Quantitative HPLC-MS/MS determination of Nuc, the active metabolite of remdesivir, and its pharmacokinetics in rat.

Remdesivir is a nucleotide analog prodrug that has received much attention since the outbreak of the COVID-19 pandemic in December 2019. GS-441524 (Nuc) is the active metabolite of remdesivir and plays a pivotal role in the clinical treatment of COVID-19. Here, a robust HPLC-MS/MS method was developed to determine Nuc concentrations in rat plasma samples after a one-step protein precipitation process. Chromatographic separation was accomplished on Waters XBrige C18 column (50 × 2.1 mm, 3.5 µm) under gradient elution conditions. Multiple reaction monitoring transitions in electrospray positive ion mode were m/z 292.2 → 163.2 for Nuc and 237.1 → 194.1 for the internal standard (carbamazepine). The quantitative analysis method was fully validated in line with the United States Food and Drug Administration guidelines. The linearity, accuracy and precision, matrix effect, recovery, and stability results met the requirements of the guidelines. Uncertainty of measurement and incurred sample reanalysis were analyzed to further ensure the robustness and reproducibility of the method. This optimized method was successfully applied in a rat pharmacokinetics study of remdesivir (intravenously administration, 5 mg kg-1). The method can act as a basis for further pharmacokinetic and clinical efficacy investigations in patients with COVID-19. Graphical abstract.

Recent caffeine ingestion reduces adenosine efficacy in the treatment of paroxysmal supraventricular tachycardia.

OBJECTIVES: Caffeine, an adenosine receptor blocker, should theoretically reduce adenosine efficacy in the treatment of paroxysmal supraventricular tachycardia (SVT). We aimed to determine the effect of recent caffeine ingestion on the likelihood of reversion of SVT with adenosine. METHODS: This was a multicenter, case-control study of adult patients with SVT treated with adenosine between September 2007 and July 2008. The primary endpoint was reversion to sinus rhythm (SR) after a 6-mg adenosine bolus, as a function of recent (within 2, 4, 6, and 8 hours) caffeine ingestion. Caffeine ingestion data were collected using a self-administered questionnaire. RESULTS: Of 68 patients enrolled, 52 (76.5%, 95% confidence interval [CI] = 64.4% to 85.6%) reverted after a 6-mg adenosine bolus. There were no significant differences in age, sex, or daily caffeine ingestion between patients who did and did not revert (p > 0.05). However, as a group, patients who did not revert had recently ingested significantly more caffeine (p < 0.05). If caffeine had been ingested less than 2 or 4 hours before the adenosine bolus, the odds of reversion to SR were significantly reduced (odds ratio [OR] = 0.18, 95% CI = 0.04 to 0.93; and OR = 0.14, 95% CI = 0.04 to 0.49, respectively). If caffeine had been ingested less than 6 or 8 hours before the adenosine, the odds of reversion were not reduced (OR = 0.31, 95% CI = 0.09 to 1.02; and OR = 0.31, 95% CI = 0.09 to 1.08, respectively). CONCLUSIONS: Ingestion of caffeine less than 4 hours before a 6-mg adenosine bolus significantly reduces its effectiveness in the treatment of SVT. An increased initial adenosine dose may be indicated for these patients.

[Quantitative analysis of adenosine and cordycepin in Cordyceps sinensis and its substitutes with LC-MS-MS].

OBJECTIVE: To develop a LC-MS-MS method for determination of adenosine and cordycepin in Cordyceps sinensis and it's substitutes. METHOD: The sawple was extracted with. 90% methanol. Multi-reactions monitoring (MRM) technique was adopted. RESULT: The regression equations and coefficients were Y = 89.04X + 506.85 (r = 0.999 7) for adenosine, Y = 99.66X + 1 251.34 (r = 0.998 8) for cordycepin respectively. The linear range was 5.0-1 000.0 microg x L(-1) for adenosine and cordycepin. The limits of detection (LOD) were 0. 44 microg x L(-1) for adenosine and 0.31 microg x L(-1) for cordycepin, respectively. The average recoveries of adenosine and cordycepin were 98.1% and 97.9%, respectively. CONCLUSION: The method was highly sensitive, selective and fast, which can be used for the determination of adenosine and cordycepin in C. sinensis and it's substitutes. This method can also be applied for the quality control of the medicinal materials.

Estandarizacion de la tecnica de adenosina deaminasa, un aspecto critico en la ayuda diagnostica para tuberculosis / Standardization of the adenosina technique deaminasa, a critical aspect in the aid diagnoses for tuberculosis

La tuberculosis es un problema de salud pública y se hace necesario contar con métodos de laboratorio que permitan apoyar su diagnóstico oportuno. A pesar de las diferencias reportadas en cuanto a sensibilidad y especificidad de la técnica para la determinación de la enzima adenosina deaminasa, su medición se ha utilizado en el diagnóstico de tuberculosis extrapulmonar, ya que participa principalmente en el catabolismo de las purinas en la diferenciación y proliferación linfocítica que se presentan como respuesta ante antígenos micobacterianos. El objetivo de este estudio fue estandarizar la técnica para la medición de adenosina deaminasa en líquidos de cavidades estériles, así como analizar los factores que podrían afectar el resultado final. Con este fin se recolectaron 100 muestras de líquidos de cavidades estériles, con diferentes aspectos y condiciones de almacenamiento. Para su análisis se empleó la técnica que recomienda el Instituto Nacional de Salud, institución que realizó el control externo del proceso. Se logró estandarizar la técnica con resultados similares a los obtenidos en el Instituto Nacional de Salud. Se concluyó que el utilizar muestras almacenadas por más de 24 horas a temperaturas inferiores a -20°C, turbias o purulentas de las que se aisle algún microorganismo diferente a Mycobacterium tuberculosis, o que presenten algún grado de hemólisis afectan los valores finales de la enzima y podrían aumentar el porcentaje de falsos positivos para tuberculosis.Palabras clave: adenosina deaminasa, tuberculosis, técnica, muestras, control de calidad.

UW is superior to Celsior and HTK in the protection of human liver endothelial cells against preservation injury.

Celsior solution (CS), a new preservation solution in thoracic organ transplantation, was evaluated for its efficacy in cold preservation of human liver endothelial cells (HLEC) and was compared to University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). HLEC cultures were preserved at 4 degrees C in CS, UW, and HTK, for 2, 6, 12, 24, and 48 hours, with 6 hours of reperfusion. Levels of lactate dehydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and adenosine 5'-triphosphate (ATP) were measured after each interval of ischemia and the respective phase of reperfusion. Preservation injury of HLEC as measured by LDH release, intracellular ATP level, and MTT reduction were overall significantly (P > CS > HTK.

Preservation of rat livers by cold storage: a comparison between the University of Wisconsin solution and Hypothermosol.

OBJECTIVES: The University of Wisconsin solution (UW) is the gold standard for cold storage (CS) of donor livers. However, UW contains the colloid Hydroxyethyl starch (HES), which may cause perfusion deficits due to its high viscosity. Recently, a new CS preservation solution, Hypothermosol (HTS), was introduced which contains the less viscous colloid Dextran. The aim of this study was to assess HTS as a cold storage solution for preservation of the liver. METHODS: In an isolated perfused rat liver model, hepatocellular damage was assessed after 24 hours of CS. Liver enzymes were measured during reperfusion with Krebs-Henseleit Buffer. Bile was collected during reperfusion as a parameter of liver function. RESULTS: CS using HTS showed a significant decrease of ALT and LDH levels (as compared to UW) at all time points during reperfusion. For LDH these results where most pronounced at t=10 min (84 +/- 7.09 vs 113 +/- 7.57: p < 0.05) and t=30 min (149.2 +/- 9.68 vs 194 +/-6.52: p< 0.05). Regarding liver function, more bile was produced after 24 hours CS in HTS, but this did not reach statistical significancy. CONCLUSIONS: Cold storage preservation of rat livers using Hypothermosol results in equal or even better preservation as compared to cold storage using UW.

Comparison of Celsior and UW solution in experimental pancreas preservation.

BACKGROUND: The University of Wisconsin solution (UW) is the gold standard for pancreas preservation. Celsior (CEL) was formulated specifically for heart preservation. Recently, experimental and clinical experience has been reported on the application of CEL to abdominal organs. In this animal study, pancreas preservation with CEL was compared with that in UW solution. PATIENTS AND MATERIALS: Heterotopic, allogeneic pancreaticoduodenal transplantation was performed in female Göttingen Minipigs (n = 12 donors, n = 12 recipients). The grafts were flushed and stored for 6 h at 4 degrees C in UW or CEL. The recipients were randomized into two groups receiving either UW (n = 6)- or CEL (n = 6)-preserved grafts with a follow-up of 5 days. Blood flow (laser Doppler), partial oxygen tension, histological changes, endothelin-1 (plasma, immunohistochemistry), lipase, amylase, trypsinogen activation peptide, and C-reactive protein (CRP) were measured. RESULTS: Partial oxygen tension was lower in the CEL group (P < 0.05). However, blood flow did not differ between UW- and CEL-preserved organs. The histomorphologic analysis of the pancreatic grafts revealed significantly less edema in the UW-preserved organs. Serum levels of amylase, lipase, CRP, and TAP taken from the central venous blood were comparable in the two groups, except for higher amylase values 36 h after reperfusion in the CEL group compared to the UW group (P < 0.05). Likewise, TAP taken from the portal venous effluent of the graft was found to be higher in the CEL group than in UW (P < 0.05). Endothelin-1 serum levels rose significantly during reperfusion without differences between the two groups. ET-1 immunohistochemistry revealed increased local ET-1 during reperfusion in all grafts. However, the ET-1 immunostaining in the CEL group was more pronounced than that in the UW group (P < 0.05). CONCLUSIONS: Our results suggest that CEL solution is not as effective in preventing pancreatic ischemia/reperfusion damage as the standard UW solution in experimental pancreas transplantation. Increased ET-1 immunostaining and reduced p(ti)O(2) in the CEL group indicate increased microcirculatory damage in the CEL group.

Improved preservation solutions for organ storage: a dynamic study of hepatic metabolism.

BACKGROUND: Organ cold storage times may be extended by modifications to organ preservation solutions. METHODS: Three preservation solutions were investigated for their ability to maintain viable hepatic bioenergetics in stored pig livers: modified University of Wisconsin (mUW); mUW+adenosine (1.34 g/L), and mUW+ iloprost (10(-8)mol/L), a prostacyclin analogue. Using human liver retrieval and storage techniques, pig livers were stored on ice for either 2 or 16 hr, after which phosphorus-31 spectra were collected every 2 min during the period of cold ischemia and hypothermic reperfusion (HtR). During HtR, metabolite concentration changes associated with phosphomonoesters, inorganic phosphate, gamma-nucleotide triphosphate (NTP), and beta-NTP were measured for all solutions. RESULTS: After a 2-hr storage, beta-NTP regeneration in mUW+iloprost produced +57.7% (P<0.01) more beta-NTP, at a faster initial rate of +66.3% (P<0.001), compared with mUW, and mUW+adenosine regenerated +35.6% (P<0.05) more beta-NTP, compared with mUW. Storage for 16 hr did not slow the rates of regeneration, and the total NTP produced during the course of the experiment remained unchanged for the respective preservation solutions. Cessation of HtR invoked a net accumulation of nucleotide diphosphate, indicating differential kinetics of adenine nucleotide hydrolysis. CONCLUSION: This large animal model study suggests significant improvements to human organ preservation solutions using prostacyclin analogues and adenosine with respect to hepatic bioenergetics.

Determination of plasma adenosine by high-performance liquid chromatography with column switching and fluorometric detection.

A new method for the rapid determination of plasma adenosine concentrations was developed by using high-performance liquid chromatography with a column switching technique and fluorometric detection. Several "stop solutions" were used to prevent the enzymatic degradation and cellular uptake and release of adenosine in blood samples. Red blood cells and certain denatured proteins were separated by centrifugation. Subsequently, the supernatant was transferred directly into autosample vials and adenosine was reacted with chloroacetaldehyde to form a strong fluorescent, 1-N6-ethenoadenosine. The adenosine derivative was injected directly and separated on a shielded hydrophobic phase column coupled with a C18 reverse-phase column using a column switching valve. Macromolecules and other interfering substances were excluded by the shielded hydrophobic phase column and bypassed to waste. Then, the adenosine derivative and other retained compounds were switched onto the reverse-phase column for further separation and subsequently to the fluorescence detector. The system reduces the analysis time and contamination of the column and hence allows a shorter cleanup time and a longer column lifetime. Adenosine as low as 30 fmol (signal-to-noise ratio, S/N = 3) can be detected by this method. The percentage of recovery of adenosine in plasma treated with adenosine deaminase was above 90%. This method is very rapid (without tedious sample preparation) and sensitive for determining adenosine in canine blood and should prove to be useful in analyzing the effects of ischemia and reperfusion on arterial and coronary venous adenosine concentrations in blood or perfusate samples released from the ischemic or hypoxic myocardium.

Evaluation of preservation conditions and various solutions for small bowel preservation.

The aim of the study was to delineate the most optimal preservation conditions for small bowel grafts. Established preservation solutions such as EuroCollins, University of Wisconsin, histidine-tryptophane-ketoglutarate-Brettschneider, phosphate-buffered sucrose (PBS 140), and 3 new solutions--extracellular fluid (ECF), lactobionate fructose, and a modified lactobionate fructose solution--were compared with saline to determine the most optimal solution for the intestine. Recipient survival, standard histology, and glutaminase activity were used to assess the degree of injury encountered after 12 hr of preservation followed by transplantation. To evaluate the various preservation conditions, ECF was used at pH 6.8 (original ECF). Grafts were preserved most optimally when a vascular washout after the cold storage period was omitted and when topical rewarming of the graft with 37 degrees C saline before reperfusion was performed. Graft survival was not significantly different after preservation with any solution tested (50-83%). Highest graft survival (83%) was achieved with lactobionate fructose and PBS140. Histologic evaluation 20 min after reperfusion revealed minor differences between most groups; a slight advantage was observed for PBS140-preserved grafts. Mucosal glutaminase activity of PBS140-preserved grafts was significantly higher 20 min after reperfusion compared with any other solution evaluated, indicating a superior graft function. These data indicate that different preservation conditions have a great impact on postoperative graft survival and that PBS140 might be preferable to any of the other preservation solutions tested.