Jourr~rrlof‘Clzron~uiogrupl~_~. 577 Bionwiiwl

(I 992)

77-85

Applic~c~fiom

Elsevier Science PublishersB.V., Amsterdam

CHROMBIO.

63 17

Determination of tannic acid and its phenolic me-cabolites in biological fluids by high-performance liquid chromatography

(First received

December

10th. 1991: revised manuscript

received

February

7th. 1992)

ABSTRACT A method

for the identification

liquid chromatography

and ellagic acid, were successfully and 4-0-methylgallic

abomasal

extracted

fluid after abomasal

into which it was administered. phenolic

of tannic acid and its phenolic

Tannic acid and four phenolic compounds. from the biological

acid were found in the sheep urine

ellagic acid in abomasal plasma.

and determination

was developed.

The concentrations

in bioligical

gallic acid, 4-0-methylgallic

of tannic

address:

Nattonal

037%4347’92/$05.00

This method could be applied to measurement

apart

4-0-methylgallic

acid

and gallic acid and

from the abomasal

fluid

acid and ellagic acid in

of other hydrolysable

tannins

and their

materials.

Research

Centre

for Environ-

The University of Queensland, 39 KesPlains, Queensland 4108. Australia.

c

4-0-methylgallic

Gallic acid, pyrogallol

acid and ellagic acid in plasma,

acid, gallic acid, pyrogallol.

Tannins found in plants are polyphenolic polymers with molecular masses ranging from 500 to 3000. Tannins are generally divided into hydrolysable and condensed tannins. They are widely used in the brewery industry and leather tanning

mental Toxicology, sels Road. Coopers

fluids by high-performance

fluids by using ethyl acetate at acidic conditions.

INTRODUCTION

Present

in biological

dosing of tannic acid. Tannic acid was found in the plasma

fluid and urine were measured.

metabolites

mctabolites

namely gallic acid, pyrogallol,

1992 Elsevier Science Publishers

industry because of their protein-precipitating property [ 1,2]. However, high contents of hydrolysable tannins in certain species of plants. such as oak (Quercu.s spp.) [3] and yellow-wood (Tcrminalia ohlongata), can cause poisoning in animals if large amounts of these plants are ingested [4,5]. In order to study the pathogenesis of hydrolysable tannin toxicity, a reliable method for determining tannic acid (TA) and its phenolic metabolites in biological materials needs to be developed.

B.V. All rights reserved

78

Various methods for determining TA and phenols in biological materials have been reported using spectrophotometry [6,7], paper chromatography [8,9] and thin-layer chromatography (TLC) [lo], but these methods either are non-specific or lack sensitivity. Gas chromatography (GC) offers high sensitivity for a wide range of phenolic compounds; however, it generally requires a derivatisation step before detection [I I .12]. Although the measurement of TA using high-performance liquid chromatography (HPLC) has previously been reported [2,13,14], TA was measured in standard solutions instead of biological materials. Due to the complex matrices of biological fluids, especially urine, it is necessary to separate TA and other phenolic compounds from the interfering substances before injection into the HPLC column. Methods for the determination of TA and its phenolic metabolites in plasma. abomasal fluid and urine obtained from a sheep given a sublethal dose of TA have been developed and are reported. EXPERIMENTAL

All chemicals are of analytical grade unless specified. TA (L.R. grade) was purchased from Ajax Chemicals (Sydney, Australia), gallic acid (GA), pyrogallol (PYR), phenol, catechol (CAT), resorcinol (RES) and ellagic acid (EA) from Sigma (St. Louis, MO, USA), 4-O-methylgallic acid (40MGA) from Spectrum Chemical (Gardena. CA. USA) and protocatechuic acid (PA) from Aldrich (Milwaukee, WI, USA). Benzoic acid (BA), 4-hydroxybenzoic acid (4HBA), phosphoric acid and ethyl acetate were obtained from British Drug House (BDH Australia, Kilsyth, Australia). Methanol (HPLC grade) was obtained from Mallinckrodt (Malhnckrodt Australia. a Division of Rhone-Poulenc, Clayton, Australia). Type I pure water (18 M&m) was obtained by passing reverse-osmosis water through a Millipore Q water purification system (Millipore, Milford, MA, USA). All stock solutions were prepared in pure methanol.

J. ZHlJ e/ rd.

c’ollw t ion qf sanzpl~~s

Blood samples were collected into heparinized blood containers (Johns Professional Products, Cheltenham, Australia) from the external jugular vein of the sheep. Plasma samples were immediately obtained by centrifugation of the blood at 1800 g for 10 min. Abomasal fluid was obtained via an abomasal fistula which was surgically inserted at least two weeks before dosing [ 151, while urine was obtained via a urethral catheter as previously described by Zhu and Filippich [16]. All samples were stored at -20°C and before being analysed defrosted at room temperature, mixed and centrifuged at 1800 g for 10 min. All samples were assayed in duplicate. E.x-traction qf’ TA, GA, PYR and 40MGA ,fiorn plu.wza To determine the optimal recovery of TA. GA. PYR and 40MGA from plasma, addition of different amounts of sulphuric acid with or without hydrolysis were evaluated (see Table 1). The sheep plasma used in this recovery study was obtained from the blood collected before TA was dosed to the animal. For TA extraction, 1 ml of plasma was spiked with 20 ~1 of TA (9.384 g/l) and 20 ,nl of PA (1.074 g/l) (internal standard), and an equal volume of 1 M. 5 A4 or concentrated sulphuric acid was added in Kimax glass culture tubes (125 mm x 16 mm) with PTFE-coated screw caps (Kimble. Vineland, NY, USA). Each tube was thoroughly mixed on a vortex mixer (Scientific Industries. Springfield, MA. USA) before extraction with ethyl acetate. In another set of tubes, 1 ml of plasma was spiked with PA and TA, and, after the addition of sulphuric acid as above, the mixture was hydrolysed in a boiling water bath for 20 min and then cooled with running tap water. Both sets of spiked samples were extracted with two 4-ml portions of ethyl acetate by mixing on a rotator mixer (Analite. Melbourne, Australia) for 30 min. After centrifugation at 1800