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Apparent positive interference from an etoposide metabolite, but not etoposide, in measuring urinary vanillylmandelic acid.

To the editor:

For 15 years, we have determined urinary vanillylmandelic acid (VMA) by a method [1] that relies on the reaction of extracted VMA with the diazo derivative of p-nitroaniline to give a pink chromophore; the chromophore undergoes a bathochromic shift in the presence of an aprotic solvent and a base to give a blue derivative with maximum absorbance at 600 nm. Hitherto, we observed neither positive nor negative interferences.

Recently, however, we observed the development of a pink color in the final stage of the procedure, masking the blue color of VMA (Fig. 1). The urine sample in question came from a pediatric patient with neuroblastoma, who was receiving etoposide and carboplatin chemotherapy.

Etoposide is a complex organic molecule with a dimethoxyphenol residue attached to the central naphthalene ring in a trans-configuration [2]. To test the possibility of a diazo reaction with etoposide, we added increasing amounts of etoposide to a control urine and measured the apparent VMA. No increase in VMA concentration was observed over a range of etoposide concentrations from 10 to 500 mg/L of urine. This nonreactivity could be either mechanistic or related to nonextractability of the etoposide. By processing etoposide through our procedure and then measuring the absorbance at 296 nm, we found that <0.1% of the etoposide reached the final measuring step. Furthermore, reaction of etoposide with the diazo derivative added in the final step produced a yellow color rather than the pink seen in the patient's sample (Fig. 1).


Etoposide undergoes extensive hepatic metabolism and excretion by the kidneys [3]. Allen et al. [4] isolated the major urinary metabolite of etoposide and identified it by mass spectrophotometry as 4-demethylepipodophyllic acid. The features of this compound make it a good candidate for reaction with a diazo derivative: an aromatic phenolic residue as possible site for reaction and a carboxyl group to carry the molecule through the extraction steps of our procedure. Other metabolites excreted in smaller amounts, i.e., picroetoposide and the aglycone, are for purely structural reasons not expected to react positively in the assay. The patient's urine gave a normal VMA spectrum 10 days after therapy with etoposide was stopped.

Several metabolites as well as xenobiotics interfere with many diazo methods used in the determination of VMA [5]. The reaction of a diazo derivative with carboplatin is highly unlikely, this simple organo-metallic compound being devoid of activated aromatic residue. Further work is under way to isolate and test the major metabolite.

We recommend that VMA analysis by use of any diazo derivative such as ours or some other [6] be interpreted with caution in patients receiving etoposide. Alternatively, methods with high resolving powers such as thin-layer chromatography [7] or HPLC [8] should be considered.


[1.] Habbal MZ. Simple, non-chromatographic, highly specific method for the determination of urinary vanilmandelic acid. US Patent 4,341,528, issued July 27, 1982.

[2.] Issell BF, Rudolph AR, Louie AC. Etoposide (VP-16-213): an overview. In: Issell BF, Muggia FM, Carter SK, eds. Etoposide (VP-16), current status and new developments. New York: Academic Press, 1984:6-8.

[3.] Craven PJ, Allen ML. PEPEG, a new antineoplastic epipodophyllotoxin. Clin Pharmacol Ther 1975;18:221-6.

[4.] Allen LM, Marcks C, Craven PJ. 4-Demethylepipodophyllic acid-9-(4,6-aethylidene-R-o-glucopyranoside), the major urinary metabolite of VP-16-213 in man. Proc Am Assoc Cancer Res 1976;17:6.

[5.] Habbal ZM. Catecholamines and derivatives. Chap. 26 in: Race GJ, ed. Laboratory medicine, Vol. 1, 11th ed. Philadelphia: JB Lippincott, 1985:1-44.

[6.] Gumboldt G. Colorimetric method for the rapid and quantitative determination of 4hydroxy-3-methoxymandelic acid (vanilmandelic acid) in human urine [Letter]. Clin Chem 1977;23: 1949-50.

[7.] Agbaba D, Stojanov M, Rajacic S, ZivanovStakic D, Majkic-Singh N. Densitometric determination of urinary 4-hydroxy-3-methoxymandelic acid (vanillylmandelic acid). Clin Chem 1993;39:2500-3.

[8.] Ong CN, Lee BL, Ong Hy, Jaecob E. Simple and rapid liquid-chromatographic method for the simultaneous measurement of vanillylmandelic acid and homovanillic acid [Tech Brief]. Clin Chem 1987;33:1262-3.

Zouheir M. Habbal (1) * Samar Muwakkitz

(1) Clin. Chem. Lab. Dept. of Lab. Med. (2) Dept. of Pediatr. School of Med. American Univ. of Beirut P.O. Box 113-6044 Beirut, Lebanon

* Address correspondence to this author at: American Univ. of Beirut Med. Center, 850 3rd Ave., 18th Floor, New York, NY 10022.
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Title Annotation:Letters
Author:Habbal, Zhouheir M.; Muwakkit, Samar
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Feb 1, 1997
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