Aims Hydroxychloroquine (HCQ) is a safe drug currently prescribed for the treatment of chronic autoimmune disorders. However, severe cases have been reported after massive overdoses in humans. A sensitive and specific LC-MS/MS method was developed for the simultaneous quantitation of HCQ and its major metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ) and bisdesethylchloroquine (BDCQ) in human whole blood. The assay was used to the quantitation of HCQ and its metabolites in one severe non-fatal poisoning. Relevance of HCQ monitoring is further discussed in order to evaluate its contribution to the management of acute poisonings. Case report and analytical procedure A 25-year-old woman was admitted to the intensive care unit (ICU) 1.5 h after the ingestion of 6 g of HCQ in a suicidal attempt. She developed life-threatening cardiovascular failure, which required emergent extracorporeal life support. Blood samples were regularly collected and the timecourse of HCQ and metabolites concentrations was studied from 4 hours after HCQ ingestion up to recovery. Briefly, 400 μL of HCQ-D4 in methanol were mixed to 100 μL of samples and centrifuged for 10 minutes at 13000 rpm. Hundred L of supernatant were then mixed to 100 μL of acidified water and 10 μL were injected in the LC-MS/MS system. Analytes were eluted on a 50 × 3 mm 3 μm Hypersil Gold aQ column (Thermo, USA). Detection was achieved using a Quantum Ultra triple-quadrupole mass spectrometer (Thermo, USA) with an electrospray ionization source in positive ion mode, using multiple reaction monitoring. Results The assay, fully validated according to European Medicines Agency's guidelines, was linear in a dynamic range of 25–2000 ng/mL for all compounds and was suitable for drug monitoring and overdoses with levels of HCQ up to 20000 ng/mL after appropriate dilution. It was successfully used to measure HCQ and its metabolites in the whole blood samples of our female poisoned patient. Blood HCQ concentration was 19500 ng/mL on ICU admission and slowly decreased to 2225 ng/mL on day 9. Blood concentrations of the metabolites were significantly less elevated than HCQ and decreased from 1123 to 182, 465 to 109 and 91 to 36 ng/mL for DHCQ, DCQ and BDCQ, respectively. All measurements of HCQ and its metabolites were modeled simultaneously using ADAPT5 software. Half-life of HCQ (8.7 h) was lower to those reported in the literature (Jordan et al , Clin Toxicol, 1999), which could be explained by alterations in its pharmacokinetics related to the extracorporeal circulation. Conclusions and perspectives A selective and sensitive LC-MS/MS method was developed to quantify HCQ and its metabolites in human whole blood. The assay was suitable to cases of overdoses with expected elevated concentrations and used to the study of HCQ toxicokinetics after a massive ingestion. A short review of the cases of HCQ poisonings admitted in the ICU showed that elevated blood HCQ concentrations on admission at the early phase of the intoxication were associated with serious cardiovascular toxicity requiring intensive supportive care. Blood HCQ concentrations seem correlated with poisoning severity on ICU admission and daily monitoring of HCQ concentrations useful to optimize patient management and evaluate the final outcome. However, further studies are still needed to investigate the exact role of HCQ metabolites in acute intoxications and chronic treatments as well as the contribution of measuring their respective concentrations in blood.