VI CONGRESS OF PHARMACY IN MACEDONIA

Comparison of new developed UV/VIS-spectrophotometric and HPLC method with UV/VIS

detection for determination of Vitamin B12 in various pharmaceutical dosage forms
1 1 1 1 1 1
Tanja Bakovska *, Marina Naumoska , Marjan Piponski , Emilija Janeva , Elena Petrovska , Elena Lazarevska Todevska ,
1 1 1
Hristina Andonoska , Tatjana Rusevska , Gordana Trendovska Serafmovska
1

Pharmaceutical Company Replek Farm Ltd., Quality Control Department, Skopje, Republic of Macedonia
Presenting author e-mail address: tanja_bakovska@yahoo.com
INTRODUCTION:
Vitamin B12, with chemical name 5,6-dimethyl-benzimidazoyl cyanocobalamin is a part of the group of B-
complex vitamins. Vitamin B12 (cobalamin) functions as a coenzyme, it is involved in the red blood cells for-
mation and nervous system functioning, in DNA synthesis and regulation, and also in fatty acid metabolism
and amino acid metabolism. Vitamin B12 is synthesized only by bacteria and it is present in food bacterially
fermented or obtained from animals that has this cobalamin from their gastrointestinal microflora.

AIM OF WORK:
Development of two, different methods for assay determination of vitamin B12 in dietary supplement
or pharmaceutical dosage form, as an alternative to each other for routine analyses in quality control
laboratories in pharmaceutical/chemical industry.
Structural formula of cyanocobalamin

MATERIALS AND METHODS:

All chemicals used were of Ph.Eur. quality and the demineralized water was
with low conductivity less than 1 µS. Cyanocobalamin working standard, the
pharmaceutical dosage forms and the dietary supplements were purchased from
ReplekFarm, Skopje, Macedonia.

Spectrophotometric system:
“VARIAN Carry Win 50” UV-VIS-spectrophotometer, 1-cm quartz cell at
wavelength range 190 - 750 nm, with resolution 0.5 nm and scan rate of 300
nm/min.

HPLC system:
“Varian Pro Star” HPLC system, with Autosampler 410, Varian 325 dual-
Figure 1. Chromatogram obtained with different injection volumes 10 µl, 20 µl, 30 µl, 40 µl
channel and Varian 335 photodiode-array detector, with ternary high pressure
and 50 µl, monitored at 550 nm.
mixing pump, controlled by Galaxy software version 1.91.

RESULTS:
Spectrophotometric method
In UV and visible spectrophotometry, aqueous solutions of Cyanocobalamin exhibit
maximums at 278 nm, 361 nm, and 550 nm. The spectrophotometric method we
propose prescribes signal measurement at wavelength of 550 nm of aqueous solu-
tions with concentration ~0.02 mg/ml. Spectrophotometric method for determination
of Cyanocobalamin has low cost and acceptable specificity, but it is not suitable for
complex samples, where the sensitivity is relatively low.

HPLC method
The optimized chromatographic method uses mobile phase composed of 15% v/v
acetonitrile and 85% v/v 20 mM KH2PO4, pH = 2.5. Volume of injection used is 50
µl, with mobile phase flow rate of 1.5 ml/min and signal monitoring at 550 nm. The
tests are performed on a LiChropher RP Select B chromatographic column 125 x 4
mm, with 5 µm particles.

Figure 2. Chromatograms obtained with different injection volumes: 30 µl, 40 µl, and 50 µl
monitored at 550 nm.

CONCLUSION:

The two, developed methods for assay determination of Vitamin B12 are:
 suitable for fast, routine analysis of the active compound Cyanocobalamin in
dietary supplements or pharmaceutical dosage forms, and can be used as an
alternative to each other in quality control laboratories in pharmaceutical/
chemical industry.
 simple, fast, cost-effective, sensitive, accurate, reproducible and rugged.

We suggest the spectrophotometric method for fast, routine analyses of Vitamin

B12 in simple matrixes in industrial application. The chromatographic method
is more selective and can be used either in routine analyses or in more complex
Figure 3. UV-VIS Spectrum of Vitamin B12 monitored at wavelength range 240– 640 nm
analyses, and it can be further optimized for purpose of analysis of multivitamin
dosage forms.