As the use of liquid chromatography-mass spectrometry (LC-MS) has increased, clinical laboratories have sought to improve efficiency by employing alternative calibration strategies to reduce the number of calibration standards. While CLIA guidelines require biannual calibration at a minimum, no regulation requires that labs generate a calibration curve with every assay. This is in stark contrast to what has become common practice in clinical laboratories.

Generating a calibration curve for every batch of runs can be a significant expense, due in large part to the high cost of stable isotope-labeled internal standards (IS). One group estimated that abiding by a conventional calibration scheme at six concentration levels results in more than 5,000 calibration points per year (1). Indeed, it has been proposed that clinical laboratories employing LC-MS assays use more calibration standards than necessary (2), and that conventional calibration methods are wasteful because they do not capitalize on the rich information contained within recent calibrations (3).

Labs have recently developed several approaches to reduce the cost and burden associated with preparing several calibration standards with every batch of samples—without sacrificing analytical quality. These efforts have found that reduced calibration standard strategies not only conserve time and cost but also are more robust than conventional multi-point calibration schemes (4, 5). In fact, redundant multi-point calibration can actually result in excessive quantitative bias or the unnecessary failure of analytical runs using conventional methods (1), due in part to process changes such as the degradation of IS stock solution concentration and/or poor IS preparation (6).

One of the first alternative calibration strategies in clinical LC-MS used a single-point (linear through zero) calibration for quantifying the immunosuppressant tacrolimus (7). A subsequent study by the same group expanded the repertoire of abbreviated calibration curves and evaluated the performance of these curves in quantifying sirolimus (8). The results of this study demonstrated that single-point, linear through zero, and two-point alternative calibration schemes yielded acceptable inaccuracy (-6.7%-1.2%) and imprecision (3.7%-8.2%).

Internal Calibration

In 2007, the concept of internal calibration was introduced as a means of direct quantification in bioanalytical LC-MS methods. In this approach, labs calculate the results directly from an analyte/internal standard area ratio and a predetermined response factor (9).

The validated data indicate linearity and good precision and accuracy over the analytical measurement range. Four conditions must be met for this strategy to be successful: 1) the relative response should not be concentration dependent; 2) the relative response should be constant between batches/days; 3) the level of analyte in the IS should not be detectable; and 4) there should be no influence from naturally occurring isotopes of the analyte on the IS peak area.

Response Ratio and Response Factor Approaches

Researchers have also expanded on the concept of response ratio (RR)-based calibration by using the measurement of the RR corrected by the response factor (RF). The authors of one study compared contemporaneous RF (cRF) and sporadic RF (sRF) measurements with clinical results obtained by interpolation on a calibration curve (10). cRF and sRF calibration in a clinically validated LC-MS assay for therapeutic drug monitoring yielded results analytically and clinically comparable to those produced by interpolation with a calibration curve.

Another variation of alternative LC-MS assay calibration compares provisional RF (pRF) to a historical RF (hRF) in order to calculate a current RF (referred to as contemporaneous RF in the study conducted by Olson et al.) that relies on a weighting factor to stabilize the CRF against random fluctuations (1). This strategy is amenable to clinical settings that use the same analytes and method routinely over a prolonged period. Indeed, CLSI document C43-A2 indicates that historical calibration curves can be used if they are shown to be linear over time (11).

More Alternatives

Other published strategies employ a different tack. A particularly notable one used a single-point calibration with a calibrator close to the center of the full calibration range as a feasible alternative to full calibration (4). In this study, the authors compared the bias and precision from multiple-point and single-point calibration in six validated multi-analyte assays for quantifying drugs in human plasma. Of particular merit, this study included the retrospective analysis of assays encompassing several variables, such as various sample preparation strategies, acidic and basic analytes, and assays in which stable isotopically labeled analogs were used as IS for the majority of analytes, some analytes, or no analytes at all.

Another alternative calibration strategy that does not use stable isotope-labeled IS is termed threshold accurate calibration (TAC). In this approach, labs spike analytes of interest into the sample as IS to achieve a 100% of cut-off or threshold concentration as added standards. A TAC ratio is calculated for each analyte using the following equation: (peak area of analyte in neat sample)/(peak area of analyte in the spiked sample – peak area of analyte in neat sample). The TAC ratio is then calibrated by analyzing a specimen containing the threshold concentration of each analyte. Although this approach requires that each sample be injected twice, the method is adaptable to accurate, high-volume screening, and it normalizes matrix effects.

Deciding on the Best Strategy

Although research has shown that the analytical performance of the aforementioned alternative LC-MS calibration strategies is at least equivalent to that of conventional calibration methods, clinical laboratories should evaluate several considerations prior to deciding which strategy to adopt. The best strategy is fit-for-purpose and takes in to account factors such as a laboratory’s sample volume, the number of different assays in a laboratory’s testing menu, and the availability of dedicated LC-MS platforms.

In the current healthcare environment, laboratories would do well to improve their efficiency in ways that translate to cost-savings within the scope of laboratory operations. While alternative LC-MS calibration is one such approach, targeted cost-benefit analyses have not been conducted. Such analyses that demonstrate clear cost-savings would provide the final piece of evidence to demonstrate that alternative LC-MS calibration strategies are not simply alternative truths: They are valid, comprehensively vetted truths for clinical laboratories.

William Clarke, PhD, is an associate professor of pathology at The Johns Hopkins University School of Medicine and director of clinical toxicology at The Johns Hopkins Hospital in Baltimore. +Email: [email protected]

Stefani N. Thomas, PhD, is a research associate at The Johns Hopkins University School of Medicine in Baltimore. +Email: [email protected]


  1. Rule GS, Rockwood AL. Improving quantitative precision and throughput by reducing calibrator use in liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2016;919:55-61.
  2. Grant RP. The march of the masses. Clin Chem 2013;59:871-3.
  3. Rule GS, Rockwood AL. Alternative for reducing calibration standard use in mass spectrometry. Clin Chem 2015;61:431-3.
  4. Peters FT, Maurer HH. Systematic comparison of bias and precision data obtained with multiple-point and one-point calibration in six validated multianalyte assays for quantification of drugs in human plasma. Anal Chem 2007;79:4967-76.
  5. Tan A, Awaiye K, Jose B, et al. Comparison of different linear calibration approaches for LC-MS bioanalysis. J Chromatogr B Analyt Technol Biomed Life Sci 2012;911:192-202.
  6. Pauwels S, Peersman N, Gerits M, et al. Response factor-based quantification for mycophenolic acid. Clin Chem 2014;60:692-4.
  7. Taylor PJ, Hogan NS, Lynch SV, et al. Improved therapeutic drug monitoring of tacrolimus (FK506) by tandem mass spectrometry. Clin Chem 1997;43:2189-90.
  8. Taylor PJ, Forrest KK, Salm P, et al. Single-point calibration for sirolimus quantification. Ther Drug Monit 2001;23:726-7.
  9. 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.
  10. Olson MT, Breaud A, Harlan R, et al. Alternative calibration strategies for the clinical laboratory: Application to nortriptyline therapeutic drug monitoring. Clin Chem 2013;59:920-7.
  11. Clinical & Laboratory Standards Institute. Gas chromatography/mass spectrometry confirmation of drugs; Approved guideline C43-A2. Wayne (PA): CLSI 2010.
  12. Rosano TG, Ohouo PY, LeQue JJ, et al. Definitive drug and metabolite screening in urine by UPLC-MS-MS using a novel calibration technique. J Anal Toxicol 2016;40:628-38.