Background Hydroxychloroquine (HCQ) is the standard of care in the treatment of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and other inflammatory rheumatic diseases and potentially for the treatment in COVID-19 patients. Determination of HCQ for therapeutic drug monitoring (TDM) can be performed in whole blood (WB), serum, and plasma. Direct comparisons of WB, serum, and plasma levels of HCQ in patients with SLE have not previously been reported. We describe a method for the determination of HCQ in human blood using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and compare the suitability of the three sample matrices. Methods A method for the determination of HCQ in human blood using LC-HRMS was developed, validated, and applied for the determination of HCQ levels in WB, serum, and plasma from 26 SLE patients. The reproducibility of the method, in the three matrices, was evaluated using quality control samples and repeated preparations and measurements of patient samples. The performance of the developed method for HCQ measurement in serum was further evaluated by comparison with two previously reported extraction methods. Results The performance of the presented method demonstrated high accuracy and precision. A large range of HCQ concentrations was observed for the SLE patients in all three matrices (WB, serum, and plasma). The mean levels in WB were approximately two-fold the levels in serum and plasma (813 ng/mL compared to 436 ng/mL and 362 ng/mL, respectively). Spiked quality controls showed high reproducibility for all matrices (coefficient of variation, CV, approx. 5%), whereas in patient samples, equally high-precision was only found using WB as the matrix (CV 3%). The CV for serum and plasma was 14% and 39%, respectively. Two alternative methods applied to serum samples did not demonstrate improved precision. Conclusions A LC-HRMS method for the measurement of HCQ in human blood was developed and validated. Whole blood was found to be the superior sample matrix in terms of sample reproducibility. Thus, whole blood samples should be used for HCQ analysis when patients are monitored for HCQ treatment effects. The assay is in clinical use to monitor levels of HCQ in patients.

Objective Identifying molecular changes that contribute to the onset and progression of Huntington's disease (HD) is of importance for the development and evaluation of potential therapies. Methods We conducted an unbiased mass‐spectrometry proteomic analysis on the cerebrospinal fluid of 12 manifest HD patients (ManHD), 13 pre‐manifest (preHD), and 38 controls. A biologically plausible and significant possible biomarker was validated in samples from a separate cohort of patients and controls consisting of 23 ManHD patients and 23 controls. Results In ManHD compared to preHD, 10 proteins were downregulated and 43 upregulated. Decreased levels of proenkephalin (PENK) and transthyretin were closely linked to HD symptom severity, whereas levels of 15 upregulated proteins were associated with symptom severity. The decreased PENK levels were replicated in the separate cohort where absolute quantitation was performed. Conclusions We hypothesize that declining PENK levels reflect the degeneration of medium spiny neurons (MSNs) that produce PENK and that assays for PENK may serve as a surrogate marker for the state of MSNs in HD. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Introduction Standardized commercial kits enable targeted metabolomics analysis and may thus provide an attractive complement to the more explorative approaches. The kits are typically developed for triple quadrupole mass spectrometers using serum and plasma. Objectives Here we measure the concentrations of preselected metabolites in cerebrospinal fluid (CSF) using a kit developed for high-resolution mass spectrometry (HRMS). Secondarily, the study aimed to investigate metabolite alterations in patients with secondary progressive multiple sclerosis (SPMS) compared to controls. Methods We performed targeted metabolomics in human CSF on twelve SPMS patients and twelve age and sex-matched healthy controls using the Absolute IDQ-p400 kit (Biocrates Life Sciences AG) developed for HRMS. The extracts were analysed using two methods; liquid chromatography-mass spectrometry (LC-HRMS) and flow injection analysis-MS (FIA-HRMS). Results Out of 408 targeted metabolites, 196 (48%) were detected above limit of detection and 35 were absolutely quantified. Metabolites analyzed using LC-HRMS had a median coefficient of variation (CV) of 3% and 2.5% between reinjections the same day and after prolonged storage, respectively. The corresponding results for the FIA-HRMS were a median CV of 27% and 21%, respectively. We found significantly (p < 0.05) elevated levels of glycine, asymmetric dimethylarginine (ADMA), glycerophospholipid PC-O (34:0) and sum of hexoses in SPMS patients compared to controls. Conclusion The Absolute IDQ-p400 kit could successfully be used for quantifying targeted metabolites in the CSF. Metabolites quantified using LC-HRMS showed superior reproducibility compared to FIA-HRMS.

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