The Use of High-Performance Liquid Chromatography in Flavor Studies

Jul 23, 2009 - A review of HPLC techniques which have been used to study flavors in a wide variety of foods and flavorings is presented. Specific exam...
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6 The Use of High-Performance Liquid Chromatography in Flavor Studies R. L. Rouseff Scientific Research Department, Florida Department of Citrus, Lake Alfred, F L 33850

A review of HPLC techniques which have been used to study flavors in a wide variety of foods and flavorings is presented. Specific examples of how HPLC has been used to study nonvolatile flavor compounds in beer, beef, tea, grapefruit juice, vanilla, soy bean etc., are presented. Relative advantages and disadvantages of using HPLC vs GLC are discussed. Methods to separate specific classes of compounds such as sugars, artificial sweeteners, bitter/astringent compounds etc.are presented. Sample pretreatment, sample compatability, along with examples of auxiliary methods of peak identification and sensory evaluation are discussed. Coupled techniques such as LC/HPLC, and HPLC/GLC/MS systems are discussed using specific examples. High performance l i q u i d c h r o m a t o g r a p h y , HPLC, has been used f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f a wide v a r i e t y o f f l a v o r compounds i n f o o d s , b e v e r a g e s , f l a v o r i n g s and f r a g r a n c e s . HPLC i s u s u a l l y but not e x c l u s i v e l y used to determine h i g h l y p o l a r a n d / o r t h e r m a l l y u n s t a b l e compounds. C a p i l l a r y GLC was and s t i l l i s the method o f c h o i c e t o determine v o l a t i l e s because o f t h e e x t r e m e l y h i g h r e s o l u t i o n and h i g h peak c a p a c i t y which can be a c h i e v e d . GLC d e t e c t i o n l i m i t s a r e a l s o i n g e n e r a l much lower than t h o s e o f HPLC. T h e r e f o r e , f l a v o r c h e m i s t s have u t i l i z e d GLC a l m o s t e x c l u s i v e l y i n s t u d i e s i n v o l v i n g aroma. A n o t h e r r e a s o n why more GLC than HPLC f l a v o r s t u d i e s have been done i s because GLC i s e a s i l y c o u p l e d t o a mass s p e c t r o g r a p h , which can be used f o r r a p i d i d e n t i f i c a t i o n o f t h e chromatographic peaks. Coupled GC/MS systems have become t h e workhorse t o o l f o r most f l a v o r c h e m i s t s . The c o u p l i n g o f HPLC/MS i s much more d i f f i c u l t because o f t h e n e c e s s i t y o f removing v a s t amounts o f s o l v e n t b e f o r e t h e a n a l y t e s e n t e r t h e MS. However, t h e c o u p l i n g o f HPLC w i t h MS i s an a c t i v e r e s e a r c h area and much p r o g r e s s has a l r e a d y been made.

0097-6156/ 85/ 0289-O079$06.00/ 0 © 1985 American Chemical Society

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In the a r e a o f t a s t e , many f l a v o r - a c t i v e compounds a r e n o n - v o l a t i l e and/or thermally unstable. S i n c e most HPLC s e p a r a t i o n s o c c u r a t room t e m p e r a t u r e , the p r o b a b i l i t y o f p r o d u c i n g and a n a l y z i n g a r t i f a c t s from thermal d e g r a d a t i o n i s g r e a t l y r e d u c e d . Another major advantage o f HPLC i s i n the area o f sample compatability. S i n c e a s u b s t a n c e must be d i s s o l v e d b e f o r e i t can be t a s t e d and s i n c e the d i s s o l v i n g l i q u i d i s o f t e n w a t e r , l i q u i d chromatography has the advantage o f o f f e r i n g a d i r e c t a n a l y s i s o f a f l a v o r - a c t i v e mixture. HPLC can r e a d i l y accommodate aqueous samples because the c h r o m a t o g r a p h i c m o b i l e phase w i l l g e n e r a l l y c o n t a i n water. GLC systems f o r the most p a r t do not t o l e r a t e w a t e r . T h e r e f o r e , solvent e x t r a c t i o n s with immiscible organic solvents are required. There a r e some f l a v o r a c t i v e compounds such as p y r a z i n e s {I) which a r e d i f f i c u l t to e x t r a c t from aqueous phases f o r subsequent GLC a n a l y s i s . It i s o f t e n p o s s i b l e to d i r e c t l y i n j e c t aqueous samples i n t o HPLC s y s t e m s , thus c i r c u m v e n t i n g the problem o f e x t r a c t i n g compounds w i t h low d i s t r i b u t i o n c o e f f i c i e n t s . HPLC f r a c t i o n s can o f t e n be c o l l e c t e d f o r l a t e r s e n s o r y evaluation. The o b j e c t i v e i s to o b t a i n s e n s o r y data f o r each c h r o m a t o g r a p h i c peak and to determine which compounds a r e f l a v o r active. S n i f f e r p o r t s were used i n GLC f o r the same r e a s o n but the s e n s o r y e v a l u a t i o n had to be done " o n - t h e - f l y " w i t h d i f f e r e n t s e n s a t i o n s coming i n r a p i d s u c c e s s i o n o f each o t h e r . Sensory f a t i g u e was o f t e n a p r o b l e m . In c o n t r a s t , s e n s o r y e v a l u a t i o n s o f HPLC f r a c t i o n s can be o b t a i n e d under c o n t r o l l e d c o n d i t i o n s w i t h adequate s p a c i n g s between s a m p l e s . However, i f s e n s o r y e v a l u a t i o n i s c o n t e m p l a t e d , p a r t i c u l a r c a r e must be t a k e n i n c h o o s i n g the s o l v e n t s used i n the c h r o m a t o g r a p h y . B u f f e r s s h o u l d g e n e r a l l y be a v o i d e d as w e l l as s o l v e n t s such as a c e t o n i t r i l e which may pose a health r i s k . In p r a c t i c e t h i s u s u a l l y l i m i t s the c h r o m a t o g r a p h i c s o l v e n t system to water and e t h a n o l . There a r e many s i t u a t i o n s where HPLC can be used to advantage i n the s t u d y o f f l a v o r s . It i s the purpose o f t h i s paper to show how HPLC can and has been used t o i t s f u l l e s t advantage i n s t u d y i n g f l a v o r and to show how HPLC can be used i n c o n j u n c t i o n w i t h GLC to p r o v i d e a powerful s e p a r a t i o n s a p p r o a c h . Sweetness Sugars Sweetness i s one o f the most i m p o r t a n t f l a v o r a t t r i b u t e s . Sugars p l a y a major r o l e on the p e r c e i v e d f l a v o r o f f o o d s , beverages and f l a v o r i n g s we consume. Because the r e l a t i v e sweetness o f i n d i v i d u a l sugars v a r y , the f l a v o r c h e m i s t needs to know the s u g a r p r o f i l e o f the p r o d u c t o f i n t e r e s t . In o r d e r to c h a r a c t e r i z e the raw m a t e r i a l s t h a t go i n t o a p r o d u c t o r to c h a r a c t e r i z e the f i n a l p r o d u c t i t i s n e c e s s a r y to know the k i n d s and a b s o l u t e amounts o f i n d i v i d u a l component s u g a r s . There a r e a number o f c l a s s i c a l c o l o r i m e t r i c methods to determine the amounts o f r e d u c i n g s u g a r s . T o t a l s u g a r s a r e determined a f t e r a c i d o r enzymic h y d r o l y s i s and n o n r e d u c i n g s u g a r s a r e determined from the d i f f e r e n c e . Paper and t h i n - l a y e r chromatography have been used to determine sugars but a n a l y s i s times a r e o f t e n l o n g , p r e c i s i o n poor o r q u a n t i t a t i o n difficult. Sugars cannot be d i r e c t l y determined by GLC because they m e l t w i t h d e c o m p o s i t i o n when h e a t e d . To c i r c u m v e n t t h i s problem v o l a t i l e

6. ROUSEFF

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d e r i v a t i v e s can be formed which a r e heat s t a b l e . To m i n i m i z e f l u c t u a t i o n s i n r e a c t i o n y i e l d s an i n t e r n a l s t a n d a r d i s u s u a l l y required. However, i n a d d i t i o n to the i n c o n v e n i e n c e o f an e x t r a d e r i v a t i z a t i o n s t e p , GLC methods s u f f e r from the problem o f m u l t i p l e r e a c t i o n p r o d u c t s which can produce m u l t i p l e peaks from a s i n g l e sugar. Except f o r f i l t e r i n g , most HPLC s u g a r p r o c e d u r e s r e q u i r e v i r t u a l l y no sample p r e p a r a t i o n . T h e r e f o r e , HPLC has q u i c k l y become the method o f c h o i c e f o r d e t e r m i n i n g s u g a r s . There a r e t h r e e major c h r o m a t o g r a p h i c approaches f o r s e p a r a t i n g s u g a r s . They a r e : f o r m a t i o n o f b o r a t e - c a r b o h y d r a t e a n i o n s and s e p a r a t i o n on s t r o n g a n i o n - e x c h a n g e columns U , 3); amino- bonded s i l i c a s o r p t i o n chromatography u s i n g p r i m a r i l y a c e t o n i t r i l e as the m o b i l e phase (4> 5); and s t r o n g c a t i o n - e x c h a n g e polymers u s i n g water as the m o b i l e phase (6^ 7). Due to advantages i n s i m p l i c i t y and r e p r o d u c i b i l i t y most r e s e a r c h e r s choose one o f the l a t t e r two a p p r o a c h e s . C a t i o n exchange systems employ o n l y water as the m o b i l e phase a n d , t h u s , have c e r t a i n c o s t and s a f e t y advantages o v e r the amino system which employs p r i m a r i l y a c e t o n i t r i l e . The two systems have o p p o s i t e e l u t i o n o r d e r s , so o f t e n the c h o i c e o f which system to use i s based on the r e l a t i v e amounts and t y p e s o f s u g a r s which need t o be analyzed. D i f f e r e n t i a l r e f r a c t o m e t e r s (RI d e t e c t o r s ) have t r a d i t i o n a l l y been used to measure sugar c o n c e n t r a t i o n s as they e l u t e from the column. However, t h e s e d e t e c t o r s a r e v e r y i n s e n s i t i v e , n o n - s p e c i f i c and r e q u i r e t h a t o n l y i s o c r a t i c s e p a r a t i o n s be employed. To remedy t h i s problem numerous p o s t column r e a c t i o n (PCR) systems have been developed ( 8 - 1 0 ) . These systems are h i g h l y s e n s i t i v e and h i g h l y s p e c i f i c , however, they can o n l y d e t e c t r e d u c i n g s u g a r s . Further, PCR systems r e q u i r e a d d i t i o n a l equipment such as e x t r a pumps and heated r e a c t i o n chambers. One o f the more p r o m i s i n g d e t e c t o r s i s the r e c e n t l y d e v e l o p e d p u l s e d amperometric d e t e c t o r (11). This d e t e c t o r i s h i g h l y s p e c i f i c and h i g h l y s e n s i t i v e . Sugars ( r e d u c i n g and n o n - r e d u c i n g ) can now be d e t e c t e d a t the ppm l e v e l . Several i n v e s t i g a t o r s (12_ 13) have used t h i s d e t e c t o r to determine low l e v e l s u g a r s and sugar a l c o h o l s i n a v a r i e t y o f p r o d u c t s . Shown i n F i g u r e 1 i s the sugar chromatogram from f l a v o r e d p o t a t o c h i p s . s

Nonsugar sweeteners A r t i f i c a l sweeteners such as s a c c h a r i n and aspertame a r e r e a d i l y a n a l y z e d u s i n g HPLC w i t h minimum sample t r e a t m e n t . Many low c a l o r i e s o f t d r i n k s c o n t a i n both a r t i f i c i a l sweeteners because o f c o s t and s t o r a g e s t a b i l i t y a d v a n t a g e s . The t a s t e o f aspertame i s c l o s e s t to t h a t o f s u g a r and i s thus g e n e r a l l y preferred. However aspertame i s more c o s t l y and h y d r o l y z e s a t the low pH o f s o f t d r i n k s . R e c e n t l y Tsang e t a l . (14) d e v e l o p e d a method to q u a n t i f y both a r t i f i c a l sweeteners i n s o f t d r i n k s . The r e v e r s e phase method was a l s o used to s e p a r a t e and i d e n t i f y f o u r s t o r a g e breakdown p r o d u c t s . Aspertame i s q u i t e r e a c t i v e w i t h a l d e h y d e s , which a r e the p r i n c i p a l f l a v o r compounds i n some s o f t d r i n k s . Thus the use o f HPLC to determine the s t a b i l i t y and f l a v o r e f f e c t s i n low c a l o r i e s o f t d r i n k s w i l l be e s s e n t i a l f o r f l a v o r c h e m i s t s working i n this area. HPLC has a l s o been used i n the s e a r c h f o r o t h e r n o n c a l o r i c s u g a r s u b s t i t u e s , s i n c e many o f t h e s e s u b s t a n c e s e x h i b i t many o f the same d e t e r m i n a t i o n problems as s u g a r s . An example i s the r e c e n t

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F i g u r e 1. E x t r a c t from f l a v o r e d p o t a t o c h i p s . Sugars s e p a r a t e d as a n i o n s by i o n exchange chromatography w i t h a sodium h y d r o x i d e e l u e n t and a p u l s e d amperometric d e t e c t o r . L a c t o s e c o n c e n t r a t i o n i n the e x t r a c t i s 70 ppm. "Reproduced w i t h p e r m i s s i o n from R e f . 13 C o p y r i g h t 1983, ' M a r c e l D e k k e r V

6. ROUSEFF

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work by Makapugay e t a l (15) i n d e t e r m i n i n g the i n t e n s e l y sweet t r i t e r p e n e g l y c o s i d e , m o g r o s i d e V i n Lo Han Kuo f r u i t from C h i n a . The i s o l a t i o n and i d e n t i f i c a t i o n o f the sweet component o f t h i s f r u i t i s q u i t e r e c e n t (16) and no p r e v i o u s a n a l y t i c a l methods had been r e p o r t e d . Thus i f t h i s compound shows promise as a sugar s u b s t i t u t e HPLC w i l l p l a y an i m p o r t a n t r o l e i n d e t e r m i n i n g the p u r i t y o f s o l u t i o n s used i n t a s t e t h r e s h o l d t e s t s , l e v e l s i n f o r m u l a t i o n s and i t s s t o r a g e s t a b i l i t y . Bitterness B i t t e r n e s s i s an i n t e r e s t i n g f l a v o r . I t can be caused by a h o s t o f s t r u c t u r a l l y d i s s i m i l a r compounds. On one hand t h e r e i s a n a t u r a l r e v u l s i o n o f t h i n g s b i t t e r perhaps due t o a n a t u r a l d e f e n s e mechanism as many n a t u r a l t o x i n s a r e b i t t e r . On t h e o t h e r hand a l i m i t e d amount o f b i t t e r n e s s i s o f t e n d e s i r a b l e to produce a c l e a n t a s t e o r something w i t h a b i t o f " b i t e . " T h e r e f o r e , i t has become n e c e s s a r y to be a b l e to m o n i t o r b i t t e r n e s s by d e t e r m i n i n g which compounds a r e b i t t e r and d e v e l o p i n g methods to q u a n t i f y t h o s e compounds. HPLC has been used e x t e n s i v e l y i n a c h i e v i n g t h e s e t w i n goals. It can r e a d i l y f r a c t i o n a t e a p r o d u c t a l l o w i n g one t o o r g a n o l e p t i c a l l y determine which f r a c t i o n i s b i t t e r and f i n a l l y to s e p a r a t e and i d e n t i f y the s p e c i f i c b i t t e r compound(s). Hop b i t t e r a c i d s . Hops a r e used i n the brewing o f beer to p r o v i d e b i t t e r n e s s and aroma. Hop components a l s o c o n t r i b u t e t o f l a v o r and foam s t a b i l i t y . Humulimic a c i d s from hops a r e the components l a r g e l y r e s p o n s i b l e f o r t h e b i t t e r n e s s o f beer ( 1 7 ) . A variety of c h r o m a t o g r a p h i c approaches have been proposed f o r the a n a l y s i s o f t h e s e compounds, they i n c l u d e : i o n exchange (18^, 1 9 ) , normal phase based on s i l i c a (20) , p a i r e d i o n r e v e r s e phase (21_, 22) and r e v e r s e phase ( 2 3 , 2 4 ) . They have been used t o e v a l u a t e the q u a l i t y and q u a n t i t y o f beer b i t t e r n e s s . C o r r e l a t i o n s between o r g a n o l e t i c b i t t e r n e s s o f i n d i v i d u a l components and t h e b i t t e r n e s s o f beer t o the amounts o f t h e s e components i n beer have been e s t a b l i s h e d ( 2 5 ) . HPLC has been used to a s s e s s the f r e s h n e s s o f hops and to s t a n d a r d i z e the use o f hop e x t r a c t s i n b e e r . Precise, specific a n a l y t i c a l t e c h n i q u e s such as HPLC a r e r e q u i r e d t o determine the e f f e c t s o f newer p r o c e s s i n g t e c h n i q u e s . In s t u d y i n g super c r i t i c a l f l u i d e x t r a c t i o n s o f hops w i t h C 0 Anderson (26) used HPLC to m o n i t o r the b i t t e r hop a c i d s . 2 i

Amino a c i d s and p e p t i d e s . S e v e r a l amino a c i d s and p e p t i d e s a r e bitter. These compounds a r e i m p o r t a n t f l a v o r components i n t h e i r own r i g h t and have been i m p l i c a t e d as p r e c u r s o r s f o r o t h e r f l a v o r - a c t i v e r e a c t i o n p r o d u c t s . T r a d i t i o n a l l y they a r e s e p a r a t e d using ion-exchange chromatography. S i n c e t h e y do not c o n t a i n a s t r o n g chromaphore, t h e y a r e d e r i v a t i z e d a f t e r column s e p a r a t i o n to form p r o d u c t s which can be e a s i l y d e t e c t e d and q u a n t i f i e d . Many d e r i v a t i z i n g agents have been d e v e l o p e d . Spackman and coworkers (27) used n i n h y d r i n and t h i s i s s t i l l the post column c h e m i s t r y employed by most commercial amino a c i d a n a l y z e r s . With the advent o f HPLC more e f f i c i e n t s e p a r a t i o n s (28^, 29) have been d e v e l o p e d which g r e a t l y reduced a n a l y s i s t i m e . Recently, fluorometric r e a g e n t s such as o - p h t h a l a d e h y d e (ΟΡΑ) (30) have been used to

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Increase s e n s i t i v i t y . U n f o r t u n a t e l y , ΟΡΑ r e a c t s o n l y w i t h p r i m a r y amines. T h e r e f o r e , the a d d i t i o n o f o x i d i z i n g agents such as sodium h y p o c h l o r i t e (31) i s n e c e s s a r y to produce p r i m a r y amines from s e c o n d a r y amines such as p r o l i n e . S e v e r a l precolumn d e r i v a t i z a t i o n t e c h n i q u e s a r e a v a i l a b l e f o r t h o s e who wish to t r a d e e x t r a sample p r e p a r a t i o n time f o r the expense and maintenance o f p o s t column pumps and r e a c t o r s . The more p o p u l a r d e r i v a t i v e s a r e d a n s y l - ( 3 2 ) , 0 P A - ( 3 3 ) , PTH-(34) and P I T C - ( 3 5 ) amino a c i d s . There a r e problems and l i m i t a t i o n s w i t h some o f t h e s e s y s t e m s , however, a n a l y s i s time i s o n l y 15-25 m i n . compared to 90-240 m i n . o f the i o n - e x c h a n g e p o s t column s y s t e m s . U n f o r t u n a t e l y , most f l a v o r r e s e a r c h e r s have been slow t o u t i l i z e the speed and s e n s i t i v i t y t h a t HPLC o f f e r s . Although b i t t e r amino a c i d s and p e p t i d e s have been r e p o r t e d i n cheese and c a s e i n ( 3 6 , 37) and i n y o g u a r t (38) a l l the a n a l y s e s were done by t r a d i t i o n a l methods. C i t r u s l i m o n o i d s and f l a v a n o n e g l y c o s i d e s . Limonoids a r e Z^c f u s e d r i n g a l i p h a t i c compounds c o n t a i n i n g a f u r a n r i n g and two l a c t o n e r i n g s a t o p p o s i t e ends o f the m o l e c u l e . Limonoids a r e found i n a l l citrus cultivars. The l i m o n o i d i n g r e a t e s t abundance i s u s u a l l y the bitter limonin. Other l i m o n o i d s a r e found i n lower c o n c e n t r a t i o n s and f o r the most p a r t a r e non b i t t e r . B i t t e r n e s s has been a major f l a v o r problem i n the c i t r u s i n d u s t r y f o r y e a r s . Thus i t has beome n e c e s s a r y to d e v e l o p s e n s i t i v e a n a l y s e s which c o u l d s e p a r a t e and q u a n t i f y the b i t t e r l i m o n o i d s from the complex c i t r u s m a t r i x . Since l i m o n o i d s a r e t h e r m a l l y u n s t a b l e , HPLC i s the t e c h n i q u e o f c h o i c e and s e v e r a l HPLC p r o c e d u r e s have been d e v e l o p e d ( 3 £ , 4 0 ) . The p r o c e d u r e p r o b a b l y i n most w i d e s p r e a d use (41) u t i l i z e s a cyano column i n the normal phase mode. The b i t t e r l i m o n i n and n o m i l i n a r e w e l l r e s o l v e d from the n o n b i t t e r obacunone and d e o x y l i m o n i n and o t h e r c i t r u s components. However, even though the a c t u a l chromato­ graphic a n a l y s i s i s r e l a t i v e l y r a p i d the o v e r a l l a n a l y s i s i s very time consuming because l i q u i d - l i q u i d e x t r a c t i o n s a r e r e q u i r e d and the sample p r e p a r a t i o n i s l e n g t h y . A d i r e c t i n j e c t i o n r e v e r s e phase HPLC p r o c e d u r e (42) has been r e c e n t l y d e v e l o p e d which g r e a t l y reduces o v e r a l l a n a l y s i s time as no s o l v e n t e x t r a c t i o n i s r e q u i r e d . Flavanone g l y c o s i d e s a r e C , h e t e r o c y c l i c r i n g systems w i t h a d i s a c c h a r i d e c o n s i s t i n g o f g l u c o s e and rhamnose a t t a c h e d a t the 7 position. These compounds a r e b i t t e r o n l y i f the rhamnose i s a t t a c h e d 1-2 to the g l u c o s e and n o n b i t t e r i f a t t a c h e d 1 - 6 . To f u r t h e r c o m p l i c a t e the problem not a l l the b i t t e r forms a r e e q u a l l y bitter. In most c i t r u s c u l t i v a r s e i t h e r t h e b i t t e r o r n o n b i t t e r form a r e e x c l u s i v e l y p r e s e n t . G r a p e f r u i t i s an e x c e p t i o n t o t h i s g e n e r a l r u l e as both forms a r e p r e s e n t i n s i g n i f i c a n t amounts. F i s h e r and Wheaton (43) d e v e l o p e d an i s o c r a t i c p r o c e d u r e f o r the s e p a r a t i o n o f the b i t t e r n a r i n g i n from i t s non b i t t e r i s o m e r , narirutin. S i n c e a t l e a s t two o t h e r b i t t e r - n o n b i t t e r i s o m e r i c p a i r s a r e known to e x i s t i n g r a p e f r u i t a g r a d i e n t s e p a r a t i o n has been d e v e l o p e d ( 4 4 ) . The b i t t e r n a r i n g i n and the n o n b i t t e r n a r i r u t i n a r e the major f l a v a n o n e g l y c o s i d e s i n g r a p e f r u i t and a r e well r e s o l v e d . The non b i t t e r h e s p e r i d i n i s w e l l s e p a r a t e d from the b i t t e r n e o h e s p e r i d i n but h e s p e r i d i n w i l l sometimes appear as a s h o u l d e r o f the much l a r g e r n a r i n g i n peak as column e f f i c i e n c y decreases with age. The s e p a r a t i o n between the 5

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85

b i t t e r p o n c i r i n and the n o n b i t t e r didymin i s not as good but adequate. A l l the b i t t e r f l a v a n o n e g l y c o s i d e s i n g r a p e f r u i t can be q u a n t i f i e d and t h e i r r e l a t i v e c o n t r i b u t i o n to j u i c e b i t t e r n e s s can be e s t i m a t e d . HPLC A n a l y s i s o f

F l a v o r s and E s s e n t i a l

juice

Oils

The s p i c y hot f l a v o r o f r e d peppers i s due t o c a p s a i c i n and r e l a t e d capsaicinoids. S p i c e m a n u f a c t u r e r s seek to produce f l a v o r i n g s o f consistent flavoring strength. In the c a s e o f ground r e d pepper s p i c e i t i s n e c e s s a r y to q u a n t i f y the major r e d pepper heat principles. An e s t i m a t e o f the s p i c e s ' " h o t n e s s " can then be e s t i m a t e d i n a n o n s u b j e c t i v e manner as the r e l a t i v e s e n s o r y " h o t n e s s " f o r each c a p s a i c i n o i d has a l r e a d y been e s t a b l i s h e d . Since c a p s a i c i n o i d s cannot be d e t e r m i n e d u s i n g GLC w i t h o u t d e r i v a t i z a t i o n , s e v e r a l HPLC methods have been d e v e l o p e d ( 4 5 , 4 6 ) which a r e based on underivatized direct i n j e c t i o n s . One o f the most r e c e n t methods ( 4 7 ) i s based on the use o f a r e v e r s e phase C-18 column w i t h common s o l v e n t s i n the i s o c r a t i c mode. A l l the major c a p s a i c i n o i d s c o u l d be s e p a r a t e d from r e d pepper e x t r a c t s i n 25 m i n . To a i d l a b o r a t o r i e s which do not p o s s e s s c a p a i c i n o i d s t a n d a r d s the a u t h o r s determined c a p a i c i n o i d r e s p o n s e times and c o n c e n t r a t i o n r e s p o n s e f a c t o r s r e l a t i v e to a c o m m e r c i a l l y a v a i l a b l e m a t e r i a l o f s i m i l i a r structure,N-vanillyl-n-nonamide. S i n c e many f l a v o r a c t i v e compounds a r e not c o m m e r c i a l l y a v a i l a b l e , the use o f c o m m e r c i a l l y a v a i l a b l e s t a n d a r d s o f s i m i l a r s t r u c t u r e i n a n a l y t i c a l methods s h o u l d be encouraged. Thus t h i s method i s w e l l s u i t e d f o r r o u t i n e a n a l y s i s . V a n i l l a e x t r a c t i s used to f l a v o r a wide v a r i e t y o f foods and i s very expensive. Herrman and S t o c k l i (48) d e v e l o p e d an HPLC method to determine the major components o f v a n i l l a e x t r a c t s . This method can be used to h e l p d i f f e r e n t i a t e n a t u r a l v a n i l l a e x t r a c t s from cheaper s u b s t i t u t e s . Shown i n F i g u r e 2a i s a s e p a r a t i o n o f s t a n d a r d s o f the major components i n v a n i l l a . Shown i n F i g u r e 2b i s the chromatogram from a t y p i c a l v a n i l l a e x t r a c t . I t s h o u l d be noted i n F i g u r e 2b t h a t n a t u r a l v a n i l l a e x t r a c t s a p p a r e n t l y l a c k e t h y l v a n i l l i n , a commonly used f l a v o r s u b s t i t u t e . E x t r a c t s o f l i c o r i c e r o o t have a l s o been used as a f l a v o r i n g agent. H i r a g a e t a l . (49) among o t h e r s have d e v e l o p e d an HPLC s e p a r a t i o n f o r the c o n s t i t u e n t s i n l i c o r i c e . Because the numerous components i n l i c o r i c e v a r y c o n s i d e r a b l y i n p o l a r i t y , a r e v e r s e phase C-18 column w i t h a w a t e r / a c e t o n i t r i l e g r a d i e n t was employed. N i n e t e e n components were i d e n t i f i e d . However, a t h r e e hour e x t r a c t i o n c o u p l e d to the 60 m i n . a n a l y s i s time p r o b a b l y makes t h i s p r o c e d u r e too l e n g t h y f o r r o u t i n e u s e . I f o n l y a few s p e c i f i c components need be q u a n t i f i e d , the a n a l y s i s time can be c o n s i d e r a b l y s h o r t e n e d . Dong and D i C e s a r e (50) d e v e l o p e d an h i g h speed i s o c r a t i c r e v e r s e phase s e p a r a t i o n f o r 2,5-dimethyl-4-hydroxy-3(2H)-furanone. T h i s compound i s commonly known as p i n e a p p l e ketone and can be q u a n t i f i e d i n food f l a v o r i n g p r o d u c t s u s i n g one o f the new 3 x 3 , C-18 columns i n as l i t t l e as 2 minutes. Other workers (51) have used r a p i d g r a d i e n t s (6 m i n . ) t o s e p a r a t e c a f f e i n e , t r i g o n e l l e and theobromine i n c o f f e e and cocoa products. I t has o f t e n been s t a t e d t h a t HPLC l a c k s the r e s o l v i n g power t o produce a d e t a i l e d s e p a r a t i o n f o r complex m i x t u r e such as

86

C H A R A C T E R I Z A T I O N A N D M E A S U R E M E N T O F FLAVOR C O M P O U N D S

• xt.

0.4

3

0.2

"Sin

20

î S

ï

5

5

Figure 2a. Chromatogram from l O u l o f a s t a n d a r d m i x t u r e . Peaks: 1 = 2.5ug o f 4 - h y d r o x y b e n z y l a l c o h o l ; 2 = 2.5ug o f v a n i l l y l a l c o h o l ; 3 = 2.5ug o f 4 - h y d r o x y b e n z o i c a c i d ; 4 = 0.5ug o f 4 - h y d r o x y b e n z a l d e h y d e ; 5 = 2.5ug o f v a n i l l i n ; 6 = 2.5ug o f e t h y l v a n i l l i n ; 7 = 0.5ug o f c o u m a r i n .

ROUSEFF

HPLC in Flavor Studies

•it. I

0.4

40.2

JU min

20

t5

10

Figure 2b. Chromatogram from l O u l o f v a n i l l a e x t r a c t . "Reproduced w i t h p e r m i s s i o n from R e f . 48 C o p y r i g h t 1980, 'Elsevier'."

88

CHARACTERIZATION AND MEASUREMENT OF FLAVOR COMPOUNDS

essential o i l s . The chromatograms shown i n F i g u r e 3 d e v e l o p e d by T h i e s (52) f o r l a v e n d e r and peppermint o i l a r e t y p i c a l . Usually o n l y 2-3 components a r e s e p a r a t e d to produce a " f i n g e r p r i n t " which can be used t o c h a r a c t e r i z e the p r o d u c t . However, S c o t t and Kucera (53) have demonstrated i n p r i n c i p l e t h a t h i g h e f f i c i e n c y m i c r o b o r e côTumns can produce s e p a r a t i o n s comparable to c a p i l l a r y GLC separations. Shown i n F i g u r e 4 i s one such s e p a r a t i o n f o r cinnamon essential o i l . T h i s d e t a i l e d s e p a r a t i o n was a c h i e v e d w i t h a 1mm column 14 meters l o n g . However, the s e p a r a t i o n took o v e r 20 h to complete which i s u n r e a l i s t i c a l l y l o n g . T h e r e f o r e , at l e a s t in terms o f the p r e s e n t t e c h n o l o g y , d e t a i l e d s e p a r a t i o n s o f e s s e n t i a l o i l s a r e p r o b a b l y b e s t done u s i n g c a p i l l a r y GLC. HPLC as a F l a v o r Research Tool I f one i s i n t e r e s t e d i n a s p e c i f i c compound o r group o f r e l a t e d compounds, HPLC i s an e x c e l l e n t t o o l to s t u d y f l a v o r changes i n d i f f e r e n t p r o d u c t s o r from d i f f e r e n t p r o c e s s e s . HPLC i s g e n e r a l l y r e g a r d e d as p o s s e s s i n g v e r y l i m i t e d a b i l i t y to s e p a r a t e l a r g e numbers o f compounds w i t h c o n s i d e r a b l y d i f f e r e n t p o l a r i t i e s . Thus most HPLC p r o c e d u r e determine compounds o f a r a t h e r narrow p o l a r i t y range. T h i s i s a d i s a d v a n t a g e when s t u d y i n g f l a v o r systems because one o f t e n does not know what i s p r e s e n t , o r what f l a v o r a c t i v e compounds might be f o r m e d . However, Q u r e s h i et a l . ( l _ , 5 4 ) have shown t h a t HPLC can be used to s u c c e s s i v e l y and s y s t e m a t i c a l l y s e p a r a t e a l a r g e number o f f l a v o r a c t i v e compounds o f v e r y d i f f e r e n t polarity. They d e v e l o p e d an HPLC p r o c e d u r e u t i l i z i n g a s i n g l e C18 column w i t h a s e r i e s o f i s o c r a t i c o r g r a d i e n t runs to s u c c e s s i v e l y s e p a r a t e p u r i n e s , p y r i m i d i n e s , n u c l e o s i d e s , n u c l e o t i d e s , p o l y p h e n o l s and pyrazines in beer. U s i n g t h i s system t h e y were a b l e to q u a n t i f y 58 compounds i n worts and beers a t f i v e d i f f e r e n t s t a g e s o f b r e w i n g . About 30 a d d i t i o n a l peaks were r e s o l v e d but not i d e n t i f i e d . Beers u s i n g m a l t s form f o u r d i f f e r e n t b a r l e y s had d i f f e r e n t q u e r c e t i n t o c a t e c h i n (Q/C) r a t i o s . Shown i n T a b l e I a r e the polyphenol v a l u e s f o r two canned beers (one American and one E n g l i s h ) and a b o t t l e d American b e e r . Whereas t h e Q/C r a t i o i s r e l a t i v e l y s i m i l a r f o r the two American beers (2.3 vs 3.1) the r a t i o was c o n s i d e r a b l y lower i n the E n g l i s h beer. Other compounds such as k o j i c a c i d , f e r u l i c a c i d , p - c o u m a r i c a c i d and c i n n a m i c a c i d a l s o showed c o n s i d e r a b l e differences. U n f o r t u n a t e l y the r e l a t i v e c o n t r i b u t i o n o f each o f the 58 compounds to beer f l a v o r i s known o n l y f o r a few. There a r e a l s o many compounds which were s e p a r a t e d by not i d e n t i f i e d which might be flavor active. A d d i t i o n a l work needs to be done to determine the r e l a t i v e c o n t r i b u t i o n o f the i d e n t i f i e d compounds t o beer f l a v o r and t o i d e n t i f y t h o s e unknown compounds which a r e f l a v o r a c t i v e . In a n o t h e r thorough f l a v o r study Wu e t a l . ( 5 5 ) determined both t h e v o l a t i l e and n o n v o l a t i l e f l a v o r compounds found i n mushroom blanching water. They used HPLC to determine such non v o l a t i l e f l a v o r components as s u g a r s , amino a c i d s and n u c l e o t i d e s . The f r e e amino a c i d s were a n a l y z e d t o determine i f t h e y might be i n v o l v e d i n any thermal r e a c t i o n s which might produce Amadori compounds o r S t r e c k e r a l d e h y d e s which i n t u r n would produce aroma components. The a u t h o r s were i n t e r e s t e d i n e v a l u a t i n g t h r e e d i f f e r e n t p r o c e s s i n g t e c h n i q u e s to r e c o v e r the f l a v o r s i n mushroom b l a n c h i n g w a t e r .

6. ROUSEFF

HPLC in Flavor Studies

peppermint oil

lavender oil

20min

Figure 3. Chromatogram o f l a v e n d e r and peppermint o i l s . Peaks: A and Ε = unknowns, Β = l i n a l o o l , C = l i n a l y l a c e t a t e , G = m e t h y l acetate. "Reproduced w i t h p e r m i s s i o n from R e f . 52 C o p y r i g h t 1984, 'Spinger-Verlag'."

Figure 4. Chromatogram o f a cinnamon e s s e n t i a l o i l . Column, 14m χ 1mm I . D . ; s o l v e n t e t h y l a c e t a t e : heptane ( 5 : 9 5 ) ; e f f i c i e n c y = 510,000 t h e o r e t i c a l p l a t e s . "Reproduced w i t h p e r m i s s i o n from R e f . 53 C o p y r i g h t 1 9 7 9 , ' E l s e v i e r . " 1

90

C H A R A C T E R I Z A T I O N A N D M E A S U R E M E N T O F FLAVOR C O M P O U N D S

Table

Compound

K o i j i c acid G a l l i c acid 3,4-Dihydroxy benzoic a c i d Quercetin p-hydroxy benzoic a c i d Catechln V a n i l l i c acid Ferulic acid Caffeic acid Gentisic acid Syringic acid Chlorogenic acid Epicatechin p-Coumaric a c i d Rutin S a l i c y l i c acid Sinapic acid Cinnamic a c i d

I.

Polyphenols

i n Commercial

Beers

Retention Time (min)

CE

Concentration CA

4.9 6.5

78.2 20.8

29.8 29.2

28.6 26.8

10.4 14.2

12.8 36.4

19.0 125.0

18.4 80.8

16.3 19.4 21.9 24.1 25.4 26.9 27.8 28.9 30.5 36.4 42.9 43.9 48.2 72.5

15.9 53.0 0.8 20.8 8.0 3.0 7.0 4.0 24.0 1.6 2.7 3.1 3.9 26.1

12.0 55.0 0.5 4.5 7.0 2.8 5.0 2.0 13.8 6.0 6.0 1.1 0.7 3.3

12.0 26.0 0.3 2.2 2.0 4.6 3.5 3.4 8.5 7.0 6.0 1.0 0.7 3.5

A l l v a l u e s c a l c u l a t e d from data o b t a i n e d CE = canned E n g l i s h beer CA = canned American beer BA = b o t t l e d American beer Data from R é f . 1.

at

280

nm.

(ug/mL) BA

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G l u c o s e was the o n l y major sugar and IMP and GMP were the o n l y major n u c l e o t i d e s f o u n d . A s e n s o r y e v a l u a t i o n o f the d i f f e r e n t p r o c e s s e d p r o d u c t s i n d i c a t e d a p r e f e r e n c e f o r the drum d r i e d p r o d u c t o v e r the freeze or spray d r i e d product. T h i s p r e f e r e n c e c o u l d not be e x p l a i n e d from s u g a r o r n u c l e o t i d e v a l u e s and the amino a c i d data was i n c o n c l u s i v e . S i n c e the a u t h o r s have amassed such a l a r g e data pool f o r both v o l a t i l e and n o n v o l a t i l e compounds i t i s u n f o r t u n a t e t h a t some form o f data a n a l y s i s such as m u l t i v a l e n t statistical a n a l y s i s was not a p p l i e d so as to d e t e r m i n e which compounds were p r i m a r i l y r e s p o n s i b l e f o r the perceived f l a v o r p r e f e r e n c e . Aqueous tea e x t r a c t s d e t e r i o r a t e r a p i d l y d u r i n g s t o r a g e . They d e v e l o p an u n d e s i r e a b l e t a s t e commonly d e s c r i b e d as " f l a t " and c o n c u r r e n t l y t u r n dark brown i n c o l o r . Roberts e t a l . (56) used a m o d i f i c a t i o n o f the HPLC p r o c e d u r e o f H o e f l e r and Coggon~T57) to s e p a r a t e the p o l y p h e n o l s i n tea to s t u d y t h i s p r o b l e m . The e f f l u e n t from the r e v e r s e phase a c e t o n e g r a d i e n t was m o n i t o r e d a t 380 nm. T h i s wavelength was chosen so t h a t o n l y o x i d i z e d p o l y p h e n o l s and no u n o x i d i z e d p o l y p h e n o l s would be d e t e c t e d . T h i r t e e n peaks were r e s o l v e d ; ten were i d e n t i f i e d as t h e a f l a v i n s and t h r e e as thearubigins. In comparing chromatograms o f f r e s h l y brewed t e a s w i t h t e a s o f 48 and 72 h many new peaks were o b s e r v e d i n the thearubigin region. They c o n c l u d e d t h a t t h e dark c o l o r and o f f f l a v o r were due t o the f o r m a t i o n o f o x i d i z e d and p o l y m e r i z e d p o l y p h e n o l i c d e r i v a t i v e s o f the t h e a r u b i g i n t y p e . In a r e a s o n a b l e but s t i l l s p e c u l a t i v e c o n c l u s i o n they reasoned t h a t t h e t h e a r u b i g e n s were formed from t h e u n o x i d i z e d and p a r t i a l l y o x i d i z e d compounds which c o n t r i b u t e g r e a t l y to the " b r i s k " t a s t e o f t e a . Thus the l o s s o f t h e s e compounds upon s t a n d i n g produced a " f l a t " t a s t i n g b e v e r a g e . It would appear t h a t t h e a u t h o r s have i n d i r e c t e v i d e n c e to s u p p o r t t h e i r h y p o t h e s i s but would need to d e v i s e a way to i d e n t i f y and m o n i t o r the l o s s o f the compounds r e s p o n s i b l e f o r the " b r i s k " t a s t e i n o r d e r to s t r e n g t h e n t h e i r h y p o t h e s i s . LC/HPLC When d e a l i n g w i t h complex f l a v o r systems t h e r e a r e c e r t a i n advantages to c a r r y i n g out p r e l i m i n a r y 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 s t o o b t a i n s e v e r a l l e s s complex f r a c t i o n s . This allows one t o b e g i n w i t h r e l a t i v e l y l a r g e amounts o f s a m p l e . Large amounts o f sample a r e o f t e n r e q u i r e d when i d e n t i f y i n g t r a c e components. High performance p r e p a r a t i v e l i q u i d chromatography can o f t e n be employed to r a p i d l y f r a c t i o n a t e the s a m p l e . Maximum s e p a r a t i o n e f f i c i e n c y can be o b t a i n e d i f d i f f e r e n t c h r o m a t o g r a p h i c systems a r e used i n the p r e p a r a t i v e and a n a l y t i c a l s e p a r a t i o n s . For example, normal phase chromatography c o u l d be used f o r the i n i t i a l f r a c t i o n i z a t i o n and r e v e r s e phase chromatography used f o r t h e anlaytical separation. Kubeczka (58) u t i l i z e d t h i s g e n e r a l approach t o s e p a r a t e a t e r p e n e m i x t u r e i n t o oxygenated t e r p e n e s , monoterpene hydrocarbons and s e s q u i t e r p e n e hydrocarbons f r a c t i o n s b e f o r e a f i n a l a n a l y t i c a l separation. He used a C-18 r e v e r s e phase system o p e r a t e d i n the s e m i p r e p a r a t i v e mode to o b t a i n h i s p r e l i m i n a r y s e p a r a t i o n s . The sample f r a c t i o n s were then a n a l y z e d by l i q u i d - s o l i d chromatography u s i n g d e a c t i v a t e d s i l i c a gel (4.8% w a t e r / n - p e n t a n e ) a t -15 C. E i g h t monoterpenes were w e l l s e p a r a t e d w i t h i n ten m i n . U s i n g the

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same c h r o m a t o g r a p h i c system e i g h t s e s q u i t e r p e n e s c o u l d be r e s o l v e d from numerous i m p u r i t i e s / t r a c e components i n a n a t u r a l mixture w i t h i n 18 m i n . The compound(s) i n each peak were f u r t h e r identified u s i n g s p e c t r o s c o p i c methods. A n a l y t i c a l HPLC r e s u l t s were compared w i t h t h o s e from GLC f o r each f r a c t i o n and were i n good agreement. C l a s s i c a l l i q u i d - l i q u i d , l i q u i d - s o l i d and f r a c t i o n a l c r y s t i l l i z a t i o n can a l s o be used b e f o r e p r e p a r a t i v e and a n a l y t i c a l HPLC t o c o n c e n t r a t e t r a c e f l a v o r a c t i v e components. Huang et a l . (59) used t h e s e c l a s s i c a l t e c h n i q u e s to i s o l a t e a n o n v o l a t i l e b i t t e r / a s t r i n g e n t f r a c t i o n from soy f l o u r . The b i t t e r f r a c t i o n was f u r t h e r s e p a r a t e d u s i n g p r e p a r a t i v e HPLC. Seven peak f r a c t i o n s were s e p a r a t e d and the f l a v o r c h a r a c t e r i s t i c s o f each c o l l e c t e d peak determined. The r e v e r s e phase a n a l y t i c a l s e p a r a t i o n o f the b i t t e r peak f r a c t i o n produced s i x w e l l r e s o l v e d p e a k s . These peaks were f u r t h e r c o l l e c t e d and i d e n t i f i e d u s i n g UV and NMR s p e c t r o s c o p y . O n l y t h r e e o f t h e s e peaks were b i t t e r . The t h r e e b i t t e r peak components were i d e n t i f i e d as s p e c i f i c i s o f l a v o n e s . O f t e n when d e a l i n g w i t h f l a v o r m i x t u r e s c o n t a i n i n g compounds w i t h l a r g e m o l e c u l a r weight d i f f e r e n c e s , gel p e r m e n t a t i o n , GPC, ( s i z e e x c l u s i o n ) chromatography can be used to s e p a r a t e the sample into simpler f r a c t i o n s . An example o f t h i s approach i s the work o f A l a b r a n (60) i n i s o l a t i n g and i d e n t i f y i n g b e e f f l a v o r p r e c u r s o r s i n i n d i v i d u a l GPC f r a c t i o n s from raw b e e f . HPLC was used to s e p a r a t e and i d e n t i f y the i n d i v i d u a l components. The compounds were f u r t h e r i d e n t i f i e d u s i n g IR and NMR s p e c t r o s c o p y . HPLC/GLC/MS The use o f HPLC need not be l i m i t e d t o the a n a l y s i s o f n o n v o l a t i l e f l a v o r components. HPLC can be c o u p l e d w i t h GC/MS to form an e x t r e m e l y powerful s e p a r a t i o n s y s t e m . The unique s e p a r a t i o n p r o p e r t i e s o f HPLC can be used t o s e p a r a t e complex f l a v o r m i x t u r e s t o s i m p l i f y l a t e r GC/MS s e p a r a t i o n and i d e n t i f i c a t i o n . This approach can be somewhat t e d i o u s to use i n t h a t a s e p a r a t e GC/MS run must be made f o r each HPLC f r a c t i o n - but f o r v e r y complex f l a v o r systems t h e r e i s o f t e n no o t h e r c h o i c e . Grob et a l . (61) d e v i s e d a d i r e c t t r a n s f e r f o r HPLC f r a c t i o n s i n t o a c a p i l l a r y GC to make t h i s a p p r o a c h more c o n v e n i e n t to u s e . They demonstrated the p r a c t i c a l w o r k a b i l i t y o f the system by s e p a r a t i n g and i d e n t i f y i n g two components i n a t o o t h p a s t e u s i n g both HPLC/GC and HPLC/GC/MS. In a v e r y d e t a i l e d s t u d y t e r Heide and coworkers (62) used HPLC t o f r a c t i o n a t e a rum f l a v o r e x t r a c t i n t o 57 components. Each o f t h e s e components were f u r t h e r a n a l y z e d by c a p i l l a r y GC/MS. Over 400 compounds were i d e n t i f i e d o f which 214 had never been r e p o r t e d b e f o r e as o c c u r i n g i n rum. Thus w i t h o u t the s e p a r a t i o n power from t h e c o m b i n a t i o n o f HPLC/GC/MS t h i s complex f l a v o r system c o u l d not have been c h e m i c a l l y c h a r a c t e r i z e d to the degree i t has i n t h i s work. Yamaguchi et a l . (63) used the c o m b i n a t i o n o f HPLC/GC/MS t o s e p a r a t e the components i n a model f l a v o r s y s t e m . However a f t e r HPLC f r a c t i o n i z a t i o n and b e f o r e GC/MS a n a l y s i s each f r a c t i o n was organoleptically analyzed. There a r e p r o b a b l y many o t h e r f l a v o r systems which c o u l d b e n e f i t from a n a l y s i s u s i n g the c o m b i n a t i o n o f HPLC/GC/MS.

6. ROUSEFF

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Literature Cited 1.

Qureshi, Α. Α.; Burger, W. C.; Prentice, M. J. Am. Soc. Brew. Chem. 1979, 37, 153-60. 2. Mopper, K. Anal. Biochem. 1973, 56, 440. 3. Mopper, K. Anal. Biochem. 1980, 52, 2018. 4. Schwarzenbach, R. J. Chromatogr. 1976, 117, 206. 5. Wheals, Β. B.; White, P. C. J. Chromatogr. 1979, 176, 421. 6. Goulding, R. W. J. Chromatogr. 1975, 103, 229. 7. Hokse, H. J. Chromatogr. 1980, 189, 98. 8. Mopper, K.; Degens, Ε. T. Anal. Biochem. 1972, 45, 147. 9. Kidby, D. K.; Davidson, D. J. Anal. Biochem. 1973, 55, 321. 10. Honda, S.; Matsuda, Y.; Takahashi, M.; Kakehi, K.; Ganno, S. Anal. Chem. 1980, 52, 1079. 11. Hughes, S.; Johnson, D. C. Anal. Chem. Acta. 1981, 132, 115. 12. Hughes, S.; Johnson, D. C. J. Agric. Food Chem. 1982, 30, 712. 13. Rocklin, R. D.; Pohl, C. A. J. Liq. Chromatogr. 1983, 6, 217. 14. Tsang, W. S.; Clarke, Μ. Α.; Parrish, F. W. J. Agric. Food Chem. 1985, 33, 734. 15. Hussein, M. M.; D'Amelia, R.P.; Manz, A. L.; Jacin, H.; Chen, W.T.C. J. Food Sci. 1984, 49, 520. 16. Makapugay, H. C.; Nanayakkara, N. P. D.; Soejarto, D. D.; Kinghorn, A. D. J. Agric Food Chem. 1985, 33, 348. 17. Takemoto,T; Arihara, S.; Nakajima, T.; Okuhira, M. Yakugaku Zasshi 1983, 103, 1151. 18. Schulze, W. G.; Ting, P. L.; Goldstein, H. In "Liquid Chromatographic Analysis of Foods and Beverages"; Charalambous, G., Ed.; Academic: New York, 1977; Vol. 2, p. 442. 19. Siebert, K. J. J. Am. Soc. Brew. Chem. 1976, 34, 79-90. 20. Slotema, F. P.; Verhagen, L. C.; Verzela, M. Brauwissenschaft 1977, 30, 145-9. 21. Schwarzenbach, R. J. Am. Soc. Brew. Chem. 1979, 37, 180-4. 22. Whitt, J. T.; Cuzmer, J. J. Am. Soc. Brew. Chem. 1979, 37, 41-6. 23. Verzele, M.; Dewaele, C. J. Am. Soc. Brew. Chem. 1981, 39, 67-9. 24. Verzele, M.; De Potter, M. J. Chromatogr. 1978, 166, 320-6. 25. Schulze, W. G.; Ting, P. L.; Henchel, L. Α.; Goldstein, H. J. Am. Soc. Brew. Chem. 1981, 39, 12-15. 26. Kowaka, M.; Kokubo, E. J. Am. Soc. Brew. Chem. 1977, 35, 16-21. 27. Anderson, B. M. J. Chromatogr. 1983, 262, 448-50. 28. Spackman, D. H.; Stein, W. H.; Moore, S. Anal. Chem. 1958, 30, 1190. 29. Benson, J. R.; Hare, P. E. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 619-22. 30. Klapper, D. G. In "Methods in Protein Sequence Analysis"; Elzinga, M., Ed; Humana Press: Clifton, New Jersey, 1982; p. 509. 31. Roth, M. Anal. Chem. 1971, 43, 880-2. 32. Felix, A. M.; Jerkelson, G. Arch. Biochem. Biophys. 1973, 157, 177-182.

94

33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.

63.

CHARACTERIZATION AND MEASUREMENT OF FLAVOR COMPOUNDS Englehart, H.; Asshauer, J.; Neue, U.; Weigand, N. Anal. Chem. 1974, 46, 336. Haag, Α.; Langer, K. Chromatographia 1974, 7, 659. Cohen, S. Α.; Jarvin, T. L.; B1dlingmeyer, B. A. American Laboratory 1984, 51, 48-59. Lowrie, R. J.; Lawrence, R. C. N. Z. J. Dairy Sci. Technol. 1972, 7, 51-3. Okhrimenko,O.V.; Chebotarev, Α. I. Izu Vyssh. Uchebn. Zaved., Pishah, Tekhnol 1975, 6, 33-5. Renz, U.; Puhan, Z. Milchwissenschaft 1975, 30, 265-71. Fisher, J. F. J. Agric. Food Chem. 1973, 23, 1199. Fisher, J. F. J. Agric. Food Chem. 1978, 26, 497. Rouseff, R. L.; Fisher, J. F. Anal. Chem. 1980, 52, 1228. Shaw, P. E.; Wilson, C. W. J. Food Sci. 1984, 49, 1217. Fisher, J. F.; Wheaton, T. A. J. Agric. Food Chem. 1976, 24, 898. Rouseff, R. L. Abstracts 1981 Pittsburgh Conference, 1981, p. 364. Sticher, O.; Soldati, F.; Joshi, R. K. J Chromatogr. 1978, 166, 221. Johnson, E. L.; Majors, R. E.; Werum, L.; Reiche, P. In "Liquid Chromaotgraphic Analysis of Food and Beverages"; Charlambous, G.,Ed.; Academic Press: New York, 1979; Vol. 1, p. 17. Hoffman, P. G.; Lego, M. C.; Galetto, W. G. J. Agric. Food Chem. 1983, 31, 1326. Herrman, Α.; Stockli, M. J. Chromatogr. 1982, 246, 313. Hiraga, Y.; Endo, H.; Takahashi, K.; Shibata, S. J. Chromatogr. 1984, 292, 451. Dong, M. W.; DiCesare, J. L. Food Technol. 1983, 37, 58. Trugo, L. C.; Macrae, R.; Dick, J. J. Sci. Food Agric. 1983, 34, 300. Thies, W. Fresenius Z. Anal. Chem. 1984, 318, 249. Scott, R. P. W.; Kucera, P. J. Chromatogr. 1979, 169, 51. Qureshi, Α. Α.; Prentice, N.; Burger, W. C. J. Chromatogr. 1979, 170, 343. Wu, C. C.; Wu, J. L.; Chen, C. C.; Chou, C. C. In "The Quality of Foods and Beverages"; Charlambous, G.,Ed.; Academic Press: New York, 1981, p.133. Roberts, G. R.; Fernando, R, S. S.; Ekanayake, A. J. Food Sci. Technol. 1981, 18, 118. Hoefler, A. S.; Coggon, P. J. Chromatogr. 1976, 129, 460. Kubeczka, Κ. H. In "Flavor '81: 3rd Weurman Symposium"; Schreuer, P.,Ed.;Walter de Gruyter: Berlin, 1981; p. 345. Huang, A. S.; Hsieh, O. Α.; Chang, S. S. J. Food Sci. 1981, 47, 19. Alabran, D. M. J. Agric. Food Chem. 1982, 30, 486. Grob, K.; Fromlich, D.; Schilling, B.; Neukom, H. P.; Nageli, P. J. Chromatogr. 1984, 295, 55. ter Heide, R.; Schaap, H.; Wobben, H. J.; de Valois, P. J.; Timmer, R. In "The Quality of Foods and Beverages"; Charalambous, G.,Ed.; Academic Press: New York, 1981, p.183. Yamaguchi, K.; Satoru, M.; Akiyoshi, Α.; Shibamoto, T. In "Liquid Chromatogragraphic Analysis of Foods and Beverages"; Charalambous, G., Ed.; Academic: New York, 1979; Vol. II, p.303.

RECEIVED September 16, 1985