Ultra-high throughput dual channel liquid chromatography with tandem mass spectrometry for quantification of four immunosuppressants in whole blood for therapeutic drug monitoring.

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is the golden standard for immunosuppressants analyses, where optimising throughput by parallel chromatography can reduce costs and turnaround time. We aimed to double our system throughput using a dual LC-MS/MS setup. Therefore, two independent UPLC systems were hyphenated to one triple quadrupole MS, with staggered injections from one autosampler on alternating columns. The method simultaneously measured the analytes tacrolimus, sirolimus, everolimus, and cyclosporin A in whole blood using isotope dilution, with a run time of 1.5 min. Using the dual LC-MS/MS system, net run-to-run time improved from 2.3 to 0.98 min per injection, where throughput increased from 26 to 61 injections per hour. For Performance Qualification, 1101 clinical samples were measured on the dual LC-MS/MS system in addition to the standard system, during a period of one month, and the results were compared using Passing Bablok regression and Bland Altman analysis. There was excellent agreement for all four analytes, with regression slopes of 0.98-1.02x and intercepts of -0.11-0.88 µg/L. Minor bias was demonstrated between the systems with mean differences from -0.93 to 1.43%. In conclusion, the throughput was doubled and idle MS time was reduced with good agreement to the standard system. Currently, the method is applied for clinical routine with frequent peak intensities of >180 injections per day.

Expeditious quantification of plasma tacrolimus with liquid chromatography tandem mass spectrometry in solid organ transplantation.

Traditionally, tacrolimus is assessed in whole blood samples, but this is suboptimal from the perspective that erythrocyte-bound tacrolimus is not a good representative of the active fraction. In this work, a straightforward and rapid method was developed for determination of plasma tacrolimus in solid organ transplant recipients, using liquid chromatography tandem mass spectrometry (LC-MS/MS) with heated electrospray ionisation. Sample preparation was performed through protein precipitation of 200 µl plasma with 500 µl stable isotopically labelled tacrolimus I.S. in methanol, where 20 µl was injected on the LC-MS/MS system. Separation was done using a chromatographic gradient on a C18 column (50 × 2.1 mm, 2.6 µm). The method was linear in the concentration range 0.05-5.00 µg/L, with within-run and between-run precision in the range 2-6 % and a run time of 1.5 min. Furthermore, the method was validated for selectivity, sensitivity, carry-over, accuracy and precision, process efficiency, recovery, matrix effect, and stability following EMA and FDA guidelines. Clinical validation was performed in 2333 samples from 1325 solid organ transplant recipients using tacrolimus (liver n = 312, kidney n = 1714, and lung n = 307), which had median plasma tacrolimus trough concentrations of 0.10 µg/L, 0.15 µg/L and 0.23 µg/L, respectively. This method is suitable for measurement of tacrolimus in plasma and will facilitate ongoing observational and prospective studies on the relationship of plasma tacrolimus concentrations with clinical outcomes.

Continuous versus intermittent infusion of cefotaxime in critically ill patients: a randomized controlled trial comparing plasma concentrations.

BACKGROUND: In critical care patients, reaching optimal ß-lactam concentrations poses challenges, as infections are caused more often by microorganisms associated with higher MICs, and critically ill patients typically have an unpredictable pharmacokinetic/pharmacodynamic profile. Conventional intermittent dosing frequently yields inadequate drug concentrations, while continuous dosing might result in better target attainment. Few studies address cefotaxime concentrations in this population. OBJECTIVES: To assess total and unbound serum levels of cefotaxime and an active metabolite, desacetylcefotaxime, in critically ill patients treated with either continuously or intermittently dosed cefotaxime. METHODS: Adult critical care patients with indication for treatment with cefotaxime were randomized to treatment with either intermittent dosing (1 g every 6 h) or continuous dosing (4 g/24 h, after a loading dose of 1 g). We defined a preset target of reaching and maintaining a total cefotaxime concentration of 4 mg/L from 1 h after start of treatment. CCMO trial registration number NL50809.042.14, Clinicaltrials.gov NCT02560207. RESULTS: Twenty-nine and 30 patients, respectively, were included in the continuous dosing group and the intermittent dosing group. A total of 642 samples were available for analysis. In the continuous dosing arm, 89.3% met our preset target, compared with 50% in the intermittent dosing arm. Patients not reaching this target had a significantly higher creatinine clearance on the day of admission. CONCLUSIONS: These results support the application of a continuous dosing strategy of ß-lactams in critical care patients and the practice of therapeutic drug monitoring in a subset of patients with higher renal clearance and need for prolonged treatment for further optimization, where using total cefotaxime concentrations should suffice.

Dried Blood Spot Analysis for Therapeutic Drug Monitoring of Clozapine.

BACKGROUND: Schizophrenia is a psychiatric disorder that affects approximately 0.4%-1% of the population worldwide. Diagnosis of schizophrenia is based primarily on Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria. Clozapine is an antipsychotic drug that is mainly used in the treatment of schizophrenia patients who are refractory or intolerant to at least 2 other antipsychotics. Due to the high variability in pharmacokinetics of clozapine, therapeutic drug monitoring (TDM) is highly recommended for clozapine therapy. OBJECTIVE: To develop and clinically validate a novel sampling method using dried blood spot (DBS) to support TDM of clozapine and norclozapine. METHODS: From June 2014 to September 2014, 15 schizophrenia patients (18-55 years) treated with clozapine were included. Plasma, DBS samples made from venous samples (VDBS), and finger prick DBS (DBS) samples were obtained before administration and 2, 4, 6, and 8 hours after clozapine intake. The study was repeated in 6 Russian patients for external validation. Passing-Bablok regression and Bland-Altman analysis were used to compare the DBS, VDBS, and plasma results for clozapine and norclozapine. RESULTS: The DBS validation results showed good linearity over the concentration time curve measured for clozapine and norclozapine. The accuracy and between- and within-day precision variation values were within accepted ranges. Different blood spot volumes and hematocrit values had no significant influence on the results. The DBS samples were stable at 20°C and 37°C for 2 weeks and at -20°C for 2 years. The mean clozapine and norclozapine DBS/plasma ratios were, respectively, 0.80 (95% CI, 0.76 to 0.85) and 1.063 (95% CI, 1.027 to 1.099) in Dutch patients. The mean clozapine DBS/DPS ratio in Russian patients was 0.70 (95% CI, 0.64 to 0.76). CONCLUSION: DBS analysis is a reliable tool for blood sampling and performing TDM of clozapine and norclozapine in daily practice and substantially extends the opportunities for TDM of clozapine.

Pharmacokinetics of Levofloxacin in Multidrug- and Extensively Drug-Resistant Tuberculosis Patients.

Pharmacodynamics are especially important in the treatment of multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB). The free area under the concentration time curve in relation to MIC (fAUC/MIC) is the most relevant pharmacokinetic (PK)-pharmacodynamic (PD) parameter for predicting the efficacy of levofloxacin (LFX). The objective of our study was to assess LFX PK variability in M/XDR-TB patients and its potential consequence for fAUC/MIC ratios. Patients with pulmonary M/XDR-TB received LFX as part of the treatment regimen at a dose of 15 mg/kg administered once daily. Blood samples obtained at steady state before and 1, 2, 3, 4, 7, and 12 h after drug administration were measured by validated liquid chromatography-tandem mass spectrometry. The MIC values of LFX were determined by the agar dilution method on Middlebrook 7H10 and the MGIT960 system. Twenty patients with a mean age of 31 years (interquartile range [IQR] = 27 to 35 years) were enrolled in this study. The median AUC0-24 was 98.8 mg/h/liter (IQR = 84.8 to 159.6 mg/h/liter). The MIC median value for LFX was 0.5 mg/liter with a range of 0.25 to 2.0 mg/liter, and the median fAUC0-24/MIC ratio was 109.5 (IQR = 48.5 to 399.4). In 4 of the 20 patients, the value was below the target value of ≥100. When MICs of 0.25, 0.5, 1.0, and 2.0 mg/liter were applicable, 19, 18, 3, and no patients, respectively, had an fAUC/MIC ratio that exceeded 100. We observed a large variability in AUC. An fAUC0-24/MIC of ≥100 was only observed when the MIC values for LFX were 0.25 to 0.5 mg/liter. Dosages exceeding 15 mg/kg should be considered for target attainment if exposures are assumed to be safe. (This study has been registered at ClinicalTrials.gov under registration no. NCT02169141.).

Determination of bedaquiline in human serum using liquid chromatography-tandem mass spectrometry.

Bedaquiline, a diarylquinoline for the treatment of multidrug-resistant tuberculosis (TB), relies on exposure-dependent killing. As data on drug exposure in specific populations are scarce, pharmacokinetic studies may be of interest. No simple and robust validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been reported to date. Therefore, a new method using a quadrupole mass spectrometer was developed for analysis of bedaquiline and N-monodesmethyl bedaquiline (M2) in human serum, using deuterated bedaquiline as the internal standard. The calibration curve was linear over a range of 0.05 (lower limit of quantification [LLOQ]) to 6.00 mg/liter for both bedaquiline and M2, with correlation coefficient values of 0.997 and 0.999, respectively. The calculated accuracy ranged from 1.9% to 13.6% for bedaquiline and 2.9% to 8.5% for M2. Within-run precision ranged from 3.0% to 7.2% for bedaquiline and 3.1% to 5.2% for M2, and between-run precision ranged from 0.0% to 4.3% for bedaquiline and 0.0% to 4.6% for M2. Evaluation of serum concentrations in a patient receiving bedaquiline showed high levels at the end of treatment, reflecting accumulation of the drug. More observational pharmacokinetic data are needed to relate altered drug concentrations to clinical outcome or adverse drug effects. A simple LC-MS/MS method to quantify bedaquiline and M2 levels in human serum using a deuterated internal standard has been validated. This method can be used in clinical studies and daily practice.

Clinical validation of the analysis of fluconazole in oral fluid in hospitalized children.

Fluconazole is a first-line antifungal agent for the treatment and prophylaxis of invasive candidiasis in pediatric patients. Pediatric patients are at risk of suboptimal drug exposure, due to developmental changes in gastrointestinal and renal function, metabolic capacity, and volume of distribution. Therapeutic drug monitoring (TDM) can therefore be useful to prevent underexposure of fluconazole in children and infants. Children, however, often fear needles and can have difficult vascular access. The purpose of this study was to develop and clinically validate a method of analysis to determine fluconazole in oral fluid in pediatric patients. Twenty-one paired serum and oral fluid samples were obtained from 19 patients and were analyzed using a validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) method after cross-validation between serum and oral fluid. The results were within accepted ranges for accuracy and precision, and samples were stable at room temperature for at least 17 days. A Pearson correlation test for the fluconazole concentrations in serum and oral fluid showed a correlation coefficient of 0.960 (P < 0.01). The mean oral fluid-to-serum concentration ratio was 0.99 (95% confidence interval [CI], 0.88 to 1.10) with Bland-Altman analysis. In conclusion, an oral fluid method of analysis was successfully developed and clinically validated for fluconazole in pediatric patients and can be a noninvasive, painless alternative to perform TDM of fluconazole when blood sampling is not possible or desirable. When patients receive prolonged courses of antifungal treatment and use fluconazole at home, this method of analysis can extend the possibilities of TDM for patients at home.

Simultaneous quantification of anidulafungin and caspofungin in plasma by an accurate and simple liquid chromatography tandem mass-spectrometric method.

INTRODUCTION: Echinocandins are a valuable addition for the treatment of invasive fungal infections, as they are efficacious, demonstrate low toxicity, and have limited drug-drug interactions. In specific clinical situations when altered pharmacokinetics can be expected or dosing guidelines are conflicting, it may be useful to measure concentrations. For this purpose, a liquid chromatography tandem mass-spectrometric method to measure anidulafungin and caspofungin in ethylenediaminetetraacetic acid plasma was developed. METHODS: The method was developed on a Thermo Fisher TSQ Quantum LC-MS/MS. For separation, a BetaBasic C4 (100 mm × 3.0 mm; 5 µm) analytical column was used. Sample preparation consisted of protein precipitation directly in the autosampler vial. The internal standard aculeacin A is structurally related, not used in humans, and commercially available. The method was validated according to the guidelines for bioanalytical method validation of the Food and Drug Administration. RESULTS: The method was accurate (bias ranging from -3.0% to 1.9%) and precise (within-run and between-run coefficients of variation of 2.2% to 7.7% and 1.6% to 9.0%, respectively). All calibration curves were linear over a range of 0.5-10.0 mg/L for anidulafungin and 0.1-20.0 mg/L for caspofungin, and if necessary, samples can be diluted 10-fold. The samples were stable for 3 freeze-thaw cycles, with a bias ranging from 0.6% to 11%. The maximum bias from the worst storage condition, 72 hours at room temperature, was -14.7%. In patient samples, anidulafungin peak concentrations ranged from 2.8 to 8.6 mg/L (n = 20) and trough concentrations ranged from 1.0 to 4.7 mg/L (n = 79). The measured caspofungin concentrations ranged from 1.9 to 7.3 mg/L (n = 20). CONCLUSIONS: The method developed has a straightforward sample preparation and uses a structural analog as the internal standard. This method has been applied successfully for the measurement of anidulafungin and caspofungin concentrations in patient samples, both for clinical practice and for research.

Dried blood spot analysis suitable for therapeutic drug monitoring of voriconazole, fluconazole, and posaconazole.

Invasive aspergillosis and candidemia are important causes of morbidity and mortality in immunocompromised and critically ill patients. The triazoles voriconazole, fluconazole, and posaconazole are widely used for the treatment and prophylaxis of these fungal infections. Due to the variability of the pharmacokinetics of the triazoles among and within individual patients, therapeutic drug monitoring is important for optimizing the efficacy and safety of antifungal treatment. A dried blood spot (DBS) analysis was developed and was clinically validated for voriconazole, fluconazole, and posaconazole in 28 patients. Furthermore, a questionnaire was administered to evaluate the patients' opinions of the sampling method. The DBS analytical method showed linearity over the concentration range measured for all triazoles. Results for accuracy and precision were within accepted ranges; samples were stable at room temperature for at least 12 days; and different hematocrit values and blood spot volumes had no significant influence. The ratio of the drug concentration in DBS samples to that in plasma was 1.0 for voriconazole and fluconazole and 0.9 for posaconazole. Sixty percent of the patients preferred DBS analysis as a sampling method; 15% preferred venous blood sampling; and 25% had no preferred method. There was significantly less perception of pain with the DBS sampling method (P = 0.021). In conclusion, DBS analysis is a reliable alternative to venous blood sampling and can be used for therapeutic drug monitoring of voriconazole, fluconazole, and posaconazole. Patients were satisfied with DBS sampling and had less pain than with venous sampling. Most patients preferred DBS sampling to venous blood sampling.