Alternative Calibration Strategies for the Clinical Laboratory: Application to Nortriptyline Therapeutic Drug Monitoring

M.T. Olson, A. Breaud, R. Harlan,N. Emezienna, S. Schools, A.L. Yergey, and W. Clarke

June 2013

www.clinchem.org/content/59/6/920.full

© Copyright 2013 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry

Background

Use of LC-MS/MS is growing in the clinical laboratory

LC-MS/MS instrument is expensive, so a single instrument often must be used for multiple quantitative assays

Additional challenges include assay throughput and meeting turnaround time goals for some assays

© Copyright 2009 by the American Association for Clinical Chemistry

Background

A major driver behind LC-MS/MS assay cost and time is the generation of a calibration curve with every assay run

CLIA requires linearity proof biannually Practice of measuring calibration line with every

assay is unique to mass spectrometry Practice of measuring calibration line with every

assay originated when the instruments were less stable than they are now

© Copyright 2009 by the American Association for Clinical Chemistry

Background Mass spectrometry quantification works on the

“response ratio”; the peak area for the analyte divided by the peak area for the internal standard

In example below, analyte area is twice the internal standard. Internal standard concentration is known, so analyte concentration is twice the internal standard.

© Copyright 2009 by the American Association for Clinical Chemistry

Background

Instead of making a calibration curve, the authors used the response ratio

There is a correction for day to day differences, the “response factor” (RF) which is the response ratio of a 1:1 analyte:internal standard mixture

RF can be measured with each batch: contemporaneous RF or cRF

Alternatively, RF can be measured with QC failures or after instrument manipulation: sporadic RF or sRF

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

The equations

ƒ is the response factor (RF)This is a well behaved assay, there should not be an intercept (k=0).

Rearranging…

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Figure 1. Nortriptyline concentrations were measured for 68 Patients on 16 days over a 2 month period. As these specimens were obtained for routine clinical therapeutic drug monitoring, the standard clinical protocol was observed (shaded box). Both the cRF and sRF schemes were performed on each specimen.

Experimental Design

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 2. The dot-dash blue line representing the calibration curve with an internal standard concentration of 36ng/mL shows the most overlap with the solid unity line. The highest proportional and constant error is seen with 6ng/mL.

Results: Is the Assay Well Behaved? (i.e., is k=0? Is ƒ=1?)

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 3. The dot-dash blue line representing the calibration curve with an internal standard concentration of 36ng/mL shows the most overlap with the solid unity line. The highest proportional and constant error is seen with 6ng/mL.

Results: Does this work?

Graph A shows patient results at the four internal standard concentrations plotted against calibration curve results

Best agreement is seen with 36 ng/mL

Bland-Altman (Graph B) shows mean bias of 3.69% for cRF versus calibration curve with a range of -15.8 to 23.2%

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Results: Is there clinical impact?

Table 1. If the cRF numbers had been used, they would have resulted in the reporting of 3 concentrations in different clinical categories than were resulted using the calibration curve in the existing protocol. If the sRF numbers had been used, 4 results would have been in different clinical categories.

© Copyright 2009 by the American Association for Clinical Chemistry

Discussion Measurement of multiple analytes with a single

LC-MS/MS assay is commonplace

Done to minimize calibration and QC No clinical benefit in the context of prescription drugs

with no abuse potential

Multiplexing risks ion suppression and augmentation for coeluting analytes

cRF or sRF calibration can cut time spent calibrating instrument and may make single analyte assays more feasible

© Copyright 2009 by the American Association for Clinical Chemistry

Discussion

LC-MS/MS assays function best when analyte concentration is close to internal standard concentration (A/IS ~ 1)

For clinical values for this assay (mostly in 100-150 ng/ml range) the ideal internal standard concentration was 36 ng/mL

Thus, selection of internal standard for an assay should reflect expected analyte concentrations in the patients

© Copyright 2009 by the American Association for Clinical Chemistry

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