5-S-cysteunyldopa differentiates patients with primary and metastatic mel- anoma from patients with dysplastic nevus syndrome and normal subjects. JAm. Acad.
creases rapidly after blood sampling, to >7.4, through loss of CO2 when specimens are exposed to ambient air; maximum pH ranges were >7.7(6). In normally collected human sera, pH values after freezing and lyophilization were respectively 8.18 and 9.05 (7). In practice, the pH of human serum samples is always higher than the pH of most control sera. As shown in Table 1, the largest Underestimations of urea were in the sera with the lowest pH. Ranking the pH values of control materials (24 samples used in the Belgian EAQ 1990-1992) vs relative recoveries calculated on the median peer group values for the Ektachem user group, compared with the recoveries obtained with the “wet” methods, confirms our experiments. Our observations indicate that the buffer capacity of the control sera exceeds that of the Kodak slides, thus preventing the reaction from occurring at the optimum conditions. The low pH effect in the control sera can be neutralized by reconstituting the control sera with 100 mmol/L sodium bicarbonate. We recommend the use of EQA control sera with a buffer capacity comparable with human serum or with pH >7.4 if these sera are intended to be used in an EQA scheme that includes the Kodak Ektachem urea method. References 1. Wisser H, Ratge D, Knoll E. Die quantitative Analytik mit der Ektachem-Mehrschichtflhntechnik. Lab Med 1988;12:30110. 2. Wisser H. Klunisch-chemische Analytik
mit der Mehrschichtfllmtechnik. Z Anal
Chem
Fresenius
1986;324:242-3. 3. KOlpmann WR, Maibaum P, Sonntag 0, Schumann G, Siekmann L. Analysis with the Kodak-Ektachem. Accuracy control using reference method values and the influence of protein concentration. Part II. Substrates. J Clin Chem Cliii Biochem 1990; 28:835-43. 4. Knoll E, Hafner F, Dettmer K, Wisser H. The determination of calcium, glucose, urea and uric acid using the Kodak Ektachem multilayer film technology: an evaluation. J Clin Chem Cliii Biochem 1982;20:491-8. 5. Malavasi B, Catapano A, Galli G, Franzini C. A collaborative trial for the evaluation of blood cholesterol measurements in clinical laboratories in Italy. Eur J Clin Chem Cliii Biochem 1992;30:157-61. 6. Libeer JC. External quality assessment in clinical laboratories; European perspectives today and tomorrow [PhD thesis]. Antwerp, Belgium: University of Antwerp, l993:630pp. 7. Rej R, Jenny RW, Bretaudiere J-P. Quality control in clinical chemistiy: characterization of reference materials. Talanta
1984;31:851-62.
Jean-Claude
Libeer’
Dept. of Clin. Pathol. Inst. of Hygiene and Epidemiol. J. Wytsmanstraat 14 B-i 050 Brussels, Belgium Walter
Cooreman
Lab, of Clin. Pathol. St. Augustinusziekenhuis Oosterveldlaan 24 B-2610 Wilrijk, Belgium Lieve
Theunis
Lab. of Gun. Pathol. Jan Palfijnziekenhuis Lange Bremstraat 70 B-2i 70 Merksem, Belgium
1Author
Improved
for correspondence.
HPLC DeterminatIon In Serum
of
5-S-Cystelnyldopa
To the Editor: In recent years, 5-S-cysteinyldopa (5-S-CD), a precursor of the reddishbrown pheomelanin (1), has drawn much
attention.
Urinary
excretion
of
5-S-CD has been studied extensively as a tumor marker for metastatic melanoma (2), and plasma (or serum) concentrations of 5-S-CD have been shown to reflect the progression of malignant melanoma (3). We previously measured serum concentrations and urinary excretion rates of 5-S-CD in healthy Japanese (4). We are now studying the clinical significance of 5-S-CD in malignant melanoma, and, to cope with the increasing need to analyze 5-S-CD in serum, we have improved our original method for HPLC determination of 5-S-CD (5). By washing alumina with a phosphate buffer, pH 4.0, before the elution of 5-S-CD with HCIO4, which removes not only compounds with longer retention times but also other impurities, we found it possible to shorten the time for each chromatographic run from 90 miii to 45 miii. Further improvement was achieved by replacing, as internal standard, 2-methyl-3-(3,4-dihydroxyphenyl)-DL-alanine (a-methyl-DOPA) with 5-S-cysteinyl-a-methyldopa (MeCD). For the HPLC analysis we used the following equipment (all from Jasco, Tokyo, Japan): a PU-980 inteffigent pump; an 851-AS inteffigent sampler; an 840-EC electrochemical detector with a potential of + 750 mV vs a Hg! Hg2C12 reference electrode; and a 4.6 mm (i.d.) x 150 mm Catecholpak C18 reversed-phase column, 7-am particle CLINICAL
size. The mobile phase contained 12 g of 85% phosphoric acid, 10 g of methanesulfonic acid, and 0.1 mmol of Na2EDTA per liter of water, the pH being adjusted to 3.10 with 5 mol/L NaOH. The column temperature was maintained at 35#{176}Cwith a Jasco 860-CO column oven; the flow rate was 0.7 mL/min. Ion-spray mass spectra were measured with a PE SCIEX API Ill Biomolecular Mass Analyzer (Perkin-Elmer, Ontario, Canada) by K. Chitani, Fujita Health University. Blood samples were drawn by venipuncture into plain evacuated tubes (heparun not added) and allowed to coagulate. The serum was separated by centrifugation and stored at - 30#{176}C until analysis, within 2 months. a-Methyl-L-DOPA, L-cysteine, and mushroom tyrosinase (2230 kU/g) were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol (HPLC grade), Na2EDTA, and all other chemicals (analytical grade) were obtained from Wako Pure Chemicals (Osaka, Japan). 5-S-CD was prepared as described earlier (5). Alumina was obtained from Merck (Darmstadt, Germany) and used after washing with HC1. Milhi-Q II system (Millipore, Bedford, MA) ultrapure water was used throughout this study. Stock solutions of 5-S-CD (5 pmol/L) and of the internal standard Me-CD (15 mol/L) were prepared in 0.1 mol/L HC1 containing 1 gIL each Na2S2O5 and Na2EDTA, and stored at -30#{176}Cfor as long as 4 months until use. Tenfold-diluted working standards (5-S-CD, 0.5 pmoI/L, and Me-CD, 1.5 moJJL) were prepared in the same solvent, divided into small portions (-100 jL), and stored at -30#{176}Cuntil use (within 3 weeks). Me-CD was prepared by adding 31 mg of mushroom tyrosinase to a solution of 0.5 mmol of a-methyl-L-DOPA and 1 mmol of L-cysteine in 50 mL of 0.05 mol/L sodium phosphate buffer, pH 6.8, and separated from the isomers by column chromatography on Dowex 50W-x2 resin. The purity of Me-CD was judged by HPLC, ‘H nuclear magnetic resonance (NMR), 13C NMR, and ion-spray mass spectrometry. We modified our original method for extracting 5-S-CD from serum (5) as follows. In a conical plastic 1.5-mL tube we placed 50 mg of acid-washed alumina and 200 pL of a solution of 10 g!L each of Na2S2O5 and Na2EDTA. Either 500 L of serum or 10 pL of a standard solution (0.5 jmol/L 5-S-CD in 0.1 mollL HC1) plus 500 L of 10 g’L Na5EDTA was added to the tube. The standard was prepared in duplicate. We then added 10 L of internal standard, 1.5 pmol/L Me-CD in 0.1 mob’L HC1 containing Na2S2O5 and Na2EDTA (1 CHEMISTRY,
Vol. 40, No. 3, 1994
495
g’L each), followed by 700 jiL of 1.5 mol/L Tris-HC1 buffer containing 20 g/L Na2EIYFA, pH 8.6, to adsoch 5-S-CD onto the alumina. The mixture was immediately shaken for 5 mm on an EM-36 microtube mixer (Taitec, Koshigaya, Japan). After centrifugation the aqueous layer was removed by aspiration,andthealumunawaswashedtwice with 1 mL of water. Then, 500 L of 0.1 moJJL potassium phosphate buffer, pH 4.0, was added to the tube, the mixture was shaken for 2 miii, the aqueous layer was removed by aspiration, and the alumina remaining was washed with 1 mL of water. 5-S-CD was then eluted with 100 iL of 0.4 mol/L HC1O4 by shaking for 2 mm. After centrifugation, 30 zL of supernate was injected into the chromatograph. 5-S-CD was quantified from peak-height ratios between 5-S-CD and Me-CD for sample and standard. The detection limit for 5-S-CD in serum was
