Zlatuse Clark (Presenter)
Bio: Currently an R&D Scientist at ARUP Laboratories. Ph.D. in Analytical Chemistry, Brigham Young University, Provo, UT. B.S. in Analytical Chemistry, Masaryk University, Brno, Czech Republic.
Authorship: Zlatuse D. Clark (1), Elizabeth L. Frank (2), Frederick G. Strathmann (2)
(1) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108; (2) Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112
13C-labeled analogs are generally considered superior internal standards (IS) to deuterated analogs. Recently, we replaced a commercially available d5-IS in our dilute-and-shoot based method for urinary 5-hydroxyindoleacetic acid (HIAA) with a custom-synthesized HIAA-13C6 IS. How much has this change improved the quality of the assay? While HIAA-d5 necessitated a quadratic curve fit, HIAA-13C6 facilitated a highly linear calibration response, increasing method robustness. Method comparison showed a very good agreement between the HIAA-d5 and HIAA-13C6 based methods. A post-column infusion study of the 1.7% specimens with concentration discrepancies >±20% revealed discrepancies in quantitation due to differential signal suppression. Signal suppression simulation by post-column infusion of increasing concentrations of caffeine showed that HIAA-13C6 allows for more consistent quantitation.
While deuterium labeled analogs are the most widely used type of stable isotope-labeled internal standards (IS) in LC-MS/MS, they do have some disadvantages. Due to differences in physicochemical properties, the interactions of the deuterated IS with the mobile and stationary phases differ from those of the non-labeled analyte, resulting in differences in retention times. The degree of the analyte-internal standard separation depends on the size of the molecule, the number of deuterium labels and their position on the molecule, and on chromatographic conditions. The larger the separation, the lower the deuterated analog’s ability to compensate for matrix effects. Also, some commercially available deuterated analogs may suffer from deuterium-hydrogen exchange during the analysis, which limits their utility as IS. It is generally accepted that 13C-labeled analogs make better ISs than deuterated ones. As the difference between a 13C atom and a 12C atom is minimal, 13C-labeled ISs co-elute with their respective analytes and thus better compensate for matrix effects. Additionally, they do not suffer from label instability.
Recently, we developed a dilute-and-shoot based method for urinary 5-hydroxyindoleacetic acid (HIAA) using a commercially available HIAA-d5 IS. Although great care was taken to optimize chromatography for HIAA to elute outside of major suppression zones, differential suppression between the analyte and its deuterated IS and consequent inaccurate quantitation remain a possibility, especially in a method without a sample cleanup. Also, the calibration curve displayed significant non-linearity which necessitated a quadratic fit. To resolve these issues, we decided to invest in a custom synthesized 13C-labeled IS.
To discuss pros and cons of 13C-labeled IS for the analysis of HIAA, a serotonin metabolite used to assess overproduction of the parent amine due to carcinoid tumor.
As expected, the HIAA-13C6 IS facilitated a highly linear calibration response. The increased linearity prompted the investigation of the extension of the analytical measurement range (AMR) from 0.5–100 mg/L to 0.5–200 mg/L with the intent to reduce the number of dilution re-analyses for specimens >100 mg/L. The extended calibration curve was highly linear: r2 > 0.999, all residuals < ±3%. However, the IS peak area in the 200 mg/L calibration standard was below or very close to the 50% of median IS peak area acceptance limit with the IS at 5 mg/L. To determine whether this problem could be remedied by increasing the IS concentration, the extended set of calibration standards and a set of controls were analyzed with HIAA-13C6 concentrations at 5, 10, 20, 40 and 80 mg/L. It was found that the IS concentration would have to be increased 8-fold in order to ensure that the 200 mg/L calibration standard IS peak area passes the acceptance criteria. As the analyte and the IS are measured in mg/L in this assay, the increase in cost of the 13C-labeled IS, from approximately $3,000/year at the 5 mg/L concentration to approximately $18,000/year at the 40 mg/L concentration, would be prohibitive. Hence the decision was made not to extend the AMR.
The HIAA-13C6 method was compared with the current HIAA-d5 method for the Quantitative Urinary HIAA by LC-MS/MS assay using 354 specimens. Deming regression equation, standard error, and correlation coefficient were: y = 1.015x–0.109; Sy/x = 0.993; R = 0.9980; %Bias = 0.18%.
The agreement between the HIAA-d5 based method and the new method was surprisingly good. The HIAA-13C6 based concentrations were within ±10% of the HIAA-d5 based values for 91.5% of the 354 analyzed specimens. Deviations > ±20% (21–36%) were found in only 6 specimens (1.7%). A post-column infusion experiment was performed to determine whether the larger deviations were due to differential signal suppression of the analyte vs the HIAA-d5 IS by sample matrix. Suppression zones affecting analyte and internal standard to different degrees were obvious in most of the samples with larger deviations. However, all 6 of these specimens had concentrations below the medical decision point, and hence clinical outcomes were not affected.
Signal suppression simulation
Signal suppression of the analyte and IS by matrix was simulated by a post-column infusion of caffeine at several different concentrations (0, 20, 100, 200, 500, 1000, and 2000 mg/L). Two sets of pooled specimens with HIAA concentrations of 0.8, 1.5, 5.5, 17, and 50 mg/L were prepared. One set was analyzed using the HIAA-d5 IS; the other using the HIAA-13C6 IS.
The experiment showed that while the signal of the analyte and of both internal standards was progressively more suppressed with increasing caffeine concentration as expected, analyte concentrations were less affected by suppression when using the HIAA-13C6 IS.
SUMMARY AND CONCLUSIONS
While a linear calibration curve response certainly contributes to method robustness, the method comparison data indicated that the quadratic curve performed satisfactorily. The laboratory did not have to drop a calibration point during the lifetime of the HIAA-d5 based assay, which is the main concern when using non-linear calibration curves. Placement of a Quality Control sample near the ULOQ allowed the laboratory to control the entire AMR despite non-linearity.
While the 13C-labeled IS eliminates differential suppression between analyte and IS, and thus increases measurement accuracy, our method comparison experiment showed that the HIAA-d5 based method actually performed quite well in terms of accuracy.
Signal suppression simulation
The signal suppression simulation experiment showed that the 13C-labeled IS allows for more consistent quantitation at increasing signal suppression. It could also be used to set wider IS peak area acceptance criteria and specifically tailor these to each assay, instead of using the more generally accepted 50–200% of IS peak area median criteria. Consequently, this could present significant savings to the laboratory, as fewer samples would fail the IS acceptance criteria and have to be re-analyzed.
References & Acknowledgements:
Dr. Frederick Strathmann
Dr. Elizabeth Frank
IsoSciences: Scott Landvatter Ph.D., Rich Tyburski
Analytic Biochemistry lab:
ARUP Institute for Clinical and Experimental Pathology®
IP Royalty: no
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