fish method quantification mononucleotides
TRANSCRIPT
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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
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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-
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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)
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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-