Population Pharmacokinetics of Intravenous Salbutamol in Children with Refractory Status Asthmaticus.

BACKGROUND: Intravenous salbutamol is used to treat children with refractory status asthmaticus, however insufficient pharmacokinetic data are available to guide initial and subsequent dosing recommendations for its intravenous use. The pharmacologic activity of salbutamol resides predominantly in the (R)-enantiomer, with little or no activity and even concerns of adverse reactions attributed to the (S)-enantiomer. OBJECTIVE: Our aim was to develop a population pharmacokinetic model to characterize the pharmacokinetic profile for intravenous salbutamol in children with status asthmaticus admitted to the pediatric intensive care unit (PICU), and to use this model to study the effect of different dosing schemes with and without a loading dose. METHODS: From 19 children (median age 4.9 years [range 9 months-15.3 years], median weight 18 kg [range 7.8-70 kg]) treated with continuous intravenous salbutamol at the PICU, plasma samples for R- and S-salbutamol concentrations (111 samples), as well as asthma scores, were collected prospectively at the same time points. Possible adverse reactions and patients' clinical data (age, sex, weight, drug doses, liver and kidney function) were recorded. With these data, a population pharmacokinetic model was developed using NONMEM 7.2. After validation, the model was used for simulations to evaluate the effect of different dosing regimens with or without a loading dose. RESULTS: A two-compartment model with separate clearance for R- and S-salbutamol (16.3 L/h and 8.8 L/h, respectively) best described the data. Weight was found to be a significant covariate for clearance and volume of distribution. No other covariates were identified. Simulations showed that a loading dose can result in higher R-salbutamol concentrations in the early phase after the start of infusion therapy, preventing accumulation of S-salbutamol. CONCLUSIONS: The pharmacokinetic model of intravenous R- and S-salbutamol described the data well and showed that a loading dose should be considered in children. This model can be used to evaluate the pharmacokinetic-pharmacodynamic relationship of intravenous salbutamol in children, and, as a next step, the effectiveness and tolerability of intravenous salbutamol in children with severe asthma.

High-throughput quantification of ten antiarrhythmic drugs in human plasma using UPLC-MS/MS.

In this paper we present an FDA validated method to analyze ten antiarrhythmic drugs (atenolol, bisoprolol, carvedilol, diltiazem, flecainide, lidocaine, metoprolol, propranolol, sotalol and verapamil). A simple and fast sample preparation protocol with protein precipitation followed by ultra performance liquid chromatography (UPLC) for chromatographic separation and mass spectrometric detection applying electrospray ionization (ESI+) and selected reaction monitoring mode (MS/MS) was used. Only 50 µl plasma sample is needed for the simultaneous quantification of all compounds within a 5 min run-to-run analysis time. Sotalol-D6, carvedilol-D5 and verapamil-D6 were used as internal standards. The method was validated according to the FDA guidelines. Correlation coefficients were higher than 0.998 for all compounds. Intra- and interday accuracies were within 15 CV(%) for all analytes. The method is currently successfully applied for routine analysis in our hospital.

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 13 C2 H4 -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.

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 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 Phosphatidylethanols in Whole Blood as a Proxy for Chronic Alcohol Consumption, Using Ultra Performance Convergence Chromatography Tandem Mass Spectrometry.

BACKGROUND: Detection of alcohol consumption after a longer period can be useful in certain patient groups. To monitor chronic alcohol consumption, a novel analytical method for the quantification of phosphatidylethanols (PEths) was developed and validated using ultra performance convergence chromatography-tandem mass spectrometry. METHODS: The main phosphatidylethanols like palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPEth), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanol, and 1,2-dioleoyl-sn-glycero-3-phosphoethanol were analyzed using a simple and fast sample preparation protocol followed by chromatographic separation using ultra performance convergence chromatography, a novel kind of supercritical fluid chromatography. Mass spectrometric detection was conducted by applying negative electrospray ionization and multiple reaction monitoring mode. Only 50 µL of whole blood is needed for the simultaneous quantification of all 3 compounds within 5-minute run-to-run analysis time. POPEth-d5 was applied as internal standard. RESULTS: The method was validated according to the Food and Drug Administration guidelines. Correlation coefficients were higher than 0.995 for all 3 compounds. Intraday and interday inaccuracies were <15% for all analytes in the established linear range. Intraday and interday imprecision were <15% for all analytes. Lower limit of quantification for 1,2-dioleoyl-sn-glycero-3-phosphoethanol, palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanol, and POPEth are, respectively, 3, 6, and 6 mcg/L. Sample stability at -80°C was 1 year. Extracts were stable for 1 day in the autosampler and 2 days at 2-8°C in a closed Eppendorf tube. Samples were tested after 3 freeze-thaw cycles and considered stable. Patient samples have been analyzed with this new method. In a cohort of 248 pregnant women, 17 patients (6.9%) scored positive for PEth. CONCLUSIONS: The described method is suitable for the simultaneous quantitative analysis of the most abundant PEth homologues. Major advantages are low LLOQs, minimal sample volume and clean-up, and a short run time. The method is now available to monitor alcohol consumption in patients and has been incorporated in clinical practice and research.

Dried Blood Spots Combined With Ultra-High-Performance Liquid Chromatography-Mass Spectrometry for the Quantification of the Antipsychotics Risperidone, Aripiprazole, Pipamperone, and Their Major Metabolites.

BACKGROUND: Risperidone, aripiprazole, and pipamperone are antipsychotic drugs frequently prescribed for the treatment of comorbid behavioral problems in children with autism spectrum disorders. Therapeutic drug monitoring (TDM) could be useful to decrease side effects and to improve patient outcome. Dried blood spot (DBS) sample collection seems to be an attractive technique to develop TDM of these drugs in a pediatric population. The aim of this work was to develop and validate a DBS assay suitable for TDM and home sampling. METHODS: Risperidone, 9-OH risperidone, aripiprazole, dehydroaripiprazole, and pipamperone were extracted from DBS and analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry using a C18 reversed-phase column with a mobile phase consisting of ammonium acetate/formic acid in water or methanol. The suitability of DBS for TDM was assessed by studying the influence of specific parameters: extraction solution, EDTA carryover, hematocrit, punching location, spot volume, and hemolysis. The assay was validated with respect to conventional guidelines for bioanalytical methods. RESULTS: The method was linear, specific without any critical matrix effect, and with a mean recovery around 90%. Accuracy and imprecision were within the acceptance criteria in samples with hematocrit values from 30% to 45%. EDTA or hemolysis did not skew the results, and no punching carryover was observed. No significant influence of the spot volume or the punch location was observed. The antipsychotics were all stable in DBS stored 10 days at room temperature and 1 month at 4 or -80°C. The method was successfully applied to quantify the 3 antipsychotics and their metabolites in patient samples. CONCLUSIONS: A UHPLC-MS/MS method has been successfully validated for the simultaneous quantification of risperidone, 9-OH risperidone, aripiprazole, dehydroaripiprazole, and pipamperone in DBS. The assay provided good analytical performances for TDM and clinical research applications.

High-throughput quantification of 8 antihypertensive drugs and active metabolites in human plasma using UPLC-MS/MS.

BACKGROUND: To assess drug adherence of patients with hypertension, an analytical method was developed and validated using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The method includes eight frequently prescribed antihypertensive drugs from four classes and their active metabolites: angiotensin converting enzyme inhibitors enalapril and perindopril (active metabolites respectively enalaprilate and perindoprilate), angiotensin II receptor blockers losartan (with the active metabolite losartan carboxylic acid) and valsartan, calcium channel blockers amlodipine and nifedipine and diuretics hydrochlorothiazide and spironolactone (with the active metabolite canrenone). METHODS: The antihypertensive drugs were analyzed using a simple and fast sample preparation protocol with protein precipitation followed by chromatographic separation using a gradient elution on a reversed phase column. Mass spectrometric detection was conducted by applying both positive and negative electrospray ionization (ESI+/ESI-) and selected reaction monitoring mode (MS/MS). Only 50µl of plasma sample is needed for the simultaneous quantification of all 12 compounds within 6min run-to-run analysis time. Enalapril-d5 was applied as internal standard for all compounds except hydrochlorothiazide (internal standard: Hydrochlorothiazide-13C,d2). RESULTS: The method was validated according to FDA guidelines. Matrix effects were examined using the method of Matuszewski. Correlation coefficients were higher than 0.995 for all compounds. Intra- and inter-day accuracies were <15% for all analytes except spironolactone (-16.8%) in the established linear range. Intra- and inter-day precision were <15% for all analytes. As a result of the lower sensitivity of hydrochlorothiazide, the lowest three calibration levels were excluded. DISCUSSION/CONCLUSIONS: The described method is suitable for the simultaneous quantitative analysis of the most commonly used antihypertensive drugs and their corresponding active metabolites. Major advantages are minimal sample volume and clean up and a short runtime. The method is now available to monitor drug adherence of patients with resistant hypertension in our hospital.

Simultaneous determination of nine ß-lactam antibiotics in human plasma by an ultrafast hydrophilic-interaction chromatography-tandem mass spectrometry.

Contemporary ß-lactam antibiotic dosing is debatable in severely ill patients, since the occurrence of pathophysiological changes in critical illness can result in great inter-individual variability. Therapeutic drug monitoring (TDM) is a commonly used dosing strategy to optimize exposure and thereby minimize toxicity and maximize the efficacy. Currently, TDM of ß-lactam antibiotics is rarely performed, due to poor availability in clinical practice. We describe an ultrafast Hydrophilic-Interaction Chromatography (HILIC) based UPLC-MS/MS method for the determination of amoxicillin, benzylpenicillin, cefotaxime, cefuroxime, ceftazidime, flucloxacillin, imipenem, meropenem and piperacillin in human plasma. This method involves simple sample preparation steps and was comprehensively validated according to standard FDA guidelines. For all analytes, mean accuracy and precision values were within the acceptance value. The lower and upper limits of quantification were found to be sufficient to cover the therapeutic range for all antibiotics. Finally, the method was successfully applied in a large pharmacokinetic study performed in the intensive care setting, and the feasibility of the analytical procedure was demonstrated in routine clinical practice. To the best of our knowledge, we report here the first HILIC-based UPLC-MS/MS assay for the determination of ß-lactam antibiotics in human plasma. This simple, sensitive and ultrafast assay requires small-volume samples and can easily be implemented in clinical laboratories to promote the TDM of ß-lactam antibiotics.

Bioequivalence study of an extemporaneously prepared oral solution of amlodipine suitable for use in pediatric patients compared to commercial tablets.

OBJECTIVE: Amlodipine, a long-acting dihydropyridine calcium channel blocker, is frequently prescribed to pediatric patients. To date, no suitable pediatric formulation has been available. In this study, an amlodipine oral solution was developed and tested for bioequivalence to tablets in healthy adult volunteers. METHODS: This study was designed as an open-label, single-dose, twosequence, two-period, crossover trial to assess the bioequivalence of a newly developed amlodipine besylate oral solution 0.5 mg/mL compared to Norvasc® 5 mg tablets. 13 adult subjects (mean [standard deviation] age of 23.2 [3.6] years, weight 71.5 [7.7] kg) were included and blood samples were collected for 72 hours. Amlodipine plasma levels were determined using a validated UPLC-MS/MS assay. Non-compartmental pharmacokinetic parameters were compared between the formulations according to European Medicines Agency (EMA) bioequivalence guidelines. RESULTS: The 90% confidence intervals of the test/reference ratios of the geometric means for the primary pharmacokinetic parameters AUC(0-72) (88.24 - 104.37%) and C(max) (99.00 - 121.40%) were within the acceptance range of 80.00 - 125.00% for bioequivalence. Mean (SD) AUC(0-72) was 102.7 (26.8) (26.8) µg × h/L for the solution and 108.2 (30.6) µg × h/L for the tablet. Mean (SD) Cmax of the solution was 3.11(1.06) µg/L with a median (IQR) t(max) of 4.0 (2.6 - 7.5) hours. Mean (SD) C(max) of the tablet was 2.91 (0.84) µg/L with a median (IQR) tmax of 6.0 (4.0 - 14.0) hours. Intrasubject coefficients of variation were 10.2% (AUC(0-72)) and 12.4% (C(max)). CONCLUSIONS: The formulations are bioequivalent according to EMA guidelines. This warrants further study of our novel amlodipine oral solution in pediatric patients.

Identification and quantification of the antipsychotics risperidone, aripiprazole, pipamperone and their major metabolites in plasma using ultra-high performance liquid chromatography-mass spectrometry.

The antipsychotics risperidone, aripiprazole and pipamperone are frequently prescribed for the treatment in children with autism. The aim of this study was to validate an ultra-high performance liquid chromatography-mass spectrometry method for the quantification of these antipsychotics in plasma. An ultra-high performance liquid chromatography-mass spectrometry assay was developed for the determination of the drugs and metabolites. Gradient elution was performed on a reversed-phase column with a mobile phase consisting of ammonium acetate, formic acid in methanol or in Milli-Q ultrapure water at a flow rate of 0.5 mL/min. The method was validated according to the US Food and Drug Administration guidelines. The analytes were found to be stable enough after reconstitution and injection of only 5 µL improved the accuracy and precision in combination with the internal standard. Calibration curves of all five analytes were linear. All analytes were stable for at least 72 h in the autosampler and the high quality control of 9-OH-risperidone was stable for 48 h. The method allows quantification of all analytes. The advantage of this method is the combination of a minimal injection volume, a short run-time, an easy sample preparation method and the ability to quantify all analytes in one run. Copyright © 2015 John Wiley & Sons, Ltd.

Development and Validation of an HPLC-UV Assay to Quantify Plasma Levels of Sulfametrol: A Preferential Antibiotic in Children.

BACKGROUND: Sulfonamides in combination with trimethoprim are frequently used antibiotics. They work synergistic. In infections with Pneumocystis jiroveci or Stenotrophomonas maltophilia, higher dosages are indicated than in other infections. Therapeutic drug monitoring (TDM) is warranted to assure the efficacy while limiting toxicity. Although trimethoprim in combination with sulfamethoxazole is the most common combination with established TDM target concentrations, the intravenous formulation is not suited for children because of its additives ethanol and propylene glycol to increase solubility. An alternative can be sulfametrol in combination with trimethoprim. When sulfametrol was introduced in the hospital, there was a need for a TDM method for sulfametrol. METHODS: A High Pressure Liquid Chromatography-Ultraviolet (HPLC-UV) detection method for sulfametrol determination in plasma was developed and validated according to the International Conference on Harmonization guidelines. Linearity, limit of detection, lower limit of quantification, recovery, process efficiency, selectivity, within-run precision, between-run precision, and sample stability were tested. RESULTS: All tested parameters met the required criteria. For linearity, r was 0.9948, lower limit of quantification was 10 mg/L, and limit of detection was 6 mg/L. Recovery was 100.4% and process efficiency 94.4%. Selectivity was met with no interfering peaks at the retention time of 4.2 minutes. Between-run precision and within-run precision were evaluated by replicating quality control levels, resulting in a within-run relative average standard deviation of 0.8% and a between-run relative standard deviation of 2.3%. Recovery of the samples after storing 8 days was 101.9% and recovery of already tested vials was 98.8% after 48 hours. CONCLUSIONS: In conclusion, an HPLC-UV method for sulfametrol determination in human plasma was developed and validated. The method is fast, accurate, reproducible, and has a short analysis time. It is now being used in routine TDM in our clinic.