Purpose: Total ceramide (11 isomers, CER) and total sphingomyelin (17 isomers, SPM) are surrogate biomarkers in human blood for monitoring patients with Niemann-Pick diseases when they have enzyme replacement therapy (Figure 1). The two bioanalytical methods used for several clinical trials in the sponsor’s lab were transferred to Covance. Both methods were cross-validated at Covance and met precision and accuracy acceptance criteria using purified porcine CER or SPM as reference standards. However, bridging normal reference ranges from the same 100 human individual blood samples showed significant difference between the two labs for total CER and total SPM. Systematic troubleshooting with innovative approaches were conducted, and results showed that the different isomer patterns between porcine and human analytes caused the problem and modification of instrument collision energy to match isomer patterns of porcine CER and SPM in DBS standards between labs are necessary to obtain comparable human reference values between labs.
Methods: Dried blood spot (DBS) samples were punched into a 96-well plate and soaked in the solvent with IS and the supernatant was transferred to a clean plate for LC–MS/MS analysis. Sample analyses were performed on Sciex API 4000 with Aquity UPLC, by using isocratic mobile phase and a reverse phase HPLC column. MRM mode was used for CER (11 isomers and its IS for 12 transitions), and SPM (17 isomers and its IS for 18 transitions), respectively. Stock solutions for CER and SPM were shared by the two labs. Mass spectrometer’s parameters, including collision energy and decluttering potential, were followed the sponsor’s parameters first and adjusted for all isomer patterns in DBS standards until the patterns to match the sponsor’s lab.
Results: Precision and accuracy of both assays met the validation acceptance criteria when standards and QCs shared the same porcine reference standard for each assay (Tables 1 and 2) and After the completion of the majority of validation parameters, reference values from 100 healthy donors were evaluated and showed significant differences between the two labs when the same stock solutions and same instrument parameters were shared. For DBS CER, the sponsor’s lab result was 12.1 ± 3.43 µg/mL (Mean ± SD), while Covance’s original result was 17.3 ± 4.23 µg/mL (Mean ± SD), Figure 2A. For DBS SPM, the sponsor’s lab result was 1240 ± 161 µg/mL (Mean ± SD), while Covance’s original result was 2314 ± 237µg/mL (Mean ± SD),Figure 2C.
Our investigation showed that the different values between labs were not caused by any lab errors but the differences in isoform patterns between natural purified porcine CER or SPM as reference standard and endogenous human CER and SPM as analytes. For SPM, adjustment of collision energy was sufficient to match isoform patterns in DBS standards between labs, the comparable results were obtained (Figures 2B & 2D). The final SPM results became 1096 ± 109 µg/mL (Mean ± SD), which is similar to the sponsor’s results (Figure 3A). For CER, because the reference standard’s isoform pattern has significantly different in C18:H2O and C18:0 than endogenous CER from humans, adjustment of collision energy and decluttering potential to match sponsor standard’s isoform pattern was not enough. The same lot of pre-spotted human DBS cards with low endogenous CER was also required for standard curve preparation. The final CER result was 12.1 ± 3.40 µg/mL (Mean ± SD), which matched results from the sponsor’s lab (Figure 3B).
Conclusion: The results of total sphingolipids in human blood can be significantly different between two labs due to isoform pattern mismatch between animals (for standards) and humans (unknown). Adjusting instrument parameters could lead to isoform pattern similarity and comparable reference ranges between labs.
Allena J. Ji– Biomarkers and Clinical Bioanalyses-Boston, Sanofi, Framingham, MA, USA
Yi Zhu– Biomarkers and Clinical Bioanalyses-Boston, Sanofi, Framingham, MA, USA
Mona Hdeib– Biomarkers and Clinical Bioanalyses-Boston, Sanofi, Framingham, MA, USA
Troy Voelker– Covance-SLC,UT
Scott Reuschel– Covance-SLC, UT