BACKGROUND: Matrix effects in HPLC-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS)(3) can cause differences in the ionization of an internal standard (IS) compared with the analyte of interest. Unless sample cleanup or chromatographic conditions eliminate or minimize ion suppression or enhancement, variability in interpatient matrices may cause erroneous results. A stable isotope-labeled IS can be used to minimize analytical interpatient variation.METHODS: We used protein precipitation and HPLCESI-MS/MS to quantify sirolimus (SIR) with both desmethoxyrapamycin (DMR) and deuterium-labeled sirolimus (SIR-d(3)) as the IS to analyze a range of whole-blood and extraction-matrix samples, and to estimate recovery, matrix effects, process efficiency, and interpatient variation. We also analyzed a series of blood samples from 72 patients taking SIR, including externa proficiency-testing samples, with these ISs.RESULTS: The range of interpatient assay imprecision (CV) values for the SIR assay was consistently lower with SIR-d(3) (2.7%-5.7%) than with DMR (7.6%9.7%). The results obtained with the 2 different ISs for the patient samples showed a linear relationship, but the results were higher with DMR as the IS than with SIR-d(3)CONCLUSIONS: The use of SIR-d(3) as the IS in the high-throughput HPLC-ESI-MS/MS assay of SIR yielded improved results compared with the use of DMR. SIR-d(3) appears to be less affected by differences in the ionization of SIR and its IS caused by the variability of interpatient matrices. The IS-related difference in SIR estimation needs further investigation.