Short Communications

Reverse Phase Ion Pair Separation of Nucleotides and Related Products in Fish Muscle

John Murray* and Alexander B. Thomson Ministry of Agriculture, Fisheries and Food, Torry Research Station, PO Box 31, 135 Abbey Road, Aberdeen A69 8DG, UK

Key Words:

Liquid chromatography, HPLC Reverse phase mode Ion pairing Nucleotides Fish

1 Introduction

The nucleotides and their breakdown products in fresh and spoiling fish muscle have been well characterized [1,2] and hypo- xanthine (Hx), the end product of a series of enzymic reactions in most fish, has beenexaminedforitsusefulnessasaspoilage index in a large number of species [3]. However, a distinct improvement in estimation of quality would result from the separation and quan- titation of all products of nucleotide degradation, provided this could be done in a reasonable time.

Reverse phase isocratic systems currently exist which allow similar separations [4] but resolution is relatively poor and elution times for inosine are large. Recently, reverse phase ion pair systems [5,61 have been demonstrated which offer greatly improved flexibility compared with those without such pairing agents. One of these methods examined by us [6], using Hypersil ODS as the support as a substitute for Supelcosil ODS, proved

unsatisfactory in that variable retention times and resolution of peaks were noted even after lengthy (3h) equilibration times with the stated eluent. Additionally the method as published did not allow the separation of inosine monophosphate (IMP) and Hx, which, while not a prerequisite of that work, is essential for our pur- poses since these are major products of fish muscle nucleotide catabolism. The system reported here has allowed us to separate nucleotides and related productsfrom fresh and aged fish muscle. Equilibration times are such that constant retentionvaluesforindi- vidual components are obtained 1 h after initial passage of the eluate.

2 Experimental

Hypersil ODS (5 pm) slurried in isopropanol was used to packa20x 0.46 cm analytical column and a 3 x 0.46 cm guard column using hexane as the pumping solvent.The columns were coupled using 0.1 5 mm id tubing. The liquid chromatograph was composed of a Waters pump, model 450UVdetector, and a U6K injector. Quanti- tation of eluted peaks was performed by a Spectra Physics model 4100 integrator coupled to the detector set at 254 nm.The eluate which was passed through a 0.45 pm membrane filter comprised 0.1 M KH2P04, adjusted to pH 7, containing 10% VIV methanol and 2.0 mM tetrabutylammonium hydrogen sulfate. Flow rate was 1.9 mllmin at 2OOC. Nucleotides and related products were obtained from Sigma and homarine from Aldrich.

A

i .01 AU

Figure 1

Integrator traces of A) standard mixture which elutes in the order homarine, Hx, IMP, inosine, AMP, ADP, and ATP. The 6 pl injection contained approximately 0.75 nanomoles of each except homarine (1.3 nanomoles), B) trout extract equivalent to 0.259 mg fish.

0 1983 Dr. Alfred Huethig Publishers Journal of High Resolution Chromatography Ei Chromatography Communications 209

10391 11 0392 Short Communications

Trout samples were prepared by homogenizing 10 g muscle with 50 m l O . 6 ~ perchloric acid. To 10 ml filtrate was added 10 ml0.2M KHpP04 buffer adjusted to pH 7 and containing 0.51 7~ KOH. The finalextractwhich hadthesamepH asthe bufferwas heldinicefor 1 h before filtering to remove the precipitated KC104.

3 Results and Discussion Figure 1A shows the separation of a seven component standard mixture and indicates that good resolutions of all components was obtained in a total run time of 10 min. An integrator trace from 3 pl of an extract from trout which had been held for five days in ice before sampling is shown in Figure 18. The major peaks are IMP and inosine with minor amounts of Hx, AMP, and ADP also evident. Area counts from the integrator were directly proportional to the amounts of individual standards in the range 0.2-2.5 nanornoles injected. Using the external standard technique, the integrator allows direct printout of the concentration of individual solutes provided a constant volume of extract is assayed. Examination of the data associated with Figure 16 indicates that IMP and inosine were present at 2.97 and 4.29 phdg respectively. This compares with levels of 5.56,0.35, and 1.27 W l g for IMP, inosine, and ATP respectively found in trout held 1 h postmortem in ice.

Homarine (2-carboxy-1-methylpyridinium chloride), which is present in certain shellfish, elutes early using the present system and thus does not interfere with the quantitation of the other components of interest in these species as we have found it to do using previously published methods [4].The decreased equilibra-

tion time and the constancy of retention times exhibited in this workalmost certainly results from the enhanced concentration of ion pairing agent employed although differences in the inherent characteristics of the two reverse phase silicas might explain the discrepancies noted initially at the lower ion pairing concentration (0.3 mM). We have observed that separation of the individual components of the standard mixture as shown in Figure 1A may vary slightly when using different batch lots of Hypersil ODS as supplied, but resolution can be optimized by minoradjustmentsto the ion pairing andlor methanol concentrations in the eluate. The present system should prove useful for quantitation of nucleotides and their degradation products in applications other than the investigation of fish quality noted here.

Crown Copyright Reserved

References

[l]

[2]

N. R. Jones and J. Murray, J. Sci. Food Agric. 13 (1962) 475.

B.4. Kassernsarn, B. SanzPerez, J. Murray, and N. R. Jones, J. FdSci. 28 ( 1963) 28.

J. R. Burt, Process Biochern. 12 (1977) 32

F. S. Anderson and R. C. Murphy, J. Chromatogr. 121 (1976) 251.

J. H. Knox and J. Jurand, J. Chromatogr. 203 (1981) 85.

togr. 242(1982) 119.

[3]

[4]

[5]

[6] . 0. C. /ngebretsen,A. M. Bakken, L. Segadal, and M. Farstad, J.Chrorna-

MS received: February 28,1983

Micro-HPLC-IP Detection System in Separation of Non-UV Absorbing Organic Compounds

10392

Kiyokatsu Jinno* and Shoji Nakanishi School of Materials Science, Toyohashi University of Technology, Toyohashi 440, Japan

Key Words:

Micro-HPLC

Non-UV absorbing organic compounds Size exclusion chromatography

ICP-AES

There are no refractive-index type detectors for micro-HPLC like those used for conventional HPLC. One of the best ways of detecting substances having no UV absorption in the effluent of a column would be by inductively coupled plasma atomic emission spectrometry (ICP-AES, ICP). ICP-AES isapowerful high sensitivity and high selectivity metal-specific [ l -1 01 detection technique with great potential for liquid chromtatographic separations. We have reported that the micro-HPLC technique can be utilized with a simple interface for combination with ICP-AES using aqueous andlor organic mobile phases [ l l , 121. However, the reported results were restricted to the monitoring of metallic elements.

It is the purpose of the present communication to examine the use of the micro-HPLC-ICP system for the analysis of carbon-con- taining materials by monitoring the carbon emission line [6].

A Japan Jarrell-Ash (Tokyo, Japan) ICAP-500s was used for carbon detection. The experimental conditions are as follows; frequency: 27.1 2 MHz, plasma power: 2.2 kW, coolant Ar gas flow rate: 16 I/ min, plasma Ar gas flow rate: 1 Ilmin, sample Ar gas flow rate: 0.50 Ilmin, carrier water flow rate: 0.50 mllmin. The micro-HPLC pump was a microfeeder MF-2 (Azuma Electric, Co. Ltd., Tokyo, Japan).

21 0 Journal of High Resolution Chromatography & Chromatography Communications 0 1983 Dr. Alfred Huethig Publishers