•First method for therapeutic drug monitoring in Paxlovid-treated COVID-19 patients.•Simultaneous quantification of nirmatrelvir and ritonavir (constituents of Paxlovid)•Sensitive and selective ...determination by LC-MS/MS.•Plasma sample preparation by simple protein precipitation.
Nirmatrelvir is an antiviral agent active against SARS-CoV-2, the virus causing the pandemic disease COVID-19. It is administrated in combination with the protease inhibitor ritonavir, which acts in case of COVID-19 mainly as enzyme blocking agent preventing the premature metabolic elimination of nirmatrelvir. The combination of the two drugs in separate tablets is marketed under the brand name Paxlovid® and shows good effectivity in preventing the progression of COVID-19 to severe disease state. In this work, we described a LC-MS/MS method for the simultaneous quantification of nirmatrelvir and ritonavir in human plasma of patients treated for COVID-19 with Paxlovid®. After addition of D6-ritonavir as internal standard, plasma proteins were precipitated by the addition of methanol. The analytes were separated by gradient elution on a C18-column and were detected by tandem mass spectrometry. Calibration functions were linear in the ranges of 10 – 10000 ng/mL for nirmatrelvir and 2 – 2000 ng/mL for ritonavir. Inter-day and intra-day precision and accuracy was better than 15 % in the quality control samples and better than 20 % at the LLOQ. The method was successfully applied on samples of hospitalized patients treated for COVID-19 and proved to be capable in supporting therapeutic drug monitoring (TDM).
Preclinical studies have revealed that the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), increases vascular tone in cerebral blood vessels. Marked elevations of ADMA ...blood levels were found in patients with diseases characterized by decreased cerebral perfusion, such as ischemic stroke. Arterial stiffness is an independent predictor of stroke and other adverse cardiovascular events. The aim of this study was to investigate the influence of a systemic subpressor dose of ADMA on arterial stiffness and cerebral perfusion in humans.
Using a double-blind, vehicle-controlled study design, we allocated 20 healthy men in random order to infusion of either ADMA (0.10 mg ADMA/kg per min) or vehicle over a period of 40 minutes. Arterial stiffness was assessed noninvasively by pulse wave analysis. All volunteers underwent measurement of cerebral perfusion by dynamic contrast-enhanced perfusion magnetic resonance imaging of the brain.
Infusion of ADMA significantly decreased total cerebral perfusion by 15.1+/-4.5% (P=0.007), whereas blood flow in the vehicle group increased by 7.7+/-2.8% (P=0.02). ADMA also increased arterial stiffness as assessed by measurement of the augmentation index (-12.6+/-1.9 to -9.6+/-1.5, P=0.007).
Our results document for the first time that subpressor doses of ADMA increase vascular stiffness and decrease cerebral perfusion in healthy subjects. Thus, ADMA is an important endogenous modulator of cerebral vascular tone and may be involved in the pathogenesis of cerebrovascular disease.
Endogenous methylarginines were proposed as cardiovascular risk factors more than two decades ago, however, so far, this knowledge has not led to the development of novel therapeutic approaches. The ...initial studies were primarily focused on the endogenous inhibitors of nitric oxide synthases asymmetric dimethylarginine (ADMA) and monomethylarginine (MMA) and the main enzyme regulating their clearance dimethylarginine dimethylaminohydrolase 1 (DDAH1). To date, all the screens for DDAH1 activators performed with the purified recombinant DDAH1 enzyme have not yielded any promising hits, which is probably the main reason why interest towards this research field has started to fade. The relative contribution of the second DDAH isoenzyme DDAH2 towards ADMA and MMA clearance is still a matter of controversy. ADMA, MMA and symmetric dimethylarginine (SDMA) are also metabolized by alanine: glyoxylate aminotransferase 2 (AGXT2), however, in addition to methylarginines, this enzyme also has several cardiovascular protective substrates, so the net effect of possible therapeutic targeting of AGXT2 is currently unclear. Recent studies on regulation and functions of the enzymes metabolizing methylarginines have given a second life to this research direction. Our review discusses the latest discoveries and controversies in the field and proposes novel directions for targeting methylarginines in clinical settings.
•Simultaneous quantification of ceftazidime and avibactam by LC-MS/MS.•Method capable of supporting therapeutic drug monitoring in human plasma.•Human plasma and dried blood spots (DBS) as ...matrix.•Improved stability of ceftazidime and avibactam in DBS versus plasma.
Ceftazidime is an established third-generation cephalosporin antibiotic frequently administered to intensive care patients. To overcome drug resistance of pathogens, it is combined with the newly developed non-ß-lactam ß-lactamase inhibitor avibactam under the brand name Zavicefta®. To facilitate therapeutic drug monitoring (TDM), we developed a method for the simultaneous quantification of these substances by LC-MS/MS. A problem for TDM is the low stability of the analytes in plasma, requiring transport times of less than 6 h at 23 °C. Thus, we evaluated dried blood spots (DBS) as matrix for better stability. For both analytes, stable isotope labelled internal standards were applied. Plasma samples were prepared by protein precipitation, DBS by liquid extraction. The chromatographic separation took place on a polar-modified C18 column, and detection was achieved by tandem mass spectrometry with ESI ionization in positive mode for ceftazidime and negative mode for avibactam. Calibration was linear in the ranges of 5 – 100 µg/mL for ceftazidime and 1.25 – 25 µg/mL for avibactam in plasma and 2.5 – 50 µg/mL and 0.625 – 12.5 µg/mL in DBS, respectively. Precision was better than 7 % and accuracy better than 10% for plasma as well as for DBS. The stability of ceftazidime and avibactam was better in DBS than in plasma or full blood, extending maximal transport times at 23 °C from 6 h in plasma or full blood to 24 h for DBS samples. However, robust estimation of plasma concentrations from DBS measurements requires validation by future clinical studies.
Asymmetrical dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. It is formed by protein arginine N-methyltransferases (PRMTs), which utilize S-adenosylmethionine as methyl ...group donor. ADMA plasma concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and producing proatherogenic changes of endothelial cell function. Four different isoforms of human PRMTs have been identified. Because the release of ADMA from human endothelial cells is increased in the presence of native or oxidized LDL cholesterol, we investigated the potential involvement of PRMT activity and gene expression in this effect. We found that the production of ADMA by human endothelial cells is upregulated in the presence of methionine or homocysteine and inhibited by either of the methyltransferase inhibitors S-adenosylhomocysteine, adenosine dialdehyde, or cycloleucine. This effect is specific for ADMA but not symmetrical dimethylarginine. The upregulation of ADMA release by native and oxidized LDL is abolished by S-adenosylhomocysteine and by the antioxidant pyrrollidine dithiocarbamate. Furthermore, a methyl-(14)C label is transferred from S-adenosylmethionine to ADMA but not symmetrical dimethylarginine, in human endothelial cells. The expression of PRMTs is upregulated in the presence of native or oxidized LDL. Our data suggest that the production of ADMA by human endothelial cells is regulated by S-adenosylmethionine-dependent methyltransferases. This activity is upregulated by LDL cholesterol, which may be due in part to the enhanced gene expression of PRMTs. In concentrations reached by stimulation of methyltransferases (5 to 50 micromol/L), ADMA significantly inhibited the formation of (15)N-nitrite from L-guanidino-(15)N(2)arginine. These findings suggest a novel mechanism by which ADMA concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and atherosclerosis.
•First LC-MS/MS quantification method for upadacitinib in human plasma.•Pexidartinib used as internal standard.•Fast and easy sample preparation.•Application of the method in TDM of rheumatoid ...arthritis patients.
Upadacitinib is a selective janus-kinase-1 inhibitor effective in the treatment of autoimmune related diseases like rheumatoid arthritis or psoriatic arthritis. Here, we described a LC-MS/MS method for the quantification of upadacitinib in human plasma applicable for therapeutic drug monitoring. Pexidartinib was used as internal standard. Plasma samples were prepared by acidic protein precipitation and the analytes were separated on a C-18 reversed phase column. Detection took place by tandem mass spectroscopy after ionization in the positive mode and collision induced fragmentation at m/z 381 → 256, 213 for upadacitinib and m/z 418 → 258, 165 for pexidartinib. The calibration function was linear in the range of 0.15 – 150 ng/mL. Precisions and accuracies were better than 10% in intra- as well as inter-day evaluations. The method was applied in therapeutic drug monitoring of patients undergoing treatment for rheumatoid arthritis with the standard dose of 15 mg upadacitinib extended release formulation once daily. At steady state, we found trough levels of 4.13 (3.51 – 11.0) ng/mL, which is comparable to values found in healthy volunteers receiving the same dose (2.8 ± 1.2 ng/mL).
► A quantification method for l-arginine and its metabolites ADMA and SDMA is presented. ► A very easy and fast sample preparation is proposed. ► Stable isotope labeled I.S.(s) are used for each ...l-arginine, ADMA and SDMA. ► Very good performance regarding precision and accuracy is achieved due to the I.S.(s). ► Matrix independency is achieved due to the I.S.(s).
The amino acid l-arginine and its metabolites ADMA and SDMA are important markers for a range of diseases in humans. Increased levels of ADMA and SDMA in plasma point to endothelial dysfunction, hypertension, renal impairment and other pathological states. We present here a method to quantify l-arginine, ADMA and SDMA in human plasma, which is suitable to support clinical research in this field. Sample preparation consisted only of protein precipitation and the analytes were separated using a silica based HILIC column. The analytes were detected by ESI MS/MS, providing high selectivity and sensitivity. The calibration functions were linear in the ranges of 7.5–150μmol/l for l-arginine, 0.15–3μmol/l for ADMA and 0.2–4μmol/l for SDMA. These ranges cover the concentrations encountered in healthy and pathological human plasma. The method employs 13C6-arginine, D7-ADMA and, for the first time in LC–MS/MS, D6-SDMA as internal standards for l-arginine, ADMA and SDMA. Therefore, matrix independency and a high intra-day precision of 0.82% for l-arginine, 2.12% for ADMA and 2.83% for SDMA, were achieved at basal plasma concentrations. The respective inter-day precision values were 4.01% for l-arginine, 3.77% for ADMA and 3.86% for SDMA.
Uremia occurs if the kidney loses the ability to eliminate toxic compounds at a sufficient rate into the urine. In 1970, N-N, N-G- and N-G,N׳-G-dimethyl-arginine (asymmetric dimethylarginine ADMA and ...symmetric dimethylarginine) were isolated from human urine. It was anticipated that both substances might be important in the pathophysiology and for the diagnosis of various pathologic states. It took 22 years, however, before this idea materialized when it was found that ADMA, which is increased in hemodialysis patients, inhibits the synthesis of the endothelial-derived relaxing factor, identified as nitric oxide. ADMA correlates with traditional and nontraditional cardiovascular risk factors and is a strong predictor of cardiovascular events and death in both patients with chronic kidney disease and in the general population. It also seems to mediate adverse cardiovascular effects of drugs such as proton pump inhibitors. To date, we have no specific pharmacologic therapy at hand to neutralize the deleterious effects of ADMA, curbing the enthusiasm for this marker and mediator of cardiovascular disease.
Background Homoarginine ( hA rg) has been shown to be cardioprotective in a model of ischemic heart failure; however, the mechanism remains unknown. hA rg can inhibit tissue-nonspecific alkaline ...phosphatase ( TNAP ), an enzyme that promotes vascular calcification. We hypothesized that hA rg will exert beneficial effects by reducing calcification in a mouse model of coronary artery disease associated with TNAP overexpression and hypercholesterolemia. Methods and Results TNAP was overexpressed in the endothelium in mice homozygous for a low-density lipoprotein receptor mutation (wicked high cholesterol WHC allele). WHC and WHC -endothelial TNAP mice received placebo or hA rg supplementation (14 mg/L in drinking water) starting at 6 weeks of age simultaneously with an atherogenic diet. Outcomes were compared between the groups after 4 to 5 weeks on treatment. Experiments were performed in males, which presented a study limitation. As expected, WHC -endothelial TNAP mice on the placebo had increased mortality (median survival 27 days, P<0.0001), increased coronary calcium and lipids ( P<0.01), increased left ventricular end-diastolic diameter ( P<0.0001), reduced ejection fraction ( P<0.05), and increased myocardial fibrosis ( P<0.0001) compared with WHC mice. Contrary to our hypothesis, hA rg neither inhibited TNAP activity in vivo nor reduced coronary artery calcification and atherosclerosis in WHC -endothelial TNAP mice; however, compared with the placebo, hA rg prevented left ventricular dilatation ( P<0.01), preserved ejection fraction ( P<0.05), and reduced myocardial fibrosis ( P<0.001). Conclusions The beneficial effect of hA rg supplementation in the setting of calcified coronary artery disease is likely due to its direct protective actions on the myocardial response to the ischemic injury and not to the inhibition of TNAP activity and calcification.
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