Revival of physostigmine – a novel HPLC assay for simultaneous determination of physostigmine and its metabolite eseroline designed for a pharmacokinetic study of septic patients
-
Nadine Pinder
,
Johannes B. Zimmermann
Abstract
Background: Physostigmine, commonly used as an antidote in anticholinergic poisoning, is reported to have additional pharmacological effects, such as activation of the cholinergic anti-inflammatory pathway in sepsis models. Due to the narrow therapeutic range of physostigmine and its metabolite eseroline, however, the plasma concentrations of these substances need to be determined so as to understand their effect and ensure safety in the treatment of septic patients.
Methods: To determine physostigmine and its metabolite eseroline, a rapid and sensitive high performance liquid chromatography (HPLC) method has been developed and validated. Spiked plasma samples were cleaned up and concentrated using a simple liquid-liquid extraction (LLE) procedure with N-methylphysostigmine as internal standard. Separation was achieved using reversed-phase HPLC on a Kinetex C18 column with gradient elution and fluorescence detection (254 nm excitation/355 nm emission).
Results: LLE produced clean extracts and a mean recovery of 80.3% for eseroline and 84.9% for physostigmine. The HPLC assay revealed a limit of detection (LOD) of 0.025 ng/mL and a lower limit of quantification (LLOQ) of 0.05 ng/mL for both analytes. Linearity was observed at 0.05–10.0 ng/mL (r2>0.999). Intra- and inter-day precision ranged from 0.7% to 6.6%, and intra- and inter-day accuracy 97.5%–110.0%.
Conclusions: The presented method is useful for human drug level monitoring of physostigmine and eseroline in accordance with current guidelines. Remarkably low plasma concentrations can be quantified after LLE with gradient elution and fluorescence detection, making this a suitable method for pharmacokinetic studies in a clinical setting.
Acknowledgments
The authors would like to thank Dr. Jürgen Burhenne, Heidelberg, and Prof. Frieder Kees, Regensburg, for kindly sharing their expertise in HPLC and liquid-liquid extraction procedures.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Financial support: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Articles in the same Issue
- Frontmatter
- Editorials
- Once upon a time: a tale of ISO 15189 accreditation
- A new integrated tool for assessing and monitoring test comparability and stability
- Liver-FibroSTARD checklist and glossary: tools for standardized design and reporting of diagnostic accuracy studies of liver fibrosis tests
- Reviews
- Thromboembolic risk in hematological malignancies
- A review of the cut-off points for the diagnosis of vitamin B12 deficiency in the general population
- Opinion Paper
- Permissible limits for uncertainty of measurement in laboratory medicine
- EFLM Position Paper
- Flexible scope for ISO 15189 accreditation: a guidance prepared by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO)
- Genetics and Molecular Diagnostics
- Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses
- Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma
- General Clinical Chemistry and Laboratory Medicine
- The Empower project – a new way of assessing and monitoring test comparability and stability
- Comparison of four automated serum vitamin B12 assays
- Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points
- INR vs. thrombin generation assays for guiding VKA reversal: a retrospective comparison
- Determination of dabigatran in plasma, serum, and urine samples: comparison of six methods
- Simple high-throughput analytical method using ultra-performance liquid chromatography coupled with tandem mass spectrometry to quantify total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine
- Revival of physostigmine – a novel HPLC assay for simultaneous determination of physostigmine and its metabolite eseroline designed for a pharmacokinetic study of septic patients
- Relationship between antiphosphatidylserine/prothrombin and conventional antiphospholipid antibodies in primary antiphospholipid syndrome
- Reference Values and Biological Variations
- Relevance of EDTA carryover during blood collection
- Reference intervals for renal injury biomarkers neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in young infants
- Cardiovascular Diseases
- NT-proBNP levels and their relationship with systemic ventricular impairment in adult patients with transposition of the great arteries long after Mustard or Senning procedure
- Letters to the Editors
- Troponin T measured with highly sensitive assay (hsTnT) on admission does not reflect infarct size in ST-elevation myocardial infarction patients receiving primary percutaneous coronary intervention
- Analytical challenges related to the use of biomarker ratios for the biological diagnosis of Alzheimer’s disease
- Serum brain injury biomarkers as predictors of mortality after severe aneurysmal subarachnoid hemorrhage: preliminary results
- Tumor markers assay by the Lumipulse G
- Real-world costs of laboratory tests for non-small cell lung cancer
- Impact of stopping vitamin K antagonist therapy on concentrations of dephospho-uncarboxylated Matrix Gla protein
- Practicability of fetal scalp blood sampling during labor using microtubes and a point-of-care (POC) lactate testing device: difficulty assessment, sampling time and failure rates
- Establishing objective analytical quality requirements in the IgE specific assay: a message in a bottle
- Bacteria on a peripheral blood smear as presenting sign of overwhelming post-splenectomy infection in a patient with secondary acute myeloid leukemia
