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doumaZofCkmmato~~pky,183(1980)83-86 BiomedicaIAppiiccztions &vier Scientific Publishing Company. Amsterdam -Printed in The Netheriads

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HARRY MORRI§ON*, DAVID AVNIR and TKOMAS ZARRELLA

(Fiit received November ZOtb, 1979; revised manmript received February 6tb, 1980)

There has recently been a s~b&anM effort devoted to the chromatographic detection of urocanic acid (UA), a major metabolite of hi&dine, and a naturally 06 cur&g sunscreen EI-71. Of the several reported h&h-performance liquid chromatographic (HPLC) methods [S-11], none has described the resolution of UA into its E (tmns) and 2 (cis) isomers, though such isomeriza- tion is critical to the function of UA as a sunscreen 1121. We report here on two HPLC systems that resolve UA isomers, one an ion-paired, reversed-phase (for recent reviews, see re&. 13 and 14) procedure (method A) with a detection limit better than those reported for unresolved UA, and the other a “normal”- phase procedure (method B).

Ut-ocanic acid (UA 1

EXPERXMENTAL

HPLC was -performed using a composite system consisting of a Waters Model 6OOQA solvent delivery system, a Rheodyne 70-10 sample injection valve with a 2O+tl loop, a Laboratory Data Control UV monitor with 254~mn detector and a Hewlett-Packard 338OA electronic integrator. Method A employed a- 250 X 4.2 mm I.D. Waters yBon&p& Cl8 (IO pm particle size) column: the eluent was 24 mg of titmbuQ&umnotiunx sulfate, 3.36 g of NaI&PO,+ - E&O and 8.00 g of Na,Hpo, in 11 of distilled (from EcMno,) water_ The flow-rate of the pH 7.3- 7.4 solution was 2.0 ~&/min; Method B used a 250 X 4.2 mm I.D. Whatman

03784347/80/9000_0000~~02_2561980ELse~ierScientific PuMsbingCampany

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Pa&Ml (I1 pm particle size) column with 2% cont. (58%) ammonia in l-propa- nol at I_ ml/& as eluent,

E-UA was from AM&h (Milwaukee, WI, U.S_A_) and TR~S recrystallized from water to give crystals of the dehydrate. After wssg with acetone and vacuum drying at 45% for several hours, the zzelting point was 239°C (uncorr.) (Et. 218’C [IS], 228OC [IS] _ M-Iistidine - i-iCl was corn Sigma (St. Loti, MO; U.S.A.).

Z-UA was prepared by irradiating a slurry of 5-4 g of E-UA in 2 1 of Water brought to pK 9 with potassium hydroxide_ The slurry was photolyzed in a Rayonet Reactor (New England Ultraviolet Company) at 310 nm for 4 days at 30°C with occasional stirring. During this time the excess potassium salt of E-UA slowly went into solution_ The resulting UA mixture (E/Z = 3:7) is v&ually at the photostationary state 1121. ‘I’& solution was lyophilized, the residue dissolved in a little water, snd the pH adjusted to 9 with glacial acetic acid- This solution was &omatographed on a 23 X 3.5 cm column of BIo-Rad AC l-X8 (acetate), ZOHOO mesh, in 0.0125 M acetic acid [7]. El&on with 300 ml of 0.0125 M acetic acid was followed by 500 ml of 0.025 M and 1200 ml of 0.1 M acetic acid. Z-UA was ah&ad first and appeared after ea._ 1000 ml of ehxent. (In order to avoid 2 + E isomer&&ion by the slightly acidic eluent, the eluent +as trapped in receiving flasks cooled with methanol-dry ice.) The combined fractions were lyophihzed to yield a fluffy white solid which was washed with hexane and dried over phosphorus pentoxide at 65°C. The yield xas 2.4 g (45%), m.p. 178-180°C (uncorr.) (lit. l75-176°C [15] ); elemental anslysis confirmed that this Z-UA is r&f a hydrate (compare E-UA above).

A sample of sweat was obtained by diluting several drops of tmsion-pro- duced sweat from the epidermis on the back of the hand with water_

RESULTS AND DISCUSSION

The separation of an artificial mixture of Z- and E-UA using method A is illustrated in Fig. 1 (curve a). Retention times are E-UA 3.0 min, and Z-UA 4.7 min. Fig. 1 (curve b), a chromatogram of human sweat, illustrates tie sensitivity of the technique. We determined that using a 2O_ccl injector volume, the detection limit for E-UA is 1 ng and for Z-UA, 3 ng (3 X LO-‘&f and 1 X 10-6M,respectively)_

Our quantitative analyses were for solutions 6 X 10e4 M to 6 X 10m3 M in UA. The response of the W detector was found to be linear over most of this range (6 X 10 4 M to 1.5 X 10m3 M) for both the E and 2 isomers (ccmelation coefficients were 0.924 and 0.997, respe&ively). The calibration curves should be freshly de&xmined or checked, however, since they are quite susceptible to small changes in flow-rate, column deterioration, ee. At higher concen&ations there are large deviations from the Beer-Lambert Law within the detector, so that soMions of more than 2 X 10-j M are best d&&cd before analysis. Alter- natively, one may use known concentrations of E- or Z-UA which are within 5% of the unknown. The precision of multiple HPLC analy~ces was usually L-2%. Areas, rather thvl peak heights, should be used for guantitation be-

Fig_ 1. HPLC analysis of E- and Z-UA wing method A: (a) synthetic mixture; (b) sweat from the back of the band.

cause the shapes of the peaks are dependent on the solvent used to dissolve the I sample (for example, *l&O vs. I-&O).

Method B complements method A in that the elution sequence is reversed; the retention times are Z-UA 2.5 min, and E-UA 3.8 min. Method _A success- fully resolves histkline from UA at a flow-rate of 1 ml/min_ Retention times are hi&dine 3.3 min, ,FZ-UA 4.5 min, and Z-UA 8.9 min_ The limit of de’xxtion for histidme is 0.5 pg_ Reversed-phase coiumns are subject to deterioration when used with buffer salts; for appropriate precautions, see ref. 17. When the cohunn deteriorates partially, a soap chromatography [lS] technique usjng hexadecylkimethylammonium bromide in place of Mrabutylammonium sul- fate will give increased resolution: E-UA 2 mm, Z-UA 8 min, at 2 ml/min.

ACKNOWLEDGEMENTS

This investigation was supported by Public Health Service Research Grant No. IROICAI8267 from the National Cancer Institute.

REFERENCES

1 R.Schoen,Morzatschr. EGnderheilkd.,l24(1976)639 (C.A.,86(1977)102672p). 2 R. Schoen, Z.Klin.Chem_Klin. Biochem.,l4 (1976) 501(C.A.,86(1977)27381n). 3 J-L_ Dhondt, B. Csrtigny and JP. Fzriaux, Clh Chem. Acta, 50 (1974) 297. 4 AS. S&xi, hdii J. Med. Res.. 6L (Y-973) 1222. 5 J_N.A Van-BaIgaoy and E. Roberts, Biochem. Pluxmacol., 22 (1973) 1405.

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