Analysis of ATP and Its Breakdown Products
by Reversed-Phase HPLC
AKIRA WATANABE, EISAKU
ABSTRACT
Analytical conditions for the quantitative determination of adenosine
5’-triphosphate(ATP), adenosine 5’-diphosphate(ADP), inosine 5’-
monophosphate(IMP), adenosine 5’-monophosphate(AMP), hypoxan-
thine(Hyp), xanthine(Xan), inosine(Ino) and adenosine(Ado) in meat
extracts by reversed-phase high-performance liquid chromatography
(HPLC) were examined. A commercial ODS column with a 5-urn
particle diameter was used, and expeimental parameters affecting the
separation were discussed. Peaks in chromatograms of meat extracts
were identified by retention time, co-injection with standards, absor-
bance ratios and the enzymatic peak shift method. The procedure
proposed was adaptable as an indication of freshness of meat.
INTRODUCTION
IT HAS BEEN well-known that ATP in muscle disappears
rapidly after slaughter. The pathway of degradation of ATP in
meat was as follows: ATP + ADP --$ AMP --f IMP --, ino-
sine(Ino) -+hypoxanthine(Hyp) (Teraskai et al., 1965). There
are many reports for the analysis of nucleotides, nucleosides
and bases by HPLC (Hartwick and Brown, 1976; Krstulovic
et al., 1977; Hartwick et al., 1979; Ramos and Schoffstall,
1983; Crescentini and Stocchi, 1984). However, the study on
the simultaneous separation of these compounds was limited
(Lang and Rizzi, 1986). On the other hand, ATP, ADP, AMP,
IMP, Ino, and Hyp were used for the determination of fresh-
ness in fish meat, i.e., K value (Saito et al., 1959). As an
indication of freshness of animal meat, Nakatani et al, (1986)
proposed the ‘Ko” value in which adenosine(Ado) and xan-
thine(Xan) were included in the “K” equation. Chromato-
graphic separations of ATP and its breakdown products (ATP-
related compounds) in meat have been reported of fish (Ryder,
1985), chicken (Kitada et al., 1983) and beef (Currie et al.,
1982). In these reports, however, Xan was not identified. In
the present study, the suitable conditions for the separation of
ATP-related compounds in meat by HPLC were examined and
index of freshness was calculated.
MATERIALS 8z METHODS
Instrumentation
The chromatographic analysis was performed on a Mode1 LC-4A
liquid chromatograph equipped with a Model SPD-2AS UV spectro-
photometer for monitoring at 254nm and a Model CR-2A integrator
(Shimadzu Corporation, Japan). For monitoring at a wavelength of
280nm, another Model 870-UV spectrophotometer and a Model DSL-
300 Integrator (Japan Spectroscopic Co. Ltd., Japan) were used. A
microparticle reverse-phaseShimpak CLC-ODS(M) (4.6mm x 15cm)
column from Shimadzu Corporation (Japan) was used.
Reagents
Reagent-gradeATP, ADP, AMP, IMP and Ado were obtained from
Boehringer Mannheim GmbH (West Germany); other nucleotides
(GMP, ITP and IDP) were from Sigma (St. Louis, MO). Ino, Hyp
and Xan were from Wako Pure Chemical Industries Ltd. (Japan) and
The authors are with the Tohoku National Agricultural Experi-
ment Station, 4 Akahira, Shimo-kuriyagawa, Morioka, lwate Pre-
fecture 020-O 1, Japan.
in Beef
TSUNEISHI and YUJI TAKIMOTO
uridine(Urd) from Nakarai Chemicals Ltd. (Japan). Tyrosine(Sr),
phenylalanine(Phe) and tryptophan(Trp) were from Takara Kohsan
Co., Ltd. (Japan). Uric acid (Uric), XOD (Xanthine oxidase, EC
1.2.3.2), AP (phosphatase, alkaline, EC 3.1.3.1) and NP (nucleoside
phosphorylase, EC 2.4.2.1) were obtained from Sigma (St. Louis,
MO). Analytical reagent grade potassium dihydrogen phosphate
(KH,PO,) and HPCL reagent grade methanol were used for the mobile
phase.
Chromatographic procedure
After preliminary experiments, the following solutions and condi-
tions were employed as the standard procedure for the analysis of
ATP-related compounds. The first eluant was O.lM KH2P0., (buffer-
1); the second was O.lM KHZPO, containing lO%(V/V) methanol
(buffer-2). The pH of these buffers was adjusted to 4.0 with H,P04
before HPLC analysis. For the first 5-min, 100% of the first buffer
was run, followed for the next 25-min by a linear gradient from 0%
to 50% of buffer-2, and then buffer-2 was brought up to 100% in lo-
min. During the last lo-min, the gradient was held and returned to
buffer-l in I-min. The initial condition was restored in about 15min.
The flow rate was l.OmL/min, and the column temperature was 24-
28°C. This procedure was referred to as method A. In this procedure,
however, the separation of IMP or ATP was unsatisfactory. Therefore,
0.02M KH,P04, adjusted to pH 4.8 with KOH, was used in buffers
1 and 2 instead of O.lM KH,PO,, pH 4.0, for the separation of ATP
and IMP. This procedure was referred to as method B.
Extraction procedure
A JapaneseShorthorn (26 months old) was stunned by pole-axe and
slaughtered by bleeding. Within 2 hr several samples, each 3-5g, from
the rib-eye muscle were placed in small vinyl bags. These samples
were stored at 4°C for various periods and then kept at -80°C until
the time of extraction. The meat samples (ca 3g) were homogenized
in 20 mL l.ON cold perchloric acid (PCA) and then centrifuged at
100000 x g for 15 min. The precipitate was extracted again. The
supernatantswere combined and neutralized with the addition of solid
potassium carbonate and centrifuged (10000 x g, 10 min). The final
volume was adjusted to 50 mL and filtered through a 0.45 urn mem-
brane filter; 20 uL was injected.
Peak identification
Assignment of the peaks was achieved by retention time analysis
and co-injection with standards. Absorbance ratios (254nm/280nm)
and enzymatic reactions were used to confirm the purity of ATP-
related compounds.
Comparison of the absorbance ratio of the standards to that of ten-
tatively identified compounds was useful for indirect confirmation of
the purity of the compounds because if the peak were completely
separated from other compounds the ratio should coincide with that
of the standard (Krstulovic et al., 1977). Enzymatic reactions with
XOD, NP, and AP were used to confirm directly the purity of ATP-
related compounds. XOD was used for the identification of Xan and
Hyp becausethis enzyme specifically catalyzed the conversion of Xan
and Hyp to uric acid. [no is also converted to uric acid by NP and
XOD. The specific enzymes for identifying ATP, ADP, AMP and
IMP were not available, however, the nonspecific enzyme AP was
used for the idcntificdtion of such components after confirming that
other nuclcotides were not eluted in the same positions as ATP, ADP,
AMP and IMP. These enzymatic reactions were performed according
to the method of Bergmeyer(1985). XOD(lOunits/mL) or NP(42units/
mL) was introduced in the meat extract at the ratio of 1:3 or 1:20,
Volume 54, No. 5, 79894OURNAL OF FOOD SCIENCE- 7 7 69
 HPLC ANALYSIS OF ATP-RELATED COMPOUNDS. .
respectively, and incubated at 25°C for 10 min. AP (lO%mits/mL) was IMP were separated most satisfactorily at pH 4.0. IMP was,
added to the meat extract at a ratio of 1:500 and incubated at 37°C however, eluted in the same position as Uric and Tyr, which
for 3 hr. The procedures of these enzymatic reactions were referred were possibly present in meat extract. IMP was separated from
to as the peak shjft method. other compounds at pH 4.5 (Fig. l[a]). But if meat contained
a large amount of IMP, Uric, ADP and Tyr were not separated
Calculation from the IMP peak. If 0.02M KH,PO, pH 4.5-5.0 were used
As retention time was affected by the line length of the HPLC
system, the following capacity factor was used to evaluate the
separations, where k’ = (retention volume -void volume)/void vol-
ume).
The index of freshness (KJ of animal meats (Nakatani et al., 1986)
was calculated as follows;
I& (%) = ((Ino + Hyp + Xan)/
(ATP + ADP + AMP + IMP + Ado + ho + Hyp -t Xan)) x 100
rai
RESULTS & DISCUSSION
Effect of pH, KH,PO,, and methanol concentration on the
separation of ATP-related compounds by HPLC
Different concentrations of KH,PO, have been used in the
mobile phase for reverse-phase HPLC analysis of mucleosides , 1

and bases. For example, Hartwick et al. (1979) employed 4

0.005M KH,?POI, pH 5.0 and Krustulovic et al. (1977) used 9
0.02M KH,PO,. In our preliminary experiments, the separa-
tion of Hyp, Xan and Urd was not affected by KH2P04 con-
centration. However, when the concentration of KH2P04 was
below O.O2M, elution time of some nucleotides was not re- cbl
producible, probably by increased interaction between nucleo-
tides and the residual silanol in the column at lower concentration
of KH,PO,.
To observe the effect of pH changes in the eluant solution on
the separation, the pH of the buffer containing O.lM Kl&PO,,
was varied in the range 3.5-5.0 by the addition of H,PO, or
KOH. Under the isocratic HPLC condition, the effect of pH
change on the capacity factor (k’) of ATP-related and other com-
pounds is shown in Fig. l[a]. ATP-related compounds were sep- 9
arated in the pH 4.0 to 4.5 region. In this region, ADP and

7 7 Phe
cc1
Pbe
1 6 6 8 IO
.7
5 5
Urd
j 59
4 Urd 4 z
k’ k’
3 3
Cd3
2 6
17 1IO
1 3- i h
8 16 U 32 40
C I I 1 I
3.5 4.0 4.5 5.0
PH
TIMEhin.)
Cal 0.1 M KHzPOb Cbl 0.02M KHzPOL, Fig. 2. - HPLC chromatograms at ATP-related compounds in meat
extract obtained by method A. [a] Chromatogram of meat ex-
Fig. 1. -Effect of pH and KHPO, concentration in the mobile tract at 4-hr after slaughter. /bJ Chromatogram of meat extract
phase on the capacity factors (k’) of ATP-related and other com- at 4°C &days after slaughter. [cJ Chromatogram of meat extract
pounds in HPLC analysis. Capacity factor (k’) = (retention vol- shown in [bJ, treated with XOD. fdJ Chromatogram of meat ex-
ume - void volume)Jvoid volume; l ATP; q ADP; A IMP; tract Shown in [bJ, treated with AP. The components identified
* Hyp; + Xan; 0 AMP; l Other compounds (Phe, Urd, Tyr and are (1) ATP; (2) ADP; 13) GMP; (4) IMP.: (5) Hyp; (6) Xan; (7)
Uric). Urd; (8) AMP; /9) lno; ll0) NAD.

1170-JOURNAL OF FOOD SCIENCE-Volume 54, No. 5, 1989

as the mobile phase, IMP was separately eluted from uric acid Hyp, Ino, and NAD were observed and Ado which would be
and Tyr (Fig. l[b]). However, complete separation was not eluted at 38-min was not detected in the chromatogram ob-
attained at pH 4.5, because the IMP peak’s area was much tained from the meat extract at 4 hr after slaughter and analysis
larger than that of Uric in the meat extract. The retention times by method A (Fig. 2[a]). Xan (peak No. 6), Urd (peak No. 7)
of ADP and IMP were also close at pH 5.0, hence, complete and peak No. 3 estimated from retention times as GMP were
separation was not achieved when a large amount of IMP was additionally observed in the chromatogram of the extract from
in the meat extract. pH 4.8 was considered most suitable, but the meat stored at 4°C for 6-days and analyzed by method A
a slight modification of pH was required with the column con- (Fig.-2[b]).This meat extract was used for evaluation of purity
dition becausethe capacity factor seemedto be affected by the of each peak by the peak shift method (Fig. 2[c] and 2[d]),
percentage of residual silanol in the column packing agent; because all break-down products from ATP were observed at
residual silanoi seemed to increase with an increase in the this stage. On the other hand, the purity of these ATP-reaited
column running time. compounds in meat extract was estimated by calculating the
Addition of methanol to the buffer did not fundamentally absorbance ratios of ATP, ADP, AMP, IMP, Hyp, Xan and
improve the spearation of ATP-related compounds. But the Ino at all stages, and the ratios at three stages are shown in
times until all compounds were eluted from the column became Table 1.
much longer, if methanol was not in the buffer. Addition of The ratios of Hyp, Xan and Ino showed no appreciablechange
lO%(V/V) methanol to the buffer was preferable. From these during storage and were comparable to those of each standard
experiments on the parameters influencing the separation, the (Table l[a]). Moreover, Xan and Hyp peaks completely dis-
chromatographic conditions as described in the Materials and appeared by the XOD treatment (Fig. 2[c]). In this chroma-
Method were selected. togram, an increase of peak No.2 was observed, possibly due
to the production of ADP from ATP during treatment, because
Analysis of ATP-related compounds in meat extract
peak No.l(ATP) decreased. The Ino peak also disappeared
with the NP and XOD treatments. These results indicated that
t$e;;;mponents were not involved in the peaks of Xan, Hyp
The chromatogram of ATP-related compounds in a PCA
extract of meat as analyzed by method A is shown in Fig. 2. Appreciable changes of the absorbanceratios were not ob-
The chromatogram obtained by method B is shown in Fig. 3. served in ADP and AMP but those of ATP and IMP consid-
The peaks were tentatively identified on the basis of retention erably changed (Table l[a]). Though the specific enzymes for
times and co-injection with standards. ATP, ADP, AMP, IMP, ATP, ADP, and AMP and IMP were not available, the non-
specific enzyme AP was used for the peak shift method be-
cause ITP, IDP and GMP, which were possibly in the meat
extract, were not eluted at the same retention times as the ATP-
related compounds. The resulting chromatogram is shown in
Fig. 2[d]. By the enzymatic reaction, the last peak which seemed
to be Ado was produced from ATP, ADP and AMP, and an
increaseof peak No. 9 meant that Ino was produced from IMP.
In this chromatogram, peak No. 2(ADP) and No. 8(AMP)
observed in Fig. 2[b] completely disappeared.Hence, the ADP
and AMP peak contained no other components.
Peak No. l(ATP) in Fig. 2[b] also completely disappeared
by treatment with AP, but the absorbance ratios appreciably
increasedduring storage (Table l[a]). Hence, it was concluded
that some other unknown nucleotides possibly overlapped the
ATP peak. When the elution system was changed to method
B (Fig. 3), the ratios remained constant during storage (Table
l/b]), and peak No. 1 (ATP) in Fig. 3 completely disappeared
by treatment with Al?. Therefore, 0.02M KH,PO, was consid-
ered suitable as the mobile phase for the separation of ATP.
The absorbanceratios of IMP (peak No.4 in Fig. 2)) in the
45 meat analyzed by method A was considerably lower than that
of the standard and decreasedgradually with storage time (Ta-
9 ble l[a]). Moreover, a small unknown peak “a” which had
the same retention time as IMP still remained after treatment
with AP (Fig. 2[d]), Uric and Tyr were eluted in the same
position as IMP under the same condition (Fig. l[a]). There-
CbJ
Table 1 -Absorbance ratios (254 nm/280 nm) of ATP-related compounds
and those in meat extract at 4 hr. bdays, and 10 days after slaughter
Compound Standard 4 hr 6 days 10 days
10 [a] Method A
ATP 5.86 5.76 6.55 7.66
IL ADP 6.10 6.01 5.98 6.20
I I I I IMP 7.85 7.05 6.45 5.54
0 i 16 24 32 40 HVP
Xan
16.88
1.64
16.51
1.70
14.40
1.53
16.00
1.59
AMP 6.17 6.31 5.92 6.20
TIME(min.) In0 7.73 7.10 7.70 7.03
Ado 6.36 N.D.’ N.D. N.D.
Fig. 3. - HPLC chromatograms for quantitative analysis of ATP
and IMP in meat extract by method B. [a/ Chromatogram of [b] Method B
meat extract at 4-hr after slaughter. fbJ Chromatogram of meat ATP 6.48 6.30 6.15 N.D.
extract stored at 4°C for 6-days after slaughter, The peak num- IMP 7.88 7.98 7.90 7.70
bers indicate the same components as Fig. 2. aN.D. = not detected

Volume 54, No. 5, 1989-JOURNAL OF FOOD SCIENCE- 1171

HPLC ANALYSIS OF ATP-RELATED COMPOUNDS. , ,
Table Z-Changes in content of ATP-related compounds and index of in muscle. IMP increased rapidly to the highest amount one
freshness lK, value1 durina storaae of beef at 4°C lumolesla raw meatl day after slaughter, then decreased gradually. ATP and ADP
Compound 4 hr 19 hr 2 days 6 days 10 days decreased rapidly within 2 days. AMP remained almost con-
ATP 3.10 0.35 0.25 0.25 trace stant throughout the storage period except for a slight decrease
AOP 1.20 0.35 0.19 0.21 0.38 after 10 days. NAD was observed as a single peak well sep-
IMP 2.27 4.53 4.26 3.01 2.18
WP 0.24 0.35 0.46 1.08 1.54
arated from other components in the HPLC chromatogram (peak
Xan 0.05 0.06 0.12 0.32 0.46 No.10 in Fig. 2), but the measurement of naturally occurring
AMP 0.16 0.18 0.15 0.17 0.10 NAD was impossible because NADH was oxidized to NAD
In0 0.24 0.33 0.49 0.94 1.20 by PCA used in the extraction step. Nakatani et al. (1986)
Ado N.D.8 N.D. N.D. N.D. N.D.
proposed that the K. value was a useful freshness index of
Total 7.26 6.15 5.92 5.98 5.86 meat when the accumulation of Xan was determined. A linear
Ko W 7.4 12.1 18.0 39.1 54.5 increase in I& value was observed with storage period. Hence,
a N.D. = Not detected this HPLC method was also useful for the evaluation of beef
freshness.

fore, these components were probably in the IMP peak. Peak REFERENCES
“a” in the IMP area was less than 1.7%, which may be dis-
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regarded. However, these components should be separatedfrom V f?H Verlagsgesellschaft mbH, Weinheim.
the IMP peak because they tend to increase with storage time. Crescent+, G. and Stocchi, V. 1984. Fast reversed-phase high-perform-
This problem was overcome using conditions specified for ance liqurd chromatographic determination of nucleotides in red blood
cells. J. Chromatogr. 290: 393.
method B. Absorbance ratios for IMP measured by method B Currie, R. W., Sporns, P., and Wolfe, F. H. 1982. Method for the analysis
were almost equal to the standard (Table l[b]). of ATP metabolites in beef skeletal muscle bv HPLC. J. Food Sci. 47:
1226.
Currie et al. (1982) reported analysis of ATP, ADP, AMP, Hartwick, R. A. and Brown, P. R. 1976. Evaluation of microparticle chem-
IMP, ITP, IDP, NAD, Ino, Hyp and adenylosuccinic acid in ically bonded reversed- base packings in the high-pressure liquid chro-
beef skeletal muscle by HPLC using an anion exchange col- F7\ygraphic analysm o P nucleosides and their bases. J. Chromatogr. 126:
umn. However, IDP and ADP did not show baseline separa- Hartwick, R. A., Grill, C. M., and Brown, P. R. 1979. Prediction of retention
tions, and ITP interfered with the separation of ATP. Using times of the nucleosides and bases on reverse phase high performance
liquid chromatography during gradient elution. Anal. Chem. 51: 34.
our HPLC condition, ITP and IDP were eluted prior to the Kitada, Y., Hasuike, A., Sasaki,.M., Tanigawa, K., Horiuchi, R., and Yuba,
ATP peak without interfering in the separation of ATP-related H. 1983. Analysis and behavmur of ATP related substances in chicken
muscie. Nippon Shokuhin Kogyo Gakkaishi (Jpn. Sot. Food Sci. Tecnol.)
compounds. In the preliminary experiment using a standard, 30: 151.
adenylosuccinic acid was eluted between the AMP and Ino Krstulovic, A. M., Brown, P. R., and R&e, D. M. 1977. Identification of
peaks by method B, though it was eluted in the same position nucleosides and bases m serum and plasma samples by reverse-phase
high performance liquid chromatography. Anal. Chem. 49: 2237.
as NAD using method A. However, this component was not Lang, H. R. M. and Rizzi, A. 1986. Se aration of purine bases, nucleosides
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phase and anion-exchange high-performance \. rqurd chromatography,
In conclusion, by using two methods, the accurate deter- J.Chromatogr. 356: 115.
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pounds of beef and rabbit muscles and a new index of freshness of muscle.
Method A enabled the separation of ATP, AMP, Ino, Hyp, Agric.Biol.Chem. 50: 1751.
and Xan. Method B was especially useful for the determination Ramos, D. L. and Schoffstall,, A. M. 1983. Reversed-phase high-perform-
of IMP and ATP. ance liquid chromatographm separation of nucleosides and mucleotides.
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index of freshness (&) measured by the HPLC method dis- MS received 10/28/88; revised 2/l/89; accepted Z/4/89.
cussed above on the cattle’s rib-eye muscle stored at 4°C are
shown in Table 2. The contents of Ino, Hyp and Xan increased
gradually with the storage of beef. Nakatani et al. (1986) have The authors express their sincere appreciation to Dr. T. Ito (Faculty of Agriculture,
reported no increase on Ino in beef after 6-days storage. These Tohoku Univ.,
Ltd.. Morioka.
Japan) for his helpful comments and thank A. Tanifuji
Jaoan) for mmolement of wlumne.
(Naruse Kikai

differences were possibly due to the difference of NP activity

7 172-JOURNAL OF FOOD SCIENCE-Volume 54, No. 5, 1989