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Liquid chromatography--tandem mass spectrometry work flow for parallel quantification of methotrexate and other immunosuppressants.

To the Editor:

Methotrexate is a potentially toxic folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. After high doses (0.035-12 g/[m.sup.2]) are administered, methotrexate concentrations in the plasma or serum are carefully monitored so that the patient can be rescued, if necessary, with the proper dose of leucovorin, a folic acid analog that bypasses the important enzymes inhibited by methotrexate (1).

Recently, the manufacturer of the fluorescence polarization immunoassay we use for monitoring (TDX platform; Abbott Laboratories) announced its intention to move the assay to a different proprietary platform. In addition, the immunoassay is known to be nonspecific. For example, the assay strongly cross-reacts with diamino-[N.sup.10]-methylpteroic acid, a minor (<5%) endogenous methotrexate metabolite and the major (>98%) product of pharmacologic inactivation of methotrexate with car-boxypeptidase [G.sub.2] (2). As an alternative to reagent-dependent proprietary methods, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, with an improved specificity, can be used to measure methotrexate (2,3).

Many laboratories, including our own, have replaced immunoassays for other immunosuppressants, which can also suffer from interferences, with laboratory-developed tests that use LC-MS/ MS (4). In contrast to methotrexate, the number of samples processed for other immunosuppressants with the immunosuppressant assay is high enough to support the LC-MS/MS infrastructure in many laboratories. To avoid adding staff and instrumentation for a new methotrexate assay, we developed a method to quantify methotrexate and other immunosuppressants in the same assay.

Our current immunosuppressant work flow involves precipitating protein from whole blood with acetonitrile containing 100 mmol/L ZnS[O.sub.4] (from Thermo Fisher and Sigma-Aldrich, respectively) and LC-MS/MS analysis with a C18 column and a Quattro Micro mass spectrometer (Waters). Quantification is performed with cyclosporin D as an internal standard for cyclosporin A (Eton Biosciences) and ascomycin (Calbiochem) as an internal standard for sirolimus and tacrolimus (Eton Biosciences) (4). As a first step to determine whether we could simply add methotrexate, we identified the optimal multiple reaction monitoring transitions (455 [right arrow] 308 and 458 [right arrow] 311, [[M+H].sup.+] molecular ions) for methotrexate (MP Biochemicals) and its deuterated internal standard, methotrexate-[d.sup.3] (Toronto Research Chemicals), respectively. Unfortunately, methotrexate was not retained by the C18 solid phase. We tested other chemistries and determined that a Supelco C8 column (5 [micro]m, 2 cm X 2.1 mm; Sigma-Aldrich) could resolve methotrexate and the other immunosuppressants and that one chromatographic method worked for all of the analytes of interest [0-0.4 min: 50% to 100% methanol in 2 mmol/L ammonium acetate, 1 mL/L formic acid, and water (J.T. Baker); 0.4-1.0 min: 100% to 50% methanol; and 1.0-3.5 min: equilibration and needle wash]. Unfortunately, the ZnSO4 in the proteinprecipitation reagent, which is important for low imprecision and low limits of detection in the immunosuppressant assay, greatly increased the limit of detection for methotrexate. This effect may be due to inadequate chelation of the [Zn.sup.2+] cation, which is likely accomplished by heme in whole-blood lysates (5). After further experimentation, we determined that we could use our current precipitating reagent for the immunosuppressants and a 50-50 mixture (by volume) of methanol (Fisher Scientific) and acetonitrile as the precipitating reagent for methotrexate. With 2 independent sample-preparation reagents, we used a separate 6-point calibration curve for each sample type.

In the final assay for methotrexate, serum (100 /[micro]L) was added to 400 /[micro]L precipitation reagent. After vortex-mixing for 5 min and centrifugation for 10 min at 13 000g, we injected 20 / [micro]L of the supernatant into the LC-MS/MS system. For methotrexate, the assay was linear from 0.01 / mol/L to 10 [micro]mol/L. The intraassay CV (n = 20) was 1.6% and 3.5% at 0.77 jumol/L and 0.08 [micro]mol/L methotrexate (UTAK Laboratories), respectively, and 14% at 0.01 [micro]mol/L (in-house control material). The interassay CV (n = 21) with new lots of control materials was 3.0%, 4.1%, and 14% at 0.74, 0.07, and 0.01 [micro]mol/L, respectively, and 2.0%, 4.5%, and 3.9% at 14.4,0.59, and 0.10 [micro]mol/L (pooled patient samples). Fig. 1 shows a favorable comparison for methotrexate measured with the LC-MS/MS method relative to measurements made via the T[D.sub.X] immunoassay (n = 30). For the other immunosuppressants, the assay was linear from 15 [micro]g/L to 1500 [micro]g/L (cyclosporine) and from 1 [micro]g/L to 40 [micro]g/L (others). Intra- and interassay CVs were < 9.5% for each analyte.


Postextraction addition experiments (extracted water vs extracted whole blood or serum) to characterize ion suppression (n = 20) demonstrated < 10% mean ion suppression or enhancement for all analytes and internal standards except for cyclosporin D (46% suppression). To ensure that ion suppression did not compromise the accuracy or imprecision of an assay that uses an analog internal standard, we demonstrated good recovery of each analyte in the 20 samples (range, 84%-102%), found no correlation between ion suppression and recoveries for any molecule (P [greater than equal to] 0.25), and observed a substantial improvement in the consistency of cyclosporin A recovery across the 20 samples ([CV.sub.peak area] = 16%) when normalized by cyclosporin D ([CV.sub.response] = 6.6%). These results suggest that using analogs as internal standards to quantify immunosuppressants and other analytes can be appropriate in the clinical laboratory; however, given the ion suppression differences between analyte and internal standard for cyclosporin A, isotope-labeled internal standards may be of advantage.

In conclusion, we have validated a parallel work flow for daily therapeutic drug monitoring of methotrexate and other immunosuppressants via the use of a single liquid chromatography program. This approach can be adopted to extend the utility of existing mass spectrometric instrumentation.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 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; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: AN. Hoofnagle Clinical Chemistry, AACC.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: Waters.

Expert Testimony: None declared.


(1.) Widemann BC, Adamson PC. Understanding and managing methotrexate nephrotoxicity. Oncologist 2006;11:694-703.

(2.) Kumar VS, Law T, Kellogg M. Liquid chromatography-tandem mass spectrometry (LCMS-MS) method for monitoring methotrexate in the setting of carboxypeptidase-G2 therapy. Methods Mol Biol 2010;603:359-63.

(3.) Steinborner S, Henion J. Liquid-liquid extraction in the 96-well plate format with SRM LC/MS quantitative determination of methotrexate and its major metabolite in human plasma. Anal Chem 1999;71:2340-5.

(4.) Streit F, Armstrong VW, Oellerich M. Rapid liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of sirolimus, everolimus, tacrolimus, and cyclosporin A in whole blood. Clin Chem 2002;48: 955-8.

(5.) Hover CG, Kulkarni AP. A simple and efficient method for hemoglobin removal from mammalian tissue cytosol by zinc sulfate and its application to the study of lipoxygenase. Prostaglandins Leukot Essent Fatty Acids 2000;62:97-105.

Hari Nair [1]

Lisa Lawrence [1]

Andrew N. Hoofnagle [1], [2]*

[1] Departments of Laboratory Medicine and

[2] Medicine University of Washington Seattle, WA

* Address correspondence to this author at:

Department of Laboratory Medicine

University of Washington

Seattle, WA 98195-7110

Fax 206-598-6189


Previously published online at DOI: 10.1373/clinchem.2011.175067
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Title Annotation:Letters to the Editor
Author:Nair, Hari; Lawrence, Lisa; Hoofnagle, Andrew N.
Publication:Clinical Chemistry
Article Type:Report
Geographic Code:1USA
Date:May 1, 2012
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