Ascorbic Acid - ACS Publications - American Chemical Society

9 Determination of Ascorbic A c i d and Dehydroascorbic A c i d HOWERDE E. SAUBERLICH, MARTIN D. GREEN, and STANLEY T. OMAYE Downloaded by UNIV OF GUELPH LIBRARY on May 31, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch009

1

U . S . Department of A g r i c u l t u r e - S E A Western H u m a n Nutrition Research Center and Letterman A r m y Institute of Research, Presidio of San Francisco, CA 94129

Advantages and limitations of commonly used and recently developed methods for the analysis of ascorbic acid and dehydroascorbic acid in foods and biological samples have been reviewed. Various procedures based on titrimetric, spectrophotometric, or fluorometric principles have been used for this purpose. Depending upon the procedure selected, dehydroascorbic acid, hydroascorbic acid, or total ascorbic acid levels may be measured. Although often quite accurate, these techniques can be laborious and time-con suming. Recently, the usefulness of high performance liquid chromatography (HPLC) in the measurement of ascorbic acid in multivitamin products has been extended to foods and biological materials, including plasma, and liver, brain, and adrenal glands. Advantages of the technique include fast analysis times, high sensitivity, and minimum sample preparation.

There are many methods for determining the α-ketolactone, L-ascorbic acid (1-threo-2,4,5,6-pentohexane-2-carboxylic acid lactone), activity in animal tissue extracts and fluids (1,2) and food extracts (3,4). With the exception of outdated bioassays, most of the analytical procedures used for the measurement of ascorbic acid fall into two categories: (i) the determination of the reduced form of ascorbic acid, usually based upon the oxidation-reduction properties of the vitamin; or (ii) the determination of total ascorbic acid based upon the oxidation of ascorbic 1

Current address: U.S. Department of Agriculture-SEA Western Regional Re search Center, Berkeley, CA 94710. This chapter not subject to U.S. copyright. Published 1982 American Chemical Society. In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

200

ASCORBIC

a c i d f o l l o w e d b y t h e f o r m a t i o n of a h y d r a z o n e or

fluorophor

ACID

(5).

The

s i t u a t i o n r e g a r d i n g c h e m i c a l analyses for the v i t a m i n r e m a i n s d y n a m i c , v e r y m u c h l i k e the s e a r c h for the b i o c h e m i c a l m e c h a n i s m of a c t i o n of ascorbic acid.

T h e complex biological relationship between

the

com

p o u n d ^ ) possessing v i t a m i n C a c t i v i t y , as w e l l as the c h e m i c a l s i m i l a r i t y of these c o m p o u n d s to others t h a t are i n a c t i v e , has m a d e t h e existence of a single, s i m p l e , a n d specific m e t h o d close to i m p o s s i b l e .

T h i s has

l e d to a p r o l i f e r a t i o n of m e t h o d p a p e r s t h a t has c o n t i n u e d to the present. Recently,

due

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chromatography L-ascorbic

to

the advancements

(HPLC),

i n h i g h performance

quantitative measurements

of

liquid

unmodified

a c i d a n d its m e t a b o l i t e s h a v e b e c o m e possible.

Soon

the

m e a s u r e m e n t of L - a s c o r b i c a c i d a n d L - d e h y d r o a s c o r b i c a c i d a n d other ascorbate

metabolites

simultaneously should

be

forthcoming.

These

m e t h o d s w i l l be of p a r t i c u l a r interest i n research, since recent suggest

a b i o l o g i c a l significance f o r

ascorbate m e t a b o l i t e s ( 6 , 7 , 8 , 9 ) .

dehydroascorbic

findings

acid and

other

I n the f o l l o w i n g p a r a g r a p h s v a r i o u s

t e c h n i q u e s often u s e d to m e a s u r e a s c o r b i c a c i d c o n t e n t w i l l be b r i e f l y r e v i e w e d a n d some of the m o r e recent d e v e l o p m e n t s i n h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c t e c h n i q u e s u s e d i n a s c o r b i c a c i d analysis w i l l be explored.

Bioassays Bioassays h a v e the d i s t i n c t a d v a n t a g e of m e a s u r i n g the s u m m a t i o n of c h e m i c a l entities t h a t possess o n l y v i t a m i n C a c t i v i t y a n d

exclude

m a t e r i a l d e v o i d of v i t a m i n C a c t i v i t y . A t the p r e s e n t t i m e , bioassays are u s e d o n l y o n o c c a s i o n i n c o m p a r a t i v e studies to e s t a b l i s h the b i o l o g i c a l specificity of c h e m i c a l s a n d i n the d e t e r m i n a t i o n of the a n t i s c o r b u t i c a c t i v i t y of i n d i v i d u a l p r o d u c t s . H o w e v e r , bioassays are t i m e - c o n s u m i n g , expensive, a n d l a c k p r e c i s i o n ; therefore, t h e i r a p p l i c a b i l i t y is l i m i t e d . Rats c a n n o t be u s e d as test a n i m a l s because of t h e i r a b i l i t y to synthesize the v i t a m i n ; h o w e v e r , g u i n e a p i g s w i t h t h e i r h i g h r e q u i r e m e n t for v i t a m i n C h a v e b e e n p r o v e d satisfactory. U n f o r t u n a t e l y , t h e r e are n o m i c r o b i o l o g i c a l organisms t h a t h a v e a n absolute r e q u i r e m e n t for a s c o r b i c a c i d t h a t c a n be u s e d as the basis f o r a bioassay. O n e of the first bioassay m e t h o d s u s e d d e f i n e d the a m o u n t of test m a t e r i a l just sufficient to p r e v e n t s c u r v y i n the g u i n e a p i g as e q u i v a l e n t to one S h e r m a n u n i t or 0.5-0.6 m g a s c o r b i c a c i d ( 1 0 ) .

T e s t a n i m a l s are

f e d a b a s a l d i e t c o n t a i n i n g a l l k n o w n n u t r i e n t s except a s c o r b i c a c i d a n d s u p p l e m e n t e d w i t h g r a d e d a m o u n t s of the test s a m p l e . A t t h e e n d of 6 - 1 0 w e e k s , the degree of p r o t e c t i o n against s c u r v y is d e t e r m i n e d b y autopsy

findings

a n d s u r v i v a l rates ( 1 1 ) .

d e n t a l h i s t o l o g y as a n e n d p o i n t (4,12,13).

S e v e r a l bioassays h a v e u s e d A f t e r 2 weeks on a given

In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

9.

SAUBERLICH E T A L .

201

Determination of Ascorbic Acid

b a s a l d i e t , s u p p l e m e n t e d w i t h g r a d e d levels of t h e test substance o r ascorbic a c i d , t h e g u i n e a p i g s are k i l l e d , t h e l o w e r jaws r e m o v e d a n d sections m a d e of t h e d e c a l c i f i e d incisors. T h e degree of p r o t e c t i o n is assessed

by microscopic

e x a m i n a t i o n f o r h i s t o l o g i c changes

s u c h as

d i s o r g a n i z a t i o n of t h e odontoblasts, the w i d t h of i r r e g u l a r i t y , a n d t h e structure of t h e d e n t i n e a n d t h e degree of c a l c i f i c a t i o n of t h e p r e d e n t i n e . T h e r e is also a s i m p l e c u r a t i v e m e t h o d b a s e d o n w e i g h t changes i n g u i n e a p i g s d u r i n g s c u r v y (14).

A q u a n t i t a t i v e bioassay b a s e d

upon

s e r u m levels of a l k a l i n e phosphatase has b e e n w o r k e d o u t f o r ascorbate Downloaded by UNIV OF GUELPH LIBRARY on May 31, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch009

a c t i v i t y (15).

I n this m e t h o d , t h e l e v e l of s e r u m a l k a l i n e p h o s p h a t a s e

i n t h e test a n i m a l is first r e d u c e d 1-5 u n i t s b y ascorbate d e p l e t i o n , a n d t h e n t h e test s a m p l e is a d m i n i s t e r e d at v a r y i n g ascorbic a c i d levels. T h e details a n d t h e n u m e r o u s p r e c a u t i o n s that one s h o u l d t a k e i n bioassays h a v e b e e n r e v i e w e d elsewhere

(4).

I n 1931, t h e u n i t of v i t a m i n

C

a d o p t e d w a s t h e a c t i v i t y i n 0.1 m L of f r e s h l y s q u e e z e d l e m o n juice. S u b s e q u e n t l y , one I n t e r n a t i o n a l U n i t ( I . U . ) o r o n e U . S . P . X I V u n i t of v i t a m i n C w a s a d o p t e d as t h e a n t i s c o r b u t i c a c t i v i t y of 0.05 m g of a s c o r b i c a c i d , t h e a p p r o x i m a t e a m o u n t i n 1 m L of l e m o n j u i c e .

Therefore, 1 g

of a s c o r b i c a c i d is e q u i v a l e n t t o 20,000 I . U .

Chemical and Physical Methods of Analysis D i r e c t spec-

Optical Absorbance and Spectrophotometry Methods.

t r o p h o t o m e t r i c m e t h o d s i n v o l v i n g l i g h t a b s o r p t i o n h a v e some l i m i t e d value for very h i g h potency

material.

T h e absorbance

s p e c t r u m of

a s c o r b i c a c i d i n n e u t r a l aqueous solutions has a p e a k v a l u e at 265 n m w i t h E b e t w e e n 7500 a n d 16,650 as r e p o r t e d i n t h e l i t e r a t u r e . differences are d u e to n o n a n a e r o b i c c o n d i t i o n s (16,17).

The

The maximum

is s h i f t e d t o w a r d s 245 n m i n a c i d i c solutions. D e h y d r o a s c o r b i c a c i d is t r a n s p a r e n t i n t h e r e g i o n of 230 n m t o 280 n m , b u t has a w e a k a b s o r p t i o n , Emax =

720 at 300 n m (18).

A basic d r a w b a c k to t h e successful a p p l i

c a t i o n of s p e c t r o p h o t o m e t r i c m e t h o d s to the e s t i m a t i o n of a s c o r b i c a c i d is t h a t t h e w e l l - d e f i n e d a b s o r p t i o n b a n d i n t h e U V r e g i o n of t h e s p e c t r u m is subject to interference f r o m m a n y substances, w h i c h w o u l d present a p r o b l e m w h e n a p p l i e d to f o o d a n d tissue extracts. Colorimetric

Methods.

T h e most

frequently

used

colorimetric

m e t h o d s h a v e b e e n r e c e n t l y r e v i e w e d b y O m a y e et a l . ( 5 ) .

Several

m e t h o d s of analyses are b a s e d u p o n t h e fact that a s c o r b i c a c i d a n d d e h y d r o a s c o r b i c a c i d possess c e r t a i n c h e m i c a l p r o p e r t i e s c h a r a c t e r i s t i c of

sugars s u c h as f o r m a t i o n o f osazones a n d c o n v e r s i o n to f u r f u r a l .

C o l o r i m e t r i c d e t e r m i n a t i o n of f u r f u r a l , a n a n i l i n e d e r i v a t i v e , has b e e n u s e d to a l i m i t e d extent f o r t h e e s t i m a t i o n of a s c o r b i c a c i d i n c e r t a i n materials. T h e s e m e t h o d s h a v e g e n e r a l l y b e e n f o u n d to b e u n s a t i s f a c t o r y


202

ASCORBIC

for t h e m e a s u r e m e n t of a s c o r b i c a c i d i n f o o d .

ACID

F o r some t i m e , m e t h o d s

for t h e d e t e r m i n a t i o n of a s c o r b i c a c i d b a s e d o n t h e r e d u c t i o n of 2,6dichlorophenolindophenol

o r t h e f o r m a t i o n of a c o l o r e d d i n i t r o p h e n y l -

h y d r a z i n e d e r i v a t i v e b y the v i t a m i n , w e r e the most satisfactory. A l t h o u g h introduced

i n 1927 ( 1 9 , 2 0 ) , d i c h l o r o p h e n o l i n d o p h e n o l

has r e m a i n e d

u s e f u l because ascorbate is essentially t h e o n l y substance i n a c i d extracts that reduces t h e i n d o p h e n o l at p H 1 to 4 t o t h e colorless leuco f o r m . F o r h i g h s e n s i t i v i t y a n d specificity the d i n i t r o p h e n y l h y d r a z i n e m e t h o d , w h e r e t h e 2- a n d 3-carbon

keto g r o u p of d i k e t o g u l o n i c a c i d forms a bis-2,4-


d i n i t r o p h e n y l h y d r a z o n e , w a s often u s e d . T h e osazone rearranges i n a c i d to f o r m a stable r e d p r o d u c t . T h i o s u l f a t e ; c e r t a i n m e t a l ions, f o r e x a m p l e , c o p p e r a n d i r o n ; a n d reductones

m a y interfere.

Depending upon the

a n a l y t i c a l c o n d i t i o n s used, fructose, glucose, a n d g l u c u r o n i c a c i d m a y also interfere i n t h e d i n i t r o p h e n y l h y d r a z i n e m e t h o d t o y i e l d h i g h values T h e d i c h l o r o p h e n o l i n d o p h e n o l m e t h o d measures o n l y r e d u c e d w h i l e the d i n i t r o p h e n y l h y d r a z i n e m e t h o d w i l l measure

(21).

ascorbate,

dehydroascorbic

a c i d a n d t o t a l ascorbic a c i d , w i t h t h e difference reflecting t h e r e d u c e d a s c o r b i c a c i d (22,23).

D e h y d r o a s c o r b i c a c i d c a n b e r e d u c e d to a s c o r b i c

a c i d b y agents s u c h as 2 , 3 - d i m e r c a p t o p r o p a n o l

( B A L ) permitting the

m e a s u r e m e n t of t o t a l a s c o r b i c a c i d w i t h d i c h l o r o p h e n o l i n d o p h e n o l ( 2 4 ) . B e c a u s e of t e c h n i c a l reasons, t h e p r o c e d u r e appears to h a v e l i m i t e d use. Several automated procedures serum have been described

T h e colorimetric methods lactone r i n g of ascorbic

f o r t h e m e a s u r e m e n t of ascorbic a c i d i n

(25,26,27,28). often p r o v i d e measures to s t a b i l i z e t h e

acid from

hydrolysis b y decreasing the p H .

A l t h o u g h t h e d r y p u r e crystals of a s c o r b i c a c i d are stable o n t h e exposure to a i r a n d l i g h t at r o o m t e m p e r a t u r e f o r l o n g p e r i o d s of t i m e ,

aqueous

solutions of the v i t a m i n are o x i d i z e d o n exposure t o a i r , a l k a l i , a n d c e r t a i n traces of metals (1). occurring

oxidative

B e l o w p H 4.0, ascorbic a c i d a n d its b i o l o g i c a l l y product,

dehydroascorbic

a c i d , are stable.

Once

d e h y d r o a s c o r b i c a c i d has b e e n o x i d i z e d to d i k e t o g u l o n i c a c i d a n d other compounds,

its v a l u e as a n a n t i s c o r b u t i c agent has b e e n lost.

I n vivo,

d e h y d r o a s c o r b i c a c i d is r e d u c e d to ascorbic a c i d ; h o w e v e r , f u r t h e r o x i d a t i o n is i r r e v e r s i b l e . The

use of m e t a p h o s p h o r i c

a c i d solutions f o r t h e e x t r a c t i o n of

a s c o r b i c a c i d f r o m p l a n t a n d a n i m a l tissues w a s first p r o p o s e d i n 1935 (29).

M e t a p h o s p h o r i c a c i d , a l o n g w i t h t r i c h l o r o a c e t i c a c i d , r e m a i n as

the reagents of choice.

Besides t h e decreased t e n d e n c y f o r h y d r o l y s i s of

the lactone r i n g , m e t a p h o s p h o r i c a c i d i n h i b i t s t h e c a t a l y t i c o x i d a t i o n of ascorbic a c i d b y m e t a l catalysts, s u c h as c o p p e r a n d i r o n ions, a n d i t inactivates t h e enzymes t h a t o x i d i z e ascorbic a c i d . O x i d a t i o n of a s c o r b i c a c i d , w h i c h a p p a r e n t l y is t h e r e s u l t of t h e a c t i o n of o x y h e m o g l o b i n , m a y o c c u r w h e n a n i m a l tissues a r e g r o u n d w i t h m e t a p h o s p h o r i c a c i d . T h i s


9.



203

o x i d a t i o n is p r o p o r t i o n a l to t h e b l o o d content of t h e tissue, b u t is n o t a serious o b j e c t i o n except i n the case of w h o l e b l o o d or i s o l a t e d r e d b l o o d cells.

T o a c e r t a i n extent, t h e p r o b l e m of o x i d a t i o n b y o x y h e m o g l o b i n

c a n b e r e d u c e d b y p r i o r t r e a t m e n t of b l o o d samples w i t h c a r b o n m o n oxide ( 3 0 ) . O t h e r c o l o r i m e t r i c m e t h o d s i n c l u d e t h e official m e t h o d of t h e U n i t e d States P h a r m a c o p e i a , w h i c h is a n i o d o m e t r i c d e t e r m i n a t i o n (31). V a r i o u s t i t r i m e t r i c p r o c e d u r e s h a v e b e e n d e s c r i b e d (32).

R e c e n t l y , a sensitive

rate assay m e t h o d f o r t h e d e t e r m i n a t i o n of a s c o r b i c a c i d w a s d e s c r i b e d


that u s e d a s t o p p e d - f l o w a p p a r a t u s (33).

Several methods based u p o n

c o u p l i n g a s c o r b i c a c i d to d i a z o n i u m c o m p o u n d s (34,35).

have been

proposed

T h e d e e p b l u e d e r i v a t i v e is d e t e r m i n e d c o l o r i m e t r i c a l l y . A

q u a n t i t a t i v e r e a c t i o n b e t w e e n selenious a c i d ( 1 m o l ) a n d ascorbic a c i d (2 m o l ) to f o r m s e l e n i u m has b e e n r e p o r t e d

(36).

S t a b l e selenious

c o l l o i d s c a n b e f o r m e d w h e n f o o d extracts c o n t a i n i n g a s c o r b i c a c i d are t r e a t e d w i t h selenious a c i d , a n d t h e r e s u l t i n g t u r b i d i t y is p r o p o r t i o n a l to t h e a s c o r b i c

a c i d content.

T h e r e is also a c h e m i c a l test f o r t h e

d e t e r m i n a t i o n of a s c o r b i c a c i d that d e p e n d s u p o n t h e r e d u c t i o n of f e r r i c i o n to ferrous i o n b y a s c o r b i c a c i d f o l l o w e d b y t h e d e t e r m i n a t i o n of t h e ferrous i o n as t h e r e d orange a - a ' - d i p y r i d y l c o m p l e x .

I n t h e presence of

o r t h o p h o s p h o r i c a c i d at p H 1—2, other r e d u c i n g or i n t e r f e r i n g m a t e r i a l s are i n h i b i t e d . usage

T h i s s i m p l e m e t h o d is fast a n d has g a i n e d c o n s i d e r a b l e

(5,37,38).

Attempts have been

made

to a d a p t t h e c e n t r i f u g a l a n a l y z e r to

provide a n automated method for determining ascorbic a c i d i n serum a n d u r i n e ( 3 9 ) . T h e m e t h o d is b a s e d o n t h e r e d u c t i o n o f f e r r i c i r o n b y a s c o r b i c a c i d , p r o d u c i n g d e h y d r o a s c o r b i c a c i d , a n d t h e f o r m a t i o n of a c o l o r b e t w e e n t h e r e s u l t i n g ferrous i o n a n d t h e c h r o m o g e n i c

reagent,

ferrozine [3-(2-pyridyl)-5,6-bis-(4-phenylsulfonic acid)-l,2,4-triazine d i s o d i u m salt]

(40).

A l t h o u g h t h e m e t h o d w a s r e p o r t e d to b e h i g h l y

p r e c i s e a n d specific, a d d i t i o n a l v a l i d a t i o n a p p e a r s necessary. E n z y m e m e t h o d s u s i n g a s c o r b i c a c i d oxidase h a v e n o t b e e n w i d e l y u s e d b u t several versions of reagents b a s e d o n d i a z o t i z e d n i t r o a n i l i n e s h a v e b e e n r e p o r t e d (41). T h e r e a c t i o n is c o m p l i c a t e d a n d t h e a s c o r b i c a c i d m o l e c u l e is p a r t l y d e s t r o y e d i n f o r m i n g t h e c o l o r e d p r o d u c t . c h r o m a t o g r a p h i c s e p a r a t i o n stage m u s t p r e c e d e t h e c o l o r

A

development

r e a c t i o n (42). T h e p r o c e d u r e is t i m e - c o n s u m i n g a n d t h e r e is n o p r o v i s i o n for r e d u c i n g a n y d e h y d r o a s c o r b i c a c i d i n t h e s a m p l e extract to a s c o r b i c acid. Fluorometric Methods. determination

of a s c o r b i c

O n e of t h e most specific m e t h o d s f o r t h e acid

a n d its b i o l o g i c a l l y a c t i v e

p r o d u c t , d e h y d r o a s c o r b i c a c i d , is t h e

fluorometric

oxidation

method introduced b y

D e u t s c h a n d W e e k s (43), w h i c h is a n official A O A C m e t h o d (44).


I t is

204

ASCORBIC

ACID

b a s e d o n t h e o x i d a t i o n of a s c o r b i c a c i d t o d e h y d r o a s c o r b i c a c i d a n d t h e c o n d e n s a t i o n of d e h y d r o a s c o r b i c form the

fluorophor,

a c i d w i t h orrTio-phenylenediamine to

quinoxaline. Deutsch a n d Weeks

(43) r i g o r o u s l y

e x a m i n e d t h e s e n s i t i v i t y a n d specificity of t h e m e t h o d a n d c o n c l u d e d t h a t t h e p r o c e d u r e w a s s u i t a b l e f o r samples c o n t a i n i n g l a r g e a m o u n t s of r e d u c i n g substances or h i g h l y c o l o r e d m a t e r i a l s . F o r t h e i n i t i a l o x i d a t i o n of ascorbic a c i d to d e h y d r o a s c o r b i c a c i d , v a r i o u s c h e m i c a l o x i d a n t s , s u c h as i o d i n e , f e r r i c y a n i d e , c h l o r a m i n e - T , 2 , 6 - d i c h l o r o i n d o p h e n o l , m e t h y l e n e blue, N-bromosuccinimide, a n d charcoal ( N o r i t ) have been reported i n Downloaded by UNIV OF GUELPH LIBRARY on May 31, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch009

the l i t e r a t u r e . S e v e r a l p r o b l e m s h a v e b e e n e n c o u n t e r e d w h e n c h a r c o a l w a s u s e d to o x i d i z e a s c o r b i c a c i d present i n t h e extracts f r o m meats, d a i r y p r o d u c t s , a n d other c o m p l e x m i x t u r e s of foods ( 4 5 ) . T h e a n a l y t i c a l results w e r e affected b y t h e grades of t h e c h a r c o a l u s e d a n d b y t h e m e t h o d of a c t i v a t i o n . T h i s is u n d e r s t a n d a b l e , since t h e c a t a l y t i c p e r f o r m ance of t h e c h a r c o a l i n m a n y redox reactions d e p e n d s u p o n t h e presence of u n s a t u r a t e d sites o n t h e c a r b o n surfaces, w h i c h c a n v a r y f r o m source to source.

I V - B r o m o s u c c i n i m i d e has b e e n r e p o r t e d to serve as a r e p l a c e

m e n t f o r N o r i t i n the s m o o t h o x i d a t i o n of ascorbic a c i d to d e h y d r o a s c o r b i c acid i n an automated

fluorometric

assay of t o t a l v i t a m i n C i n f o o d

p r o d u c t s ( 4 5 ) . I t appears to serve as a n o x i d i z i n g agent that is selective (46, 47). T h e reagent is i m m u n e to reductones a n d r e d u c t i c a c i d s , w h i c h are g e n e r a l l y present i n fruits a n d vegetables. S e v e r a l adaptations of t h e b a s i c

fluorometric

m e t h o d are n o w a v a i l

a b l e f o r specific a p p l i c a t i o n to p l a s m a (48), a n d to f o o d extracts

(49,50).

A s p e c t r o p h o t o f l u o r o m e t r i c assay p r o c e d u r e also has b e e n d e v i s e d u s i n g D E A - S e p h a d e x c o l u m n t o separate e r y t h o r b i c

(isoascorbic

acid) a n d

ascorbic a c i d ( 5 1 ) . T h i s is o f p a r t i c u l a r i m p o r t a n c e since e r y t h o r b i c a c i d , a v e r y c o m m o n f o o d a d d i t i v e , has b e e n suggested to b e a n antagonist of a s c o r b i c a c i d (52,53,54).

I n g e n e r a l , t h e spectrofluorometric assay p r o

cedures i n v o l v e measurements of

fluorescence

o n solvent extracts of t h e

a c i d i f i e d samples at 3 6 5 - 3 4 8 n m a n d 4 3 5 - 4 5 0 n m , as t h e w a v e l e n g t h s o f m a x i m u m excitation and emission, respectively Chromatographic Methods.

(48,50,51).

T h e methods mentioned for ascorbic

a c i d analysis suffer f r o m l a c k of specificity to v a r y i n g degrees.

Several

attempts h a v e b e e n m a d e to correct this b y t h e a d d i t i o n o f m a s k i n g agents o r t h e use of c o l u m n a n d / o r t h i n l a y e r c h r o m a t o g r a p h y . A l t h o u g h combining

chromatographic

described

above complicates

separations

with

t h e analyses

p e n s a t e d f o r b y t h e i n c r e a s e d specificity.

the analytical

methods

c o n s i d e r a b l y , this is c o m I n m a n y cases, p a r t i c u l a r l y

n a t u r a l p r o d u c t s , w h e r e interference is h i g h , t h e use of a c h r o m a t o g r a p h i c separation

is u n a v o i d a b l e .

T h e selection o f t h e specific

method

or

c o m b i n a t i o n of m e t h o d s to b e u s e d d e p e n d s u p o n a v a r i e t y o f factors i n c l u d i n g t h e i n f o r m a t i o n d e s i r e d a n d t h e n a t u r e of t h e s a m p l e .


9.

205



M a n y p u b l i c a t i o n s h a v e dealt w i t h modifications of t h e w o r k o f M a p s o n a n d P a r t r i d g e ( 5 5 ) as a p p l i e d to t h e q u a l i t a t i v e a n d q u a n t i t a t i v e d e t e r m i n a t i o n of ascorbic a c i d a n d v a r i o u s b r e a k d o w n p r o d u c t s ( 8 ) b y paper chromatography.

T h e l o c a t i o n of a s c o r b i c a c i d o n t h e c h r o m a t o -

grams c a n b e r e v e a l e d b y several d e v e l o p m e n t dichlorophenolindophenol,

agents i n c l u d i n g 2,6-

a m m o n i a c a l s i l v e r n i t r a t e t e t r a z o l i u m salts,

iodine vapor, ammonium molybdate, and molybdophosphoric acid. Other c h r o m a t o g r a p h i c m e t h o d s , s u c h as c o l u m n a n d t h i n l a y e r c h r o m a t o g r a p h y h a v e b e e n t r i e d o u t i n a s c o r b i c a c i d studies (56,57,58). S u c h c h r o m a t o Downloaded by UNIV OF GUELPH LIBRARY on May 31, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch009

g r a p h i c p r o c e d u r e s h a v e b e e n v e r y v a l u a b l e i n s p e c i a l investigations s u c h as t h e o c c u r r e n c e of b r e a k d o w n p r o d u c t s or metabolites of t h e v i t a m i n Chromatographic procedures

(9,59). firmatory

are also u s e f u l to p r o v i d e

con

e v i d e n c e w h e n testing f o o d f o r w h i c h t h e specificity of t h e

m e t h o d is not k n o w n . A s m e n t i o n e d before, t h e isomer of a s c o r b i c a c i d , erythorbic acid

(isoascorbic

a c i d ) , is p a r t i c u l a r l y difficult to i d e n t i f y

w h e n present i n foods. Interference b y isoascorbic a c i d i n a s c o r b i c a c i d analyses has b e e n c o r r e c t e d b y c h r o m a t o g r a p h i c means

(60).

S e v e r a l investigators have r e p o r t e d that a s c o r b i c a c i d c a n b e a n a l y z e d by g a s - l i q u i d chromatography following conversion of the parent c o m p o u n d to its t r i m e t h y l s i l y l ether (61-66). found

to b e r e l i a b l e a n d to p r o d u c e

obtained b y colorimetric procedures.

T h e procedures

results c o m p a r a b l e

have

been

with

those

I n most cases, h o w e v e r ,

m e n t of o n l y t h e r e d u c e d f o r m of t h e v i t a m i n is p o s s i b l e

measure

(67).

One

m e t h o d is suitable f o r m i c r o a n a l y t i c a l w o r k a n d has t h e a d v a n t a g e t h a t several other c a r b o h y d r a t e s a n d c a r b o h y d r a t e d e r i v a t i v e s c a n b e m e a s u r e d s i m u l t a n e o u s l y i n t h e same extract (67). T h e recent d e v e l o p m e n t of c o m m e r c i a l H P L C

systems h a s p r o v i d e d

a p o w e r f u l i n s t r u m e n t a t i o n f o r t h e separation, c h a r a c t e r i z a t i o n , i d e n t i f i c a t i o n , a n d q u a n t i t a t i o n of m i n u t e amounts of essential d i e t a r y c o m p o n ents (68,69).

Developments i n hardware a n d packings for H P L C

have

o v e r c o m e t h e p r o b l e m s o f n o n r e p r o d u c i b l e b e h a v i o r a n d l o w efficiency separations p r e v i o u s l y associated

with column

chromatography

(70).

H P L C has a l r e a d y b e e n a p p l i e d to t h e q u a n t i t a t i v e analysis of analgesics, pesticides, a n d fat-soluble v i t a m i n s w i t h p r e c i s i o n a n d a c c u r a c y a n d a m i n i m u m of s a m p l e c l e a n - u p .

Such instrumentation provides a rapid,

accurate, a n d sensitive t e c h n i q u e subnanomole

f o r the s e p a r a t i o n a n d analysis of

q u a n t i t i e s of a w i d e r a n g e of c o m p l e x

high-molecular-

w e i g h t , n o n v o l a t i l e , t h e r m a l l y l a b i l e , c o m p o u n d s t h a t are v i t a l f o r m e t a b o l i c a n d n u t r i t i o n a l studies. S e v e r a l reports h a v e d e s c r i b e d t h e use of H P L C i n t h e analyses of ascorbic a c i d i n foods a n d v i t a m i n p r o d u c t s (71, 72, 73, 74) a n d i n tissue samples

(75).

P r o c e d u r e s v a r y i n t h e t y p e of c o l u m n ,

d e t e c t i o n systems a n d means o f s t a b i l i z a t i o n o f extracts.

mobile-phase,

Reversed-phased,


206

ASCORBIC

ACID

B o n d a p a k C c o l u m n s ( W a t e r Associates, M i l f o r d , M a s s a c h u s e t t s ) w i t h fixed

w a v e l e n g t h detector ( 2 5 4 n m ) a n d t h e s a m p l e o r standards sta

b i l i z e d i n 0 . 8 % m e t a p h o s p h o r i c a c i d h a v e b e e n u s e d w i t h u r i n e samples (76).

I n t h a t s t u d y 0 . 8 % m e t a p h o s p h o r i c a c i d s e r v e d as t h e m o b i l e

phase. O t h e r m o b i l e phases, s u c h as m e t h a n o l - w a t e r ( 5 0 : 5 0 ) o r a m m o n i u m salts i n m e t h a n o l - w a t e r , w e r e t r i e d b u t r e s u l t e d i n a s c o r b i c a c i d values t h a t w e r e t o o h i g h w h e n c o m p a r e d w i t h t i t r i m e t r i c measurements ( 7 6 ) . A s i m i l a r p r o c e d u r e , s u b s t i t u t i n g a n i o n exchange c o l u m n , has b e e n u s e d f o r m u l t i v i t a m i n p r o d u c t analyses ( 7 7 ) . W i t h t h e a d d i t i o n of elec Downloaded by UNIV OF GUELPH LIBRARY on May 31, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch009

t r o c h e m i c a l d e t e c t i o n , l i q u i d c h r o m a t o g r a p h y analysis of a s c o r b i c a c i d becomes q u i t e specific a n d sensitive (77,78,79).

T h e s e c o u p l e d detector

systems ( l i q u i d c h r o m a t o g r a p h y e l e c t r o c h e m i c a l detector, L C E C )

have

been

acid

a p p l i e d w i t h excellent success

content i n food

to t h e analyses of a s c o r b i c

a n d a n i m a l tissues

(78,79).

F i g u r e 1 represents a

c h r o m a t o g r a m f o r h u m a n u r i n e o b t a i n e d w i t h t h e use of H P L C a n d a n a m p e r o m e t r i c e l e c t r o c h e m i c a l detector

(72).

T h e ascorbic

acid peak

represents 19 n g . F i g u r e 2 represents t h e analysis of a s c o r b i c a c i d i n a m o u s e b r a i n tissue extract also e m p l o y i n g H P L C a n d a n e l e c t r o c h e m i c a l detector ( 7 5 ) . T h e ascorbic a c i d p e a k corresponds to a p p r o x i m a t e l y 15 ng.

A l t h o u g h t h e a b o v e p r o c e d u r e s f o r t h e analysis of a s c o r b i c a c i d

content b y H P L C h a v e b e e n v e r y u s e f u l f o r d e t e r m i n i n g t h e r e d u c e d

URIC ACID

ASCORBIC ACID

Figure 1. Analysis of ascorbic acid in urine employing HPLC and an amperometric electrochemical detector (72): column, Zipax SAX, 2.1 mm X 50 cm glass; mobile phase, 0.05M acetate buffer, pH 4.75; flow rate, 0.33 mL/min.

4 ELUTION

TIME

1

O

(Minutes)


9.



207


2 , 5 - D I H Y D R O X Y B E N Z O I C ACID

I

I

I

I

L

0

2

4

6

8

ELUTION TIME (Minutes)

Figure 2. Analysis of ascorbic acid in a mouse brain tissue extract employing HPLC and an electrochemical detector. (Reproduced, with permission, from Ref. 75. Copyright 1975, Pergamon Press, Inc.) f o r m of t h e v i t a m i n , t h e y w e r e n o t u s e f u l f o r d e t e r m i n i n g other forms of ascorbate.

Recently, conditions were described for the h i g h performance

l i q u i d c h r o m a t o g r a p h i c s e p a r a t i o n of a s c o r b i c a c i d f r o m d e h y d r o a s c o r b i c a c i d w h e n i n p u r e solutions ( S O ) . D e h y d r o a s c o r b i c a c i d w a s m o n i t o r e d at 228 n m a n d a s c o r b i c a c i d a t 268 n m ( F i g u r e 3 ) . U n f o r t u n a t e l y , t h e m i n i m u m d e t e c t i o n l i m i t s w e r e 500 n g p e r i n j e c t i o n f o r d e h y d r o a s c o r b i c a c i d c o m p a r e d w i t h 10 n g p e r i n j e c t i o n f o r ascorbic a c i d . F i n l e y a n d Duang

(81)

h a v e also d e s c r i b e d r e c e n t l y a h i g h p e r f o r m a n c e

liquid

c h r o m a t o g r a p h i c m e t h o d that w i l l separate a n d estimate a s c o r b i c a c i d , dehydroascorbic acid, a n d 2,3-diketogulonic a c i d i n fruit a n d vegetable extracts. S u b s e q u e n t methods f o r m e a s u r i n g t h e three forms of ascorbate i n a n i m a l tissue extracts s h o u l d b e soon f o r t h c o m i n g . Other Chemical and Physical Methods.

P o l a r o g r a p h y has b e e n

t r i e d i n s p e c i a l i n v e s t i g a t i o n s , s u c h as studies of t h e b o u n d f o r m of ascorbic a c i d . B u t because of l i m i t e d specificity, t h e p r o c e d u r e has n o t seen w i d e a p p l i c a t i o n (82,83).

A s c o r b i c a c i d is o x i d i z e d at t h e d r o p p i n g

m e r c u r y electrode, t h e basis of t h e p o l a r o g r a p h i c d e t e r m i n a t i o n . D e h y d r o a s c o r b i c a c i d is n o t m e a s u r e d , h o w e v e r , since i t is n o t r e d u c i b l e at t h e d r o p p i n g m e r c u r y electrode. M a s o n et a l . (84) h a v e d e v e l o p e d a m e t h o d for the d e t e r m i n a t i o n of ascorbic a c i d b a s e d o n e l e c t r o c h e m i c a l o x i d a t i o n at t h e t u b u l a r c a r b o n electrode t h a t has b e e n m o d i f i e d to measure w a t e r -



208

ASCORBIC

Figure 3. Simultaneous analysis for ascorbic acid and dehydroascorbic acid with the use of a gradient analysis HPLC method. The minimum detectable quantities were 10 ng/ injection for ascorbic acid and 500 ng/injection for dehydroascorbic acid (80): column, LiChrosorb NH , 10 ixm; mobile phase, 0.005M KH2PO4, pH 3.5 and CH3CN; detection, ascorbic acid, 268 nm and dehydroascorbic acid, 228 nm. (Reproduced, with permission, from Hewlett-Packard.)

ACID

2

soluble

vitamins

1

1

1

0 2 4 ELUTION TIME (Minutes)

(thiamin, riboflavin, pyridoxine,

nicotinamide,

and

ascorbic a c i d ) i n p h a r m a c e u t i c a l p r e p a r a t i o n s . Other methods

for the d e t e r m i n a t i o n of a s c o r b i c

acid include a

q u a l i t a t i v e spot test ( 8 5 ) a n d h i g h v o l t a g e electrophoresis

(86).

Applications Blood and Animal Tissues.

T h e most c o m m o n l y u s e d a n d p r a c t i c a l

p r o c e d u r e for e v a l u a t i n g v i t a m i n C n u t r i t i o n a l status is t h e m e a s u r e m e n t of s e r u m ( p l a s m a ) ascorbic

a c i d do

levels of ascorbic a c i d ( 8 7 ) . not

necessarily i n d i c a t e

L o w p l a s m a levels of

scurvy, although

scorbutic

patients i n v a r i a b l y have l o w or no p l a s m a a s c o r b i c a c i d , b u t c o n t i n u e d l o w levels of p l a s m a ascorbate

of less t h a n 0.10

e v e n t u a l l y l e a d to signs a n d s y m p t o m s

of s c u r v y .

mg/100

mL

would

I n general, serum

ascorbic a c i d concentrations are u s u a l l y m o r e reflective of recent intakes rather t h a n of t o t a l b o d y stores

(88).

W h o l e b l o o d a s c o r b i c a c i d values m a y be a less sensitive i n d i c a t o r of v i t a m i n C n u t r i t u r e t h a n s e r u m o r p l a s m a levels of t h e v i t a m i n because the v i t a m i n C content i n erythrocytes n e v e r falls to the l o w levels f o u n d i n s e r u m o r p l a s m a (89,90).

A l s o there are n o w e l l - e s t a b l i s h e d classifi

cations a v a i l a b l e r e l a t i n g b l o o d v i t a m i n C values to the n u t r i t i o n a l status of this v i t a m i n i n a p o p u l a t i o n

(88).

L e u k o c y t e a s c o r b i c a c i d c o n c e n t r a t i o n s are g e n e r a l l y c o n s i d e r e d p r o v i d e a better reflection of tissue stores t h a n other b l o o d S u p p o r t i n g e v i d e n c e for this b e l i e f i n c l u d e s observations

to

components. s u c h as:

l e u k o c y t e ascorbate levels d r o p s l o w l y d u r i n g a s c o r b i c a c i d

(i)

deficiency,


9.

209



r e a c h i n g zero just before the onset of c l i n i c a l s y m p t o m s of s c u r v y

(91);

( i i ) l e u k o c y t e ascorbate levels correlate w e l l w i t h a s c o r b i c a c i d r e t e n t i o n o n diets w i t h a fixed, i n a d e q u a t e l e v e l of a s c o r b i c a c i d ( 9 2 ) ; ( i i i ) studies c o r r e l a t i n g p l a s m a ascorbate levels w i t h l e u k o c y t e ascorbate levels s u g gest t h a t t h e l e u k o c y t e levels reflect the a m o u n t of a s c o r b i c a c i d for storage w h i l e p l a s m a levels reflect its m e t a b o l i c t u r n o v e r rate ( 9 3 , 9 4 ) ; a n d ( i v ) d i r e c t e v i d e n c e i n d i c a t e s t h a t l e u k o c y t e ascorbate levels reflect total b o d y ascorbate p o o l better t h a n a n y other b l o o d c o m p o n e n t

(95).

P r o m p t s t a b i l i z a t i o n of a s c o r b i c a c i d is e s p e c i a l l y i m p o r t a n t i n t h e Downloaded by UNIV OF GUELPH LIBRARY on May 31, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch009

case of p l a s m a or s e r u m samples. M e t a p h o s p h o r i c a c i d is often u s e d f o r this p u r p o s e because it also serves as a p r o t e i n p r e c i p i t a n t . S u c h p r o p e r ties are d e s i r a b l e i n the i n a c t i v a t i o n of oxidase a n d the c a t a l y t i c effect of copper.

O x a l i c a c i d is a n a t t r a c t i v e s t a b i l i z e r for a s c o r b i c a c i d analysis

because of its l o w e r cost a n d greater s t a b i l i t y ; h o w e v e r , i t is not a p r o t e i n p r e c i p i t a n t , therefore, it has a l i m i t e d use f o r the e x t r a c t i o n of a n i m a l tissues. T h e use of e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A ) i n a d d i t i o n to t h e m e t a p h o s p h o r i c a c i d has b e e n r e c o m m e n d e d ( 9 6 ) .

E D T A would

chelate d i v a l e n t cations, a n d a s t u d y has s h o w n i t w i l l s t a b i l i z e a s c o r b i c a c i d i n the presence of c o p p e r for several days (96).

P e r c h l o r i c a c i d has

b e e n u s e d also b u t because of its i n h e r e n t dangerous p r o p e r t i e s its use is g e n e r a l l y a v o i d e d .

T r i c h l o r o a c e t i c a c i d a n d E D T A also seem a p p r o

p r i a t e extractants for ascorbate i n p l a n t m a t e r i a l s ( 9 7 ) . A s n o t e d earlier, p l a s m a f r o m b l o o d

samples m u s t b e

promptly

s t a b i l i z e d a n d , i f necessary, the a c i d i f i e d samples m a y b e stored f r o z e n at — 6 5 ° C .

B e c a u s e of the existence of o x y h e m o g l o b i n i n w h o l e b l o o d

or r e d c e l l suspensions, some c o n s i d e r a t i o n m u s t be g i v e n to i n a c t i v a t e o x y h e m o g l o b i n or use a n assay for t o t a l ascorbic a c i d content.

With

respect to tissue analysis, some d i s c r e t i o n m u s t be c o n s i d e r e d as to the degree of b l o o d c o n t a m i n a t i o n . Foods.

T h e d i s t r i b u t i o n of a s c o r b i c a c i d w i t h i n one i n d i v i d u a l f r u i t

or v e g e t a b l e

or

between

v a r i o u s foods

is often

extremely variable.

Significant difference c a n b e f o u n d i n t h e s k i n as c o m p a r e d w i t h t h e p u l p of f r u i t .

S e e d - c o n t a i n i n g tissues s h o w s t r i k i n g changes i n c o n c e n

t r a t i o n of ascorbic a c i d d u r i n g m a t u r a t i o n , b u t i n storage organs s u c h as potatoes a n d leaves, the average l e v e l r e m a i n s r e l a t i v e l y constant t h r o u g h out the g r o w t h p e r i o d . Post-harvest storage w i l l affect the v i t a m i n content of the r a w f r u i t or v e g e t a b l e c o m m e n s u r a t e w i t h the t i m e a n d t e m p e r a t u r e of storage, extent of c e l l u l a r tissue d a m a g e d , a n d the presence of ascorbic a c i d oxidase. T e m p e r a t u r e changes, s l i c i n g , c u t t i n g , or b r u i s i n g of fruits a n d vegetables, s u c h as is l i k e l y to o c c u r i n p r o c e s s i n g , c a n a l l c o n t r i b u t e to ascorbate loss. S i g n i f i c a n t losses also o c c u r w i t h c o o k i n g because

of

the t e m p o r a r i l y a c c e l e r a t e d a c t i o n of e n z y m e s . I n these instances, extrac t i o n takes p l a c e a n d t h e c o n c e n t r a t i o n of v i t a m i n i n the l i q u o r a p p r o x i -


210

ASCORBIC

ACID

mates that of the tissues. H o w e v e r , w h e n f r u i t is b o i l e d w i t h sugar, as in

the m a k i n g of

j a m , the v i t a m i n C

content

is r e m a r k a b l y

stable.

F r e e z i n g is a g o o d m e t h o d of p r e s e r v i n g f r u i t s a n d vegetables o n l y after p r o p e r p r e c a u t i o n s h a v e b e e n t a k e n to b l a n c h a n d r e m o v e e n z y m e s t h a t m i g h t o x i d i z e ascorbic a c i d .

A l s o t h a w i n g of the f o o d before

cooking

m a y result i n progressive loss of the v i t a m i n , e s p e c i a l l y i f e n z y m e s

are

present. F o r ascorbic a c i d analysis, m e t a p h o s p h o r i c a c i d is v e r y u s e f u l i n the i n a c t i v a t i o n of t h e c a t a l y t i c effect of ascorbic

a c i d oxidase

as w e l l as


other c a t a l y t i c o x i d i z i n g agents discussed p r e v i o u s l y . F o o d s s u c h as f r u i t s and

vegetables

dehydroascorbic

also h a v e a t e n d e n c y

to h a v e

a larger proportion

a c i d t h a n a n i m a l tissues; c o n s e q u e n t l y ,

methods

of

that

assay for o n l y the r e d u c e d f o r m of ascorbate m a y p r o v i d e m i s l e a d i n g l o w values. Pharmaceuticals.

I n commerce, ascorbic

s i v e l y b y synthesis ( 9 8 ) .

a c i d is p r o d u c e d

exclu

B e c a u s e o f its r a t h e r p u r e n a t u r e a n d h i g h

concentrations i n v i t a m i n - m u l t i v i t a m i n tablets, analysis b y

conventional

or s o p h i s t i c a t e d p r o c e d u r e s c a n be p e r f o r m e d easily. T h e U S P p r o v i d e s a reference s t a n d a r d of L - a s c o r b i c a c i d for assay purposes.

The

methods

u s e d c a n be chosen f r o m the m a n y discussed above. T h e m e t h o d officially a p p r o v e d b y the A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s is t h e m i c r o fluorometric

A

procedure developed by Deutsch and Weeks

Method for

in Biological

the Determination Tissues by

Introduction. methods

of Ascorbic

Acid

HPLC

A s n o t e d e a r l i e r , s e v e r a l investigators h a v e r e p o r t e d

for t h e d e t e r m i n a t i o n of ascorbic

using H P L C

(44).

(71-75).

acid i n various

substances

D i f f e r e n t groups of investigators h a v e

employed

a v a r i e t y of c o l u m n s a n d e l u t i o n c o n d i t i o n s to a c h i e v e the s e p a r a t i o n of ascorbic

from

exchange samples

i n t e r f e r i n g substances.

columns

have been

u s e d i n these

Both

reversed-phase

u s e d to a c h i e v e

ascorbic

a c i d assays

an H P L C

have

and

assay.

represented

ion The

mainly

n o n m a m m a l i a n m a t e r i a l s . I n some instances, the samples h a v e r e q u i r e d v a r i o u s p r e - c o l u m n treatments. A m o n g the d e s i r a b l e characteristics of a n HPLC

a n a l y t i c a l assay are m i n i m a l s a m p l e h a n d l i n g a n d m o d i f i c a t i o n

p a r t i c u l a r l y w i t h r e g a r d t o c o m p l e x b i o l o g i c a l tissues.

A s i m p l e assay

u s e d to d e t e r m i n e the ascorbic a c i d content of s e r u m , l i v e r , b r a i n , a n d a d r e n a l g l a n d of the g u i n e a p i g is d e s c r i b e d i n the f o l l o w i n g section. Methods. of 3 %

A l l tissues w e r e c o l l e c t e d a n d s t a b i l i z e d b y t h e a d d i t i o n

metaphosphoric

a c i d i n the r a t i o of

1 p a r t tissue to 3 parts


9.

Determination


metaphosphoric acid ( w / v ) .

of Ascorbic

211

Acid

S a m p l e s w e r e t h e n c e n t r i f u g e d at 3 X

10

3

g to r e m o v e p r e c i p i t a t e d p r o t e i n s . Samples w e r e t h e n t r a n s f e r r e d to c l e a n tubes, f r o z e n , a n d stored at — 6 5 ° C u n t i l assayed. S a m p l e s w e r e a n a l y z e d on

commercially

Associates).

available H P L C

columns

and

equipment

(Waters

S a m p l e s w e r e i n j e c t e d d i r e c t l y i n t o the c o l u m n i n 1 fxh

aliquots u s i n g a n auto-injector

(Waters Associates).

N o deterioration

i n c o l u m n p e r f o r m a n c e w a s o b s e r v e d over a 6 - m o n t h i n t e r v a l . F l o w rate was m a i n t a i n e d at 1 m L / m i n u n d e r a l l c o n d i t i o n s t h r o u g h o u t the assays. A s c o r b i c a c i d w a s m e a s u r e d i n U V a b s o r b a n c e u n i t s at 254 n m a n d Downloaded by UNIV OF GUELPH LIBRARY on May 31, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch009

q u a n t i t a t e d b y m e a s u r i n g p e a k heights. S t a n d a r d curves w e r e p r e p a r e d b y the a d d i t i o n of k n o w n a m o u n t s of ascorbic a c i d to solutions of metaphosphoric

acid.

I n the d e v e l o p m e n t

of

3%

this assay, the use

of

p e r c h l o r i c a c i d w a s a v o i d e d w h i l e t r i c h l o r o a c e t i c a c i d w a s f o u n d to b e u n s u i t a b l e as a s t a b i l i z e r because of its U V characteristics. S t a n d a r d curves w e r e p r e p a r e d for e a c h assay.

T h e final assay as a d a p t e d u s e d

0 . 1 2 5 % citrate m a d e f r o m t h e t r i s o d i u m salt at p H 7.3 as t h e eluant. Results.

I n the d e v e l o p m e n t

of this assay, s e v e r a l

reverse-phase

c o l u m n s as w e l l as the /JPorasil c o l u m n w e r e e x a m i n e d for t h e i r separatory ability.

F r o m a m o n g the f o l l o w i n g c o l u m n s the P o r a s i l c o l u m n

was

j u d g e d as g i v i n g the best e l u t i o n p a t t e r n w i t h p l a s m a : jaBondapak C N , /xBondapak p h e n y l , /xBondapak C i . A l t h o u g h r e t e n t i o n times for b o t h 8

m e t a p h o s p h o r i c a c i d a n d a s c o r b i c a c i d r e m a i n e d a p p r o x i m a t e l y t h e same ( T a b l e I ) , the q u a l i t y of the c h r o m a t o g r a m w a s j u d g e d s u p e r i o r for the /xPorasil c o l u m n .

T h i s d e c i s i o n w a s b a s e d o n o v e r a l l p e a k shape a n d

s y m m e t r y as w e l l as baseline s t a b i l i t y .

Table I. Effect of Column Types on the H P L C Retention Time of Ascorbic A c i d Dissolved in 3 % Metaphosphoric A c i d Retention Column

Type

/xBondapak C N /xBondapak p h e n y l ^Bondapak Cis /xPorasil

Ascorbic

Acid

Time

(min)

a

Metaphosphoric

2.67 2.68 2.36 2.31

Acid

2.09 2.15 1.84 1.84

° E l u t i o n buffer was 0.125% citrate, t r i s o d i u m salt, p H 7.3. used are stated i n text. V a l u e s are the average of three trials.

A l l other c o n d i t i o n s

S u b s e q u e n t to the selection of a n a p p r o p r i a t e c o l u m n v a r i o u s c h a r a c teristics of the buffer w e r e e x a m i n e d . B o t h p H a n d buffer s t r e n g t h w e r e varied a n d retention time was measured.

T h e results are p r e s e n t e d i n

T a b l e I I . G e n e r a l l y i t was f o u n d t h a t a decrease i n buffer strength or a n


212

ASCORBIC

ACID


Table II. Effect of Various Concentrations of Citrate Buffer and p H on H P L C Retention Time and Chromatographic Pattern for Ascorbic A c i d and Metaphosphoric A c i d Buffer Concentration (% Citrate)

pH

Ascorbic Acid

1.000 0.500 0.125 0.062

7.3 7.3 7.3 7.3

2.73 2.68 2.20 1.94

2.47,2.52 (2) 2.20,2.36 (2) 1.78 (1) 1.68 (1)

1.000 0.500 0.125 0.062

5.5 5.5 5.5 5.5

3.10 2.83 2.47 2.26

2.68 (1) 2.57, 2.73 (2) 1.99,2.15 (2) 1.78,1.89 (2)

1.000 0.500 0.125 0.062

3.0 3.0 3.0 3.0

3.10 3.10 2.83 3.10

2.78 2.73 2.26, 2.20

Retention Time

(min)'

Metaphosphoric

(1) (1) 2.41 (2) (2)

°A11 values are the average of three trials. V a l u e s i n parentheses indicate the n u m b e r of peaks observed for metaphosphoric acid. A n a l y t i c a l conditions e m p l o y e d : /xPorasil c o l u m n ; flow rate, 1.0 m L / m i n ; sample size, 1 fiL.

increase i n p H decreased the retention time.

F u r t h e r , t h e n u m b e r of

peaks o b s e r v e d for m e t a p h o s p h o r i c a c i d v a r i e d f r o m 2 to 1 w i t h changes i n buffer s t r e n g t h or p H . A t p H 3.0, a n y changes i n buffer s t r e n g t h c a u s e d a shift i n baseline. T o d e t e r m i n e the s t a b i l i t y of ascorbic a c i d u n d e r t h e v a r i o u s c o n centrations of

c i t r a t e buffer

a n d p H , the

following experiment

was

p e r f o r m e d w i t h the use of a d o u b l e w a v e l e n g t h , d o u b l e b e a m , spectro photometer ( P e r k i n - E l m e r 557). V a r i o u s c o n c e n t r a t i o n s of a s c o r b i c a c i d (0.2, 2.0, a n d 20 pM)

were

i n c u b a t e d i n a cuvette w i t h the v a r i o u s buffers l i s t e d i n T a b l e I I a n d m o n i t o r e d at 254 n m . T h e c o n c e n t r a t i o n of a s c o r b i c a c i d w a s

selected

to a p p r o x i m a t e the s a m p l e a s c o r b i c a c i d c o m i n g i n t o contact w i t h a buffer v o l u m e d e t e r m i n e d b y its t i m e i n t r a n s i t t h r o u g h t h e c o l u m n . N o detect a b l e losses

were

observed

over

the 5 - m i n

incubation time for

any

c o n c e n t r a t i o n of a s c o r b i c a c i d u n d e r a n y c o n c e n t r a t i o n of citrate buffer or p H . T o q u a n t i t a t e the a s c o r b i c content of v a r i o u s tissues, a s t a n d a r d curve (3.0-50 / x g / m L ascorbic acid) was prepared i n 3 %

metaphosphoric

for e a c h a n a l y t i c a l r u n . A t y p i c a l s t a n d a r d c u r v e is s h o w n i n F i g u r e 4. C o r r e l a t i o n coefficients of 0.998 or b e t t e r w e r e c o n s i s t e n t l y o b t a i n e d for the s t a n d a r d c u r v e . P o o l e d s e r u m samples w e r e u s e d to m e a s u r e d a y - t o d a y a n d w i t h i n - r u n p r e c i s i o n . T h e coefficient of v a r i a t i o n for w i t h i n - r u n


9.

SAUBERLICH ET

AL.

213


20.0

18.0


16.0

14.0

12.0

E c

10.0

8.OH

6.0^

4.0

2.0A

0.0 10

20

30

40

50

60

ASCORBIC ACID, >xg/ml Figure 4. HPLC standard calibration curve obtained for ascorbic acid dissolved in 3% metaphosphoric acid; see text for conditions used; for ascorbic acid: y = 0.3547x + 0.620, r = 0.9983.


214

ASCORBIC

d e t e r m i n a t i o n s w a s 0 . 7 8 % a n d for d a y - t o - d a y d e t e r m i n a t i o n s w a s

ACID

4.6%.

P r e c i s i o n d a t a are p r e s e n t e d i n T a b l e I I I . A s c o r b i c a c i d a d d e d to s e r u m samples w a s r e c o v e r e d i n the r a n g e of 9 0 - 9 3 % . T h e a s c o r b i c a c i d contents of s e r u m , l i v e r , a d r e n a l g l a n d , a n d b r a i n w e r e d e t e r m i n e d i n t w o groups of g u i n e a p i g s . O n e g r o u p of g u i n e a p i g s was f e d a n ascorbic a c i d a d e q u a t e g u i n e a p i g c h o w diet ( R a l s t o n P u r i n a N o . 5 0 2 2 ) , w h i l e the second g r o u p w a s f e d the R e i d - B r i g g s ( 9 9 ) v i t a m i n C deficient diet. A f t e r a n 18-day f e e d i n g p e r i o d , the a n i m a l s w e r e s a c r i ficed

by

decapitation


centrifuged.

and

trunk blood

was

collected,

chilled, and

T h e s e r u m f r a c t i o n w a s t h e n s t a b i l i z e d b y the a d d i t i o n of

3 parts ( v / v )

3%

m e t a p h o s p h o r i c a c i d , c e n t r i f u g e d at 4 ° C to

remove

the p r e c i p i t a t e , t r a n s f e r r e d , a n d stored at — 6 5 ° C u n t i l assayed. tissues w e r e

homogenized

in 3%

metaphosphoric

All

a c i d to a c h i e v e

a

f o u r f o l d d i l u t i o n ( w / v ) , c e n t r i f u g e d at 4 ° C , t r a n s f e r r e d , a n d s t o r e d at — 6 5 ° C u n t i l assayed. T h e results are p r e s e n t e d i n T a b l e I V . A l l g u i n e a p i g s f e d the ascorbate deficient R e i d - B r i g g s diet c o n t a i n e d s i g n i f i c a n t l y less ascorbic a c i d i n e a c h tissue e x a m i n e d w h e n c o m p a r e d to a n i m a l s f e d the ascorbate a d e q u a t e c h o w diet. T h e greatest percentage decrease i n ascorbic a c i d content w a s f o u n d i n the a d r e n a l g l a n d ( 9 2 . 4 % ) by brain ( 6 6 . 9 % ) , liver ( 5 6 . 9 % ) , and serum ( 4 3 . 5 % ) .

followed

Figures

5-8

d e p i c t t y p i c a l H P L C c h r o m a t o g r a m s of a s c o r b i c a c i d extracts of s e r u m , liver, adrenal gland, a n d brain, respectively. Discussion.

The

method

presented

provides

a fast

and

repro

d u c i b l e means of d e t e r m i n i n g the ascorbic a c i d content of v a r i o u s tissues. I n a d d i t i o n to a h i g h rate of s a m p l e h a n d l i n g (12 s a m p l e s / h f o r c o m p l e x tissues s u c h as l i v e r , b r a i n , a n d a d r e n a l g l a n d a n d 15 s a m p l e s / h

for

p l a s m a ) , the m e t h o d r e q u i r e s a m i n i m u m of s a m p l e p r e p a r a t i o n a n d is p r a c t i c a l for r o u t i n e analysis of b i o l o g i c a l samples. method

utilizes equipment

F u r t h e r m o r e , the

a v a i l a b l e to e a c h l a b o r a t o r y w i t h a

rudi

m e n t a r y H P L C system.

Table III.

Precision Data for H P L C of Ascorbic A c i d

Parameter W i t h i n R u n (n = M e a n ± s.d. C.V., % D a y - t o - D a y (n = M e a n ± s.d. C.V., %

Determination

Ascorbic Acid

(mg/dL)

a

10) 8.5 ± 0.067 0.78 10) 13.8 db 0.63 4.6

° P o o l e d guinea p i g serum samples were e m p l o y e d . A n a l y t i c a l conditions e m p l o y e d : c o l u m n , /xPorasil; eluant, 0.125% citrate t r i s o d i u m salt, p H 7.3; flow rate, 1.0 m L / m i n ; sample size, 1 /xL.


9.

SAUBERLICH ET

AL.

215



0.0075-1

m-PHOSPHORIC 0.0050-

ACID

E c to CN

u z

Ascorbic Acid - ACS Publications - American Chemical Society

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