Quantification of vancomycin and clindamycin in human plasma and synovial fluid applying ultra-performance liquid chromatography tandem mass spectrometry

Periprosthetic joint infection is a challenging infection involving the joint prosthesis and adjacent tissue, such as synovial fluid, synovial tissue, and bone tissue. The current treatment consists of multiple surgical revisions and long-term antibiotic therapy. Treatment failure can cause poor functional outcome and reduced quality of life. Further research on the extent of antibiotic penetration into the infected tissues is of great importance. Our work aimed to develop and validate a novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of the commonly administered antibiotics vancomycin and clindamycin in plasma and synovial fluid. An extraction procedure consisting of zinc sulfate precipitation and dilution with eluent was used for both analytes. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 C18 column (1.8 µm, 2.1 × 100 mm), and quantification was carried out by a Waters Xevo TQ-S micro mass spectrometer. Stable isotope-labeled vancomycin-d10 served as internal standard. The method validation was performed based on the guidelines of the EMA and FDA. The calibration curves were linear over the range of 0.5–50 mg/L, with a coefficient of determination above 0.990. The validation results for precision and accuracy, specificity, matrix effects and stability were all within the acceptance range. An accurate and rapid method for the simultaneous quantification of vancomycin and clindamycin in human plasma and synovial fluid on the UPLC-MS/MS was developed, optimized and validated. The analysis has a run time of 5.2 min and 50 µL sample volume is needed. This developed method was successfully applied in eight patients with PJI and is suitable to determine the exposure of antibiotics in plasma and synovial fluid in patients during current PK/PD studies.</p

Failure of target attainment of beta-lactam antibiotics in critically ill patients and associated risk factors: a two-center prospective study (EXPAT)

Background: Early and appropriate antibiotic dosing is associated with improved clinical outcomes in critically ill patients, yet target attainment remains a challenge. Traditional antibiotic dosing is not suitable in critically ill patients, since these patients undergo physiological alterations that strongly affect antibiotic exposure. For betalactam antibiotics, the unbound plasma concentrations above at least one to four times the minimal inhibitory concentration (MIC) for 100% of the dosing interval (100%ƒT>1–4×MIC) have been proposed as pharmacodynamic targets (PDTs) to maximize bacteriological and clinical responses. The objectives of this study are to describe the PDT attainment in critically ill patients and to identify risk factors for target non-attainment. Methods: This prospective observational study was performed in two ICUs in the Netherlands. We enrolled adult patients treated with the following beta-lactam antibiotics: amoxicillin (with or without clavulanic acid), cefotaxime, ceftazidime, ceftriaxone, cefuroxime, and meropenem. Based on five samples within a dosing interval at day 2 of therapy, the time unbound concentrations above the epidemiological cut-off (ƒT > MICECOFF and ƒT > 4×MICECOFF) were determined. Secondary endpoints were estimated multivariate binomial and binary logistic regression models, for examining the association of PDT attainment with patient characteristics and clinical outcomes. Results: A total of 147 patients were included, of whom 63.3% achieved PDT of 100%ƒT > MICECOFF and 36.7% achieved 100%ƒT > 4×MICECOFF. Regression analysis identified male gender, estimated glomerular filtration rate (eGFR) ≥ 90 mL/min/1.73 m2 , and high body mass index (BMI) as risk factors for target non-attainment. Use of continuous renal replacement therapy (CRRT) and high serum urea significantly increased the probability of target attainment. In addition, we found a significant association between the 100%ƒT > MICECOFF target attainment and ICU length of stay (LOS), but no significant correlation was found for the 30-day survival. Conclusions: Traditional beta-lactam dosing results in low target attainment in the majority of critically ill patients. Male gender, high BMI, and high eGFR were significant risk factors for target non-attainment. These predictors, together with therapeutic drug monitoring, may help ICU clinicians in optimizing beta-lactam dosing in critically ill patients

Simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram in plasma using liquid chromatography–tandem mass spectrometry

A simple and specific UPLC–MS/MS method was developed and validated for simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and its active metabolite keto-doxapram. The internal standard was fentanyl-d5 for all analytes. Chromatographic separation was achieved with a reversed-phase Acquity UPLC HSS T3 column with a run-time of only 5.0 min per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate or formic acid in Milli-Q ultrapure water or in methanol with a total flow rate of 0.4 mL min−1. A plasma volume of only 50 μL was required to achieve adequate accuracy and precision. Calibration curves of all five analytes were linear. All analytes were stable for at least 48 h in the autosampler. The method was validated according to US Food and Drug Administration guidelines. This method allows quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram, which is useful for research as we

Clinical Validation of a Dried Blood Spot Assay for 8 Antihypertensive Drugs and 4 Active Metabolites

BACKGROUND: Drug nonadherence is one of the major challenges faced by resistant hypertension patients, and identification of this problem is needed for optimizing pharmacotherapy. Dried blood spot (DBS) sampling is a minimally invasive method designed to detect and determine the degree of nonadherence. In this study, a DBS method for qualifying 8 antihypertensive drugs (AHDs) and 4 active metabolites was developed and validated using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). METHOD: The DBS assay was validated analytically and clinically, in accordance with FDA requirements. Analytical validation was accomplished using UHPLC-MS/MS. For clinical validation, paired peak and trough levels of DBS and plasma samples were simultaneously collected and comparatively analyzed using Deming regression and Bland-Altman analyses. All concentrations below the set lower limit were excluded. Deming regression analysis was used to predict comparison bias between the collected plasma and DBS samples, with DBS concentrations corrected accordingly. RESULTS: The UHPLC-MS/MS method for simultaneously measuring 8 AHDs and their metabolites in DBS, was successfully validated. With Deming regression no bias was observed in N = 1; constant bias was seen in N = 6 and proportional bias in N = 11 of the AHDs and metabolites. After correction for bias, only one metabolite (canrenone) met the 20% acceptance limit for quantification, after Bland-Altman analyses. In addition, amlodipine, valsartan, and [enalaprilate] met the 25% acceptance limit. CONCLUSIONS: A novel DBS assay for simultaneously qualifying and quantifying 8 AHDs and their metabolites, has been successfully developed and validated. The DBS assay is therefore a suitable method to detect drug nonadherence. However, with the exception of canrenone, the interchangeable use of plasma and DBS sa

Potentially clinically relevant concentrations of Cefazolin, Midazolam, Propofol, and Sufentanil in auto-transfused blood in congenital cardiac surgery

Background: Use of donor blood in congenital cardiac surgery increases the risk for post-operative morbidity and mortality. To reduce the need for allogenic blood transfusion a technique for peri-operative mechanical red cell salvage is applied. Blood from the operation site is collected in a reservoir, processed, passed through a lipophilic filter and returned to the patient. Influence of this cellsaver system on coagulation, fibrinolysis and inflammatory markers is known. To our knowledge no studies have been performed on the effects of autotransfusion on drug concentrations. A clinically relevant drug dose could potentially be returned to the patient through the auto-transfused blood, leading to unwanted drug reactions post-operatively. We aimed to measure drug concentrations in blood salvaged from the operation site and in the auto-transfused blood to determine if a clinically relevant drug dose is returned to the patient. Methods: The study was performed at the Department of Cardiothoracic Surgery of a tertiary university hospital. Blood samples were taken from the reservoir, after processing before the lipophilic filter, the auto-transfused blood, and the waste fluid. Samples were stored at -80 C and drug concentration for sufentanil, propofol, midazolam and cefazolin were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Drug concentrations measured in the reservoir and the auto-transfused blood were compared and the relative reduction was calculated for each patient. Results: Blood samples were taken from 18 cellsaver runs in 18 patients, age 0-13years. Drug concentrations in the reservoir were comparable to concomitant concentrations in the patient. For sufentanil 34% (median, IQR 27-50) of drug concentration was retained from the reservoir in the auto-transfused blood, for midazolam 6% (median, IQR 4-10), for cefazolin 5% (median, IQR 2-6) and for propofol 0% (median, IQR 0-0) respectively. Conclusion: Depending on the drug, up to 34% of the drug concentration salvaged from the operation site is returned to the patient through autotransfusion, potentially causing unwanted drug reactions post-operatively. Additionally, influence of a cellsaver system should be considered in pharmacological research during and after congenital cardiac surgery and could result in dose adjustments in the postoperative phase

Antihypertensive drug concentration measurement combined with personalized feedback in resistant hypertension:a randomized controlled trial

Background:Adherence to antihypertensive drugs (AHDs) is crucial for controlling blood pressure (BP). We aimed to determine the effectiveness of measuring AHD concentrations using a dried blood spot (DBS) sampling method to identify nonadherence, combined with personalized feedback, in reducing resistant hypertension.Methods:We conducted a multicenter, randomized, controlled trial (RHYME-RCT, ICTRP NTR6914) in patients with established resistant hypertension. Patients were randomized to receive either an intervention with standard of care (SoC) or SoC alone. SoC consisted of BP measurement and DBS sampling at baseline, 3 months (t3), 6 months (t6), and 12 months (t12); AHD concentrations were measured but not reported in this arm. In the intervention arm, results on AHD concentrations were discussed during a personalized feedback conversation at baseline and t3. Study endpoints included the proportion of patients with RH and AHD adherence at t12.Results:Forty-nine patients were randomized to receive the intervention+SoC, and 51 were randomized to receive SoC alone. The proportion of adherent patients improved from 70.0 to 92.5% in the intervention+SoC arm (P = 0.008, n = 40) and remained the same in the SoC arm (71.4%, n = 42). The difference in adherence between the arms was statistically significant (P = 0.014). The prevalence of resistant hypertension decreased to 75.0% in the intervention+SoC arm (P &lt; 0.001, n = 40) and 59.5% in the SoC arm (P &lt; 0.001, n = 42) at t12; the difference between the arms was statistically nonsignificant (P = 0.14).Conclusion:Personalized feedback conversations based on DBS-derived AHD concentrations improved AHD adherence but did not reduce the prevalence of RH.</p

Highly sensitive and rapid determination of tacrolimus in peripheral blood mononuclear cells by liquid chromatography–tandem mass spectrometry

After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC–MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 μL containing 1 million PBMCs was used in combination with MagSiMUS-TDMPREP. To each sample we added 30 μL lysis buffer, 20 μL reconstitution buffer containing 13C2H4-tacrolimus as internal standard, 40 μL MagSiMUS-TDMPREP Type I Particle Mix and 175 μL Organic Precipitation Reagent VI for methanol-based protein precipitation. A 10 μL aliquot of the supernatant was injected into the UPLC–MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r2 = 0.997) over the range 5.0–1250 pg/1 × 106 PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results. A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome

Simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram in plasma using liquid chromatography-tandem mass spectrometry

Item does not contain fulltextA simple and specific UPLC-MS/MS method was developed and validated for simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and its active metabolite keto-doxapram. The internal standard was fentanyl-d5 for all analytes. Chromatographic separation was achieved with a reversed-phase Acquity UPLC HSS T3 column with a run-time of only 5.0 min per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate or formic acid in Milli-Q ultrapure water or in methanol with a total flow rate of 0.4 mL min(-1) . A plasma volume of only 50 muL was required to achieve adequate accuracy and precision. Calibration curves of all five analytes were linear. All analytes were stable for at least 48 h in the autosampler. The method was validated according to US Food and Drug Administration guidelines. This method allows quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram, which is useful for research as well as therapeutic drug monitoring, if applicable. The strength of this method is the combination of a small sample volume, a short run-time, a deuterated internal standard, an easy sample preparation method and the ability to simultaneously quantify all analytes in one run

Quantification of beta-lactam antibiotics cefuroxime and flucloxacillin in human synovial fluid, using ultra-performance convergence chromatography-tandem mass spectrometry

Total hip- and knee arthroplasty generally result in successful outcomes. A small percentage of patients however suffer from periprosthetic joint infections (PJI) postoperatively, often with severe consequences. The standard treatment of chronic PJIs consists of a staged arthroplasty exchange during which antibiotic therapy plays a crucial role. For successful antibiotic treatment, adequate concentrations at the infection site are a prerequisite. Regarding the treatment of PJIs, knowledge is lacking with respect to the relationship between administered dosages and plasma- and infection site concentrations of the antibiotics. To gain insight into the antibiotic exposure at the infection site, validated analytical methods for analysis of the antibiotics in matrices at the site of the PJI are essential. We describe a validated ultra-performance convergence chromatography-tandem mass spectrometry (UPC2-MS/MS) method for quantification of the beta-lactam antibiotics cefuroxime and flucloxacillin in synovial fluid. This method was successfully validated for antibiotic quantification in synovial fluids according to the EMA guidelines and consists of a simple sample preparation. For both antibiotics, the accuracy and precision were within requirements (RSD 0.995). The method has a run time of 4.5 min and 50 μL synovial fluid is needed and the validated method will be applied during a PK/PD study to determine the exposure of the study antibiotics in synovial fluid at the site of PJIs

Clinical Pharmacokinetics of Fosfomycin after Continuous Infusion Compared with Intermittent Infusion: a Randomized Crossover Study in Healthy Volunteers

Continuous infusion (CON) of fosfomycin has been proposed as potentially advantageous in certain clinical scenarios. However, no clinical data on the pharmacokinetics (PK) of fosfomycin after CON are available to date. This study aimed to investigate the PK of fosfomycin after CON and compare it with intermittent infusion (INT) of fosfomycin. A randomized two-way crossover study including 8 healthy male volunteers was performed. Each subject received fosfomycin as INT of 8 g over 30 min every 8 h and, separated by a washout period, as CON of 1 g/h preceded by a loading dose of 8 g over 30 min. PK sampling was performed for 18 and 24 h in the CON and INT groups, respectively. Fosfomycin was generally well tolerated. However, 2 out of 8 subjects (25%) developed thrombophlebitis at the infusion site following CON, which was prevented in the following subjects with a simultaneous coinfusion of Ringer's lactate. The steady-state maximum concentration of drug in serum (Cmax) and area under the concentration-time curve from 0 to 24 h at steady state (AUCSS,0 -24) of fosfomycin after INT were 551.5 + 67.8 mg/liter and 3,678.5 + 601.9 h · mg/liter, respectively. CON led to an average steady-state concentration of 183.8 + 35.9 mg/liter, resulting in a calculated AUCSS,0 -24 of 4,411.2 + 862.4 h · mg/liter, which was 1.2-fold higher than that with INT. CON resulted in a 100% T>MIC (time during which the drug concentration exceeds the MIC) for MICs of ≤128 mg/liter, whereas the %T>MIC for INT was only 44% for an MIC of 128 mg/liter. CON of fosfomycin led to improved PK and PK/pharmacodynamic (PD) determinants in plasma of healthy volunteers. The clinical relevance of these findings remains to be investigated in patients