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Letters to the Editor |
Department of Internal Medicine IV, Clinical Chemistry Unit, University Hospital, University of Tuebingen, Tuebingen, Germany
aAddress correspondence to this author at: Department of Internal Medicine IV, Clinical Chemistry Unit, University Hospital, University of Tuebingen, Hoppe-Seyler-Str. 3, D-72076-Tuebingen, Germany. Fax49-07071-29-4696; e-mail Erwin.Schleicher{at}med.uni-tuebingen.de.
To the Editor:
Catecholamines and their metabolites are measured for the diagnosis of pheochromocytoma(1)(2) and neuroblastoma(3) and for various other reasons(4)(5)(6). The widely used HPLC assay, which uses electrochemical detection (ECD), combines high selectivity and sensitivity(7). The internal standard, 3,4-dihydroxybenzylamine (DHBA), is added before analysis. After pretreatment with a sample clean-up column, catecholamines are separated and quantified by HPLC-ECD. Commercial methods (Chromsystems) elute norepinephrine, epinephrine, DHBA, and dopamine at 8, 9, 12–13, and 17–19 min, respectively. The next sample is injected after 25 min.
We sometimes observed an unknown peak in the plasma catecholamine chromatogram that interfered with the internal standard DHBA (Fig. 1A
). When we repeated the analysis, the spurious extra peak disappeared (Fig. 1B
). When we evaluated the medical histories of these patients, we found that for a preceding analysis they had received dipyrone orally or intravenously within 12 h before blood collection. Therefore, we hypothesized that administration of dipyrone and/or its metabolites may have led to the interference with DHBA in the subsequent analysis.
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Dipyrone (metamizole) is widely used and has effective analgesic, antipyretic, and antispasmodic properties. After oral or intravenous administration, dipyrone is rapidly hydrolyzed to the active moiety 4-methylaminoantipyrine (MAA)(8)(9). MAA is further metabolized to 4-formylaminoantipyrine (FAA) and 4-aminoantipyrine (AA), which is acetylated to 4-acetylaminoantipyrine (AAA). These 4 major metabolites account for 
60% of the administered dose excreted in urine.
To determine whether dipyrone and/or its metabolites interfere with the measurement of plasma catecholamines, we prepared solutions of dipyrone (1 mg/mL) and MAA, AA, FAA, and AAA (50 µg/mL) corresponding to 5 times the expected maximum concentration obtained after a 1-g dose of dipyrone(9). Dipyrone caused a peak at the expected retention time of dopamine (Fig. 1C
) and another broad peak in the next chromatogram at the retention time of DHBA when an aqueous injection was started after 25 min (not shown). The bioactive metabolite MAA also had a retention time of 13 min in the next analysis, and thus it interfered with the internal standard (Fig. 1D
). These findings indicate that the interfering peak is MAA, which is readily formed from the chemically labile drug dipyrone(9). AA eluted after 23 min, and no signal was observed for FAA or AAA.
To evaluate whether this interference is relevant in vivo, we studied 6 patients who routinely received 1 g of dipyrone intravenously for analgesia after surgery. Informed written consent was obtained from all participants, and the local ethics committee approved the protocol. Blood samples were obtained before drug administration and 1, 2, 4, 6, 8, 10, and 12 h after dipyrone administration. Urine was collected during the periods 0–4, 4–8, 8–12, and 12–24 h after drug administration. HPLC chromatograms of plasma catecholamines showed an extra peak after 13 min in the subsequent analysis—consistent with the elution time of the metabolite MAA—up to 7 h after dipyrone administration. No interference was detected before or 12 h after administration of dipyrone, in accordance with the plasma half-life of 3 h for MAA and 5 h for AA(8). Furthermore, we observed no interference of dipyrone with dopamine at any time point.
Urinary catecholamine measurements were not affected by dipyrone administration. To investigate this unexpected finding, we treated MAA and AA solutions according to the protocol for urine samples, including column pretreatment. HPLC-ECD analysis showed no signal in the chromatogram, but a marked disturbance was seen when the pretreatment was omitted, indicating that pretreatment eliminates the interference caused by MAA and AA. This unexpected observation may be attributable to the different binding methods: for plasma samples the relative nonspecific adsorption of catecholamines on Al2O3 is used, whereas urinary catecholamines are bound to immobilized phenylboronic acid by formation of cyclic esters. This specific interaction is possible only for cis-diol compounds and cannot occur with dipyrone or its metabolites. A previous study of pretreatment procedures revealed that the Al2O3 method is superior for plasma samples because they are less diluted (specificity was not examined)(10). Interference with HPLC-ECD-based catecholamine analysis by paracetamol(11) and labetalol(12), but not by dipyrone, has been reported.
In conclusion, we found that (a) the metabolite MAA may interfere with the next analysis if the runtime is shorter than 40 min; (b) MAA and AA do not interfere with urinary catecholamine analysis if phenylboronic acid pretreatment is used; and (c) dipyrone may interfere with plasma dopamine determination if dipyrone is administered intravenously.
Acknowledgments
The authors thank Sanofi-Aventis for providing the dipyrone metabolites, Ms. Steffi Hasanovic for excellent technical assistance, and Drs. R. Lehmann (Clinical Chemistry Unit, University of Tuebingen, Germany) and K. Gempel (Institute for Clinical Chemistry, Schwabing–City Hospital, Munich, Germany) for critical discussion. Meanwhile, Chromsystems has referred the described disturbance for plasma catecholamine analysis.
Footnotes
1 The authors contributed equally to this work. 
References
The following articles in journals at HighWire Press have cited this article:
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  A. Pfafflin, E. Schleicher, and K. Mussig 3-O-Methyldopamine (3-O-Methoxytyramine) Interferes with the Internal Standard 3,4-Dihydroxybenzylamine in a Plasma Catecholamine HPLC Method Clin. Chem., November 1, 2007; 53(11): 1976 - 1977. [Full Text] [PDF]
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