Ten-year results of an international external quality control programme for measurement of anti-tuberculosis drug concentrations.

OBJECTIVES: Participation in an external (interlaboratory) quality control (QC) programme is an essential part of quality assurance as it provides laboratories with valuable insights into their analytical performance. We describe the 10 year results of an international QC programme for the measurement of anti-tuberculosis (TB) drugs. METHODS: Each year, two rounds were organized in which serum (or plasma) samples, spiked with known concentrations of anti-TB drugs, were provided to participating laboratories for analysis. Reported measurements within 80%-120% of weighed-in concentrations were considered accurate. Mixed model linear regression was performed to assess the effect of the measured drug, concentration level, analytical technique and performing laboratory on the absolute inaccuracy. RESULTS: By 2022, 31 laboratories had participated in the QC programme and 13 anti-TB drugs and metabolites were included. In total 1407 measurements were reported. First-line TB drugs (isoniazid, rifampicin, pyrazinamide and ethambutol) represented 58% of all measurements. Overall, 83.2% of 1407 measurements were accurate, and the median absolute inaccuracy was 7.3% (IQR, 3.3%-15.1%). The absolute inaccuracy was related to the measured anti-TB drug and to the performing laboratory, but not to the concentration level or to the analytical technique used. The median absolute inaccuracies of rifampicin and isoniazid were relatively high (10.2% and 10.9%, respectively). CONCLUSIONS: The 10 year results of this external QC programme illustrate the need for continuous external QC for the measurement of anti-TB drugs for research and patient care purposes, because one in six measurements was inaccurate. Participation in the programme alerts laboratories to previously undetected analytical problems.

Standardization of evaluation method and prognostic significance of histological response to preoperative chemotherapy in high-grade non-round cell soft tissue sarcomas.

BACKGROUND: Preoperative chemotherapy is widely applied to high-grade localized soft tissue sarcomas (STSs); however, the prognostic significance of histological response to chemotherapy remains controversial. This study aimed to standardize evaluation method of histological response to chemotherapy with high agreement score among pathologists, and to establish a cut-off value closely related to prognosis. METHODS: Using data and specimens from the patients who had registered in the Japan Clinical Oncology Group study, JCOG0304, a phase II trial evaluating the efficacy of perioperative chemotherapy with doxorubicin (DOX) and ifosfamide (IFO), we evaluated histological response to preoperative chemotherapy at the central review board. RESULTS: A total of 64 patients were eligible for this study. The percentage of viable tumor area ranged from 0.1% to 97.0%, with median value of 35.7%. Regarding concordance proportion between pathologists, the weighted kappa coefficient (κ) score in all patients was 0.71, indicating that the established evaluation method achieved substantial agreement score. When the cut-off value of the percentage of the residual tumor area was set as 25%, the p-value for the difference in overall survival showed the minimum value. Hazard ratio of the non-responder with percentage of the residual tumor < 25%, to the responder was 4.029 (95% confidence interval 0.893-18.188, p = 0.070). CONCLUSION: The standardized evaluation method of pathological response to preoperative chemotherapy showed a substantial agreement in the weighted κ score. The evaluation method established here was useful for estimating of the prognosis in STS patients who were administered perioperative chemotherapy with DOX and IFO. TRIAL REGISTRATION: UMIN Clinical Trials Registry C000000096. Registered 30 August, 2005 (retrospectively registered).

Replicates Number for Drug Stability Testing during Bioanalytical Method Validation-An Experimental and Retrospective Approach.

BACKGROUND: The stability of a drug or metabolites in biological matrices is an essential part of bioanalytical method validation, but the justification of its sample size (replicates number) is insufficient. The international guidelines differ in recommended sample size to study stability from no recommendation to at least three quality control samples. Testing of three samples may lead to results biased by a single outlier. We aimed to evaluate the optimal sample size for stability testing based on 90% confidence intervals. METHODS: We conducted the experimental, retrospective (264 confidence intervals for the stability of nine drugs during regulatory bioanalytical method validation), and theoretical (mathematical) studies. We generated experimental stability data (40 confidence intervals) for two analytes-tramadol and its major metabolite (O-desmethyl-tramadol)-in two concentrations, two storage conditions, and in five sample sizes (n = 3, 4, 5, 6, or 8). RESULTS: The 90% confidence intervals were wider for low than for high concentrations in 18 out of 20 cases. For n = 5 each stability test passed, and the width of the confidence intervals was below 20%. The results of the retrospective study and the theoretical analysis supported the experimental observations that five or six repetitions ensure that confidence intervals fall within 85-115% acceptance criteria. CONCLUSIONS: Five repetitions are optimal for the assessment of analyte stability. We hope to initiate discussion and stimulate further research on the sample size for stability testing.

Eco-Friendly, Simple, Fast, and Sensitive UPLC-MS/MS Method for Determination of Pexidartinib in Plasma and Its Application to Metabolic Stability.

Pexidartinib is the first drug approved by the U.S. Food and Drug Administration specifically to treat the rare joint tumor tenosynovial giant cell tumor. In the current study, a validated, selective, and sensitive UPLC-MS/MS assay was developed for the quantitative determination of pexidartinib in plasma samples using gifitinib as an internal standard (IS). Pexidartinib and IS were extracted by liquid-liquid extraction using methyl tert-butyl ether and separated on an acquity BEH C18 column kept at 40 °C using a mobile phase of 0.1% formic acid in acetonitrile: 0.1% formic acid in de-ionized water (70:30). The flow rate was 0.25 mL/min. Multiple reaction monitoring (MRM) was operated in electrospray (ESI)-positive mode at the ion transition of 418.06 > 165.0 for the analyte and 447.09 > 128.0 for the IS. FDA guidance for bioanalytical method validation was followed in method validation. The linearity of the established UPLC-MS/MS assay ranged from 0.5 to 1000 ng/mL with r > 0.999 with a limit of quantitation of 0.5 ng/mL. Moreover, the metabolic stability of pexidartinib in liver microsomes was estimated.

Predictive Performance of Bayesian Vancomycin Monitoring in the Critically Ill.

OBJECTIVES: It is recommended that therapeutic monitoring of vancomycin should be guided by 24-hour area under the curve concentration. This can be done via Bayesian models in dose-optimization software. However, before these models can be incorporated into clinical practice in the critically ill, their predictive performance needs to be evaluated. This study assesses the predictive performance of Bayesian models for vancomycin in the critically ill. DESIGN: Retrospective cohort study. SETTING: Single-center ICU. PATIENTS: Data were obtained for all patients in the ICU between 1 January, and 31 May 2020, who received IV vancomycin. The predictive performance of three Bayesian models were evaluated based on their availability in commercially available software. Predictive performance was assessed via bias and precision. Bias was measured as the mean difference between observed and predicted vancomycin concentrations. Precision was measured as the sd of bias, root mean square error, and 95% limits of agreement based on Bland-Altman plots. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 466 concentrations from 188 patients were used to evaluate the three models. All models showed low bias (-1.7 to 1.8 mg/L), which was lower with a posteriori estimate (-0.7 to 1.8 mg/L). However, all three models showed low precision in terms of sd (4.7-8.8 mg/L) and root mean square error (4.8-8.9 mg/L). The models underpredicted at higher observed vancomycin concentrations (bias 0.7-3.2 mg/L for < 20 mg/L; -5.1 to -2.3 for ≥ 20 mg/L) and the Bland-Altman plots showed a great deviation between observed and predicted concentrations. CONCLUSIONS: Bayesian models of vancomycin show not only low bias, but also low precision in the critically ill. Thus, Bayesian-guided dosing of vancomycin in this population should be used cautiously.

Review of the Preanalytical Errors That Impact Therapeutic Drug Monitoring.

PURPOSE: Preanalytical errors comprise the majority of testing errors experienced by clinical laboratories and significantly impact the accuracy of therapeutic drug monitoring (TDM). METHODS: Specific preanalytical factors in sample timing, collection, transport, processing, and storage that lead to errors in TDM were reviewed. We performed a literature search using several scientific databases including PubMed, ScienceDirect, Scopus, Web of Science, and ResearchGate for human studies published in the English language from January 1980 to February 2021, reporting on TDM and the preanalytical phase. RESULTS: Blood collection errors (ie, wrong anticoagulant/clot activator used, via an intravenous line, incorrect time after dosing) delay testing, cause inaccurate results, and adversely impact patient care. Blood collected in lithium heparin tubes instead of heparin sodium tubes produce supertoxic lithium concentrations, which can compromise care. Specimens collected in serum separator gel tubes cause falsely decreased concentrations due to passive absorption into the gel when samples are not processed and analyzed quickly. Dried blood spots are popular for TDM as they are minimally invasive, allowing for self-sampling and direct shipping to a clinical laboratory using regular mail. However, blood collection techniques, such as trauma to the collection site, filter paper fragility, and hematocrit (Hct) bias, can adversely affect the accuracy of the results. Volumetric absorptive microsampling is a potential alternative to dried blood spot that offers fast, volume-fixed sampling, low pain tolerance, and is not susceptible to Hct concentrations. CONCLUSIONS: The identification of preanalytical factors that may negatively impact TDM is critical. Developing workflows that can standardize TDM practices, align appropriate timing and blood collection techniques, and specimen processing will eliminate errors.

Development and Validation of a Simple and Rapid Ultrahigh-Performance Liquid Chromatography Tandem Spectrometry Method for the Quantification of Hydroxychloroquine in Plasma and Blood Samples in the Emergency Context of SARS-CoV-2 Pandemic.

ABSTRACT: Therapeutic drug monitoring of hydroxychloroquine (HCQ) has been recommended to optimize the treatment of patients with COVID-19. The authors describe an ultrahigh-performance liquid chromatography tandem spectrometry method developed in a context of emergency, to analyze HCQ in both human plasma and blood samples. After adding the labeled internal standard and simple protein precipitation, plasma samples were analyzed using a C18 column. Blood samples required evaporation before analysis. The total chromatographic run time was 4 minutes (including 1.5 minutes of column equilibration). The assay was linear over the calibration range (r2 > 0.99) and up to 1.50 mcg/mL for the plasma samples (5.00 mcg/mL for the blood matrix). The limit of quantification was 0.0150 mcg/mL for plasma samples (0.05 mcg/mL blood matrix) with accuracy and precision ranging from 91.1% to 112% and from 0.750% to 11.1%, respectively. Intraday and interday precision and accuracy values were within 15.0%. No significant matrix effect was observed in the plasma or blood samples. This method was successfully applied to patients treated for COVID-19 infection. A simple and rapid ultrahigh-performance liquid chromatography tandem spectrometry method adapted to HCQ therapeutic drug monitoring in the context of SARS-CoV-2 infection was successfully developed and validated.

Performance Assessment of a Multifaceted Unfractionated Heparin Dosing Protocol in Adult Patients on Extracorporeal Membrane Oxygenator.

BACKGROUND: The use of extracorporeal membrane oxygenator (ECMO) support devices are associated with complications, including bleeding and thrombosis. Unfractionated heparin (UFH) is the gold standard anticoagulant in ECMO patients. Clinically, UFH is monitored through activated clotting time (ACT), activated partial thromboplastin time (aPTT), and anti-factor Xa assay. It is unknown which assay best predicts anticoagulation effects in adults. OBJECTIVE: To assess the correlation of UFH dosing and monitoring using an established protocol. METHODS: A pilot, prospective cohort, historically controlled study was conducted at a tertiary care hospital. Patients ≥18 years-old who received ECMO on the multifaceted anticoagulation protocol were included and compared with those on the conventional method of anticoagulation. The primary end point was to assess the correlation between UFH dose and different monitoring methods throughout 72 hours using the new protocol guided by ACT and anti-factor Xa assay. RESULTS: In each arm, 20 patients were enrolled. The study revealed that anti-factor Xa assay had the largest number of "strong" correlations 11/20 (55%), followed by both aPTT and aPTT ratio 10/20 (50%), and, finally, ACT 2/20 (10%). Concordance between anti-factor Xa assay and the other monitoring parameters in the prospective arm was generally low: 31% with aPTT ratio, 26% with ACT, and 23% with aPTT. CONCLUSION AND RELEVANCE: The adaption of a multifaceted anticoagulation protocol using anti-factor Xa assay may provide a better prediction of heparin dosing in adults ECMO patients compared with the conventional ACT-based protocol. Further studies are needed to assess the safety and different monitoring modalities.

Effectiveness of a Calculation-Free Weight-Based Unfractionated Heparin Nomogram With Anti-Xa Level Monitoring Compared With Activated Partial Thromboplastin Time.

BACKGROUND: Accurate monitoring of intravenous unfractionated heparin (UFH) is essential to mitigate the risk of adverse drug events associated with dosing errors. Although recent data support anti-factor Xa (anti-Xa) monitoring preferentially over activated partial thromboplastin time (aPTT) to improve time to therapeutic anticoagulation, the utility of incorporating anti-Xa monitoring with a calculation-free weight-based UFH nomogram has not been formally evaluated. OBJECTIVE: The primary objective of this study was to evaluate the time to therapeutic anticoagulation of a calculation-free weight-based UFH nomogram integrated with anti-Xa monitoring versus a historical control of aPTT monitoring utilizing manual dose calculations. METHODS: This was a retrospective analysis of patients with anti-Xa monitoring and a novel calculation-free weight-based UFH nomogram compared with a historical control with aPTT monitoring and manual calculations. RESULTS: A total of 103 patients in the aPTT cohort and 100 patients in the anti-Xa cohort were analyzed. The anti-Xa cohort achieved goal therapeutic target 3.8 hours sooner than the aPTT cohort (P = 0.03). Patients with anti-Xa monitoring required 1 fewer adjustment per 2.5 patient-days of UFH with the venous thromboembolism nomogram (P = 0.02). Patients in the aPTT cohort required more infusion interruptions because of supratherapeutic values (P = 0.007) and boluses because of subtherapeutic values (P = 0.044). There were no differences in rates of thromboembolism, major bleeding, or clinically relevant nonmajor bleeding between the cohorts. CONCLUSION AND RELEVANCE: This study demonstrated that anti-Xa UFH monitoring integrated with a calculation-free nomogram results in faster time to therapeutic anticoagulation and fewer dose adjustments compared with aPTT monitoring with manual calculations.

Methotrexate Monitoring in Dermatology- A Retrospective Cohort Study.

BACKGROUND AND OBJECTIVES: There are currently no evidence-based recommendations to guide lab monitoring in the first 90 days of methotrexate treatment. The purpose of this study was to determine whether certain monitoring practices or baseline patient characteristics were associated with increased risk of developing clinically meaningful lab abnormalities during the course of methotrexate treatment. PATIENTS AND METHODS: This retrospective cohort study analyzed 243 dermatologically managed patients taking methotrexate at the University of Virginia Health System. Odds ratios were used to analyze the risk of these patients developing lab abnormalities that result in a change in clinical management, referred to as clinically relevant events. Chi-square analysis was used to determine the optimal timing of methotrexate lab monitoring. RESULTS: A diagnosis of congestive heart failure (P=0.03), chronic kidney disease (P=0.03), and an initial low platelet count (P=0.008) increased the odds of developing a clinically relevant event at some point during methotrexate therapy. In the first 15 days following methotrexate initiation, only 1/114 (0.9%) lab draws resulted in discontinuation of the medicine, 1/114 (0.9%) resulted in maintenance of a stable dose, and 2/114 (1.8%) resulted in repeat laboratory testing. CONCLUSION: In the absence of concerning baseline patient characteristics, dermatologists may consider postponing initial lab monitoring until 15 days post methotrexate initiation.J Drugs Dermatol. 2021;20(3):320-325. doi:10.36849/JDD.5790.

Determination of the Peptide AWRK6 in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and Its Application to Pharmacokinetics.

AWRK6 was a synthesized peptide developed based on the natural occurring peptide dybowskin-2CDYa, which was discovered in frog skin in our previous study. Here, a quantitative determination method for AWRK6 analysis in rat plasma by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established and validated following U.S. FDA guidelines. A combination of plasma precipitation and liquid-liquid extraction was applied for the extraction. For pharmacokinetics study, the rats were administrated with AWRK6 via intraperitoneal and intravenous injection. The prepared plasma samples were separated on an ODS column and analyzed by tandem MS using precursor-to-product ion pairs of m/z: 533.4→84.2 for AWRK6 and m/z: 401.9→101.1 for internal standard Polymyxin B sulfate in multiple reaction monitoring mode. AWRK6 concentrations in rat plasma peaked at about 1.2 h after intraperitoneal injections at 2.35, 4.7 and 9.4 mg/kg bodyweight. The terminal half-life was around 2.8 h. The absolute bioavailability of AWRK6 was 50% after 3 doses via injection, and the apparent volume of distribution was 4.884 ± 1.736 L. The obtained determination method and pharmacokinetics profiles of AWRK6 provides a basis for further development, and forms a benchmark reference for peptide quantification.

Comparison of Two Immunoassay Screening Methods and a LC-MS/MS in Detecting Traditional and Designer Benzodiazepines in Urine.

Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.

Precision psychiatry in clinical practice.

The treatment of depression represents a major challenge for healthcare systems and choosing among the many available drugs without objective guidance criteria is an error-prone process. Recently, pharmacogenetic biomarkers entered in prescribing guidelines, giving clinicians the possibility to use this additional tool to guide prescription and improve therapeutic outcomes. This marked an important step towards precision psychiatry, which aim is to integrate biological and environmental information to personalise treatments. Only genetic variants in cytochrome enzymes are endorsed by prescribing guidelines, but in the future polygenic predictors of treatment outcomes may be translated into the clinic. The integration of genetics with other relevant information (e.g., concomitant diseases and treatments, drug plasma levels) could be managed in a standardised way through ad hoc software. The overcoming of the current obstacles (e.g., staff training, genotyping and informatics facilities) can lead to a broad implementation of precision psychiatry and represent a revolution for psychiatric care.Key pointsPrecision psychiatry aims to integrate biological and environmental information to personalise treatments and complement clinical judgementPharmacogenetic biomarkers in cytochrome genes were included in prescribing guidelines and represented an important step towards precision psychiatryTherapeutic drug monitoring is an important and cost-effective tool which should be integrated with genetic testing and clinical evaluation in order to optimise pharmacotherapyOther individual factors relevant to pharmacotherapy response (e.g., individual's symptom profile, concomitant diseases) can be integrated with genetic information through artificial intelligence to provide treatment recommendationsThe creation of pharmacogenetic services within healthcare systems is a challenging and multi-step process, education of health professionals, promotion by institutions and regulatory bodies, economic and ethical barriers are the main issues.

Core Recommendations for Antifungal Stewardship: A Statement of the Mycoses Study Group Education and Research Consortium.

In recent years, the global public health community has increasingly recognized the importance of antimicrobial stewardship (AMS) in the fight to improve outcomes, decrease costs, and curb increases in antimicrobial resistance around the world. However, the subject of antifungal stewardship (AFS) has received less attention. While the principles of AMS guidelines likely apply to stewarding of antifungal agents, there are additional considerations unique to AFS and the complex field of fungal infections that require specific recommendations. In this article, we review the literature on AMS best practices and discuss AFS through the lens of the global core elements of AMS. We offer recommendations for best practices in AFS based on a synthesis of this evidence by an interdisciplinary expert panel of members of the Mycoses Study Group Education and Research Consortium. We also discuss research directions in this rapidly evolving field. AFS is an emerging and important component of AMS, yet requires special considerations in certain areas such as expertise, education, interventions to optimize utilization, therapeutic drug monitoring, and data analysis and reporting.

Evaluation of Hepatitis B Reactivation Among Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Hepatitis B reactivation (HBVr) in cancer patients is a well-established complication due to chemotherapy-induced immunosuppression. Studies have reported HBVr associated with immunosuppressive medications, such as rituximab, methotrexate, and high dose steroids. There are different risks for different types of chemotherapy with rituximab carrying one of the highest risks for hepatitis B reactivation. Tyrosine kinase inhibitors (TKIs) are the standard of care in patients with chronic myeloid leukemia (CML). The risk of HBVr in chronic myeloid leukemia has been reported in many studies, but to this date, there are no clear guidelines or recommendations regarding screening and monitoring of HBV in CML patients receiving TKIs. We conducted this review to identify the risk of HBVr in patients with CML who are treated with tyrosine kinase inhibitors. We recommend testing for HBV status in patients who are to be treated with TKIs and to consider giving prophylaxis in those who are positive for HBsAg at baseline. More studies are needed to assess the risk of reactivation in patients with Hepatitis B core antibody positive receiving TKIs. Currently, monitoring such patients for reactivation may be the best strategy.

No Concluding Evidence on Optimal Activated Clotting Time for Non-cardiac Arterial Procedures.

OBJECTIVES: Heparin has a non-predictable effect in the individual patient. The activated clotting time (ACT) is used to measure the level of anticoagulation after administration of heparin. To date, appropriate heparin dose protocols and corresponding therapeutic ACT values have not been established in non-cardiac arterial procedures (NCAP). The aim of this review was to study the use of ACT monitoring during NCAP, and whether an optimal ACT could be determined based on the fewest arterial thrombo-embolic complications (ATEC) and bleeding complications. METHODS: This systematic review was performed in accordance with the PRISMA Guidelines. A systematic search was conducted in MEDLINE, EMBASE, and the Cochrane database. Any associations were evaluated between peri-procedural ACT levels and ATEC and bleeding complications detected during the same admission as the primary procedure or during 30 day follow up. Also, heparin dose protocols, peri-procedural target ACTs, different ACT devices, protamine use and pre-, peri-, and post-procedural anticoagulation therapy were evaluated. RESULTS: In total, 21 studies with 3982 patients were included, on both open and endovascular NCAP. Four studies were primarily designed to correlate peak peri-procedural ACT with clinical outcomes; however, the definitions of the results and the clinical outcomes were too heterogeneous for analysis. There was major variability in all studied aspects of ACT measurement, heparin and protamine use, and in the type of procedures in the included studies. Overall methodological quality of the included studies was poor. No randomised controlled trials were found. Studies were at a high risk of bias. CONCLUSIONS: This systematic review demonstrates a lack of data and no consensus in the literature concerning the optimal ACT, and the possible association with haemorrhagic complications and ATEC during NCAP.

Long-Term Performance of Laboratory-Developed Liquid Chromatography-Tandem Mass Spectrometry Tests and a Food and Drug Administration-Approved Immunoassay for the Therapeutic Drug Monitoring of Everolimus.

BACKGROUND: Laboratory-developed tests (LDTs) are analytical tests developed and validated "in-house" for clinical diagnosis. Regulatory agencies, such as the United States Food and Drug Administration (FDA), encourage using regulatory-approved assays rather than LDTs. In the ongoing Zortracker everolimus study, samples were provided monthly to participating clinical laboratories that conduct therapeutic drug monitoring of everolimus. This allowed for the long-term (6-year) comparison of the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays, which are LDTs, to the FDA-approved everolimus Quantitative Microsphere System (QMS). METHODS: Each laboratory received the same 3 blinded samples. LC-MS/MS and QMS assays were compared using Passing Bablok regression analysis. Data were analyzed in 12-month periods to detect trends over time. RESULTS: The slopes of the Passing Bablok regression curves remained unchanged in 2013 and 2014 (reference LC-MS/MS; test QMS: slope = 0.934 and 1.008). However, by 2016, the slope increased significantly to between 1.111 and 1.320, then dropped to 0.980 in 2017 and 0.912 in 2018, suggestive of changes in QMS bias compared with LC-MS/MS over longer periods. Outliers did not affect these results. The interlaboratory variability of LC-MS/MS and QMS remained unchanged from 2013 to 2015, with coefficients of variation of 15.3%, 18.4%, and 17.2% for LC-MS/MS, and 13.0%, 13.1%, and 15.3% for QMS, respectively, per year. At the end of the observation period, the average coefficients of variation in LC-MS/MS laboratories dropped to 14.3%, 12.6%, and 14.2%, whereas the variability in QMS laboratories was 19.5%, 13.3%, and 19.6% in 2016, 2017, and 2018, respectively. CONCLUSIONS: Initially, QMS everolimus concentrations in patient samples were comparable with those detected in LC-MS/MS laboratories. However, concentration bias of the QMS assay significantly changed within 6 years, emphasizing the need for long-term, independent performance tracking of therapeutic drug monitoring assays, including FDA-approved assays.

Comparison of 4 Commercial Immunoassays Used in Measuring the Concentration of Tacrolimus in Blood and Their Cross-Reactivity to Its Metabolites.

BACKGROUND: Therapeutic drug monitoring of tacrolimus is necessary for appropriate dose adjustment for a successful immunosuppressive therapy. Several commercial immunoassays are available for tacrolimus measurements. This study aimed at simultaneously evaluating the analytical performances of 4 such immunoassays, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a standard. For the first time, cross-reactivity to tacrolimus metabolites was assessed at concentrations frequently observed in clinical settings, as opposed to the higher concentrations tested by assay manufacturers. METHODS: An affinity column-mediated immunoassay (ACMIA), using upgraded flex reagents; released in 2015, a chemiluminescence immunoassay (CLIA), an electrochemiluminescence immunoassay (ECLIA), and a latex agglutination turbidimetric immunoassay (LTIA) were evaluated using frozen whole blood samples collected from transplantation patients. Cross-reactivities to 3 major tacrolimus metabolites (13-O-demethyl-tacrolimus [M-I], 31-O-demethyl-tacrolimus [M-II], and 15-O-demethyl-tacrolimus [M-III]) were evaluated. RESULTS: Each immunoassay correlated well with LC-MS/MS, and the Pearson's correlation coefficients (R) were 0.974, 0.977, 0.978, and 0.902 for ACMIA, CLIA, ECLIA, and LTIA, respectively. Using Bland-Altman difference plots to compare the immunoassays with LC-MS/MS, the calculated average biases were -6.73%, 6.07%, 7.46%, and 12.27% for ACMIA, CLIA, ECLIA, and LTIA, respectively. The cross-reactivities of ACMIA to the tacrolimus metabolites M-II and M-III were 81% and 78%, respectively, when blood was spiked at 2 ng/mL, and 94% and 68%, respectively, when it was spiked at 5 ng/mL. CONCLUSIONS: Each immunoassay was useful, but had its own characteristics. ACMIA cross-reactivities to M-II and M-III were much higher than the respective 18% and 15% reported on its package insert, suggesting that cross-reactivity should be examined at clinically relevant concentrations.

Determination of Perampanel in Dried Plasma Spots: Applicability to Therapeutic Drug Monitoring.

BACKGROUND: Although therapeutic drug monitoring of antiepileptic drugs is typically based on the analysis of plasma samples, alternative matrices, such as dried plasma spots (DPSs), may offer specific advantages. The aims of this work were to (1) develop and validate a bioanalytical method for the quantitative determination of the second-generation antiepileptic drug perampanel in DPSs; (2) assess short- and long-term stability of perampanel in DPSs; and (3) test the clinical applicability of the developed method. METHODS: Two hundred microliters of plasma were dispensed on a glass paper filter and dried. Glass paper filter discs were then inserted into clean tubes. After addition of the internal standard (ie, promethazine), the analytes were extracted with 5-mL methanol, dried at room temperature (23 ± 2°C), and reconstituted. Separation and quantification were achieved on 2 serial reverse-phase monolithic columns connected to an UV detector (λ = 320 nm). RESULTS: Calibration curves were linear in the validated concentration range (25-1000 ng/mL). Intraday and interday accuracy were in the range of 99.2%-111.4%, whereas intraday and interday precision (coefficient of variation) ranged from 2.8% to 8.6%. The lowest limit of quantitation was 25 ng/mL. The stability of the analyte in DPSs was assessed and confirmed under different storage conditions. Perampanel concentrations estimated in DPS samples from patients receiving therapeutic doses were equivalent to those measured in plasma samples. CONCLUSIONS: This simple method enables the quantitation of perampanel in DPSs with adequate accuracy, precision, specificity, and sensitivity. The short- and long-term stabilities of perampanel in DPSs are highly beneficial for sample shipment or storage at ambient temperature. Moreover, DPSs decreases the costs associated with storage and transportation compared with conventional wet samples.

Development and Validation of an UHPLC-MS/MS Assay for the Therapeutic Monitoring of Brivaracetam Plasma Concentrations in Patients with Epilepsy.

BACKGROUND: Brivaracetam is an antiepileptic drug used as an add-on therapy for partial-onset seizures in subjects aged 4 years and older. Owing to potential drug interactions and intersubject variability in plasma concentrations, therapeutic monitoring for brivaracetam may be useful. The aim of this study was to develop a simplified method for measuring brivaracetam plasma concentrations applicable to therapeutic drug monitoring in epilepsy. METHODS: An ultra high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated according to current guidelines for bioanalytical methods. Sample preparation (100 µL) involved only a simple precipitation step by acetonitrile. Brivaracetam-d7 was used as internal standard. The chromatographic analysis was performed by a Synergi Fusion column using 0.1% formic acid in water/acetonitrile as a binary gradient mobile phase, at a flow rate of 0.3 mL/min. Both brivaracetam and the internal standard eluted at 1.01 minutes. This method was applied to measure trough and 1-hour postmorning dose brivaracetam plasma concentrations of 11 patients with epilepsy. RESULTS: The method was validated over a concentration range of 0.10-10 mcg/mL. The mean recovery was 95%. Both intra- and inter-assay imprecision and inaccuracy were <15% for all quality control samples. The lower limit of quantitation and detection was 0.10 and 0.05 mcg/mL, respectively. No interferences or carry-over was observed. Median (25%-75% quartiles) trough and 1-hour postdosing brivaracetam plasma concentrations were 0.61 mcg/mL (0.47-0.83 mcg/mL) and 1.55 mcg/mL (1.24-2.12 mcg/mL), respectively, at a median dose of 80 mg/d (50-150 mg/d). Large, up to 8-fold, intrasubject fluctuations of brivaracetam concentrations between trough and 1-hour postdosing were observed. CONCLUSIONS: The present assay is faster and simpler than previously published analytical reports for brivaracetam in human plasma and is suitable for therapeutic drug monitoring.