MSACL 2024 Abstract

Development and Validation of a TDM Assay for Nimodipine Quantitation in CSF and Plasma

Paula R. Hoyne, Ph.D. (1), ThanhThao Tran, MT (1), Jonathan Hoyne, Ph.D. (1)
(1) Mayo Clinic, Jacksonville, FL

Jonathan Hoyne, Ph.D. (Presenter) Mayo Clinic

Presenter Bio: I have been Director of Clinical Chemistry at Mayo Clinic in Florida since 2008. I am currently the Laboratory Director of Outpatient Chemistry where I oversee automated chemistry, automated immunoassay, ELISA assays, M-protein by SPEP and IFE and small molecule quantitation and TDM by LC-MS/MS. I am the Laboratory Director of the Immunochemical Core Laboratory in Rochester, MN where I oversee a core laboratory with automated chemistry and immunoassay instrumentation as well as research immunoassay by Luminex, Meso-Scale Diagnostics and manual ELISA. We also conduct studies using small molecule LC-MS/MS and Thermo Q-Exactive Plus technologies.

From 2008 - 2022 I oversaw all aspects of the Clinical Chemistry service at our institution including the hospital lab, chemistry point of care, respiratory and blood gases, general chemistry, automated immunoassay, immunoassay by ELISA and small molecule quantitation by LC-MS/MS.

Relevant Financial Disclosures (within past 24 months, reported on Feb 20, 2024) Stock/Bonds DHR stock Using Generic Terms? YES Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Aneurysmal subarachnoid hemorrhage (aSAH) is a debilitating threat to life and neurological function. Amongst patients that reach the hospital, delayed cerebral ischemia (DCI) is a frequent negative sequalae of aSAH. Standard preventative treatment of delayed cerebral ischemia involves oral administration of 60 mg Nimodipine every 4 hours. Nimodipine is an L-type calcium antagonist that acts as a vasodilator. Equilibrium concentrations of nimodipine in CSF and serum on standard dosing are highly variable. Generally, higher concentrations of CSF nimodipine within the therapeutic range improve outcome but the correlation isn’t linear or universal. A subset of patients fails to achieve quantifiable nimodipine concentrations in CSF. Other patients experience systemic hypotension with equilibrium concentrations consistent with those that in other patients decrease risk of DCI. Systemic hypotension is a contraindication for full nimodipine dose, but lower doses are associated with an increased risk of DCI. The nature of the interpatient variability of equilibrium nimodipine concentrations is ill-understood. Previous studies suggest sex, age, body surface area, metabolic heterogeneity, location, or magnitude of initial insult play roles but these have not been interrogated in combination. Currently nimodipine dose is standardized across patients without accounting for these sources of individual variation. We developed a high sensitivity nimodipine assay which will allow us to evaluate CSF and plasma concentrations of nimodipine. We will utilize this assay to investigate the within- and between- patient variability in the relationship between dose and equilibrium plasma and CSF concentrations.

METHODS:
We developed an LC-MSMS assay for nimodipine in CSF and Plasma. We spiked nimodipine into negative patient pools to create calibrators and control materials as well as mock patient samples. After mixing of the spiked samples, we aliquoted 250 mcL of manufactured CSF or plasma samples and extracted with 500 mcL of acetonitrile + interanal standard. Nitrendipine at 1.25 ng /mL. Incubate 5 mins at RT. Vortex for 30 seconds and centrifuged at 3000g for 10 mins. 500 mcL of each extract are transferred to a 96 microwell plate and 40 mcL of sample are injected into the LC-MS/MS.

Liquid chromatography was performed with an LX-2 LC system and an HPLC autosampler at a flow rate of 0.4 mL / min.
Mobile phase A: 20 mM NH4Acetate in 50/50 MeOH:H2O + 0.1% formic acid.
Mobile phase B: 20 mM Ammonium Acetate + 0.1% formic acid in 100% MeOH.
Time
0 60 80% A 20% B
60 180 10% A 90% B Ramp
180 210 10% A 90% B
210 270 80% A 20% B Ramp
270 300 80% A 20% B

Tandem Mass Spectrometry was performed using an ABSciEx API 4500. The mass spectrometer was operated in ESI positive mode.
Nimodipine was monitored at 419.1/343.2 for the quantifier ion and 419.1/301.0
Nitrendipine was monitored at 361.2/315.0 for the quantifier ion and 361.2/329.1
LOQ is 10 pg/mL in plasma and 2 ng/mL in plasma. Plasma LOQ represents the lowest likely level in patient samples rather than an analytical limit. AMR is 10 pg - 750 pg/mL in CSF and 2 ng – 75 ng/mL in plasma. The assay is linear within these values. Intraassay and interassay CV is well below 20% at levels consistent with patient values expected from the literature.

CONCLUSION:
Sensitivity of the assay in CSF was sufficient to expect quantifiable levels in patient samples for all patients based on previously published data. Performance of the assay in plasma was consistent with published methods. Other parameters of assay performance were well within analytical expectations for a TDM assay. We expect to be able to use this assay to interrogate the variability between plasma and CSF nimodipine concentrations within patients. We will combine TDM data with pharmacogenetic, demographic, and physiologic data to attempt to derive a model of nimodipine pharmacokinetics which will allow clinically useful estimation of CSF nimodipine concentration based on measured plasma nimodipine concentrations. Alternately, we will measure CSF nimodipine concentrations for clinical evaluation of therapeutic dose within patients.