Abstract
BackgroundHydroxychloroquine (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.MethodsA 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.ResultsThe 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.ConclusionsA 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.
Highlights
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting predominantly women, with an up to 9:1 ratio in the young and middle-aged [1]
The within- and between-run accuracy and precision were within the recommended European Medicines Agency (EMA) guidelines, with bias < 15% in comparison with the nominal concentrations and CV < 5% at each of the three evaluated levels (LLOQ 8.3 ng/mL, quantification (LLOQ) 8.3 ng/mL; low (QCL) 25 ng/mL, and Quality control with high concentration (QCH) 4556.3 ng/mL) (Table 2) in order to validate the accuracy and reproducibility of the measurements within the methodological range
Hydroxychloroquine is a drug with a plasma protein binding degree estimated to 50% [31], and it has been speculated that whole blood (WB) is favorable because of the technical challenges in separating blood cells and platelets from plasma [32]
Summary
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting predominantly women, with an up to 9:1 ratio in the young and middle-aged [1]. Despite the variable clinical phenotype, antimalarials such as hydroxychloroquine (HCQ) and chloroquine phosphate are standard of care in SLE [2, 3], rheumatoid arthritis (RA) [4], or other inflammatory rheumatic diseases [5]. They have been suggested for treatment in COVID-19 patients [6]. 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. We describe a method for the determination of HCQ in human blood using liquid chromatography-highresolution mass spectrometry (LC-HRMS) and compare the suitability of the three sample matrices
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