CHIRALITY 6277-282 (1994)

Evaluation of Free D-Glutamate in Processed Foods

KIMBER L. RUNDLETT AND DANIEL W. ARMSTRONG
Department of Chemisty, University of Missouri-Rolla, Rolla, Missouri

ABSTRACT Monosodium glutamate (MSG) is added to many processed foods at signif-

icant levels for flavor enhancement. It is also naturally occurringat high levels in some foods.
The enantiomericcompositionof free glutamate in foods was examined and all foods analyzed
were found to contain D-glUtaXlXlte. The relative percent of D-glutamate in the food products
studied depended on the origin of the glutamate. Foods to which MSG was added by the
manufacturer had a high total level of MSG but a lower relative percentage of the D-enanti-
omer (usually less than 0.8%). In comparison, fermented foods tend to have high relative
levels of D-glutamate but a lower total amount of the amino acid. The relative percent of
D-glutamate in nonfermented foods containing no added MSG was also found to be low
compared to fermented products. In some cases the percent o-glutamate could be related to
the relative amounts of other food ingredients such as cheese. o 1994 Wiey-Liss, hc.

KEY WORDS: amino acids, D-glutamate, monosodium glutamate, food analysis

Most a-amino acids contain a stereogenic center and exist
as the L- and D-enantiomers. Although it has been generally
accepted that the free amino acids and proteins found in higher
organisms are composed exclusively of the L-enantiomers of
amino acids, the mirror image D-forms are known to be
present in some naturally occurring peptide antibiotics and in
the cell walls of bacteria.',' Recent studies have shown that
free a amino acids are ubiquitous (although not necessarily in
high concentrations) in the physiological fluids of humans and
rodents.s7 A variety of factors were found to affect the rela-
tive levels of amino acids present in mammals including
disease, pregnancy, sex, young age, and diet.4 Of these fac-
tors, the diet was thought to be the most important factor
governing the relative ratios of amino acid enantiomers exam-
ined in the blood and excreted in the urine of laboratory
rodents. These studies were made possible by recent ad-
vances in the analytical methodologies of enantiomericsepara-
tions and quantitation of trace levels of amino acid enanti-
omers (see the Experimental Section.)
Concurrently there has been expanded interest in the oc-
currence of D-&O acids in food and beverage products.
There have been several recent reports of the presence of
amino acids in milk products8p9and fermented foods and
beverages." In these studies, the fermented foods were
found to contain unusually high relative levels of free D-&O
acids which were attributed to the fermentation process. Sig-
nificant levels of o-alanine, ~-glutamicacid, and D-glutamine
(analyzed together as glutamic acid in these studies) and D-as-
partic acid and D-asparagine (analyzed together as aspartic
acid) were found in these products. Interestingly, these are
the predominant D-&O acids present in bacterial cell
walls.'~~ D - ~ acidO enantiomers seem to be most prevalent
in at least three types of foods: (1) highly processed foods
(that is, those which undergo extremes in pH, temperature,
etc.), l1 (2) food products exposed to microbial degradation
0 1994 Wiley-Liss, Inc.
(fermentation, aging, etc.),a'o and (3) food sources which
naturally contain amino acids (e.g., certain seafoods). ''
This present study examines the enantiomeric composition
as well as the total percent of free glutamic acid in a variety of
human food products using precolumn derivatization and
HPLC. Glutamic acid is a naturally occurring, nonessential
amino acid that is commonly used as a food additive in the form
of its monosodium salt (monosodium glutamate, MSG). MSG
is added to many processed foods at levels of 0 . 2 4 9 %by
weight to increase palatability and only the L-enantiomer pro-
cesses flavor enhancement characteristics.l3 Free glutamate
is also ubiquitous in nature; it is naturally present at relatively
high levels in foods such as tomatoes, peas, mushrooms, and
potatoes. The goal of this study is to evaluate and comparethe
enantiomeric composition of glutamate (MSG, glutamic acid)
in fermented foods, foods containing added MSG, and nonfer-
mented foods to which MSG has not been added.
MATERIALS AND METHODS
Apparatus
Enantiomeric analyses were carried out using a coupled
HPLC system described previo~sly.~ It consisted of 2 pumps
(LC-6A, Shimadzu, Kyoto, Japan), a fluorescence detector
(RF-535, Shimadzu), UV detector (SPD-2AM, Shimadzu),
switching valve (Rheodyne, Cotati, USA), and 20 p1 injection
valve (Rheodyne.) The columns used were 250 x 4.6 mm C18
and y-cyclodextrin and were supplied by Advanced Separation
Technologies, Inc. (Whippany, NJ).
Received for publication December 29,1993;accepted February 8,1994.
Address reprint requests to Dr. Daniel W.Armstrong, Department of Chem-
istry, College of Arts and Sciences. University of Missouri-Rob, 341 Schrenk
Hall,Rolla, MO 65401.
278 RUNDLETT AND ARMSTRONG

Standards and Reagents

Acetonitrile, triethylamine, glacial acetic acid, and ethyl
ether were of HPLC grade and purchased from Fisher (St.
Louis, MO). 9-Fluorenylmethyl chloroformate was purchased
from Aldrich Chemical Company (Milwaukee, WI).All other
chemicals were obtained from Sigma Chemical Company (St.
Louis, MO). Solid phase extraction cartridges (CIS, 0.5 g)
were purchased from Alltech (Deerfield, IL).

Derivatization Procedure and

Chromatographic Conditions
The enantiomers of glutamic acid were analyzed as the
9-fluroenylmethylcarbonylglycine chloride (FMOC-Gly-CI)
derivative. This reagent was prepared according to the direc-
tions given by Carpino et al. l4 Derivatization was performed
by adjusting the sample to pH 8 with 0.5% (w/v) sodium
carbonate. An appropriate amount of FMOC-Gly-CI was dis-
solved in a small amount of acetone and added to the sample.
The sample was shaken and allowed to react for 15-20 min.
Acetic acid, 5% (v/v), was added to quench the reaction, the
derivatives were extracted from the sample, and 20 p,l was
injected into the HPLC system. The eluent for the column
was acetonitrile/water/aceticacid, 300:700: 3 (v/v/v). A typical
achiral separation is shown in Figure 1. A polar organic mobile
phase was used for the eluent for the y-cyclodextrin column
consisting of acetonitrile/methanoI/acetic acidhiethylamine,
875: 125:3 12 (v/v/v/v). A UV wavelength of 266 nm was used
to monitor the eluent from the column and fluorescence
detection was used to monitor the eluent from the chiral
column. The fluorescence excitation and emission wave-
lengths used were 266 and 315 nm, respectively. Figures 2
and 3 show chiral separations of FMOC-Gly-~,L-glutamate in
1
1
minutes 2o
I

various food products after column switching.
2-Aminoadipic acid was used as an internal standard for
quantitation of MSG.The amino acids were derivatized with
9-fluorenylmethyl chloroformate (FMOC) using the same de-
rivatization procedure as that for FMOC-Gly-CI. The chro-
matographic system used for quantitation was composed of a
column and a fluorescence detector and the mobile phase
was acetonitnle/water/aceticacidhiethylamine, 300 7003: 1
(v/v/v/v). Figure 4 shows a typical separation used for quanti-
tation of MSG in a cracker sample.
It should be noted that quantitation of extremely low levels
of D-glutamic acid (<1%) in the presence of large amounts of
the L-enantiomer, or when simply determining the enantio-
meric ratio, it is often inaccurate to simply use the data from
the automated peak integrator. This is because the linear
dynamic range of the detector is sometimes exceeded for the
larger of the two peaks (see the L-enantiomers in Fig. 2).
Hence, the larger peak often must be quantatively measured
in a second determination after accurate serial dilutions.
Preparation of Food Samples
All food samples were purchased at local markets. Care
was taken to prepare and derivatize the samples immediately
upon exposure to air in order to avoid erroneous results due
to bacterial contamination.
Fig. 1. A chromatogram showing the achiral separation of FMOC-glycyl deriva-
tized worcestershire sauce on C,, column. Mobile phase: 700:3002 (v/v/v) water/
acetonitde/acetic acid. UV detection was at 266 nm and the flow rate was 1.5
drnin. The arrow designates the peak containing FMOC-GIY-D,L-GIU.
HPLC-grade water was added to all samples and the sam-
ples were sonicated for 10 min. For quantitative measure-
ments, the 2-aminoadipic acid internal standard was added
prior to sonication. Aqueous soup and cracker emulsions,
most of which contain high total amounts of MSG, were ex-
tracted with hexane [1:1 x 2 (v/v)l to remove fats and oils,
derivatized, and passed through a c18 solid phase extraction
cartridge. The amino acid derivatives were eluted with diethyl
ether, the ether was evaporated, and the samples were dis-
solved in an acetonitrile/water/aceticacid mobile phase.
Acetone and acetic acid were added to all other samples
prior to sonication in order to precipitate proteins. The sam-
ples were centrifuged at 3000 rpm for 10 min. The superna-
tant was extracted with diethyl ether [l:l X 2 (v/v)l, deriva-
tized, and the derivatives were extracted into diethyl ether
El: 1 x 2 (v/v)]. The ether was evaporated and the derivatized
amino acids were dissolved in the desired acetonitrile/water/
acetic acid mobile phase for injection. (Solid phase extraction
was not used for these samples due to fouling and capacity
problems, which gave nonreproducible results. )
 EVALUATION OF FREE D-GLUTAMATE
McCormick Flavor Enhancer (MSG)
%D = 0.22%
4 minutes
I
18
Fig. 2. A chromatogramshowing the enantioseparationof FMOC-glycylderivatives of MSG in commercialMSG products
on a y-cyclodextrin column. Mobile phase: 850:150 1 2 3 (v/v/v/v) acetonitrile/methanoVtriethylamine/acetic
cence detection: excitation wavelength is 266 nm, emission wavelength is 315 nm. Flow rates: 1.0 d m i n for McCormick
MSG and 1.5 mvmin for Sigma MSG.
RESULTS AND DISCUSSION
Table 1 lists the enantiomeric composition and total weight
percent of monosodium glutamate (MSG) in the foods studied.
As might be expected, foods containing MSG added by
the manufacturer have higher total levels of this compound
than most of the other products studied. Soy sauce, worces-
tershire sauce, and A.l. steak sauce are the only fer-
mented foods studied which appear to have total natural
MSG levels comparable to those products where it is an
additive.
While most fermented foods (to which MSG has not been
added) have lower total amounts of MSG, they have much
higher relative percents of the D-enantiomer than the other
types of foods analyzed. All of the fermented foods listed in
Table 1 contain high relative amounts of D-glutamate, usually
exceeding 5%, with Meeter’s Kraut Juice having the highest
level of 35.7%. Commercial flavor enhancers have low rela-
tive levels of D-glutamate (about 0.2%), as do the products to
which MSG has been added during processing (less than 1%).
For example, sour cream, a fermented product, has a high
relative level of D-glutamate (i.e., 5.39%), which decreases to
about 0.446% for the sour cream dips which list MSG as an
ingredient. Nonfermented products that do not have MSG
added during processing also have low relative amounts of the
D-enantiomer (less than 1.9%) compared to the fermented
foods (Table 1).
279
Sigma Monosodium Glutamate (MSG)
%D = 0.25%
v
0
minutes
18
acid. Fluores-
Nabisco Cheese Nips crackers appear to be an exception to
the aforementioned general rule in that the relative percent of
D-glutamate, (2.93%, Table 1) is high even though MSG has
been added during processing. However, cheese was previ-
ously shown to contain high relative levels of D - G (glutamic
~
acid + glutamine, analyzed together as glutamic acid), ranging
from 5 to 22%.’ Cheese is listed as the third ingredient after
flour and shortening on the food label while MSG is listed near
the end. Apparently, the large relative amount of D-glutamate
contributed by the cheese is not completely diluted by the low
levels from the added MSG. Keebler White Cheddar Crack-
ers, which have low relative amounts of the o-enantiomer,
also lists cheese and MSG as ingredients, but they contain
less cheese and more added MSG based on the relative loca-
tion of the cheese and MSG on the ingredients list. The
glutamate contribution from the cheese in these crackers ap-
pears to be neghgible compared to that from the added MSG.
It appears that the relative level of D-glutamate can often give
one a reasonable estimate of the relative amounts of certain
added ingredients (cheese and MSG, for example).
Free D-glutamate was found at finite levels in all foods
analyzed in this study. In fact, free amino acids have been
found in all physiological, biological, and “pure” commercial
. ~ ~ the effects of
samples analyzed in this I a b o r a t ~ r yWhile
D-O acid consumption on humans are not very well
known, some deleterious physiological effects of high doses of
280 RUNDLETT AND ARMSTRONG

Kikoman Soy Sauce Meeter's Kraut Juice

% D = 35.3%

L kI
i %D = 7.7%
h
Ill
- I I
0 13 0 minutes 18
minutes
Fig. 3. A chromatogram showing the enantioseparation of FMOC-glycyl derivatives of MSG in Kikoman Soy Sauce and
Meeter's Kraut Juice (fermented products) on a y-cyclodextrin column. Mobile phase: 850: 150:12:3 (v/v/v/v) acetonitrile/
methanoUtriethylamine/acetic acid. Fluorescence detection as in Figure 3. Flow rates: 3.0 d m i n for the soy sauce and 2.0
d m i n for the kraut juice.

1 2
some amino acids on lab animals have been reported:
D-serine was related to nephrotoxicity in rats15; renal and
hepatic damage in rats was associated with D-proline,16 and
D-glutamate was reported to exhibit immunosuppressive ac-
tivity in mice when administered in large doses." In addition,
for some D-enantiomers of protein amino acids, consumption
of large amounts of a amino acids was reported to interfere
with the normal metabolism of a amino acids. 17q1'

In mammals, a amino acids are metabolized mainly through

the a amino acid oxidase pathway, where a amino acids are
oxidized to or-keto acids which may subsequently undergo
stereospecific transamination to ch amino acids. l9 Of the many
amino acids studied, D-glutamate showed the slowest rate of
oxidation to an a-keto acid by mammalian D-amino acid oxi-

-
dase. l9 Therefore, compared to other D-&O acids, D-gluta-
mate probably contributes very little to the protein nutritional
value of food. It also appears that amino acids are eliminated
from the bodv via the k i d n e ~However.
.~ the human Dhvsio- . <
i/L
logical effects (if any) of frequent consumption of D-glutamate
are unknown, 0 minutes 15

ACKNOWLEDGMENTS Fie. 4. A chromatomam showing the achiral seDaration of FMOC derivatized

Support of this work by the National Institute of Health Keebler Munch 'Em Crackers with kminoadipic acid added as the internal stan-
( G NIH ~ BMT ~ 2RO1
~ G M ~ ~ ~ is~ gratefully
~ - ~ ac-~ Mdard. Peak designations: (1) MSG, (2) 2-aminoadipic acid. The flow rate was 1.5
d m i)n and fluorescence detection was used (see Fig. 3). Mobile phase: 700:300:3 1
knowledged. (vlvlviv) water/acetonitrile/aceticacid/triethylamine.
 EVALUATION OF FREE D-GLUTAMATE 281
TABLE 1. Enantiomeric composition and total percent of monosodium glutamate in food products

MSG listed
on product
Product label % MSG“ %Db nc

MSG
McCormick Flavor Enhancer Yes 99.9 *
0.26 0.08 7
Accent Flavor Enhancer Yes 99.9 0.21 f 0.06 5
Sigma Monosodium Glutamate - 99.9 *
0.23 0.05 4
Soup products
Wylers Beef Bouillon Yes 3.18 0.52 2 0.05 8
MBT Beef Broth (dried) Yes 7.53 0.82 & 0.38 8
Smack Ramen Beef Broth (dried) Yes 3.86 *
0.23 0.05 6
Wyler’s Chicken Broth (dried) Yes 5.35 0.39 & 0.07 10
Smack Ramen Chicken Broth (dried) Yes 3.36 0.52 2 0.35 6
MBT Onion Broth (dried) Yes 3.59 0.80 5 0.10 4
Campbell’s Chicken Broth (canned) Yes 0.44 0.36 f 0.04 6
Cracker products
Nabisco Chicken in a Biskit Crackers Yes 0.53 0.40 & 0.19 4
Keebler White Cheddar Crackers Yes 0.27 0.54 & 0.11 4
Nabisco Cheese Nips Crackers Yes 0.30 *
2.93 0.70 6
Keebler Munch ’Em Crackers Yes 0.42 0.40 f 0.10 4
Nabisco Sociables Crackers No“ 0.028 *
1.62 0.18 4
Sauces
Heinz 57 Sauce Nd 0.38 0.71 2 0.08 8
A. 1. Steak Sauced No 0.35 6.69 2 0.17 8
Lea and Penin’s Worcestershire Sauced Nd 0.18 5.83 f 0.09 8
French’s Yellow Mustard Nd 0.062 0.95 f 0.07 8
Griffin’s Yellow Mustard Nd 0.065 0.56 * 0.08 8
Tabasco Pepper Sauced No 0.023 5.63 f 0.74 4
Kikoman Soy Sauced No 0.94 7.93 * 0.23 4
La Choy Soy Sauced No 1.04 7.77 f 0.51 4
Vinegars
Reese Red Wine Vinegaf’ No 0.00092 12.6 & 0.80 4
Heinz Red Wine Vinegaf‘ No 0.00018 13.7 ? 1.10 4
Regina Red Wine Viegaf’ No 0.00030 *
18.1 0.70 4
Sauerkraut juice
Meeter’s Kraut Juiced No 0.058 35.7 f 0.6 3
Bush’s Sauerkrautd (drained juice) No 0.026 *
31.1 2.7 8
Del Monte Sauerkrautd (drained juice) No 0.020 21.1 2 1.4 4
Tomato products
V-8 Vegetable Juice No 0.35 0.81 f 0.08 5
Hunt’s Tomato Paste No 0.89 *
0.57 0.03 4
Contadina Tomato Paste No 1.14 *
1.10 0.20 4
Hunt’s Ketchup No 0.47 1.62 f 0.20 9
Heinz Ketchup No 0.91 *
1.52 0.08 8
Milk products
Peevley Buttermilkd No 0.022 6.17 k 0.10 7
Peevley Sour Creamd No 0.010 5.39 f 0.25 8
Dean’s Bacon & Cheddar Dipd Yes 0.15 0.37 f 0.04 5
Dean’s French Onion Dipd Yes 0.14 0.61 f 0.05 4

“%MSG = (wt. MSG/wt. sample) x 100.

b % ~= (D/D + L) x 100. The number following 2 sign is the standard deviation of n determinations of the same product.
‘Number of determinations of the same product.
dFermented or aged food product.
‘Contains hydrolyzed vegetable protein.
’Contains natural flavorings.
282 RUNDLETT AND ARMSTRONG
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