Assay For Therapeutic Drug Monitoring Research Articles

Analytical performance evaluation of two therapeutic drug monitoring assays on the Atellica CI analyzer

Analytical performance evaluation of two therapeutic drug monitoring assays on the Atellica CI analyzer

A high performance liquid chromatography-tandem mass spectrometry assay for therapeutic drug monitoring of 10 drug compounds commonly used for antimicrobial therapy in plasma and serum of critically ill patients: Method optimization, validation, cross-validation and clinical application

Background and aimsIntensive care antibiotic treatment faces challenges due to substantial pharmacokinetic differences in critically ill patients. Individualized antibiotic dosing guided by therapeutic drug monitoring (TDM) is considered to minimize the risk of treatment failure and toxicity. This study aimed to develop a valid method for simultaneous LC-MS/MS quantification of 10 drugs frequently used in intensive care antibiotic therapy for which TDM-guided dosing is recommended: piperacillin, meropenem, flucloxacillin, cefuroxime, vancomycin, colistin A and B, linezolid, ciprofloxacin and tazobactam. Methods and ResultsThorough optimization of sample preparation and chromatography resulted in a fast and simple method based on protein precipitation of 50 µL plasma or serum and gradient elution using an Acquity UPLC HSS-T3 column. Electrospray ionization-triple quadrupole mass spectrometry in dynamic multiple reaction monitoring was used for quantification, covering the therapeutic range of each drug compound. Validation following EMA and FDA recommendations, including inter-platform validation and inter-laboratory comparison, demonstrated high accuracy, precision and robustness of the new method. The assay was successfully used to monitor plasma antibiotic levels of critically ill patients (n = 35). ConclusionThe established multiplex method covers major drug classes with documented dosing challenges, provides a reliable basis for the implementation of high-throughput TDM, and its application confirmed the clinical impact of TDM in a real-world setting.

Clinical feasibility of a label-free SERS assay for therapeutic drug monitoring of methotrexate

We present a therapeutic drug monitoring (TDM) feasibility study using a label-free surface-enhanced Raman spectroscopy (SERS)-based assay for quantifying methotrexate (MTX) from samples collected from children with acute lymphoblastic leukemia undergoing high-dose MTX therapy. We show that, when combined with an appropriate sample preparation (solid phase extraction) and multivariate data analysis (partial least squares regression), the SERS assay demonstrated a good correlation with a reference chromatographic method (r = 0.889, p < 0.005). We also found that the SERS-based approach underestimated MTX concentration, with a 10% bias calculated, but both methods showed similar variability (RSD of 8.4% for HPLC and 11.6% for SERS). The presented results are strong evidence that SERS can be used for TDM of MTX, and this work brings us one step closer to implementing a SERS-based assay in clinics.

Production of antibody and development of enzyme-linked immunosorbent assay for therapeutic drug monitoring of eravacycline

Eravacycline (ERC) was approved for clinical use in 2018. It is more potent than other tetracyclines and can overcome resistance, making it an attractive option for combating multidrug-resistant bacterial infections. Intensive pharmacokinetic (PK) studies are currently being conducted to ensure the effectiveness and safety of ERC in various groups of patients, including those undergoing extracorporeal therapies. This study is the first attempt to develop a simple, efficient, and high-throughput immunoassay for quantifying ERC in human or animal serum. BSA-ERC conjugate as immunogen elicited antibody production in rabbits. Monitoring of the immune response and comparison of homologous and heterologous coating antigens allowed selection of immunoreagents and development of an assay that was selective for ERC possessing sensitivity (IC50), dynamic range (IC20-IC80) and detection limit equal to 3.3 ng/mL, 0.27–54 ng/mL and 0.09 ng/mL, respectively. The developed ELISA showed acceptable recovery of ERC (85–105 %) from rabbit and human serum in the clinically relevant concentration range of 0.1–3.0 mg/L. The method was used to quantify serum ERC concentration in the pilot PK study in Soviet chinchilla rabbits. The results were confirmed by HPLC-MS/MS.

Valproic acid monitoring: Serum prediction using a machine learning framework from multicenter real-world data

BackgroundOur study employs machine learning to predict serum valproic acid (VPA) concentrations, aiming to contribute to the development of non-invasive assays for therapeutic drug monitoring. MethodsMedical records from 2002 to 2019 were obtained from the Taiwan Chang Gung Research Database. Using various machine learning algorithms, we developed predictive models to classify serum VPA concentrations into two categories (1–50 μg/ml or 51–100 μg/ml) and predicted the exact concentration value. The models were trained on 5142 samples and tested on 644 independent samples. Accuracy was the main metric used to evaluate model performance, with a tolerance of 20 μg/ml for continuous variables. Furthermore, we identified important features and developed simplified models with fewer features. ResultsThe models achieved an average accuracy of 0.80–0.86 for binary outcomes and 0.72–0.88 for continuous outcome. Ten top features associated with higher serum VPA levels included higher VPA last and daily doses, bipolar disorder or schizophrenia spectrum disorder diagnoses, elevated levels of serum albumin, calcium, and creatinine, low platelet count, low percentage of segmented white blood cells, and low red cell distribution width-coefficient of variation. The simplified models had an average accuracy of 0.82–0.86 for binary outcome and 0.70–0.86 for continuous outcome. LimitationsThe study's predictive model lacked external test data from outside the hospital for validation. ConclusionsMachine learning models have the potential to integrate real-world data and predict VPA concentrations, providing a promising tool for reducing the need for frequent monitoring of serum levels in clinical practice.

A Single-Run HPLC-MS Multiplex Assay for Therapeutic Drug Monitoring of Relevant First- and Second-Line Antibiotics in the Treatment of Drug-Resistant Tuberculosis.

The treatment of drug-resistant Mycobacterium tuberculosis relies on complex antibiotic therapy. Inadequate antibiotic exposure can lead to treatment failure, acquired drug resistance, and an increased risk of adverse events. Therapeutic drug monitoring (TDM) can be used to optimize the antibiotic exposure. Therefore, we aimed to develop a single-run multiplex assay using high-performance liquid chromatography-mass spectrometry (HPLC-MS) for TDM of patients with multidrug-resistant, pre-extensively drug-resistant and extensively drug-resistant tuberculosis. A target profile for sufficient performance, based on the intended clinical application, was established and the assay was developed accordingly. Antibiotics were analyzed on a zwitterionic hydrophilic interaction liquid chromatography column and a triple quadrupole mass spectrometer using stable isotope-labeled internal standards. The assay was sufficiently sensitive to monitor drug concentrations over five half-lives for rifampicin, rifabutin, levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, terizidone/cycloserine, ethambutol, delamanid, pyrazinamide, meropenem, prothionamide, and para-amino salicylic acid (PAS). Accuracy and precision were sufficient to support clinical decision making (≤±15% in clinical samples and ±20-25% in spiked samples, with 80% of future measured concentrations predicted to fall within ±40% of nominal concentrations). The method was applied in the TDM of two patients with complex drug-resistant tuberculosis. All relevant antibiotics from their regimens could be quantified and high-dose therapy was initiated, followed by microbiological conversion. In conclusion, we developed a multiplex assay that enables TDM of the relevant first- and second-line anti-tuberculosis medicines in a single run and was able to show its applicability in TDM of two drug-resistant tuberculosis patients.

A supervised machine-learning approach for the efficient development of a multi method (LC-MS) for a large number of drugs and subsets thereof: focus on oral antitumor agents.

Accumulating evidence argues for a more widespread use of therapeutic drug monitoring (TDM) to support individualized medicine, especially for therapies where toxicity and efficacy are critical issues, such as in oncology. However, development of TDM assays struggles to keep pace with the rapid introduction of new drugs. Therefore, novel approaches for faster assay development are needed that also allow effortless inclusion of newly approved drugs as well as customization to smaller subsets if scientific or clinical situations require. We applied and evaluated two machine-learning approaches i.e.,a regression-based approach and an artificial neural network (ANN) to retention time (RT) prediction for efficient development of a liquid chromatography mass spectrometry (LC-MS) method quantifying 73 oral antitumor drugs (OADs) and five active metabolites. Individual steps included training, evaluation, comparison, and application of the superior approach to RT prediction, followed by stipulation of the optimal gradient. Both approaches showed excellent results for RT prediction (mean difference ± standard deviation: 2.08 % ± 9.44 % ANN; 1.78 % ± 1.93 % regression-based approach). Using the regression-based approach, the optimum gradient (4.91 % MeOH/min) was predicted with a total run time of 17.92 min. The associated method was fully validated following FDA and EMA guidelines. Exemplary modification and application of the regression-based approach to a subset of 14 uro-oncological agents resulted in a considerably shortened run time of 9.29 min. Using a regression-based approach, a multi drug LC-MS assay for RT prediction was efficiently developed, which can be easily expanded to newly approved OADs and customized to smaller subsets if required.

Surface plasmon resonance assays for the therapeutic drug monitoring of infliximab indicate clinical relevance of anti-infliximab antibody binding properties.

The therapeutic antibody infliximab (IFX) has improved the life quality of numerous autoinflammatory disease patients. However, IFX can trigger the generation ofanti-drug antibodies (ADA), whose optimal evaluation andmanagement are currently subject of controversial discussions. We present two novel surface plasmon resonance (SPR) biosensor assays for therapeutic drug monitoring of IFX and characterization of ADA and investigated the diagnostic value of ADA binding properties. IFX and ADA were quantified via developed SPR biosensor assays (IFXmon and ADAmon, respectively) and diagnostics-approved ELISA in sera from inflammatory bowel disease patients. Pre-analytic ADA enrichment with magnetic beads enabled analytical drug tolerance of the ADAmon assay. The dissociation ratio (DissR) as an index forADA:IFX binding stability was calculated from the SPR sensorgrams of ADA quantification runs. IFX levels determined by IFXmon assay and ELISA showed high agreement, whereas ADA quantification concordance between ADAmon assay and ELISA was poor. In patients, DissR was predominantly constant over time and differed significantly between therapy outcomes. A DissR cut-off of 1.524 indicated undetectable IFX levels with 71.4% sensitivity and 88.9% specificity. Additionally, the SPR reference surface was exploited as serum-individual negative control to check result plausibility within multi-sample run sequences. Overall, both SPR biosensor assays exhibited reliable quantitative performance with accuracies superior to their ELISA counterparts and precision inferior to ELISA only for ADAmon. DissR presented itself as promising ADA binding parameter and could contribute to both earlier and more tailored therapeutic decisions.

Teaching a New Dog Old Tricks: Ultrafast Liquid Chromatography/Tandem-Mass Spectrometry Analysis of Nine ß-Lactam Antibiotics for Improved Therapeutic Drug Monitoring.

Therapeutic drug monitoring (TDM) of β-lactam antibiotics provides critical knowledge in hospital intensive care unit environments to support dosing within the narrow window between therapeutic failure and toxicity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the most suitable analytical technique for these drugs; however, clinicians, patients, and laboratories would benefit from shortening the timeframe between the collection of samples and reporting of results. The authors developed a very rapid LC-MS/MS method for 9 β-lactam antimicrobial drugs on a commercial core-shell reverse-phase LC column by exploiting the performance of such stationary phase materials at a high mobile-phase linear velocity and using a simple flow split to optimize ionization conditions in the mass spectrometer ion source. The method's performance was assessed using a currently validated routine LC-MS/MS assay performed on the same instrument. Routine ß-lactam assays were reduced from >6 minutes per sample to less than 2 minutes with improved chromatographic resolution, while still maintaining acceptable analytical performance (average correlation coefficient: 0.99670, interday imprecision: 2.0%-10.8%, and bias: -1.68%), hence generating results in agreement with an existing validated method for patient and quality assurance program samples. Time-critical results, such as those for β-lactam antimicrobials, may be reported by the TDM laboratory several hours earlier than current methods allow, providing improved patient care and generating capacity on LC-MS/MS instruments for larger batch sizes and/or additional assays. The simple-to-implement technique demonstrated in this study may be applicable to other TDM assays or any LC-MS/MS method where faster turnaround times are desirable.

Evaluation of the interchangeability between the new fully-automated affinity chrome-mediated immunoassay (ACMIA) and the Quantitative Microsphere System (QMS) with a CE-IVD-certified LC-MS/MS assay for therapeutic drug monitoring of everolimus after solid organ transplantation.

This study aims to evaluate the interchangeability between the Siemens Healthineers' "EVRO" new affinity chrome-mediated immunoassay (ACMIA/EVRO) and Thermo Fisher Scientific's "EVER" Quantitative Microsphere System (QMS/EVER) with Chromsystems' CE-IVD-certified "MassTox" liquid-chromatography/tandem-mass spectrometry (LC-MS/MS) assay for the therapeutic drug monitoring of everolimus. A single lot of reagent, calibrators and controls were used for each assay. A total of 67 whole blood samples (n=67) from patients receiving solid organ transplant were analyzed (n=31 with kidney transplant and n=36 with liver transplant); Passing-Bablok regression and Bland-Altman difference plot were used to evaluate bias and individual agreement; LC-MS/MS analysis was used to measure the actual concentrations of calibrators and controls compared to the assigned value. ACMIA/EVRO did not show any systematic bias compared to LC-MS/MS (intercept=0.244ng/mL, 95% CI:-0.254 to 0.651ng/mL). Nevertheless, significant proportional bias (slope=1.511, 95% CI: 1.420 to 1.619) associated to a combined bias of 44.8% (95% CI: 41.2-48.3%) was observed. Conversely, QMS/EVER did not show any bias at both systematic (intercept=-0.151ng/mL, 95% CI:-0.671 to 0.256ng/mL) and proportional level (slope=0.971, 95% CI: 0.895 to 1.074) with a non-statistically significant combined bias of-3.6% (95% CI:-8.4-1.1%). Based on a concentration of calibrators and controls above the assigned value for both the analytical methods, in the ACMIA/EVRO a correction which was approximately one-third of the correction for the QMS/EVER was observed. ACMIA/EVRO but not QMS/EVER shows a lack of interchangeability with the CE-IVD-certified LC-MS/MS assay. We hypothesize that, as the ACMIA/EVRO uses an anti-sirolimus antibody, the under-corrected assigned value in the assay calibrators was not sufficient to reproduce the everolimus metabolites cross-reactivity occurring in real samples.

Automated HPLC-MS/MS assay for the simultaneous determination of ten plasma antibiotic concentrations

Therapeutic drug monitoring (TDM) of antibiotics (ATB) in patients with serious bacterial infections allows optimization of the efficacy of the treatment while reducing the risk of toxicity. Notably, early measurement of plasma beta-lactam concentration has been shown to be associated with reduced mortality in intensive care patients. In this context, a rapid, robust, and accurate assay method is essential for daily TDM.A fully automated procedure for quantification of the plasma concentrations of ten ATB was developed. The ATB were divided into two calibration pools, with Pool 1: aztreonam, ceftobiprole, cefoxitin, avibactam, tazobactam and Pool 2: metronidazole, ceftriaxone, daptomycin, ceftolozane, moxifloxacin. Sample preparation consisting of acetonitrile plasma protein precipitation and H20 dilution was applied to all analytes. This procedure was carried out by an automated sample preparation system directly coupled to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Since the instrument extracts sample n while sample n-1 is in the LC-MS/MS system, the delay between obtaining the results for two samples corresponds to the analytical run time, which is less than 7 min.The method was validated according to the Food and Drug Administration guidelines. The method was sensitive (lower limit of quantification 0.1–1 mg/L, depending on the ATB), accurate (intra/inter-assay bias −14.8 to 14.2 %) and precise (intra/inter-assay CVs 1.27 to 16.3 %). Application of the TDM assay was illustrated by the report of an intensive care patient treated with the ceftazidime/aztreonam/avibactam combination. Four assays were performed in 8 days with results returned within 24 h to quickly manage the dose regimen in this patient.An automated, simple, rapid, robust LC-MS/MS analysis was developed and validated for the simultaneous quantification of plasma concentrations of 10 ATB and was applied with success to perform TDM. This method provides a shorter turnaround time than classic sample batch-based analytical methods.

REverSe TRanscrIptase chain termination (RESTRICT) for selective measurement of nucleotide analogs used in HIV care and prevention.

Sufficient drug concentrations are required for efficacy of antiretroviral drugs used in HIV care and prevention. Measurement of nucleotide analogs, included in most HIV medication regimens, enables monitoring of short- and long-term adherence and the risk of treatment failure. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay rapidly infers the concentration of intracellular nucleotide analogs based on the inhibition of DNA synthesis by HIV reverse transcriptase enzyme. Here, we introduce a probabilistic model for RESTRICT and demonstrate selective measurement of multiple nucleotide analogs using DNA templates designed according to the chemical structure of each drug. We measure clinically relevant concentrations of tenofovir diphosphate, emtricitabine triphosphate, lamivudine triphosphate, and azidothymidine triphosphate with agreement between experiment and theory. RESTRICT represents a new class of activity-based assays for therapeutic drug monitoring in HIV care and could be extended to other diseases treated with nucleotide analogs.

Air contamination of therapeutic drug monitoring assay reagents results in falsely high plasma ammonia levels.

Accumulating evidence shows that contamination of blood samples by atmospheric ammonia affects blood ammonia test levels; however, reports on the effect of ammonia contamination of assay reagents are limited. Here, we aimed to clarify the effect of ammonia contamination of assay reagents, particularly the therapeutic drug monitoring (TDM) reagents, on the detection levels of blood ammonia using enzymatic assays. Ammonia gas was measured in the refrigerator compartment of the automatic analyser and the reaction tank water, probe wash water and drain outlets connected to the automatic analyser. At different time points following the closure of the cold storage, ammonia levels in quality control plasma samples were measured using three commercial assay reagents to evaluate the effect of air contamination. The distribution of evaporated ammonia in the reagent was measured using the CicaLiquid NH3 assay kit containing the assay reagent most affected by air contamination. It was confirmed that ammonia gas was generated in the cold storage of the automatic analyser. More than half of the reagents detected >0.25 ppm ammonia, and the highest concentration was detected in the TDM reagent. The ammonia levels obtained using all three reagents increased significantly after 3h of air contamination. The effect was resolved by measuring a 'dummy' sample or mixing the reagents by inversion. We demonstrated that air contamination by TDM reagents placed in cold storage could result in significantly falsely high ammonia measurements. Preventing this effect would improve the accuracy of ammonia measurements.

P241

Abstract Background In recent decades, biological drugs targeting anti-tumour necrosis factor-α (TNF-α) have represented the cornerstone in the treatment of inflammatory bowel diseases. Currently, anti- TNF-α represents a breakthrough in patient care. However, 20–50% of patients experience primary or secondary loss of response. This may be due to pharmacodynamic and/or pharmacokinetic issues, resulting in subtherapeutic drug concentrations, frequently related to the development of anti-drug antibodies (ADA). In this context, therapeutic drug monitoring (TDM) is essential to patients’ management in order to pinpoint the basis of reduced efficacy. Indeed, a fast and reliable assessments of TDM is mandatory to allow rapid intervention, to avoid toxicity and improve treatment efficacy. In this study we assess the analytical performance of a novel fully automated chemiluminescent assay in measuring biological drugs levels and ADA serum concentration and to compare its performances to the current diagnostic method (ELISA). Methods Serum levels of biological drugs (Infliximab and Adalimumab) and their respective ADA were measured in twenty infliximab (IFX)-treated IBD patients and twenty adalimumab (ADL)-treated IBD patients using both the Promonitor® ELISA kits (Grifols) and the i-TRACKER chemiluminescent (CLIA) kits (Theradiag Results The CLIA method is a random-access platform that allows the continuous loading of samples, while the ELISA is a time-consuming manual method to be planned in advance to reduce expenses. Actually, in our laboratory is performed only twice a month. A very good correlation was observed both for Infliximab (Spearman r=0.9695, 95%CI = 0,92-0,99, p&amp;lt;0.0001) and Adalimumab serum levels (Spearman r=0.9476, 95%CI = 0,86-0,98, p&amp;lt;0.0001). Among the patients with undetectable IFX serum levels, 6/8 (75%) resulted ADA positive by ELISA versus 8/8 (100%) by CLIA. Moreover, CLIA disclosed very high ADA concentration in 3 samples compared to borderline results by ELISA. Regarding ADL, all the 9 patients with undetectable serum levels presented similar ADA concentration as tested with ELISA or CLIA. Conclusion In conclusion, the i-TRACKER CLIA Infliximab and Adalimumab assays showed a very good correlation for serum-based drugs level quantification as compared to the related ELISA methods. Furthermore, a good agreement was observed for ADA analysis, with a higher sensitivity against anti-IFX antibodies by CLIA compared to ELISA. Our data clearly highlighted that a fully automated assay for TDM would certainly reduce costs and significantly improve the management of IBD patients in terms of a shorter turn-around time.

Generic, Straightforward and Robust Assay for Therapeutic Drug Monitoring of 10 Antibiotics in Plasma Using 5-Sulfosalicylic Acid Coupled to 2d- Liquid Chromatography Coupled to Mass Spectrometry (2d-Lc-Ms/Ms): Validation and One Year Review Retrospective Study Highlighting Tdm Importance

Generic, Straightforward and Robust Assay for Therapeutic Drug Monitoring of 10 Antibiotics in Plasma Using 5-Sulfosalicylic Acid Coupled to 2d- Liquid Chromatography Coupled to Mass Spectrometry (2d-Lc-Ms/Ms): Validation and One Year Review Retrospective Study Highlighting Tdm Importance

Biosensor-Enabled Multiplexed On-Site Therapeutic Drug Monitoring of Antibiotics.

Personalized antibiotherapy ensures that the antibiotic concentration remains in the optimal therapeutic window to maximize efficacy, minimize side effects, and avoid the emergence of drug resistance due to insufficient dosing. However, such individualized schemes need frequent sampling to tailor the blood antibiotic concentrations. To optimally integrate therapeutic drug monitoring (TDM) into the clinical workflow, antibiotic levels can either be measured in blood using point-of-care testing (POCT), or can rely on noninvasive sampling. Here, a versatile biosensor with an antibody-free assay for on-site TDM is presented. The platform is evaluated with an animal study, where antibiotic concentrations are quantified in different matrices including whole blood, plasma, urine, saliva, and exhaled breath condensate (EBC). The clearance and the temporal evaluation of antibiotic levels in EBC and plasma are demonstrated. Influence of matrix effects on measured drug concentrations is determined by comparing the plasma levels with those in noninvasive samples. The system's potential for blood-based POCT is further illustrated by tracking ß-lactam concentrations in untreated blood samples. Finally, multiplexing capabilities are explored successfully for multianalyte/sample analysis. By enabling a rapid, low-cost, sample-independent, and multiplexed on-site TDM, this system can shift the paradigm of "one-size-fits-all" strategy.

Evaluation of the Preanalytical Interference of Hemoglobin, Bilirubin, or Lipids in Therapeutic Drug Monitoring Assays on Beckman Coulter AU Analyzers.

The aim of this study was to evaluate the influence of hemolysis, icterus, and lipemia (HIL) interferences on 8 therapeutic drug monitoring (TDM) assays. Amikacin, carbamazepine, digoxin, lidocaine, lithium, methotrexate, phenobarbital, and theophylline were spiked in specimen pools at the clinical decision cutoff values. The interferents were spiked in vitro in specimen pools. All analytes were tested on Beckman Coulter AU analyzers. Hemolysis interference was detected in quantitative microsphere system (QMS) amikacin at 55.59 μg/mL at a concentration of 500 mg/dL hemoglobin. Icterus interference was detected in enzyme multiplied immunoassay technique amikacin at 43.62 μg/mL and in QMS amikacin at 55.59 μg/mL, at a concentration of 20 mg/dL bilirubin. Although the reference range value is recommended for clinical significance bias assessment for HIL interferences on most chemistry assays, an important investigation of the HIL interferences on TDM assays is to establish interferent thresholds at the clinical critical cutoff values.

The First WHO International Standard for Adalimumab: Dual Role in Bioactivity and Therapeutic Drug Monitoring.

The expanded availability of adalimumab products continues to widen patient access and reduce costs with substantial benefit to healthcare systems. However, the long-term success of these medicines is highly dependent on maintaining consistency in quality, safety and efficacy while minimizing any risk of divergence during life-cycle management. In recognition of this need and demand from global manufacturers, the World Health Organization (WHO) Expert Committee on Biological standardization established the WHO 1st International standard (IS) for Adalimumab (coded 17/236) in October 2019 with a defined unitage ascribed to each of the individual bioactivities evaluated in the study namely, TNF-α binding, TNF-α neutralization, complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity. For development of the IS, two candidate standards were manufactured as per WHO recommendations. Analysis of extensive datasets generated by testing of a common set of samples including the candidate standards by multiple stakeholders including regulatory agencies using their own qualified assays in a large international collaborative study showed comparable biological activity for the tested candidates for the different activities. Use of a common standard significantly decreased the variability of bioassays and improved agreement in potency estimates. Data from this study clearly supports the utility of the IS as an important tool for assuring analytical assay performance, for bioassay calibration and validation, for identifying and controlling changes in bioactivity during life-cycle management and for global harmonization of adalimumab products. In addition, in a separate multi-center study which included involvement of hospital and clinical diagnostic laboratories, the suitability of the adalimumab IS for therapeutic drug monitoring assays was examined by analysis of data from testing of a common blind coded panel of adalimumab spiked serum samples representative of the clinical scenario along with the IS and in-house standards in diverse immunoassays/platforms. Both commercially available and in-house assays that are routinely used for assessing adalimumab trough levels were included. Excellent agreement in estimates for adalimumab content in the spiked samples was observed regardless of the standard or the method with inter-laboratory variability also similar regardless of the standard employed. This data, for the first time, provides support for the extended applicability of the IS in assays in use for therapeutic drug monitoring based on the mass content of the IS. The adalimumab IS, in fulfilling clinical demand, can help toward standardizing and harmonizing clinical monitoring assays for informed clinical decisions and/or personalized treatment strategies for better patient outcomes. Collectively, a significant role for the adalimumab IS in assuring the quality, safety and efficacy of adalimumab products globally is envisaged.

Therapeutic Drug Monitoring of Second- and Third-Generation Antiepileptic Drugs.

Therapeutic drug monitoring has traditionally been widely used for first-generation antiepileptic drugs (AEDs) such as carbamazepine and phenytoin. The last 2 decades have seen the introduction of second- and third-generation AEDs (eg, lamotrigine, levetiracetam, and topiramate) into clinical practice. To use data from the College of American Pathologists Therapeutic Drug Monitoring, Extended Proficiency Testing Survey to determine the performance of assays used for therapeutic drug monitoring of newer AEDs, including comparison of enzyme immunoassay and chromatographic techniques. Six years of proficiency testing surveys were reviewed (2013-2018). Steady growth was seen in participant volumes for newer AEDs. The analytical performance of automated enzyme immunoassays for lamotrigine, levetiracetam, and topiramate was similar to that of chromatographic methods, consistent with published literature using patient samples for comparisons. The majority of participating laboratories now use enzyme immunoassays to measure levetiracetam. Survey results reflect steadily growing interest in therapeutic drug monitoring of newer AEDs. The increasing availability of robust immunoassays for new AEDs should facilitate their clinical utility, especially for clinical laboratories that do not perform chromatographic assays for therapeutic drug monitoring.

Development and Validation of UHPLC-MS/MS Assay for Therapeutic Drug Monitoring of High-dose Methotrexate in Children with Acute Lymphoblastic Leukemia.

PurposePrecise and timely detection of methotrexate (MTX) concentration played a key role in high-dose MTX individualization therapy in acute lymphoblastic leukemia (ALL) children to avoid serious adverse effects or nonresponse. This report described a sensibility and validation of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for therapeutic drug monitoring (TDM) of methotrexate concentration in children’s plasma.MethodsOne-step protein precipitation of samples was accomplished by adding 200 μL of acetonitrile to 100 μL of plasma sample. The separation of plasma samples was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution HD column with gradient elution using a mobile phase constituted of acetonitrile and 1% formic acid. The detection was executed by electrospray ionization (ESI) of triple quadrupole tandem mass spectrometer (TQMS) in the multiple reaction monitoring (MRM) mode with the transitions m/z 455.2 → 307.9 for methotrexate and m/z 458.2 → 311.2 for IS, separately. Linear concentration range of the calibration curve was 44–11,000 nmol/L and 44 nmol/L was the lower limit of quantification.ResultsThe methotrexate elution time was at 1.577 min, and the overall running time was only 3.3 min. The intra- and interday precision for all the analysis results was within 11.24%, and mean recoveries rate of methotrexate exceeded 87.98%.ConclusionThe described and fully validated UHPLC-MS/MS method was successfully applied in clinical TDM after infusion of high-dose methotrexate 1–5 g/m2 to 41 childpatients.