MSACL 2024 Abstract

Mitotane Quantification Method in Plasma Using GC-MS

Chris Thompson, Alison Lightfoot, Loralie Langman, and Paul J. Jannetto
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester MN

Chris Thompson, B.S. Biochemistry (Presenter) Mayo Clinic

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction:
Mitotane, 2,4’-Dichlorodiphenyldichloroethane (2,4’-DDD), is a chemotherapy drug used to treat adrenal cortical carcinoma. Plasma concentrations of mitotane need to be monitored continuously in the treatment of this disease to maintain the narrow established therapeutic range of 14-20 mcg/mL. Additionally, one of the main metabolites of mitotane, 2,4’-Dichlorodiphenyldichloroethylene (2,4’-DDE), could be monitored as it has the potential to provide additional value for the treatment of patients. However, the clinical utility of this metabolite requires further investigation.

Methods:
Standard stocks of 2,4’-DDD (Cat#: 32098, Restek) and 2,4’-DDE (Cat#: 32099, Restek) were purchased in methanolic solution. Standards were prepared by spiking varying levels of stock standards into Na-Heparin pooled human plasma (CA# IPLANAH100ML, Innovative Research). 200 µL of standards, controls, blanks, and patient samples were added to a 2 mL 96-well plate (CA# 96-6009, Chromtech). Next, 400 µL of internal standard (CA#’s CLM-6999-1.2, CLM-4693-1.2, Cambridge Isotope Labs) in acetonitrile was added to each well. The plate was then placed on a vortex mixer for 1 minute and centrifuged at 3500 RPM for 10 minutes. After centrifugation, the resulting supernatant is transferred to a GC-MS vial, which is sealed with a crimp cap.

The extracted samples were then analyzed using a 1 µL injection by GC-MS (Agilent GC model 6890 and MS model 5975). A J&W Scientific DB-5MS, 15m, 0.25mm I.D. capillary column with a film thickness of 1 micron was installed and used for analysis in the GC. The GC and MS methods were built using Agilent Chemstation. The GC method parameters were set at 260 °C for the inlet, 280 °C for the Aux temperature with an oven ramp from 60 °C to 320 °C over the course of 9.5 minutes. For the MS an inert electron ionization source was installed in the MS. The carrier gas used for the study was helium. Agilent Chemstation was used to build and submit each batch. Agilent Mass Hunter was used as the quantification software.

Results:
The linearity for samples (N=5) spanning the AMR (0.25-40 mcg/mL) yielded a linear regression with an R2=0.998 and slope of 0.9578 and 0.9765 for both mitotane and DDE, respectively. The intraday precision of the control levels (0.5, 5, and 30 mcg/mL) averaged a CV of less than 10%, with an N=20. The carryover for mitotane is negligible, however the carryover for the metabolite is about 200% of the LLOQ area and drops off significantly after the first 2 blanks. Patient samples were run using this method and compared to an external laboratory which used a GC-FID for their analysis. This comparison yielded a slope of 1.023 and an R2 of 0.9820 using linear regression analysis.

Conclusion:
Through development and validation of a quantitative GC-MS method for mitotane and its metabolite, we demonstrate that this method can provide accurate and precise quantitative results for therapeutic monitoring of mitotane and its metabolite DDE in patient samples. A Hamilton STAR liquid handling instrument method is being developed to automate a portion of the extraction process for implementation into the clinical lab setting.