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171
RESUMEN
Se disean dos mtodos para la extraccin de las vitaminas del complejo B en muestras de Arthrospira maxima
cubana. Para la determinacin, se emplea un sistema HPLC formado por una columna de octadecilsilano y fases
mviles de metanol - agua - cido fosfrico 0.1 mol/L (20:79.5:0.5) para el anlisis de la cianocobalamina y (30:69.5:0.5)
para el resto de las vitaminas, empleando hexanosulfonato sdico 5 mM en ambos casos. Las velocidades de flujo
son de 1 y 0.8 mL/min respectivamente. La determinacin se realiza a 362 y 270 nm. Se observ que la Arthrospira
maxima cultivada en Cuba constituye una rica fuente de vitaminas del grupo B presentando un contenido promedio
de 3.1 mg/10g de tiamina, 4.0 mg/10g de riboflavina, 9.0 mg/10g de piridoxina, 0.29 mg/10g de cianocobalamina, 21
mg/10g de cido nicotnico y 26 mg/10g de nicotinamida.
Palabras clave: vitaminas del complejo B. Mtodos de extraccin. Arthrospira mxima. Cromatografa liquida de alta
resolucin.
ABSTRACT
Two methods for the extraction and determination of B-complex vitamins in Cuban Arthrospira maxima samples are
described. High performance liquid chromatography was employed using a column of octadecylsilane and mobile phase
containing metanol-agua-0.1M, orthofosforic acid (20:79.5:0.5) were used in the analysis of cyanocobalamine and
(30:69.5:0.5) for the remaining vitamins. 5 mM sodium hexanosulfonate was used as ion pair. Flow rates were set at
1 mL/min and 0.8 mL/min and injection volumes at 20 and 100 ?L. Vitamin determinations at 362 and 270 nm using
a diode array detector produced good results. High detection values for B-complex vitamins in Cuban Arthrospira
maxima were obtained.
KEY WORDS: Extraction methods. B-complex vitamins. Arthrospira maxima. High performance liquid chromatography.
INTRODUCCION INTRODUCTION
Se conoce que en los alimentos se encuentran It is a well known fact that traditional sources
las fuentes tradicionales de obtencin de vitami- for vitamins are to be encountered in foodstuffs.
nas, sin embargo los investigadores continuamente However, researchers are continually engaged in
buscan otras fuentes no convencionales para the search for other non-conventional sources in
obtener estos componentes indispensables en la order to obtain these indispensable dietary com-
dieta. Las algas son una importante fuente de ponents. Although algae represent a major sour-
vitaminas, aunque en la actualidad son los pases ce for vitamins, currently it is mainly the Asiatic
Ars Pharmaceutica, 42:3-4; 171-183, 2001
172 FERNNDEZ-CRDENAS T, GONZLEZ-SAN MIGUEL H M y TRAVIESO L.
asiticos los que mayormente la incorporan en la countries that have incorporated them into their
alimentacin (1-3). foodstuffs1-3.
En el presente trabajo, se estudian las vitami- In the present work B-group vitamins present
nas del grupo B presentes en la Arthrospira mxima in Arthrospira maxima cultivated in Cuba are
cultivada en Cuba con el objetivo de evaluar su studied in order to evaluate the potential of this
potencial como fuente de suministro de estos alga as a source of supply for these nutrients.
nutrimentos. Este trabajo parte de estudios desa- This work represents a continuation of studies
rrollados en otros pases con semejantes prop- developed in other countries with similar objec-
sitos (4). tives4.
La Arthrospira es conocida por su elevado Arthrospira maxima is well known for its high
contenido intracelular de protenas. Tambin se intracellular protein content. Additionally, high
han reportado importantes niveles de pigmentos y levels of pigments, lipids and acidic fats have
lpidos as como cidos grasos. La pared celular been reported. The fragility of the cellular wall
de esta alga es frgil lo cual resulta favorable al of this alga proves favourable in its evaluation
evaluarla como posible fuente de vitaminas (5, 6). as a possible source of vitamins5,6.
La Association of Official Analytical Chemist The Association of Official Analytical Che-
(7) ha recomendado la determinacin de vitami- mists7 recommends the determination of B-com-
nas del complejo B mediante mtodos microbio- plex vitamins through microbiological, spectro-
lgicos, espectrofotomtricos y fluoromtricos: photometric and fluorometric methods. These
estas tcnicas tambin han sido sugeridas para la techniques have also been suggested for the
cuantificacin de vitaminas en la Spirulina pla- quantification of vitamins in Spirulina platen-
tensis (1-3). La cromatografa lquida de alta sis1-3. High performance liquid chromatography
resolucin (HPLC) ha sido la principal va de (HPLC) has provided the main method of analy-
anlisis de las mezclas de vitaminas (8-10) y sis of vitamin mixtures8-9 and more recently,
ms recientemente, la electroforesis capilar ha capillary electrophoresis has been employed to
sido empleada para llevar a cabo la separacin carry out the separation of vitamin mixtures11-14.
de mezclas de vitaminas (11-14). In both pharmaceutical preparations and in
Los detectores flluoromtricos y espectrofo- natural sources, fluorometric and spectrophoto-
tomtricos acoplados a HPLC han sido emplea- metric detectors coupled to HPLC have been
dos para el anlisis de las vitaminas B1, B2, B6, employed in the analysis of vitamins B1, B2, B6,
B12 y la nicotinamida tanto en preparaciones B12 and nicotinomide. Reversed phase chromato-
farmacuticas como en fuentes naturales; la cro- graphy has been widely used in the study of these
matografa de fase reversa ha sido muy utilizada compounds8,10,15 and in order to improve the re-
en el estudio de estos compuestos (8, 10, 15). sults of chromatographic analysis the use of io-
Para mejorar los resultados del anlisis cromato- nic pairs has been introduced16,17.
grfico se ha incorporado la utilizacin de pares The simultaneous determination of vitamins
inicos (16,17). constitutes a highly useful method of analysis
La determinacin simultnea de vitaminas and may be applied to different natural sources.
constituye una va muy favorable para el anli- The complexity of natural matrices, in particular
sis, aplicable en diferentes fuentes naturales. La algae, make the use of purification processes
complejidad de las matrices naturales, en parti- followed by exhaustive washing procedures ne-
cular las algas, hace necesario el empleo de pro- cessary in order to guarantee an adequate reco-
cedimientos de purificacin seguidos de lavados very of vitamins15,18,19. Such processes involve
exhaustivos para garantizar una adecuada recu- work in which ionic interchanges are applied, as
peracin de las vitaminas (15, 18, 19). De esta well as chemical and enzymatic treatments in
forma, se encuentran reportes en los cuales se solid phase extractions, in order to carry out the
aplica el intercambio inico, as como tratamientos preparation of the samples.
qumicos y enzimticos unidos a extraccin en The current work presents the results obtai-
fase slida para llevar a cabo la preparacin de ned employing extraction and purification pro-
las muestras. cedures for thiamine, riboflavin, pyridoxine,
En el presente trabajo se muestran los resul- cyanocobalomine, nicotinamide and nicotinic acid
tados obtenidos al emplear procedimientos de ex- in Arthrospira maxima. The identification and
traccin y purificacin de tiamina, riboflavina, quantification of the vitamins through HPLC was
piridoxina, cianocobalamina, nicotinamida y cido applied after isolating and purifiying the vitamin
nicotnico en Arthrospira maxima. La identifica- elements.
cin y cuantificacin de las vitaminas mediante
HPLC fue aplicada despus de aislar y purificar
los factores vitamnicos. EXPERIMENTAL EVIDENCE
Reactives
PARTE EXPERIMENTAL
In the study, cyanocobalamine, nicotinic acid,
Reactivos nicotinamide thiamine, riboflavin and standard
pyridoxine USP were used. The reactives used
En el estudio se utiliz cianocobalamina, ci- were: orthophosphoric acid (Fluka, Buchs, Swit-
do nicotnico, nicotinamida, tiamina, riboflavina zerland), methanol HPLC grade (Merck, Darms-
y piridoxina estndares USP. Los reactivos em- tadt, Germany), sodium hexanosulphonate and
pleados fueron: cido ortofosfrico (Fluka, Bu- sodium cyanide (BDH, Poole, UK). The water
chs, Suiza), metanol grado HPLC (Merck, Dar- was purified in a mili-Q system (Millipore, Mil-
mstadt, Alemania), hexanosulfonato de sodio y ford, MA, USES); hydrochloric acid (Panreac,
cianuro de sodio (BDH, Poole, UK). El agua Spain). Papain and diastase were employed for
fue purificada en un sistema Milli-Q (Millipore, biochemical uses (BDH, Poole, UK).
Milford, MA, USES); cido clorhdrico (Panre-
ac, Espaa). Se emple papaina y diastasa para
fines bioqumicos (BDH, Poole, UK). Chromatographic system
Fase mvil para el anlisis de Cianocoba- Mobile phase for thiamine, riboflavin, pyri-
lamina: metanol - agua - cido fosfrico 0.1 mol/ doxine, nicotinic acid, and nicotinamide: me-
L (20:79.5:0.5). Flujo, 0.8 mL/min. thanol water- phosphoric acid 0.1 mol/L
(30:69.5:0.5). Flow, 0.8 mL/min.
Fase mvil para tiamina, riboflavina, piri-
doxina, cido nicotnico, nicotinamida: meta-
nol - agua - cido fosfrico 0.1 mol/L (30:69.5:0.5)
5 mM en hexanosulfonato de sodio. Flujo, 0.8
mL/min.
Procedimiento 1 Tratamiento para el anlisis Procedure 1 Treatment for the analysis of cya-
de cianocobalamina nocobalamine
El clculo de la recuperacin se realiz me- The calculation of recovery was applied through
diante el anlisis de patrones de cada una de las the analysis of the standards of each of the vita-
vitaminas sometidos a los procedimientos extrac- mins subjected to extraction procedures( pro-
tivos (Procedimientos 1 y 2). Despus de apli- cedures 1 & 2). After the application of the
car el procedimiento correspondiente, se proce- corresponding procedure, the chromatographs were
di al anlisis cromatogrfico. analysed.
TABLE 1. The recovery percentage of the vitamin standards subjected to processes 1 & 2.
k estn entre 1-20 que es el rango establecido be between 1-20, representing established ran-
para muestras complejas. ges for complex samples.
TABLA 2.- Evaluacin del factor de capacidad (k), factor de separacin () y resolucin (Rs)
de la separacin cromatogrfica.
Vitamina k Rs
Tiamina 18.3 1.4 103
Riboflavina 13.5 1.2 40.6
Piridoxina 6.8 1.5 104
Ac. Nicotnico 4.18 1.12 7.6
Nicotinamida 2.54 1.2 86
Cianocobalamina 8.12 2.47 7.5
TABLE 2.- The evaluation of capacity factor (k), separation factor () and resolution (Rs)
of the chromatographic separation.
Vitamin k Rs
Thiamine 18.3 1.4 103
Riboflavin 13.5 1.2 40.6
Pyridoxine 6.8 1.5 104
Ac. Nicotinic 4.18 1.12 7.6
Nicotinamide 2.54 1.2 86
Cyanocobalamine 8.12 2.47 7.5
En cuanto a la separacin y posible superpo- With regard to the separation and possible
sicin de bandas, fueron evaluados los valores superimposition of bands, the values of separa-
del factor de separacin y resolucin. Para ello tion factor and resolution were determined. In
se determin la selectividad entre cada vitamina order to do so, the selectivity among each vita-
y la seal consecutiva, obtenindose en ambos min and the consecutive signal were determined.
casos valores superiores a uno por lo que puede In both cases values greater than one were obtai-
afirmarse que no hay ningn caso de solapamiento ned, allowing us to confirm that overlapping
entre las respuestas cromatogrficas. chromatographic responses did not occur.
Determinacin de las Vitaminas del Complejo B The determination of B complex vitamins in the
en las muestras de Arthrospira maxima samples of Arthrospira mxima
cercanos al de la Nicotinamida (5.4 min.), el Acido ved. Confirmation of such was achieved through
Nicotnico (4.5 min), la Piridoxina (6.9 min), la the addition of the standards of these vitamins,
Riboflavina (9.1 min) y la Tiamina (19.1 min), which resulted in the consequential increase in
lo cual fue confirmado por la adicin de patro- the chromatographic signals established for each
nes de estas vitaminas con el consecuente au- case. The spectral analysis of the chromatogra-
mento de las seales cromatogrficas sealadas phic signals showed similar spectra to those
para cada caso. El anlisis espectral de las sea- obtained for the corresponding vitamin standards.
les cromatogrficas mostr espectros similares This allowed us to confirm the presence of these
al de los patrones de las correspondientes vita- vitamin factors in samples of Arthrospira maxi-
minas, esto permiti confirmar la presencia de ma.
estos factores vitamnicos en las muestras de In the quantification of vitamin B-complexes,
Arthrospira mxima. the calibration curve method was employed. In
Para la cuantificacin de las vitaminas del the evaluation of each vitamin, the linearity of
complejo B, se emple el mtodo de curva de the analytical response was checked (Table 3).
calibrado. En la evaluacin de cada vitamina, se
comprob la linealidad de la respuesta analtica
(Tabla 3).
Parmetro y
criterios de B1 B2 B6 B12 Acido Nicotinamida
Nicotnico
aceptacin
Coef. de
correlacin 0.9967 0.9950 0.9993 0.9991 0.9911 0.9931
r 0.99
Coef. de
determinacin 0.9918 0.9954 0.9849 0.9983 0.9823 0.9825
r 0.98
2
Hiptesis
nula(ordenada t = 2.04 t = 0.07 t = 1.77 t = 1.00 t = 2.66 t = 2.95
en el origen)
texp < (3.18)
Hiptesis = 0.9978 = 0.9881 = 0.9942 = 0.9990 = 0.9961 = 0.9899
nula(pend.)
1, p << 1 p = 3.10-4 p = 4.10-4 p = 3.10-4 p = 1.10-5 p = 4.10-4 p = 8.10-4
parameter
adaptation B1 B2 B6 B12 Nicotinic Nicotinamide
acid
criteria
Coef. of
correlation 0.9967 0.9950 0.9993 0.9991 0.9911 0.9931
r 0.99
Coef. of
determination 0.9918 0.9954 0.9849 0.9983 0.9823 0.9825
r2 0.98
Null
hypothesis t = 2.04 t = 0.07 t = 1.77 t = 1.00 t = 2.66 t = 2.95
(ordered in
origin)
texp < (3.18)
Null = 0.9978 = 0.9881 = 0.9942 = 0.9990 = 0.9961 = 0.9899
hypothesis
(pend.) -4 -4 -4 -5 -4 -4
p = 3.10 p = 4.10 p = 3.10 p = 1.10 p = 4.10 p = 8.10
1, p << 1
La precisin del sistema expresada en trmi- The precision of the system expressed in ter-
nos de repetibilidad y reproducibilidad muestran ms of repeatability and reproducibility present
coeficientes de variabilidad menores del 2% (Tabla coefficients of variability lower than 2% (Table
4). 4).
30 0,80
B1 70 0,50 0,52 0,37 2,2 100,4
100 0,48
1,25 1,01
B2 5 1,50 1,48 0,68 0,14 99,8
12,5 0,54
5 1,38
B6 8,5 1,13 0,88 0,47 2,05 100,2
15 0,65
Acido 2,5 1,68
Nicotnico 7 0,78 1,13 0,77 2,1 100,7
10 0,98
1 1,01
Nicotinamida 5 1,21 1,45 0,96 2,08 100,4
10 0,64
4 0.9
B12 20 1.0 1.0 0.7 0.46 99.8
40 1.0
1,25 1,01
B2 5 1,50 1,48 0,68 0,14 99,8
12,5 0,54
5 1,38
B6 8,5 1,13 0,88 0,47 2,05 100,2
15 0,65
Nicotinic 2,5 1,68
acid 7 0,78 1,13 0,77 2,1 100,7
10 0,98
1 1,01
Nicotinamide 5 1,21 1,45 0,96 2,08 100,4
10 0,64
4 0.9
B12 20 1.0 1.0 0.7 0.46 99.8
40 1.0
Una vez evaluado el mtodo, se determin After evaluating the method, the vitamin con-
el contenido vitamnico de la Arthrospira tent of Arthrospira maxima cultivated in Cuba
maxima cultivada en Cuba (Tabla 5). Puede was determined (Table 5). It can be observed
observarse que en nuestro caso los valores se that the values obtained in this work are to be
encuentran dentro del rango reportado para otras found within the range of values reported for
especies similares (21). other similar species21.
TABLA 5.- Cuadro comparativo del contenido de vitaminas del grupo B en Arthrospira maxima cubana
y otras especies de microalgas (en mg/10g).
(a) Earthrise Farms Spirulina grown in the USA. Typical Analysis and Specifications, 1988.
(b) Spirulina Mexicana, S.A. Certificado de Calidad. 1990.
(a) Earthrise Farms Spirulina grown in the USA. Typical Analysis and Specifications, 1988.
(b) Spirulina Mexicana, S.A. certificate of Quality 1990.
CONCLUSIONES CONCLUSIONS
Los mtodos extractivos planteados permiten The extraction methods considered allow high
obtener altos porcentajes de recuperacin de las recovery percentages of the vitamins present in
vitaminas presentes en el alga sin afectar la es- algae to be obtained without affecting their sta-
tabilidad de las mismas. La cuantificacin rea- bility. The quantification carried out through the
lizada por la tcnica de HPLC propuesta, es sa- HPLC technique proposed, is both reliable and
tisfactorio y confiable. Se pudo determinar el satisfactory. The content of B-group vitamins in
contenido de vitaminas del grupo B presentes en Arthrospira maxima cultivated in Cuba, could
la Arthrospira maxima cultivada en Cuba, obser- be determined. High content values for these
vando un alto valor en el contenido de estos metabolites were observed allowing its possible
metabolitos, lo cual permite considerar su posi- use in foodstuffs, as an alternative source for
ble uso en la alimentacin, como fuente alterna- these elements, to be considered.
tiva de estos elementos.
BIBLIOGRAFIA/BIBLIOGRAPHY
1. Li, S. (1989). Cultivation and application of microalgae in Peoples Republic of China. In Algae Biotechnology
Symposium, London.
2. Osse, A.C. (1990). A utilizaao de algas marinhas como aditivo para rascoes animais. Instituto de Investigaoes
Pesqueiras, Brasil.
3. Mc Hugh, D.J. (1991). Worldwide distribution of commercial resources of seaweed including. GELIDUM Hidrobio-
loga, 221,19-29.
4. Molloy F. J. 1990. Utilized and potentially utilizable seaweed on the Namibian coast: Biogeography and accessibility.
Hydrobiologia, 204/205: 293-299.
5. Cumberbatch, J., Bentez, I., Torres, J.L., Travieso, L., Rivas, E. (1995). Secado de biomasa de Arthrospira mxima
utilizando spray dryer. I Taller Internacional de Microalgas y Plantas Acuticas (Cuba).
6. Guglielmi, G., Rippka, R., Tandeau, N. (1993). Main properties that justify the different taxonomic position of
Spirulina spp. and Arthrospira spp. Among cyanobacteria. Bulletin de lnstitute Ocanographique. Monaco.
Nmero especial 12.
7. AOAC, Association of Official Analytical Chemist. Official Methods of Analysis (1990). 15 th Ed. Washington D.C.
8. Kwok, P.C., Rose, W.P., Tabor, R., Pattison, T.S. (1981). Simultaneous determination of vitamins B1 , B2, B6, and
niacinamide in multivitamin pharmaceutical preparations by paired-ion reversed-fase high pressure liquid chroma-
tography. J. Pharm. Sci., 70, 1014.
9. Vanderslice, J-T., Brownlee, S.R., Cortissoz, M.E. (1984). Liquid Chromatographic determination of vitamin B-6 in
foods. J. Assoc. Off. Anal. Chem. 67(5), 999-1007.
10. Gennaro, M.C. (1991). Separation of agua-soluble vitamins by reversed-fase ion-interaction-reagent high performance
liquid chromatography: application to multivitamin pharmaceuticals. J. Chromatogr. 29(9), 410-415.
11. Fujiwara, S., Iwase, S., Honda, S. (1988) Analysis of agua-soluble vitamins by micellar electokinetic capillary
chromatography. J. Chromatogr. 447(1), 133-140.
12. Boso, R.L., Bellini, M.S., Miksik, I., Deyl, Z. (1995) Microemulsion electrokinetic chromatography con different
organic modifiers: separation of agua- and lipid-soluble vitamins. J. Chromatogr. A. 709(1), 11-19.
13. Hsieh, Y., Kuo, K. (1997) Separation of retinoids by micellar electrokinetic capillary chromatography. J. Chroma-
tograph. A. 761(1-2), 307-313.
14. Buskov, S., Mller, P., Sorensen, H., Sorensen, J.C., Sorensen, S. (1998) Determination of vitamins in food based
on supercritical fluid extraction prior to micellar electrokinetic capillary chromatographic analyses of individual
vitamins. J. Chromatograph. A. 802 (1), 233-241.
15. Rizzolo, A., Polesello, S. (1992). Chromatographic determination of vitamins in foods. J. Chromatograph., 624, 103-
152.
16. Lam, F.L., Molcomb, I.J., Fusaki, S.A. (1984). Liquid chromatographic assay of ascorbic acid, niacinamide, pirido-
xina, tiamina and riboflavina in multivitamin-mineral preparations. J. Assoc. Off. Anal. Chem., 67(5), 1007-1011.
17. Gonzlez, H.M., Arques, J.M., Font, G., Maes, J. (1987). Determinacin de vitaminas del grupo B mediante
cromatografa lquida de alta resolucin. An. Real Acad. Farm. , 53, 602-608.
18. Augustin J., Klein B. P., Becker D., Venuggopal P. B. (1985). Methods of vitamin assay, Wiley Interscience, USA,
p. 497-502.
19. Dalbacke, J., Dahlquist, J. (1991). Determination of vitamin B12 in multivitamin-multimineral tablets by high-
performance liquid chromatography after solid fase extraction. J.Chromatogr., 541, 383-392.
20. Quattrocchi, O.A., Abelaira, S., Laba, F. (1992) Introduccin a la HPLC. Merck Argentina (Buenos Aires), P. 40-
63.
21. Henrikson, R. 1994. Microalga Spirulina: Superalimento del futuro. Ed. Urano, EU, p.1-28.