MSACL 2016 EU Abstract

α-Methyldopa Interference in Urinary Normetanephrine Measurement by LC-MS/MS?

Jody van den Ouweland (Presenter)
Canisius-Wilhelmina Hospital, Netherlands

Authorship: J.M. van den Ouweland1, M.Mol2, L.Tax1, T. Beijers1, H. van Daal1
Departments of Clinical Chemistry1 and Internal Medicine2,

Short Abstract

α-Methyldopa is known for analytical interference in HPLC methods for metanephrine measurement, but at present it is unclear whether this also holds for LC-MS/MS. We present a case of analytical interference from α-methyldopa in our LC-MS/MS method for urinary normetanephrine analysis. A closely eluting interfering peak had been mistakenly integrated by the MS instrument software as being normetanephrine and was reported to the clinic, despite notification of an ion ratio failure. The interference could be eliminated by modification of column type and chromatographic conditions. Our finding emphasizes the importance of critical evaluation of each chromatogram and notification of ion ratio failure. Particularly, for compounds with unresolvable mass fragmentation, chromatography remains key to guarantee accurate patient results.

Long Abstract

In general, LC-MS/MS is thought to be more selective and less susceptible to analytical interferences when compared to HPLC. In urinary or plasma metanephrine analysis for diagnoses of pheochromocytoma, false-positive results could arise either from direct interference with the analytical method or pharmacological effects on the disposition of catecholamines (1).

Here, we present a case of analytical interference from α-methyldopa (Aldomet) in our LC-MS/MS method for urinary metanephrine analysis. A 36-year-old woman was referred to our hospital for diagnostic work-up of her hypertension. She had been treated successfully for hypertension during pregnancy with α-methyldopa. Post-partum, after initial normalization, her blood pressure increased despite the use of a-methyldopa. Her normetanephrine value of 19.2 umol/24h was greater than 6 times the upper reference limit (0.4-3.0 umol/24h) with metanephrine being only slightly elevated (1.5 umol/24h; reference value 0.1-1.1 umol/24h), as measured by UPLC-MS/MS (Quattro Premier XE Mass Spectrometer, Waters). Her physician reported that she was taking α-methyldopa, an indirect-acting alpha-anticholinergic receptor blocker, which is indicated treatment for gestational hypertension. α-Methyldopa is known for analytical interference in HPLC methods for metanephrine measurement, but at present it is unclear whether this also holds for LC-MS/MS. A closer inspection of the MS data made clear that a closely eluting peak had been mistakenly integrated by the MS instrument software (Quanlynx, Waters) as being normetanephrine.

Furthermore, the software normalizes the highest, in this case interfering peak within a specified Rt window to 100% resulting in a minor normetanephrine peak. In retrospect, two aspects should have arisen suspicion of assay interference. Firstly, the interfering peak displayed a slightly earlier retention time (Rt 1.30 min) when compared to the stable-isotopically labelled internal standard (d3-normetanephrine)(Rt 1.40 min), as well as to other patient samples in the series. Secondly, the software gave notification of a quantifier/qualifier ratio failure (m/z 166 > 106 (quantifier) / m/z 166 > 134 (qualifier) ratio was 1.60 (normal 0.80 (+/- 0.16)). Quantification of the true normetanephrine signal revealed a normal value of 0.7 umol/24h (ion ratio 0.83), thereby more or less excluding a diagnosis of pheochromocytoma. To confirm the use of α-methyldopa as being the cause of the interference, α-methyldopa was withheld for eight wks. and urinary metanephrines were re-analyzed. The interfering signal had disappeared, resulting in a normal normetanephrine value of 1.2 umol/24h. Urine normetanephrine analysis of a second patient on α-methyldopa showed the same interfering peak in our LC-MS/MS assay (result not shown).

α-Methyldopa (Mr 211) is known to become decarboxylated to α-methyldopamine (Mr 167) by aromatic-L-amino acid decarboxylase with subsequent oxidative hydroxylation by dopamine-β-hydroxylase to α-methylnorepinephrine (Mr 183). á-Methylnorepinephrine and normetanephrine are isomers, with the same molecular formula but different structures. In the positive mode, normetanephrine is protonated to produce the molecular ion m/z 184, with subsequent loss of water resulting in fragment m/z 166. Further loss of a methoxy group (-OCH3) in the collision cell gives the major daughter fragment m/z 134. Full scan spectra of á-methyldopa and α-methylnorepinephrine both revealed a strong m/z 166 signal, most likely to occur from in-source decarboxylation for α-methyldopa and loss of water for α-methylnorepinephrine, but collision-induced dissociation showed no marked m/z 134 or m/z 106 fragments. Subsequent normetanephrine analysis of urine samples spiked with various concentrations of either α-methyldopa or α-methylnorepinephrine (up to 0.5 mmol/L) did, however, not result in the interferent as was found in the patient taking α-methyldopa. Analytical interference from other α-methyldopa metabolites, e.g. α-methyldopamine, have not been tested and therefore cannot be ruled out.

Our LC-MS/MS method for urinary metanephrine analysis uses a HILIC analytical column and an acetonitrile/water gradient. Normetanephrine interferences were absent when urine samples were re-analysed by our plasma LC-MS/MS method, which uses a reverse-phase (T3) column and methanol/water gradient. So, analytical interference of α-methyldopa with LC-MS/MS measurement of normetanephrine appears to depend on the column type and/or chromatographic conditions. From now, we use the reverse-phase column for both urine and plasma metanephrine analysis. We believe that α-methyldopa should be added to the list of drugs that may interfere with (nor)metanephrine analysis in urine or plasma by LC-MS/MS and preferably be discontinued prior to testing (2-5). Our finding emphasizes the importance of critical evaluation of each chromatogram and notification of ion ratio failure. So, despite the alleged specificity, LC-MS/MS is not immune of analytical interferences leading to false-positive results in metanephrine measurement. Particularly, for compounds with unresolvable mass fragmentation, chromatography remains key to guarantee accurate patient results (3).


References & Acknowledgements:

1.Lenders JW, Duh QY, Eisenhofer G, Gimenez-Roqueplo AP, Grebe SK, Murad MH, Naruse M, Pacak K, Young WF Jr; Endocrine Society. Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2014;99:1915-42.

2.Wright MJ, Thomas RL, Stanford PE, Horvath AR. Multiple reaction monitoring with multistage fragmentation (MRM3) detection enhances selectivity for LC-MS/MS analysis of plasma free metanephrines. Clin Chem. 2015;61:505-13.

3. Peitzsch M, Adaway JE, Eisenhofer G. Interference from 3-O-methyldopa with ultra-high performance LC-MS/MS measurements of plasma metanephrines: chromatographic separation remains important. Clin Chem. 2015;61:993-6.

4. Petteys BJ, Graham KS, Parnás ML, Holt C, Frank EL. Performance characteristics of an LC-MS/MS method for the determination of plasma metanephrines. Clin Chim Acta 2012;413:1459-65.

5. Dunand M, Donzelli M, Rickli A, Hysek CM, Liechti ME, Grouzmann E. Analytical interference of 4-hydroxy-3-methoxymethamphetamine with the measurement of plasma free normetanephrine by ultra-high pressure liquid chromatography-tandem mass spectrometry. Clin Biochem. 2014;47:1121-3.


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