the same injection (30, 20, 168 and 10 µg/mL

THOMAS et al.: STABILITY-INDICATING HPLC METHOD FOR DETERMINATION OF VITAMINS B1, B2, B3 & B6

respectively of vitamin B1, B2, B3 and B6 for standard preparation). Repeatability of the sample measurement was carried out in six different sample preparations from same homogenous blend of marketed sample (30, 20, 168 and 10 µg/mL respectively of vitamin B1, B2, B3 and B6 for sample preparation). It showed very low relative standard deviation (RSD) of peak area for all the four vitamins. The RSD for repeatability of standard preparation is 0.60, 0.24, 0.58 and 0.31 for vitamins B1, B2, B3, and B6 respectively, whereas the RSD for repeatability of sample preparation is 1.19, 0.54, 1.27 and 0.52 for vitamins B1, B2, B3, and B6 respectively. This shows that the method is precise as relative standard deviation is below 2.0%. Table 2 represents the precision data obtained for the method.

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significant (p<0.02) difference in mean %assay was found as the calculated value of F is lower than the critical value of F. Hence, the robustness of the method is established to the extent of variations applied to the experimental conditions. Accuracy

The accuracy of the method was determined by spiking working standards of the four vitamins into the placebo at different concentration levels: 80, 100 Table 2 — Method precision for vitamins B1, B2, B3 and B6 %assay Sample

Vitamin B1 Vitamin B2 Vitamin B3 Vitamin B6

1

107.22

101.67

105.86

109.89

2

104.90

100.39

104.35

109.03

3

104.20

100.86

104.38

108.95

Intermediate precision

4

107.07

101.42

106.11

110.32

Intermediate precision of the method was determined by same sample blend by two different analysts, using different instruments, in two different labs and on different days. The results are included in Table 3. As the value of RSD is below 2% for the four vitamins, intermediate precision of the method is established.

5

107.07

101.84

107.12

109.25

6

106.12

101.52

107.59

109.00

Mean

106.10

101.28

105.90

109.40

± SD

1.28

0.55

1.35

0.56

RSD

1.2

0.54

1.27

0.52

Table 3 — Intermediate precision of method

Robustness

Robustness of the method was determined by analyzing same sample blend at normal operating conditions and also by changing some operating analytical conditions such as detection wavelength, flow rate, pH of the solution and column temperature. The parameters and results of normal operating conditions (original) against changed conditions are included in Table 4. These data were subjected to ANOVA test to see any significant difference between the data sets. No

Parameter

Normal

Changed

Laboratory

Quality assurance

Stability study

Analyst

Shino Thomas

Analyst-II

Date

08/ 01/ 07

18/ 01/ 07

HPLC system

#1

#2

% assay* Vitamin B1

106.00±1.20 (1.13) 104.00±1.40 (1.35)

Vitamin B2

101.33±0.60 (0.59)

Vitamin B3

106.00±1.36 (1.28) 104.40±1.12 (1.07)

Vitamin B6

106.66±0.66 (0.63) 113.33±2.00 (1.76)

100±1.33 (1.33)

*Data represents mean ± SD (RSD) of triplicate determinations

Table 4 — Robustness of method Operating

%assay*

condition

Vitamin B1

Vitamin B2

Vitamin B3

Vitamin B6

Normal1

104.20±1.22

104.00±0.59

109.44±1.36

110.67±0.60

Variation: low2

104.80±1.09

102.67±0.45

109.60±1.19

110.33±0.54

Variation: high3 103.80±1.21 105.33±0.56 *Data represents mean ± SD of triplicate determinations 1 Wavelength 280 nm, flow rate 1.5 mL/min, temperature 30°C and pH 3.0 2 Wavelength 275 nm, flow rate 1.3 mL/min, temperature 25°C and pH 2.8 3 Wavelength 285 nm, flow rate 1.7 mL/min, temperature 35°C and pH 3.2

109.28±1.21

110.67±0.59

INDIAN J. CHEM. TECHNOL., NOVEMBER 2008

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and 120% of target concentration of each of the vitamins. The resulting solutions were assayed in triplicate and the results obtained were compared with the expected results and expressed as percentage. The mean recoveries (%) of vitamin B1, B2, B3 and B6 were found to be 102.45 ± 2.65, 99.31 ± 0.48, 99.84 ± 1.04 and 103.37 ± 1.21 respectively which are within the acceptance limit. Analysis of the marketed formulations

The developed method was applied to the analysis of the four water soluble vitamins in three marketed products viz. Riconia (Rexin Pharmaceutical Pvt. Ltd., Baddi), Polybion (marketed by Merck) and ViSyneral Z (Aviat Healthcare Pvt. Ltd., Navi Mumbai). The results of analysis are given in Table 5. The contents of vitamins were found in the range between 80-120% with RSD less than 2% which indicates the suitability of the method for routine analysis of these vitamins in pharmaceutical dosage forms.

are given in Table 6. The stressed condition samples are evaluated relative to the control sample with respect to assay and degradation (%). The high degradation (%) of vitamin B1 (38.18 %) and vitamin B2 (42.17 %) indicate susceptibility of these vitamins to acid hydrolysis and photolytic degradation respectively. The purity angle is a measure of the spectral heterogeneity of a peak based on the comparison of spectra over the entire peak, using the spectral contrast angle. The non-ideal effects are quantified and provided Table 5 — Analysis of marketed formulations %assay* Riconia

Polybion

ViSyneral Z

Vitamin B1

91.98±0.92 (1.00)

118.7±1.2 (1.01)

118.25±1.32 (1.12)

Vitamin B2

110.4±0.60 (0.54)

82.93±0.55 (0.66)

96.4±0.45 (0.47)

Vitamin B3

100.75±1.09 (1.08)

98.23±0.87 (0.89)

103.71±1.02 (0.98)

Vitamin B6

Stability indicating property

The values of assay, degradation (%), purity angle and purity threshold with stress conditions

118.07±0.74 100.6±0.79 100.74±0.66 (0.63) (0.79) (0.66) *Data represents mean ±SD (RSD) of triplicate determinations

Table 6 — Stress study data for niacinamide, riboflavin, pyridoxine and thiamine Condition Control Sample

Acid-1 N HCl/5 mL

Alkali-1 N NaOH/5 mL

Peroxide-3%(w/v), 5 mL

Thermal deg, 105˚C/1 h

Photolytic deg, 2600 Lux/24 h

Vitamin (mg/15 mL) Niacinamide Riboflavin Pyridoxine Thiamine Niacinamide Riboflavin Pyridoxine Thiamine Niacinamide Riboflavin Pyridoxine Thiamine Niacinamide Riboflavin Pyridoxine Thiamine Niacinamide Riboflavin Pyridoxine Thiamine Niacinamide Riboflavin Pyridoxine Thiamine

Assay Degradation 26.47 3.04 1.64 5.30 23.63 2.6 1.43 3.28 26.75 3.09 1.38 4.02 26.11 3.07 1.32 4.65 21.64 2.79 1.67 3.97 27.13 1.76 1.6 4.7

Percent Angle 10.71 14.55 13.04 38.18 15.69 24.1 1.37 19.57 12.35 18.25 8.38 25.03 42.17 2.55 11.24

Purity Threshold 0.235 0.272 1.131 0.807 0.192 0.155 0.757 0.339 0.110 0.114 0.423 0.098 0.067 0.219 0.690 0.483 0.186 0.685 1.024 0.501 0.065 0.373 1.028 0.408

Purity 1.342 2.356 2.726 3.515 1.127 1.561 1.764 2.496 1.042 1.214 1.451 1.528 1.044 1.324 1.805 1.441 1.254 2.749 3.012 3.593 1.11 2.084 2.265 1.978

THOMAS et al.: STABILITY-INDICATING HPLC METHOD FOR DETERMINATION OF VITAMINS B1, B2, B3 & B6

as a value of the threshold angle. When the peak is pure, the purity angle is lower than the threshold angle as seen in Table 6. Conclusion The developed HPLC technique is precise, accurate and stability indicating. Statistical analysis proves that the method is reproducible and selective for the analysis of vitamins B1, B2, B3 and B6 in pharmaceutical liquid dosage forms. As the method estimates the vitamins in presence of their degradation products, it can be employed as a stability indicating method. Acknowledgements The authors are thankful to Ranbaxy Laboratories Ltd. for providing laboratory and instrumental facilities and also samples of the vitamins. References 1 The Merck Index, 13th edn, Monograph No. 9366 (Merck and Co., Inc., White House Station, NJ, USA), 2001, 1657. 2 The Merck Index, 13th edn, Monograph No. 8285 (Merck and Co., Inc., White House Station, NJ, USA), 2001, 1470. 3 The Merck Index, 13th edn, Monograph No. 6550 (Merck and Co., Inc., White House Station, NJ, USA), 2001, 1168. 4 The Merck Index, 13th edn, Monograph No. 8072 (Merck and Co., Inc., White House Station, NJ, USA), 2001, 1429.

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5 Indian Pharmacopoeia, 4th edn (Government of India, Ministry of Health and Family Welfare, The controller of Publications, Civil Lines, Delhi, India), 1996. 6 Marszall M L, Lebiedzinska A, Czarnowski W & Szefer P, J Chromatogr A, 1094 (2005) 91. 7 Heudi O, Kilinc T & Fontannaz P, J Chromatogr A, 1070 (2005) 49. 8 Midttun O, Hustad S, Solheim E, Schneede J & Ueland P M, Clin Chem, 51 (2005) 1206. 9 Chatzimichalakis P F, Saminidou V F, Verpoorte R & Papadovannis I N, J Sep Sci, 27 (2004) 1181. 10 Moreno P & Salvado V, J Chromatogr A, 870 (2000) 207. 11 Li K, Biomed Chromatogr, 16 (2002) 504 12 Ivanovic D, Popovic A, Radulovic D & Medenica M, J Pharm Biomed Anal, 18 (1999) 999. 13 Amin M & Reusch J, Analyst, 112 (1987) 989. 14 Kwok R P, Rose W P, Tabor R & Pattison T S, J Pharm Sci, 70 (1981) 1014. 15 Kirchmeier R L & Upton R P, J Pharm Sci, 67 (1978) 1444. 16 Naess O, Tilander T, Pedersen-Bjergaard S & Rasmussen K E, Electrophoresis, 19 (1998) 2912. 17 Fotsing L, Fillet M, Bechet I, Hubert P & Crommen J, J Pharm Biomed Anal, 15 (1997) 113. 18 Okamoto H, Nakajima T & Ito Y, J Chromatogr A, 986 (2003) 153. 19 ICH, Q1A Stability Testing of New Drug Substances and Products, Int Conf on Harmonization. Geneva, October 1993. 20 ICH, Q2B Validation of Analytical Procedure: Methodology, Int Conf on Harmonization. Geneva, November 1996. 21 United States Pharmacopoeia, XXII edn (The US Pharmacopoeial Convention, Inc. 12601 Twinbrook Parkway, Rockville MD 20852), 1990, 1566.