fish method quantification mononucleotides

7/23/2019 Fish Method Quantification Mononucleotides

1/4

1098

J.

Agric. Food

Chem.

1991, 39, 1098-1101

A

Method for the Quantitation of 5 -Mononucleotides in Foods and Food

Ingredients

Wayne W. Fish

Biosciences Branch, Phillips P etroleum Research Center, Bartlesville, Oklahoma 74004

~ ~~ ~~ ~~~~ ~~ ~ ~

A

method

is

presented

for

the

quant i ta t ion

of

the flavor-potentia t ing 5' -mononucleotides in foods

and

food ingredients. Extracted nucleotides, nucleosides, an d purines are separated by ion-pairing reversed-

phase high-performance l iquid chromatography. T he method is

not

affected b y hig h levels of sal ts in

th e sample, and recovery of 5 ' -mononucleotides after samp le trea tm ent

and

analysis

is

102

3 . In

addition

to m easurem ents of 5 '-mononucleotides in foods and food ingredients, data are presented to

i l lustrate the method's uti l i ty in m onitoring nucleotide levels during food or ingredient processing or

preparat ion.

INTRODUCTION

Like

monosodium glutamate

(MSG),

guanosine

5'-

monop hosphate (5 '-GMP) and inosine 5'-monophosphate

(5'-IMP)

are

umami substances

that

have become

im-

portant

flavor

potent ia tors

in

the

food industry. Th eir

widespread utilization

thus

necessi ta tes that they be

quantitated in

a broad spectrum

of sources from

flavor

additives such as yeast extracts to food produ cts such

as

bouillons an d sauces. Although anion-exchange chroma-

tography has been the prevailing metho d for

the

separat ion

and quanti ta t ion

of

nucleotides (HarwickandBrown,1975;

Floridi

e t

al.,

1977; McK eag

and

Brow n, 1978; Nissinen,

1980; Riss e t

al.,

1980; Freese e t al.

19841,

its applicat ion

to

flavor mononucleotides

in

foods

is

l imited

because

of

the frequent presence of interferin g levels of salts. T h e

advent

of high-performance

liquid

chromatography

( H P L C )

has

allowed

the

sepa rat ion of cel lular nucleo-

sides

and nucleotides by other methodologies such

as

reversed-phase

HPLC

(Wakizaka e t a l . , 1979; Taylor et

al.,

1981)

and

ion-pairing reversed-phase

HPLC

(Hoff-

ma n and Liao, 1977; Qu reshi et

al.,

1979; Walseth e t a l . ,

1980; Knox and Jurand, 1981; Payne

and

Ames, 1982).

Certainly, recent publicat ions in th e flavor ind ust ry [e.g.,

Ken ney (1990)] point o ut the rapidly growing importance

of

HPLC in flavor research and flavor quali ty control .

However,

I found tha t none of the published

HPLC

methods for nuc leot ide separa t ion an d q uant i ta t ion could

be direct ly applied to the extract ion a nd d etermination of

5'-mononucleotides in foodsand food ingredients. Toward

this end,

an extract ion procedure and one of the above

HPLC methodologies were m odified for application to food

produc ts ; the

results

are reported herein.

MATERIALS AND METHO DS

Materials.

All nucleotides,nucleosides, and purine bases were

purchased from Sigma (St. Louis, MO). PIC A, regular (tet-

rabutylammonium phosphate), was purchased from Waters

Associates (Milfo rd,MA). Methanol for HPL C was of Optima

grade from Fisher Scientific (Fa ir Lawn, NJ) . Water for HPLC

was produced by a Milli-Q system (Millipore/Waters). Yeast

products were obtained as samples from the supp liers; h e foods

were purchased at a local supermarket.

Standard Samples.

Stand ard nucleotides were dissolved in

0.1 M potassium phosphate buffer, pH 7.5. The true concen-

tratio n of each nucleotide in its standa rd solution was determined

by its absorbance measured in an AVIV 14DS UV-vis scanning

spectropho tometer with the use of the following molar extinc tion

coefficients at pH 7 (Beaven et al., 1955; Bock et al., 1956): 5'-

CM P, 9.0 X

l o 3

at

271

nm; 5'-AM P, 15.4 X 103 at 259 nm; 5 -

002 1-056 1 1 1439-1Q90$02.5OlO

UM P, 10.0 x 103 at 262 nm; B'-GMP, 13.7 x 103 at 252 nm; 5'-

IMP, 12.2 x 103 at 248.5 nm; and 5'-TMP, 9.6 x 108at 267 nm.

Af ter the elution position of each individual nucleotide was

determ ined, aliquota of each of the nucleotides were combined

into a mixture of the six standards. The nom inal concentration

of individual nucleotides in the m ixture of standards was 50 MM.

Chromatographic Procedure.

A Dionex BIOLC HPLC

equipped with an autosampler and a variable-wavelength detector

was used throughout. A Synchropak 250 mm X 4.6 mm RPP-

100 C-18 column (Synchrom, Lafayette, N ) was employed. Thre e

differentcolumns of this type from th e same m anufacturer gave

similar resolution among com ponents and iden tical sequence of

elution of nucleotides and nucleosides; however, the retention

times of th e compounds varied slightly from column

to

column.

Quantitative results from the th ree columns were identical.

A

flow rate of solvent through the column was maintained at 1.0

mL /min. Nucleotides were detected by their absorbance a t 254

nm. In samples of unknown composition, peaks were tentativ ely

iden tified by their retentio n time. The iden tity of each of the

tentatively identified peaks was further supported by the co-

elution of each with the authen tic compound. Instrum ent control

and data handling were accomplished hrou gh the use of Dionex

AI 400 software. The so lvent systems of Qureshi e t al. (1979)

were used in this study; he aqueous solvent, solvent

A,

contained

water/glacial acetic acid/PIC

A

in a 97.5:1.5:1.0 (v/v/v) ratio,

while the organic solvent, solvent B, consisted of methanol/glac ial

acetic acid/P IC

A

in a 97.5:1.5:1.0 ( v/ v/ v) rat io. The volume of

PIC A used in each solvent correspon ds to a con centration of 3.5

mM. Columns were washed with H2O and s tore d in methano l

when not in use.

Sample Preparation.

Samples were either yeast in some

phase of processingor a com mercial food or flavor pro duct. Thus,

the p rincipal steps of sample preparation were (i) extraction of

the nucleotides followed by (ii) acid precip itation of any

macromolecules from the extra ct with recovery of all of t he free

5'-mononucleotides, During various stages of process develop-

men t, yeast suspensions were sampled directl y and trea ted with

perchloric acid

as

described below. Food and flavor producta

were pre treate d in one of two ways. Water-solub le samples were

suspended in water at

1

g dry weight/lO mL final volume. They

were then stirred at 10

C

for

1

h before an a liquot was removed

for perchloric acid treatment.

Water-insoluble samples (the rice dish and the stuffing mix)

were treated

in

two ways. Fir st, 15 mL of H2O/g of dry sample

was adde d to each sample

so

ha t the mixture was soupy enough

tha t it could be stirred on a magnetic stirrer. The mixture was

stirred at 10 C for 1 h, a sample was centrifuged to remove

debris,and an aliquo t was removed for perchloric acid treatm ent.

Second, each food dish was prepared according to its man ufac-

ture r's directions. It was then added to a volume of water 2-5

times its prepared weight, blended for

2

min, stirred at

10 C

for

15 min, centrifuged to remove debris, and a liquoted for per-

chloric acid treatment.

1991 Am erican Chemical Society


2/4

Quantltatlon

of

5 MononucleotMes in Foods

Table

I.

Recovery of 5~-Mononucleotidesr o m Various

Samule Treatment Protocols

J Agric. Food

Chem.,

Vol. 39, No. 6, 1991

1 99

recoveryb

treatmenta

5-GMP 5-IMP

5-AMP

nucleotides in H z O l o o f 4 l o o f 2 l o o f 3

injected

directly

nucleotides added to HzO; 97 4

l o o f 5

9 5 f 4

treatment l

nucleotides added to HzO;

treatment 2d

nucleotides added

to

heated 46 10 102 5 110

broken yeast cells;

treatment

1

nucleotides added to

heated

97 4 9)

96 9)

broken yeast cella;

treatment 2

nucleotides

added

to heated 102 11) 102 11)

broken yeast

cells,

8.66

mg/mL each:

treatment

2

nucleotides

added to heated 105 6) 99 6)

broken

yeast cells, 0.05

mg/mL each; treatment

2

a

The concentration of each nucleotide

in

the

sample

before

treatment

was 0.15mg/mL unless

another concentration

isindicated.

Average

and standard deviation

of three

samples

unless a larger

number of samples is indicated . Treatment

1:

Precipitation

with

0.6 HC104and neutralization

with 1

M KHC03. Treatment

2:

Precipitation with0.6% HClOdand neutralization with 1

M

KHC03-

50 mM N a D T A . e Yeast cella

contained

endogenous

5-AMP;

recovery of

exogenously

added

5-AMP

was

99

when datawere

corrected for the amount of endogenous nucleotide.

98 12)

99 12) 99 12)

110 f e 9)

Th e perchloric acid treatm ent step was performed as follows.

A 0.7 mL volume of sample

was

added

to

0.7 mL of ice-cold 1.2

M

HC104and mixed. Th e mixture was allowed to sta nd on ice

for

5

min and th en cen trifuged on

a

bench-top centrifuge at 12000g

for 2 min

to

clarify the supernata nt. An 0.8-mL aliquot of the

supernatantwas the n adde d to0.85 mL of ice-cold1.0M KHC03/

50m M NQEDTA in

a

15-mL screw-top conical centrifuge tu be,

and the con tents were thoroughly mixed. After setting on ice

about 15 min, the sample was centrifuged

to

pellet the KC104,

and an aliquot of th e su pern atant was further d iluted with water

(if

necessary) or directly injected onto the column for analysis.

Samples were either centrifuged or filtered prior to injection. It

is important that the quantity of KHCO3 be sufficient to

completely neutralize the H C104so ha t the pH

of

the neutralized

sample isaround 6.5. Th e EDTA is necessary

to

ensure com plete

recovery of 5-GMP (see Resu lts and Discussion ).

RESULTS AND DISCUSSION

D e v e l o p m e n t of t h e M e t h od

fo r

5-Mononuc leot ide

Qu a n t i t a t i on . Th e ion -pa ir ing r e ve r se d -pha se HP LC

solvent system of Q ureshi e t a l. (1979) was chosen for our

applicat ions after i t was observed tha t th e levels of ions

in many sources for ana lys is thwarted a t tem pts to quan -

t i ta te 5-mononucleotides by ion-exchange HPL C. Fur-

thermo re, reversed-phase H PL C in the absence of an ion-

pairing agent did not provide adequate resolut ion of th e

nucleotides from food sources.

It

was also necessary to

modify th e published grad ient program of Qureshi e t a l .

(1979) to achieve comp lete separat ion of t he 5-mononu-

cleotides. With th is new gradient program, a different

elut ion sequence for t he nucleosides and nucleotides was

observed from th at rep orted earl ier (Qureshi e t a l. , 1979).

As

i l lustrated in Figure 1,a complete separat ion of the

5-mononucleotides w as achieved with t he new gradien t

program. T h e elut ion program included a 5-min wash

with solvent A after sample inject ion, a 15-min l inear

gradient to 10 solvent

B,

a 3-min isocrat ic wash at 1 0%

solvent

B, a

6-min l inear gradient to 40% solvent

B,

a

1-min isocratic wash

at 4 0 %

solvent

B,

a 4-min l inear

gradient back to 100 solvent A (ini t ia l condit ions), and

finally, a 10-min re-equil ibrat ion wash w ith 1 00% solvent

A before the next sample injection. Unlike the program

Figure

1.

Elution profile of 5-mononucleotide standard s. Th e

quantity of each nucleotide injected onto the column was 0.278

of 5-GMP, 0.585 pg of 5-IMP, and 0.415 pg

of

5-TMP. Full-

scale absorbance was

0.05

at 254 nm.

pg of 5-CM P, 0.484 pg of 5-AMP, 0.383 pg of 5-UMP,0.569 g

t

4

U

Figure

2.

Separation of purines, nucleosides, and nucleotides.

The nominalamount of each compound injected onto the column

was about 0.3

pg.

Full-scale absorbance

was

0.05

at

254 nm.

of Qureshi e t a l . (1979), this gradient program did not

require washing with methan ol every four samples. As

many as 48 h of continuous runs were made without a

noticeable effect on th e columns resolving properties; more

extended schedules of ru ns were not t ried.

On a routine, day-to-day basis, the peak areas for each

of five of th e six stan dar ds agreed to within *2.5% for

mu ltiple injections. 5-CMP, because of its location near

th e breakthrough peak was less precise (*6%). T he area

of each peak was

a

l inear function of the qu anti ty of

5-

mononucleotide injected, normally between 0.04 and 0.9

pg. These quanti t ies were of the approp riate range for

the detector sensi t ivi ty routinely employed, which was

0.05 absorbance unit ful l scale . Further more , very few

UV-absorbing compounds were observed to elute in the

region a t which 5-GMP and 5-IMP elute; this great ly

simplifies recognition and quanti ta t ion of these two flavor

mononucleotides.

As observed by oth ers (H offman and Liao, 1977;Qureshi

e t al.,

1979;

W a l se th e t

al.,1980;

nox

and

J u r a n d ,

1981;

Pay ne an d Ames, 1982), the num ber or location of

phosphate ester bonds o n a given nucleoside has

a

profound

impact on i ts re tention t ime. Th is is i l lustrated in Figure

2 by th e elut ion t imes for the pu rines, their nucleosides,

an d their various nucleoside phosphate esters. T o provide

maxim al resolution am ong th e 5-mononucleotides, som e

resolution amon g the other comp ounds was compromised

by our e lut ion program. Nevertheless, i t was st i l l possible

to identify and quan ti ta te m ost of these various nucleo-


3/4

1100

J.

Agric. FoodChem.,

Vol.

39. No.

6,

1991

Table

11.

S-Mononucleotide

Contents of Selected

Foods

and Food Ingredients

Fish

of dry product (w/w)* SD n)

product

5-AMP 5-GMP 5-IMP

A (yeast

extract)

B (yeast

extract)

C (yeast autolysate)

D

(yeast

extract)

E

(instant beef bouillon)

F (beef-flavoredbroth)

G

(gravy mix)

H

(rice

and sauce

before

prep)

I (rice

and

sauce

after

prep)

J (stuffing

mix before

prep)

K

(stuffing mix

after

prep)

a ND, none detected at

the

level

assayed.

1.64

f .15 (2)

0.39

f 0.01 (3)

0.10 f .01 (4)

0.18 f .01 (3)

ND

ND

ND

0.005f 0.001

(2)

ND

ND

ND

sides and nucleotides. Th is can prove to be very useful

in developing and monitoring produ ction processes s will

be discussed la ter.

Th e method of trea tment of th e sample before HP LC

analysis was found to be a cri t ical aspect of th e method-

ology. As i l lustrated in Table I, i t was discovered that

recoveries of 5-GMP were reduced by as much as 5 e 7 0

?4

in some samples when the acid-treated extract was

neutral ized with only KHC 03. Th is loss of 5-GMP was

a t ime-dependent phenomenon th a t occurred a fte r neu-

tralization of th e acid-treated materia l. Th e inclusion of

EDTA wi th the KHCO3 in the neut ra l iz ing solut ion

elimina ted this loss of 5-GMP. No furth er invest igations

were ini t ia ted to elucidate the compon ent(s) responsible

for th e dim inut ion of 5-GMP. As a final

test

of 5-mono-

nucleotide recovery using th e p rocedure, th e recoveries of

5-GMP and 5-IMP were determined in various yeast

samples to which known a mou nts of each of th e nucle-

ot ide s tandards had been added and which were then

subjected to our analysis procedure. T he average recovery

was 102 f 3% for 26 samples to which the 5-mononu-

cleotides had been ad ded over a 170-fold concentrat ion

range (T able I).

Applications

of

the Method

As stated earl ier, the

major ad vantage of ion-pairing reversed-phase chroma-

tography over ion-exchange chromatography is that the

functionali ty of the former is unaffected by the ionic

composit ion of th e sample. Thu s, analysis m ay be applied

direct ly to autolysates, extracts, fermentor creams, or

processed foods. Th is, of course, suggests tha t, in addition

to i ts ut i l i ty for the quanti ta t ion of flavor mononucle-

ot ides in finished products, i t may be used as a quanti ta t ive

monitor during process development or production. Such

an ap plicat ion is i l lustrated in Figure

3,

which examines

the nucleoside and nucleotide levels a t one step during

process development.

Two characterist ics of th e process are evident from t he

analysis. First , 5-mononucleotides have been produced,

and their respective levels can be quanti ta ted. Second, i t

can also be deduced t ha t a substantia l qu anti ty of 5-AMP

and 5-GMP has been dephospho rylated during a previous

step. If the causat ive agent (heat , pH , enzyme) of the

phosphate ester hydrolysis can be identified and i ts

detri me ntal action overcom e, the n t h e yield of 5-mOnO-

nucleotides would be increased by 70%.

Tab le I1 presents th e quanti ta t ive analysis of flavor-

pote ntiatin g 5-mon onucleotides from a selection of com-

mercial yeast-based flavor enhancers an d some selected

food produc ts. 5-AMP levels are included as it can serve

as a direct precursor of 5-IMP. Th e data from the samples

selected for this table i l lustrate applicat ions of the

method ology. As discussed above, yeast produ cts as flavor

potentia tors can be evaluated quanti ta t ively for their

098.5

1.79

f 0.03 (3)

0.41

.05 (3)


4/4

Quantitation

of

5-Mononucleot#es in Foods

LITERATURE CITED

Beaven, G. H.; Holiday, E. R.; Johnson, E. A. Optical properties

of nucleic acids and their components. In The Nucleic Acids;

Chargaff,E.,Davidson,J.N.,

Eds ;

Academic Press: New York,

Bock, R. M.; Ling, N. S. Ultraviolet absorption spectra of ad-

enosine-54riphosphate and related S-ribonucleotides. Arch.

Biochem. Biophys. 1966 ,62, 253-264.

Floridi, A,; Palmerin i, C. A.; Fini, C. Simultaneous analysis of

bases, nucleosides and nucleoside mono- and p olyphosphates

by high-performance liquid chromatography. J Chromatogr.

Freese, E.; Olem pska-Beer, Z.; E isenberg, M. N ucleotide com-

position of cell extracts analyzed by full-spectrum recording

in high-performance liquid chromatography.

J

Chromatogr.

1984,284,125-142.

Hartwick, R. A,; Brown, P. R. Performance of microparticle

chemically bonded anion-exchange resins in the analysis of

nucleotides.

J.

Chromatogr. 1976,112,651-662.

Hoffman,

N.

E.; Liao, J. C. Reversed phase high performance

liquid chromatographic separations of nucleotides in the

presence of solvophobic ions.

Anal. Chem. 1977, 49, 2231-

2234.

Kenney , B. F. Applications of High-Performance Liquid Chro-

matography for the Flavor Research and Quality Control

Laboratories in th e 1990s. Food Technol. 1990,44 (9),76-84.

Knox, J. H.; Juran d, J. Zwitterion-pair chromatography of nu-

cleotides and related species.

J.

Chromatogr. 1981,203,85-

92.

Matoba, T.; K uchiba, M.; Kimura, M.; Hasegawa, K. Therm al

degradation of flavor enhancers, inosine 5-monophosphate,

and guanosine 5-monophosphate in aqueous solution. J.

ood

Sci. 1988,53, 1156-1159, 1170.

McKeag, M.; Brown, P. R. Modification of high-pressure liquid

chromatographic nucleotide analysis.

J.

Chromatogr. 1978,

Nissinen,

E.

Analysis of purine and pyrimidine bases, ribonu-

cleosides, and ribonucleotides by high-pressure liquid chro-

matography. Anal. Biochem. 1980,106 ,497-505.

Payne, S. M.; Ames, B. N. A procedure fo r rapid extrac tion and

high pressure liquid chromatographic separation of the nu-

1955; Vol. 1 pp 493-553.

1977,

138,

203-212.

152, 253-254.

J. A M .

Foodchem. ,

Vol. 39, No.

6.

1991 1101

cleotides and o the r small molecules from bacterial cells. Anal.

Biochem. 1982,123,151-161.

Qureshi,

A.

A.; Pren tice, N.; Burger, W. C. Separa tion of potential

flavoring compounds by high performance liquid chromatog-

raphy. J. Chromatogr. 1979, 170, 343-353.

Riss,

T.

L.; Zorich, N. L.; Williams, M. D.; Richardson, A. A

comparisonof th e efficiency of nucleotide extraction by several

procedures and the analysis of nucleotides from extracts of

liver and isolated hepatocytes by HPL C. J Liq. Chromatogr.

Shaoul, 0 ;porns, P. Hydrolytic stability at intermediate p Hs

of the common p urine nucleotides in food, inosine-5-mono-

phosphate,

guanosine-5-monophosphate

nd adenosine-5-

monophosphate.

J.

ood Sci. 1987,52,810-812.

Taylor, M. W.; Hershey, H. V.; Lev ine, R. A.; Coy, K.; Olivelle,

S.

Improved method of resolving nucleotides by reversed-phase

high-performance liquid chromatography. J. Chromatogr.

Wakizaka, A.; Kurosaka, K.; Okuhara, E. Rapid separation of

DNA constituen ts, bases, nucleosides, and nucleotides, under

th e same chromatographic conditions using high-performance

liquid chromatographywitha reversed-phasecolumn.

J.

Chro-

matogr. 1979,162, 319-326.

Walseth, T. F.; Graff, G.; Moss, M. C., Jr.; Goldberg, N. D.

Separation of 5-ribonucleoside monophosphates by ion-pair

reversed phase high performance liquid chromatography.

Anal. Biochem. 1980,107,240-245.

Wynants, J.; VanBelle, H. Single-run high-performance liquid

chromatography of nucleotides, nucleosides, and major pu-

rine bases and its application todifferent tissue extracts. Anal.

Biochem. 1986,144, 258-266.

1980,3,133-158.

1981,219,133-139.

Received for review September 18, 1990. Revised manuscript

received January 2,1991. Accepted January

18,

1991.

RegistryNo. 5-AMP, 61-19-8; 5-GMP, 85-32-5; 5-IMP, 131-

AMP, 130-49-4;2-GMP, 130-50-7;5-ADP, 58-64-0; adenine, 73-

24-5; guanine, 73-40-5; hypoxanthine, 68-94-0; inosine, 58-63-9;

adenosine, 58-61-7; guanosine, 118-00-3.

99-7; 3-IM P, 572-47-4; 3-AMP, 84-21-9; 3-GMP, 117-68-0; 2-

fish method quantification mononucleotides

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