validation of simultaneous determination of vitamin...

International Journal of Pharmaceutical

Biological and Chemical Sciences

e-ISSN: 2278-5191

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS)

| APR-JUN 2014 | VOLUME 3 | ISSUE 2 | 27-34 www.ijpbcs.net or www.ijpbcs.com

Research Article

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VALIDATION OF SIMULTANEOUS DETERMINATION OF VITAMIN A ACETATE

AND VITAMIN E ACETATE IN MULTIVITAMIN TABLETS BY RP-HPLC

R. Prabakaran1, A. Chenthilnathan

1* and S.Vikraman

2

1Department of Pharmaceutical Chemistry, Manonmaniam Sundaranar University,

Tirunelveli – 627 012, Tamil Nadu, India.

2Apex Laboratories Private Limited, Alathur, Kancheepuram District-603110 Tamil Nadu, India.

*Corresponding Author Email: [email protected]

INTRODUCTION

Vitamin A acetate (Fig 1) chemically, (2E, 4E, 6E, 8E)-

3, 7-Dimethyl - 9- (2, 6, 6- trimethylcyclohex-1-en-1-yl)

nona-2, 4, 6, 8-tetraen-1-yl acetate, is a natural form of

vitamin A which is the acetate ester of retinol [1]. It has

potential antineoplastic and chemopreventive activities.

Vitamin E acetate (Fig. 2) chemically, [(2R) -2, 5, 7, 8-

Tetramethyl-2-[(4R, 8R) - 4, 8, 12-trimethyltridecyl]

chroman-6-yl] acetate, It is the ester of acetic acid and

vitamin E. It is often used in dermatological products

such as skin creams [2].

Figure 1: Structure of Vitamin A acetate

ABSTRACT:

A simple, efficient and reproducible RP-HPLC method for simultaneous determination of Vitamin A acetate and Vitamin E

acetate in a combined multivitamin tablet dosage form has been developed and validated. The Chromatographic Separation

was carried out on Symmetry shield RP 18 (250 × 4.6mm; 5µm) column using the mobile phase consists of Acetonitrile :

Isopropyl alcohol : n-Hexane (400:400:200). The mobile phase was flowed at the rate of 2.0 ml/min and effluent was detected

at 280 nm. The retention times of Vitamin A acetate and Vitamin E acetate were 1.182 min and 1.829 min respectively. The

method was validated according to ICH guidelines and the acceptance criteria for system suitability, specificity, linearity,

precision, robustness and ruggedness were met in all cases. The method was linear in the range of 80-120 % of the operating

concentration of Vitamin A acetate (r2 = 0.9987) and Vitamin E acetate (r2 = 0.9991). The percentage relative standard

deviation for Intermediate precision was found to be less than 2.0%. Hence, the method could be successfully applied for

routine analysis of Vitamin A acetate and Vitamin E acetate from multivitamin tablets.

KEYWORDS: Vitamin A acetate and Vitamin E acetate, Multivitamin tablets, RP-HPLC, Validation.

A. Chenthilnathan et al; VALIDATION OF SIMULTANEOUS DETERMINATION OF VITAMIN A ACETATE AND VITAMIN E …….

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS) | APR-JUN 2014 | VOLUME 3 | ISSUE 2 | 27-34| www.ijpbcs.net

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Figure 2: Structure of Vitamin E acetate

A literature survey revealed that the availability of many

methods which includes simultaneous assessment of the

status of fat soluble vitamins; retinol, α-tocopherol, 25

hydroxyvitamin D3 and 24 , 25 dihydroxyvitamin D3 in

serum of blood donors [3] and other analytical methods

[4-8]. In this present work, an attempt was made to

develop a simple, feasible and simultaneous

determination of Vitamin A acetate and Vitamin E

acetate in a combined multivitamin tablet dosage form

by RP-HPLC. The proposed method was validated in

accordance with International Conference on

Harmonization (ICH) guidelines.

MATERIALS AND METHODS

Experimental

Chemicals and reagents

Acetonitrile of HPLC grade, Isopropyl alcohol and n-

hexane were purchased from E.Merck (India) Ltd.,

Mumbai. Vitamin A acetate and Vitamin E acetate were

a gift sample by Apex Laboratories Private Limited,

Alathur, Kancheepuram District-603110 Tamil Nadu,

India. The commercially available multivitamin tablets

containing Vitamin A acetate and Vitamin E acetate

were procured from the local market.

Instrumentation and chromatographic conditions

The Chromatographic Separation was carried out on

Symmetry shield RP 18 (250 × 4.6mm; 5µm) column

using the mobile phase consists of Acetonitrile :

Isopropyl alcohol:n-Hexane (400:400:200) and filter

through 0.45µ membrane filter before use, degassed and

were pumped into the column at a flow rate of 2.0

ml/min. The detection was monitored at 280 nm. The

volume of injection loop was 20 µl prior to the injection

of the drug solution; the column was equilibrated for at

least 15 min. with the mobile phase following through

the system.

Preparation of Standard and Sample Preparations

Standard Preparation

Weighed accurately about 20 mg of Vitamin A acetate

WS and 110 mg of Vitamin E acetate WS powder in an

iodine flask and 10 ml of ammonia solution was added

and refluxed at 50 °C for 30 minutes, cooled and make

up the volume to 50 ml with diluent (mixture of

Tetrahydrofuran, n-Hexane and Methanol and in the

ratio of 400:300:300) in a volumetric flask and filtered. 2

ml of above solution was transferred into a 50 ml

volumetric flask and make up to 50 ml with diluent.

Filtered through 0.45 micron filter.

Sample Preparation

Twenty multivitamin tablets were selected, weighed and

powdered. About 945 mg of powdered sample was

weighed and transfer into an iodine flask. 10 ml of

ammonia was added and refluxed at 50°C for 30

minutes, cooled and make up the volume to 50 ml with

diluent (mixture of Tetrahydrofuran, n-Hexane and

Methanol and in the ratio of 400:300:300) in a

volumetric flask and filter. 5 ml of filtrate was

transferred into a 100 ml volumetric flask and make up

the volume with diluents. Filtered through 0.45 micron

filter.



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RESULTS AND DISCUSSION

All of the analytical validation parameters for the

proposed method were determined according to

International Conference on Harmonization (ICH)

guidelines [9].

Specificity and System Suitability

The specificity of the HPLC method is illustrated in Fig.

3 where complete separation of Vitamin A acetate and

Vitamin E acetate were noticed in presence of excipients

used in the tablet formulation. The retention time of

sample and standard peaks were comparable. In addition,

there were no any interfering peaks eluted at the

retention time of analytes from the blank and placebo

solution. In peak purity analysis with PDA detector,

purity angle was always less than purity threshold for the

analyte. This showed that the peaks of analyte were pure

and excipients in the formulation do not interfere with

the analyte. The results were shown in Table No.1. The

resolutions between the peaks were found more than 2.0.

Hence the peaks were well separated. The results were

given in the Table No.2.

Table 1: Specificity for Vitamin A acetate and Vitamin E acetate

Vitamins Type Retention Time

(min) Area Purity angle Purity threshold

Vitamin A acetate

Standard 1.182 125395 1.236 1.754

Sample 1.165 126350 1.127 1.526

Vitamin E acetate

Standard 1.892 215388 2.024 7.516

Sample 1.814 216170 1.205 9.256

Table 2: System Suitability of Vitamin A acetate and Vitamin E acetate

S.No Vitamin A acetate Vitamin E acetate Resolution

1 125395 215388

3.6

2 125980 215302

3 125634 215234

4 125901 215654

5 125324 216789

Average 125647 215673

SD 293.005 643.701

% RSD 0.2 0.3



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Figure 3: Typical HPLC Chromatogram of Sample Multivitamin Tablets

(Vitamin A acetate and Vitamin E acetate)

Linearity and Range

The linearity of this method was determined at five

concentration levels from 80% -120% of the operating

concentration for Vitamin A acetate and Vitamin E

acetate and it was shown in the table 3 and 4. The plot of

peak area of each sample against respective

concentration of Vitamin A acetate and Vitamin E

acetate were found to be linear (figure 4 and 5) in the

range of 80% - 120% of the operating concentration.

Correlation co –efficient of the standard curve was found

to be 0.9987 and 0.9991 for Vitamin A acetate and

Vitamin E acetate respectively. It observed that

correlation coefficient and regression analysis are within

the limits.

Table 3: Linearity of Response for Vitamin A acetate

Linearity Level % Concentration Area

1 80 100851

2 90 114263

3 100* 124465

4 110 136551

5 120 148213

Slope 1170.1200

Intercept 7856.6000

Correlation Coefficient 0.9994



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Table 4: Linearity of Response for Vitamin E acetate

Linearity Level % Concentration Area

1 80 173219

2 90 194085

3 100* 215421

4 110 235468

5 120 259564

Slope 2140.7300

Intercept 1478.4000

Correlation Coefficient 0.9996

Figure 4: Linearity of Response for Vitamin A acetate

Figure 5: Linearity of Response for Vitamin E acetate

y = 1170.1x + 7856.6

R2 = 0.9987

0

20000

40000

60000

80000

100000

120000

140000

160000

0 20 40 60 80 100 120 140

Concentration (µg/ml)

Are

a

y = 2140.7x + 1478.4

R2 = 0.9991

0

50000

100000

150000

200000

250000

300000

0 20 40 60 80 100 120 140

Concentration (µg/ml)

Are

a



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Precision

The precision of an analytical procedure expresses the

closeness of agreement between a series of

measurements obtained from multiple sampling of the

homogenous sample under the prescribed conditions.

Reproducibility

Examines the precision between laboratories and is often

determined in collaborative studies. Reproducibility data

for Vitamin A acetate and Vitamin E acetate were shown

in Table 5 and 6. This indicated that method was highly

precise.

Table 5: Precision - Reproducibility for Vitamin A acetate

S.No. Sample Name Area Drug Recovery (%)

1. Sample -1 125865 102.1

2. Sample -2 125772 102.0

3. Sample -3 126589 101.7

4. Sample -4 125547 102.8

5. Sample -5 125864 102.1

6. Sample -6 126854 102.9

Mean 102.3

Standard deviation 0.42

RSD % 0.4

Table 6: Precision - Reproducibility for Vitamin E acetate


1. Sample -1 218412 106.2

2. Sample -2 216542 105.3

3. Sample -3 217956 106.0

4. Sample -4 216542 105.3

5. Sample -5 219832 106.9

6. Sample -6 217235 105.6

Mean 105.9


RSD % 0.6

ROBUSTNESS

Measure of methods capacity to remain unaffected by

small, but deliberate variation in method.

Change in the ratio of solvents in the mobile phase

(±2.0%)

Three sample preparations were analyzed as per the

methodology by changing the ratio solvents in the

mobile phase by means of ±2.0.The robustness data

Vitamin A acetate and Vitamin E acetate, by changing

the ratio of solvents in the mobile phase. It was shown in

Table No.7. It was observed that there were no marked

changes in the chromatograms and the % difference in

assay value between the Precision and Robustness

studies were found not more than 2.0% which

demonstrates that the proposed method is robust.



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Table 7: Robustness - Change in the ratio of solvents in the mobile phase (±2.0%)

High Organic - Acetonitrile: Isopropyl alcohol: n-Hexane (410:400:190)

Low Organic - Acetonitrile: Isopropyl alcohol: n-Hexane (390:400:210)

RUGGEDNESS

Six sample preparations were analysed as per the

methodology by a different analyst on a different

instrument on a different day. The ruggedness data of

Vitamin A acetate and Vitamin E acetate were shown in

Table no. 8 and 9. It was observed that there were no

marked changes in the chromatograms, which

demonstrates that the proposed method is rugged.

Table 8: Ruggedness data for Vitamin A acetate


1. Sample -1 125787 102.9

2. Sample -2 126213 102.4

3. Sample -3 126145 102.4

4. Sample -4 125465 101.8

5. Sample -5 126532 102.7

6. Sample -6 124985 101.4

Mean 102.2


RSD % 0.5

Table 9: Ruggedness data for Vitamin E acetate


1. Sample -1 216289 106.2

2. Sample -2 217521 106.8

3. Sample -3 217023 106.6

4. Sample -4 215986 106.1

5. Sample -5 216523 106.3

6. Sample -6 216452 106.3

Mean 106.4


RSD % 0.3

Vitamins Method % Label Claim Precision Result % Difference

Vitamin A acetate Low Organic 101.8

102.3 0.5

High Organic 101.5 0.8

Vitamin E acetate Low Organic 106.2

105.9 0.3

High Organic 106.8 0.9



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CONCLUSION

The Proposed study describes a simple, feasible and

sensitive reverse-phase high-performance liquid

chromatographic method for the quantitative

determination of Vitamin A acetate and Vitamin E

acetate in a combined multivitamin tablet dosage form.

The method was validated as per ICH guidelines and

found to be simple, specific, linear and precise.

Therefore the proposed method can be successfully used

for the routine analysis of Vitamin A acetate and

Vitamin E acetate in solid dosage form without

interference.

ACKNOWLEDGEMENTS

The authors are thankful to the management of Apex

Laboratories Private Limited, Alathur, Kancheepuram

District-603110, Tamil Nadu, India, for providing the

necessary facilities to carry out for the research work.

REFERENCES

1. Machlin, L.J. ed. Handbook of Vitamins, Marcel

Dekker, Inc., New York, 1991.

2. http://www.seacretspa.com/Vitamin-E.

3. Mata-Granados JM, Luque de Castro MD, Quesada

Gomez JM, 2008, Inappropriate serum levels of

retinol, α-tocopherol, 25 hydroxyvitamin D3 and

24,25 dihydroxyvitamin D3 levels in healthy Spanish

adults. Simultaneous assessment by HPLC. Clinical

Biochemistry, 9:676-80.

4. Eduardo Paredes, Salvador E. Maestre, M Soledad

Prats, José L. Todolí, 2008, Single-injection

calibration approach for high performance liquid

chromatography. Journal of Chromatography A,

185:178-84.

5. Nina Hermans, Paul Cos, Dirk Vanden Berghe,

Arnold J. Vlietinck, Tess de Bruyne, 2005, Method

development and validation for monitoring in vivo

oxidative stress: evaluation of lipid peroxidation and

fat soluble vitamin status by HPLC in rat plasma.

Journal of Chromatography B, 822:33-39.

6. J.M. Quesada, J.M. Mata-Granados, M.D. Luque de

Castro, 2004, Automated method for the

determination of fat-soluble vitamins in serum. The

Journal of Steroid Biochemistry and Molecular

Biology, 89-90:473-77.

7. S Casal, B Macedo, M.B.P.P Oliveira, 2001,

Simultaneous determination of retinol, β-carotene

and α-tocopherol in adipose tissue by high-

performance liquid chromatography. Journal of

Chromatography B: Biomedical Sciences and

Applications, 763:1-8.

8. P Salo-Väänänen, V Ollilainen, P Mattila, K

Lehikoinen, E Salmela-Mölsä, V Piironen, 2000,

Simultaneous HPLC analysis of fat-soluyble vitamins

in selected animal products after small-scale

extraction. Food Chemistry, 71:535-43.

9. ICH guidelines, analytical method validation (Q3).

Geneva, July 2000.

*Corresponding author address:

A. Chenthilnathan

Assistant Professor &Head i/c

Department of Pharmaceutical Chemistry

Manonmaniam Sundaranar University,

Tirunelveli – 627 012, Tamil Nadu

Email: [email protected]

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