Letters to the Editor Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: C. Ding, equities in Sequenom. Honoraria: None declared. Research Funding: G.S.H. Yeo, Bench-toBedside grant (09/1/50/19/622) from BMRCNMRC; C. Ding, Bench-to-Bedside grant (09/ 1/50/19/622) from BMRC-NMRC. Expert Testimony: None declared. Patents: S. Jin, G.S.H. Yeo, and C. Ding have filed patent applications on fetal nucleic acids in maternal plasma for noninvasive prenatal diagnosis. Some of these patents have been licensed to Sequenom, Inc. Acknowledgments: We thank Michelle Lin and Li Ping Yap for assistance in sample collection.

References 1. Agarwal A, Sayres LC, Cho MK, Cook-Deegan R, Chandrasekharan S. Commercial landscape of noninvasive prenatal testing in the United States. Prenat Diagn 2013;33:521–31. 2. Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE, Neveux LM, Ehrich M, et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med 2011;13:913–20. 3. Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 2009;10:R25. 4. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A 2008;105: 16266 –71. 5. Chen EZ, Chiu RW, Sun H, Akolekar R, Chan KC, Leung TY, et al. Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing. PLoS One 2011;6:e21791.

Shengnan Jin2 Yen Ching Lim2 Desmond P.Y. Ng2 Hai Yang Law3 Kenneth Y.C. Kwek3 George S.H. Yeo3 Chunming Ding2* 2

Growth, Development and Metabolism Program Singapore Institute for Clinical Sciences Agency for Science, Technology and Research Singapore 3 KK Women’s and Children’s Hospital Singapore

Singapore Institute for Clinical Sciences Agency for Science, Technology and Research 30 Medical Drive, Singapore 117609 Fax ⫹65-6778-4193 E-mail [email protected]

Previously published online at DOI: 10.1373/clinchem.2013.216838

Response Factor–Based Quantification for Mycophenolic Acid To the Editor: In contrast with chemistry or immunoassay analyzers, quantification for liquid chromatographytandem mass spectrometry (LCMS/MS)1 assays is mostly done by time-consuming multipoint calibration in each run (CR) (1 ). Few reports on alternative quantification methods have been published, with most of them being impractical for clinical purposes (1–3 ) and none of them covering a large period of time (1– 4 ). We evaluated the performance of quantification based on an experimentally derived response factor (RF) used for 1 year without change (4 ) for mycophenolic acid (MPA) compared to CR. We also examined the variables influencing RF quantification performance. MPA was extracted by protein precipitation using a reagent containing deuterated internal standard (IS) and analyzed on an Alliance HPLC 2795 separations module coupled to a Quattro Micro tandem MS (both Waters Corp.). From June 2012 to May 2013, the first 10 patient samples, QCs of each run, and external QCs were quantified by CR and RF. CR

1

*Address correspondence to this author at: Growth, Development and Metabolism Program

692 Clinical Chemistry 60:4 (2014)

Nonstandard abbreviations: LC-MS/MS, liquid chromatography-tandem mass spectrometry; CR, quantification by calibration to each run; RF, response factor; MPA, mycophenolic acid; IS, internal standard; RR, response ratio.

was performed using a weighted (1/x) linear regression of 7 calibrators. RF quantification was performed by multiplying the observed response ratio (RR) (area analyte/area IS) by an RF, determined as the slope of a linear regression model, which plotted the theoretical concentration MPA as a function of the RR (data from the interassay variability experiment). Both methods were analytically validated, and no substantial difference between RF and CR quantification was observed. Of the 2170 patient samples quantified by CR and RF, 193 (8.9%) showed a difference ⱖ15%. The majority of the differences (171/193) occurred during the period of January 15, 2013, to February 6, 2013. In the first run of this period, a sudden and major decrease (⫺33.5%) of IS area with a stable MPA area (2.7%) was observed for QCs. This occurred together with the introduction of a new lot of IS solution, pointing to a bad preparation of the new lot (Fig. 1). As pipette control results were fine, this was probably due to human error. For the remaining 22 samples with a difference ⱖ15%, most had a concentration ⱕ0.4 mg/L. At these low concentrations, small absolute differences yield large relative differences. Deuterated MPA was used as IS at concentration near the middle of the reported therapeutic reference interval (1.0 –3.0 mg/L) (3 ). There was no effect of the distance from the optimal RR (1.0) on the difference between RF and CR quantification. For 85 of 2170 (3.9%) samples, a discordant clinical classification would have been made using the 1.0 –3.0 mg/L reference interval. Most often this was caused by minor absolute differences (ⱕ0.2 mg/L) (43/85), likely not causing substantial changes in clinical management. More striking differences were noticed in the period using the bad IS solution (31/85). External QC

Letters to the Editor

Fig. 1. Medium and low QC results for every assay run day for quantification by RF and CR.

results were commensurate with RF and CR. Routine maintenance procedures, the annual maintenance by the manufacturer, and reagent solvent, calibrator, or column switches did not substantially impact the performance of RF quantification. Even an unplanned intervention to replace a broken switch-valve and repair the injector showed no impact on RF performance.

As already reported for nortriptyline (3 ), the short-term CV for RF quantification of MPA was slightly lower than the CV obtained by CR (3.2% vs 4.5% at 1.0 mg/L MPA). This can be explained by the variability of calibrator pipetting and the intercept using CR (1 ). RF quantification seems to give a more robust estimate of the slope and eliminate intercept variation. The long-term CV (exclud-

ing runs with bad IS lot) was slightly higher for RF quantification (7.8% for RF vs 6.5% for CR at 1.0 mg/L MPA) and was caused by the variation between lots of IS (new lot every 10 –16 assay run days). However, other IS addition protocols can yield other results. Because our LC-MS/MS method for voriconazole requires preparation of the IS-containing precipitation reagent every assay run day (4 ), Clinical Chemistry 60:4 (2014) 693

Letters to the Editor the short-term CV was also substantially higher (11.6% with RF and 6.5% with CR at 2.8 mg/L) (4 ). Our report shows that bad IS lots can be detected by standard QC procedures. In case of unexplained QC violations, the RF should be reevaluated, preferably by running a calibration curve in duplicate or triplicate (1 ). Activity-based costing (5 ), allocating direct (technician, instrument, reagent) and indirect (supervision, sample handling, overhead, administrative) costs, estimated the potential benefit for applying this principle on 3 of our LC-MS/MS assays (MPA, voriconazole, lamotrigine) at $84 700 annually, depending on the actions performed during the gained time. In conclusion, RF quantification can replace CR as long as QC protocols are used to detect shifts in RF, which are mainly caused by variations in added IS. This strategy leads to substantial reductions in cost and workload.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant con-

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tributions 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 author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: S. Pauwels, the Fund for Scientific Research, Flanders (1700314N); P. Vermeersch, the Fund for Scientific Research, Flanders (1842013N). Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared.

References 1. Nilsson LB, Eklund G. Direct quantification in bioanalytical LC-MS/MS using internal calibration via analyte/stable isotope ratio. J Pharm Biomed Anal 2007;43:1094 –9. 2. Maier B, Vogeser M. Target analyte quantification by isotope dilution LC-MS/MS directly referring to internal standard concentrations: validation for serum cortisol measurement. Clin Chem Lab Med 2012;24:833–7. 3. Olson MT, Breaud A, Harlan R, Emezienna N, Schools S, Yergey AL, Clarke W. Alternative calibration strategies for the clinical laboratory: application to nortriptyline therapeutic drug

monitoring. Clin Chem 2013;59:920 –7. 4. Pauwels S, Vermeersch P, Van Eldere J, Desmet K. Fast and simple LC-MS/MS method for quantifying plasma voriconazole. Clin Chim Acta 2012;413:740 –3. 5. Kaplan RS, Porter ME. How to solve the cost crisis in health care. Harv Bus Rev 2011;89:46 – 52, 54, 56 – 61 passim.

Steven Pauwels2,3* Nele Peersman2 Mathieu Gerits2 Koen Desmet2,3 Pieter Vermeersch2,3 2

Clinical Department of Laboratory Medicine University Hospitals Leuven Leuven, Belgium 3 Department of Cardiovascular Sciences KU Leuven Leuven, Belgium

*Address correspondence to this author at: University Hospitals Leuven Laboratory Medicine Herestraat 49 3000 Leuven, Belgium Fax ⫹32-16-34-79-31 E-mail [email protected] Previously published online at DOI: 10.1373/clinchem.2013.216671