False-Positive Carbamazepine Results by Gas Chromatography Mass Spectrometry and VITROS 5600 Following a Massive Oxcarbazepine Ingestion

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1 False-Positive Results by Gas Chromatography Mass Spectrometry and VITRS 5600 Following a Massive xcarbazepine Ingestion Uttam Garg, 1 * Leo Johnson, 1 Amy Wiebold, 1 Angela Ferguson, 1 Clint Frazee, 1 and Stephen Thornton 2 CASE DESCRIPTI A 23-month-old patient with a seizure disorder presented to the emergency department after a suspected unintentional ingestion of his seizure medication. He had significant central nervous system depression and seizure-like movements. Although at the time of presentation it was known that the patient ingested his antiseizure medication, the name of the medication was not known. Also, it was not clear if the patient coingested any other drug. Broad-spectrum urine drug screening by immunoassay and GC-MS that includes presumptive screening of >200 drugs and toxins was ordered. Additionally, volatile screening for ethanol, methanol, acetone, and isopropanol was ordered. testing that is available on a stat basis on the Vitros 5600 Chemistry Analyzer was also requested. Urine drug screening by GC-MS (GC-7890, MS- 5975; Agilent) showed the presence of carbamazepine and oxcarbazepine. concentration measured by immunoassay on the Vitros 5600 Chemistry Analyzer (rtho-clinical Diagnostics) was 7.9 μg/ml (therapeutic range, 4 12 μg/ml). Unless there was a coingestion of another drug, the patient's symptoms of central nervous system depression and seizure-like movements were not consistent with the reported carbamazepine level. Because the presence of carbamazepine was reported on both GC-MS and immunoassay methods, it was thought that the patient ingested carbamazepine. wing to the presence of oxcarbazepine on the GC-MS, it was thought that the patient also ingested oxcarbazepine. As the history became clearer, it was noted that the patient was on oxcarbazepine and did not have access to carbamazepine. Further investigation suggested that the patient ingested a large amount of oxcarbazepine, estimated at 4.5 g. This raised the question of false-positive carbamazepine results on both the immunoassay and GC-MS. To answer this question, an HPLC method with UV detection was used. The HPLC method detects carbamazepine and its metabolite 10,11-epoxide carbamazepine and oxcarbazepine and its metabolite 10,11- dihydro-10-hydroxycarbamazepine (DiCBZ). and 10,11-epoxide carbamazepine were not detected by HPLC. However, 2 large peaks corresponding to oxcarbazepine and DiCBZ were present. Concentrations of oxcarbazepine and DiCBZ were 20.4 and 49.3 μg/ml, respectively. 1 Department of Pathology and Laboratory Medicine, and 2 Division of Toxicology, Children's Mercy Hospital, Kansas City, M. *Address correspondence to this author at: Department of Pathology and Laboratory Medicine, 2401 Gillham Road, Children's Mercy Hospital, Kansas City, M Fax ; ugarg@cmh.edu. DI: /jalm American Association for Clinical Chemistry July : JALM 1 Copyright 2018 by American Association for Clinical Chemistry.

2 Table 1. Measured carbamazepine concentrations in serum samples supplemented with various concentrations of oxcarbazepine and DiCBZ. xcarbazepine added, μg/ml measured, μg/ml DiCBZ added, μg/ml measured, μg/ml 12.5 < < < < < > <3.0 These findings suggested that oxcarbazepine and/or its metabolite(s) interfere with the Vitros 5600 carbamazepine immunoassay and may produce a false-positive GC-MS drug screen. To study the interference of oxcarbazepine and DiCBZ in the Vitros 5600 immunoassay, plasma samples with different concentrations (12.5, 25, 50, 100, and 200 μg/ml) of oxcarbazepine and DiCBZ were prepared and analyzed. values for the samples supplemented with oxcarbazepine were <3, 4.3, 8.9, 16.3, and >20 μg/ ml. Measured carbamazepine concentrations in all the samples supplemented with DiCBZ were <3 μg/ml (Table 1). When urine samples supplemented with 10 μg/ml oxcarbazepine and DiCBZ were analyzed on GC-MS, a peak matching the spectrum of carbamazepine was detected in the sample supplemented with DiCBZ. The sample supplemented with oxcarbazepine did not show the carbamazepine peak. DISCUSSI and oxcarbazepine are used as monotherapy and adjunctive therapy for the treatment of partial and generalized seizures. is metabolized to an active metabolite, 10,11-epoxide carbamazepine. Although the carbamazepine metabolite is active, it is generally not monitored unless altered metabolism or clearance is suspected. Immunoassays for the measurement of carbamazepine, available on most automated chemistry analyzers, are frequently used for therapeutic drug monitoring and suspected toxicity. The therapeutic range for carbamazepine is 4 12 μg/ml. It is important to note that various immunoassays detect 10,11-epoxide carbamazepine to a different degree, ranging from negligible to >90%. Therefore, different immunoassays may give clinical results that are significantly different. These differences are apparent in renal failure, in which accumulation of the metabolite is disproportionally high. Vitros immunoassay cross-reactivity ranges from 7.1% 11.2%, depending on the concentration of the metabolite. When needed, both carbamazepine and 10,11-epoxide carbamazepine can be accurately measured by chromatographic methods (1 3). xcarbazepine is a prodrug that is almost immediately and completely metabolized by hepatic cytosolic keto-reduction to pharmacogically active DiCBZ. Because metabolism of oxcarbazepine is very rapid and the parent drug is present in a very low concentration, only DiCBZ is measured for therapeutic drug monitoring. Immunoassays are not readily available for the measurement of DiCBZ. HPLC with UV or mass spectrometry are frequently used for therapeutic drug monitoring of DiCBZ (1, 3). The therapeutic range for DiCBZ is μg/ml. and oxcarbazepine are structurally similar, and their immediate metabolites, 10,11- epoxide carbamazepine and DiCBZ, metabolize to a common metabolite 10,11-dihydroxycarbamazepine (Fig. 1). wing to these similarities, it would seem that oxcarbazepine and its metabolites may 2 JALM :01 July 2018

3 xcarbazepine H 2 H 2 H Heat in GC-MS injection port 10,11-dihydro-10-hydroxycarbamazepine (DiCBZ) H 2 H H H 2 10,11-epoxide H 2 10,11-dihydroxycarbamazepine Fig. 1. Metabolism of oxcarbazepine and carbamazepine. Both are metabolized to a common metabolite. In the GC-MS injection port, at high temperature, DiCBZ is converted to carbamazepine. interfere with carbamazepine immunoassays. However, there are no prior reports in the medical literature of false-positive carbamazepine by immunoassay due to oxcarbazepine or its metabolite. ne in vitro study, however, showed very low cross-reactivity of oxcarbazepine and DiCBZ with 2 commercial carbamazepine immunoassays (4). It is likely because oxcarbazepine is quickly metabolized to DiCBZ, and DiCBZ does not cross-react with antibody used in the carbamazepine assay. This assertion is confirmed by our in vitro studies. xcarbazepine, but not DiCBZ, produced falsepositive results by immunoassay. Drug screening is frequently performed on patients, particularly in pediatrics, presenting in the emergency department with a suspected drug overdose and symptoms of unresponsiveness, seizures, or altered mental status. Immunoassays and chromatographic techniques are frequently used for drug screening. Most hospitals use immunoassays for screening only 5 10 drugs. Immunoassays can provide rapid results but are prone to interferences. For instance, many studies have reported interference of carbamazepine and oxcarbazepine in tricyclic antidepressant immunoassays (5). Chromatographic techniques such as gas chromatography or HPLC linked to mass spectrometry can simultaneously screen for hundreds of drugs and toxins (6, 7). In general, it is thought that chromatographic techniques, particularly when linked to mass spectrometry, are very specific and free of interferences. Though mostly true, interferences and/or artifacts that can cause false-positive results with mass spectrometry do happen and have been reported (8 10). Interferences in chromatographic methods are generally because of July : JALM 3

4 structural and mass spectral similarities. For example, optical isomers must be separated by use of special columns or chiral-derivatizing agents. Drugs that are structurally similar and produce similar high molecular weight ion fragments may not be distinguishable by library matching or selected ion monitoring (9). In gas chromatography, another unique phenomenon of conversion or degradation of one compound into another can happen at high temperature in the injection port. In the 1990s, for example, several laboratories reported the presence of methamphetamine in multiple negative urine specimens. Investigations showed that it was due to the presence of high levels of pseudoephedrine or ephedrine in these samples, and derivatization with certain agents such as 4-carbethoxyhexafluorobutyryl chloride, heptafluorobutyric anhydride, and -trifluoroacetyl-l-prolyl chloride produces a methamphetamine-like substance from pseudoephedrine or ephedrine at high injection port temperatures (10). In our case, we encountered a similar situation with the conversion of DiCBZ into carbamazepine (Fig. 1). This was demonstrated by the detection of carbamazepine by GC-MS in a patient's sample that had a high level of oxcarbazepine and DiCBZ, and absence of carbamazepine by HPLC. This was further confirmed by injection of oxcarbazepine and DiCBZ in GC-MS. DiCBZ, but not oxcarbazepine, was responsible for false-positive carbamazepine on GC-MS. n the contrary, in the immunoassay, oxcarbazepine, but not DiCBZ, produced false-positive results for carbamazepine. It is likely that immunoassays will not produce false-positive results in patients taking therapeutic doses of oxcarbazepine since oxcarbazepine is quickly converted to DiCBZ, and the concentration of TAKEAWAYS Though rare, mass spectrometry methods can be susceptible to false-positive results. Despite the use of 2 independent methods, false-positive results can happen. An accurate patient history can assist in determining if a result is a true result or a spurious result that requires extra investigation. Package inserts (instructions for use) do not contain an exhaustive list of interfering substances. The absence of a compound on such a list does not preclude it acting as an interferant. oxcarbazepine is very low. However, detection of carbamazepine by GC-MS for patients taking oxcarbazepine may be a common finding due to the higher concentration of DiCBZ. Similar to our findings, falsepositive carbamazepine signals due to breakdown of DiCBZ in the GC-MS injection port has been reported (8). In conclusion, we encountered a case of falsepositive carbamazepine due to an oxcarbazepine overdose. The false-positive carbamazepine result by immunoassay was due to oxcarbazepine, and the false-positive result by GC-MS was due to DiCBZ. Toxicology and clinical chemistry laboratories should be aware of this false-positive result and consider further testing when both carbamazepine and oxcarbazepine are simultaneously detected. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: o authors declared any potential conflicts of interest. 4 JALM :01 July 2018

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