European Medicines Agency Guidelines Research Articles

Simultaneous quantitation of favipiravir and its hydroxide metabolite in human plasma and hamster matrices using a UPLC-MS/MS method.

Favipiravir, a broad-spectrum RNA-dependent RNA polymerase inhibitor, is currently being evaluated in preclinical and clinical studies for the treatment of various infectious diseases including COVID-19. We developed an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of favipiravir and its hydroxide metabolite (M1), in human and hamster biological matrices. Analytes were separated on an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8μm) after a simple protein precipitation with acetonitrile. The mobile phase consisted of water and methanol, each containing 0.05% formic acid. Experiments were performed using electrospray ionization in the positive and negative ion mode, with protonated molecules used as the precursor ion and a total run time of 6min. The MS/MS response was linear over the concentration ranges from 0.5-100 μg/ml for favipiravir and 0.25-30 μg/ml for M1. Intra- and inter-day accuracy and precision were within the recommended limits of the European Medicines Agency guidelines. No significant matrix effect was observed, and the method was successfully applied to inform favipiravir dose adjustments in six immunocompromised children with severe RNA viral infections. In conclusion, the UPLC-MS/MS assay is suitable for quantification of favipiravir over a wide range of dosing regimens, and can easily be adapted to other matrices and species.

An alternative approach to validation of liquid chromatography-mass spectrometry methods for the quantification of endogenous compounds

Despite the progress in the quantification of xenobiotics, the development and validation of methods designed for endogenous substances still remain challenging due to the natural presence of the analytes in a biological matrix, leading to the inability to obtain a blank sample. Several generally recognized procedures are described to solve this issue, like using surrogate or analyte-depleted matrices or surrogate analytes. However, the workflows used do not always meet the requirements for developing a reliable analytical method or are cost-intensive.This study aimed to design an alternative approach for preparing validation reference samples using authentic analytical standards while preserving the nature of the biological matrix and solving the problem with the inherent presence of analyzed compounds in a studied matrix. The methodology used is based on the standard-addition type procedure. However, unlike the original method, the addition is modified according to a previously measured basal concentration of monitored substances in the pooled biological sample to obtain a predefined concentration in reference samples according to the European Medicines Agency (EMA) validation guideline. The study shows the advantages of described approach on an example of LC-MS/MS analysis of 15 bile acids in human plasma and compares it with other methods commonly used in this field.The method was successfully validated according to the EMA guideline with lower limit of quantification of 5 nmol/L and linearity in the range of 5 - 2000 nmol/L. Finally, the method was used in a metabolomic study on a cohort of pregnant women (n = 28) to confirm intrahepatic cholestasis, the major liver disease observed in pregnancy.

#3207 KIDNEY FUNCTION ESTIMATORS FOR DRUG DOSE ADJUSTMENT OF DIRECT ORAL ANTICOAGULANTS IN OLDER ADULTS WITH ATRIAL FIBRILLATION

Abstract Background and Aims The Food and Drug Administration and the European Medicines Agency (EMA) recommend using the Cockcroft-Gault equation (CG) for drug dose adjustment of direct oral anticoagulant drugs (DOACs) in non-valvular atrial fibrillation (NVAF), whereas Cardiology guidelines recommend using estimated glomerular filtration rate (eGFR). This issue is of great importance in older adults, which are more prone to adverse drug reactions and have higher prevalence of atrial fibrillation as compared to younger adults, and in whom CG has worse diagnostic performance than most eGFR equations. We aimed to assess if prognosis was similar according to drug dose status using different kidney function estimators based on creatinine and/or cystatin C in older adults with NVAF. Method We used data from the Berlin Initiative Study (BIS): a population-based prospective cohort study initiated in 2009, with five biennial study visits. Participants with a history of NVAF (based on ICD-10 codes) and a dispensed prescription of DOAC four months prior to baseline or a follow-up visit according to claims data were included. Drug dose status was defined according to the EMA guidelines. CG, deindexed (in ml/min) creatinine and/or cystatin C-based CKD-EPI, and deindexed BIS 1 (creatinine-based) and BIS 2 (creatinine and cystatin C-based) equations were included. Associations between dosing status and mortality, stroke or systemic embolism, and bleeding events were assessed using marginal structural Cox models with time-varying variables and taking account of various confounders. Subgroup analyses were performed in rivaroxaban and apixaban users, but not in dabigatran and edoxaban users because of too small sample sizes. Results Two hundred twenty four patients treated with DOACs were included in this analysis (median age 87 years, median eGFRCKD-EPIcr 56 ml/min/1.73m², median follow-up length 40 months for the mortality analysis). Of those, 99 (44%) were taking rivaroxaban, 86 (38%) apixaban, 21 (9%) edoxaban, and 18 (8%) dabigatran. Using CG, 154 (69%) had appropriate DOAC dose at baseline, 52 (23%) were underdosed, and 18 (8%) were overdosed. Discrepancies were found by comparing dosing status according to CG and eGFR equations (Figure 1). During the follow-up period, 109 (14.9/100 person-years) participants died, 25 (3.6/100 person-years) experienced a stroke or systemic embolism, and 60 (9.9/100 person-years) experienced a bleeding event. Drug dose status was not significantly associated with mortality and the occurrence of stroke or systemic embolism, whatever equation was used. Underdose status was associated with a significantly lower risk of bleeding events with all the equations but overdose status was not associated with a higher risk of bleeding events (Figure 2). In subgroup analyses, drug dose status was not associated with mortality and bleeding event in apixaban users, whatever equation was used. In rivaroxaban users, underdose status was associated with a significant higher risk of death by using CG, eGFRCKD-EPIcys, and BIS 2, and a lower risk of bleeding event by using eGFRCKD-EPIcr. Conclusion In this population of very old adults with NVAF, drug dose status of DOAC was not associated with mortality or the occurrence of stroke or systemic embolism for any of the studied equations. However, underdose status was associated with bleeding events occurrence regardless of the equation used. These associations differed according to the used drug, but this should be interpreted with cautious due to small sample sizes. Our results do not allow us to provide any guidance which equation to use in this context. A study including a larger group of patients with discrepancy in dose status according to the used equations would be of great interest.

Addressing practical issues in the smooth implementation of revised guidelines for non-clinical studies of vaccines for infectious disease prevention

Herein, we investigated possible practical issues for the smooth implementation of the revised Japanese Guidelines for Non-clinical Studies of Vaccines for the Prevention of Infectious Diseases, which were raised in response to public comments on the proposed guideline revision and a gap analysis of the World Health Organization and European Medicines Agency guidelines. We identified main issues such as the non-clinical safety studies of adjuvants and evaluation of local cumulative tolerance in toxicity studies. The revised Japanese Pharmaceuticals and Medical Devices Agency (PMDA)/Ministry of Health, Labour and Welfare (MHLW) guidelines require non-clinical safety studies for vaccines containing new adjuvants, but additional safety pharmacology studies or safety studies in two animal species may be required if non-clinical safety studies raise any concerns (i.e., systemic distribution). Adjuvant biodistribution studies may aid in understanding vaccine characteristics. The evaluation of local cumulative tolerance in non-clinical studies, which was the focus of the Japanese review, can be omitted by including a warning in the package insert to avoid injection to the same site. The study's findings will be reflected in a Q&A to be released by the Japanese MHLW. We hope that this study will contribute to the global and harmonized development of vaccines.

Rapid high performance liquid chromatography method for erlotinib quantification in vitro: Application to study the effect of resveratrol on metabolism and cellular uptake of erlotinib.

Erlotinib is a selective epidermal growth factor receptor inhibitor that is used for the treatment of non-small cell lung cancer and pancreatic cancer. Its metabolism is mainly mediated by cytochrome P450 3A (CYP 3A). Resveratrol, a natural compound found in many plants and supplements, is known to inhibit CYP3A enzyme, therefore, it may act as an inhibitor for the metabolism of erlotinib. Development of a rapid high performance liquid chromatography with photodiode array detection (HPLC-PDA) method for the quantification of erlotinib in liver microsomes and cancer cells and its application to study resveratrol effect on metabolism and cellular uptake of erlotinib. HPLC-PDA was used to develop an efficient bioanalytical method with a 2.5-min runtime preceded by a simple protein precipitation step. The method was validated according to the European Medicines Agency guidelines. Erlotinib metabolic stability and resveratrol effect on erlotinib metabolite formation were evaluated in rat liver microsomes. Furthermore, the method was used to measure the intracellular concentrations of erlotinib in cancer colorectal cells and investigating resveratrol effect on the cellular uptake of erlotinib. A rapid HPLC-PDA method was developed and validated for the first time to address potential drug interaction of erlotinib with resveratrol. Resveratrol was a strong inhibitor of erlotinib metabolism in vitro with IC50 = 4.03 μM. Resveratrol, however, had no effect on erlotinib cellular uptake after 1h incubation in human colorectal cancer cells. The study suggests that resveratrol may produce a potential herb-drug interaction with erlotinib at the metabolism level and should be investigated in patients in the clinic.

In Vivo Evaluation of the Pharmacokinetic Interaction between Levothyroxine and Amiodarone in Rat Plasma: Evaluation of Importance of Therapeutic Drug Monitoring during Co-Therapy.

Amiodarone-induced thyrotoxicosis (AIT) is a common condition in patients who are receiving amiodarone for cardiac arrhythmia. This risk is elevated in iodine-deficient regions. Levothyroxine is the standard treatment for patients with hypothyroidism. This investigation is concerned with the evaluation of the possible pharmacokinetic interaction between amiodarone and levothyroxine upon co-therapy in rats and to investigate the cause of thyrotoxicosis. A selective, sensitive and precise RP-HPLC method was developed for the simultaneous determination of levothyroxine and amiodarone in rat plasma. A stationary phase of C18 Xterra RP column and a mobile phase consisting of acetonitrile: acidified water with 0.1% trifluoracetic acid (pH = 4.8) with gradient elution were used. The experiment was conducted at ambient temperature with flow rate of 1.5mL/min for the chromatographic separation and quantitation of the investigated drugs. Protein precipitation with methanol was applied for the analysis of the two drugs in rat plasma. The method was linear over concentration range of 5-200μg/mL for both levothyroxine and amiodarone. The European Medicines Agency guideline was applied for the validation of the developed bioanalytical method. The method was successfully applied to in vivo pharmacokinetic study in which levothyroxine and amiodarone were quantified in plasma of rats after receiving an oral dose of levothyroxine and amiodarone. After the calculation of the pharmacokinetic parameters, a statistical analysis was performed to elucidate the existence of significant difference between test and control groups in rats. The combination of levothyroxine and amiodarone significantly decreased levothyroxine bioavailability in rats, making the therapeutic drug monitoring mandatory in patients receiving levothyroxine and amiodarone. In addition, the increased clearance of levothyroxine upon the co-administration with amiodarone may explain the reported hypothyroidism.

Development and Validation of an Ultrasensitive LC-MS/MS Method for the Quantification of Melatonin in Human Saliva.

A growing body of literature describes the potential effects of circadian disruption on human health. Indeed, psychiatric diseases, metabolic syndrome, and cancers may be linked to disturbance of the circadian rhythm. Currently, the best practice to assess circadian rhythm is the measurement of melatonin levels. Our goal was thus to develop and validate a highly sensitive LC-MS/MS method to follow salivary melatonin levels throughout the day and night. Our method reached a lower limit of the measuring interval (LLMI) of 0.8 pg/mL. To our knowledge, it is the most sensitive method allowing quantitation of melatonin in saliva. Saliva, obtained from passive drooling or salivette, was extracted by an efficient and quick liquid-liquid extraction with no further cleanup needed. The method was validated according to the European Medicines Agency (EMA) guidelines and provided excellent results regarding accuracy, precision, linearity, selectivity, and specificity. Comparison between radioimmunoassay and our method was performed and showed differences at low levels, most likely due to cross-reactivity with other indols. To assess daytime melatonin levels in humans, salivary melatonin levels of ten volunteers were monitored throughout the day and showed lower daytime levels than reported in previous studies.

Impact of comorbidities in elderly and frail patients and response to biological therapy in psoriasis.

Impact of comorbidities in elderly and frail patients and response to biological therapy in psoriasis.

A high-throughput LC-MS/MS method for simultaneous determination of direct oral anticoagulants in human plasma.

Direct oral anticoagulants are widely used in many indications to prevent thromboembolic events. Routine therapeutic monitoring is not required; however, there is increasing evidence suggesting the benefit of plasma level measurement in some situations. In addition, laboratory monitoring might help improve patient and drug non-compliance and thus individualize therapy. In the present study, we developed a sensitive and high throughput ultra-high-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma. A one-step extraction procedure in 96-well formate for phospholipid and protein removal was used for sample pre-treatment, and analytes were separated using gradient elution over 4.2 min. Analytes were detected on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode. The method was validated according to the European Medicine Agency guideline for the selectivity, linearity and lower limit of detection, precision and accuracy, matrix effects, extraction recovery, carryover, dilution integrity and stability over a concentration range of 3.0-1000 ng/mL for all analytes. The validated method was applied to real clinical samples of patients treated with one of the drugs. Therefore, we can conclude that our method is suitable for therapeutic drug monitoring of direct oral anticoagulants. This article is protected by copyright. All rights reserved.

Characterization of famotidine API and simultaneous Quantification of its organic impurities by UPLC

To evaluate the characterization of Famotidine API (active pharmaceutical ingredient) and develop the accurate, precise, linear ultra-performance liquid chromatographic (UPLC) method for quantitating determination of organic impurities (Impurity-A, Impurity-B, and Impurity-C) in Famotidine API, pharmaceutical dosage forms and the method has been validated as per international council of harmonization (ICH) guidelines. The Famotidine API characterization was done by using spectroscopic techniques such as mass, infrared spectroscopy (IR), differential scanning calorimetry (DSC), proton nuclear magnetic resonance (1H-NMR), Carbon-13 nuclear magnetic resonance (C13-NMR), and two-dimensional nuclear magnetic resonance (2D-NMR). The organic impurities (A, B, and C) quantification was done using ACQUITY UPLC CSH C18- 100 mm × 2.1 mm, 1.7 μm column connected to ACQUITY UPLC with PDA detector. The optimized chromatographic conditions were achieved at 0.3 mL/min flow rate using a gradient system with 0.1% Tri fluoro acetic acid in water acetonitrile and Methanol as mobile phase. Three organic impurities are measured at λmax 260 nm at 45°C column temperature. Famotidine structure was confirmed by the appraised complete characterization data. The coefficient of correlation (r2) for the Impurity-A, Impurity-B, and Impurity-C was obtained not less than 0.99. The limit of detection (LOD) obtained 0.12 µg/mL and the limit of quantification (LOQ) obtained 0.4 µg/mL for each impurity concerning Famotidine test concentration. The method was fully validated as precision, accuracy, LOQ precision, LOQ accuracy, ruggedness, and robustness complying with FDA, ICH, AOAC, and European medicines agency (EMEA) guidelines. The characterization study and the proposed UPLC method were specific, accurate, precise, linear, rugged, and robust for the determination of the three organic impurities in Famotidine API and it can be implemented for its intended use in pharmaceutical industries.

A HPLC-DAD method to facilitate large-scale therapeutic drug monitoring of dalbavancin

Dalbavancin, a long-acting lipoglycopeptide antibiotic targeting susceptible Gram-positive bacteria, is WHO critically important antibiotic, increasingly used in critical situations such as osteoarticular infections. To ensure its effectiveness and its safety, the therapeutic drug monitoring (TDM) of dalbavancin is strongly recommended. In the absence of an available minimum inhibitory concentration (MIC), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommends a breakpoint of 0.125 mg/L for Staphylococcus aureus, corresponding to a trough target concentration of 25 mg/L. Nowadays, the TDM is usually performed using a high-performance liquid chromatography (HPLC) method coupled with a tandem mass spectrometry. However, this expensive and specialized equipment and reagents may be difficult to acquire for non-specialized laboratories. The use of HPLC coupled with diode array detector (DAD) facilitates TDM with a lower cost, while preserving the reliability of the results. Our aim was to provide a sensitive and specific method, relying on HPLC-DAD for extending the TDM of dalbavancin beyond non-specialized labs, therefore maximizing its efficiency and Benefit/risk ratio. Our method complied with the European Medicines Agency guidelines of bioanalytical validation. Irrespective of the concentrations of dalbavancin, the coefficient of variation < 10% confirmed the reliability of this analytical method, with a calibration curve ranging from 5 to 100 mg/L. No interferences nor carryover was observed. Our HPLC-DAD method, combined with a simple extraction, provides a widely usable, inexpensive and easy-to-implement new asset for the TDM of Dalbavancin.

Enhanced safety surveillance of GSK's quadrivalent seasonal influenza vaccine in Germany and Spain (2021/2022 season) using an electronic patient-reported outcome system for vaccine safety remote monitoring.

Seasonal influenza epidemics are managed through vaccination each winter in the European Union, to prevent infections, complications, and deaths. As circulating virus strains vary unpredictably, vaccines are reformulated annually, and their safety monitored rapidly and continuously at the start of each season, following European Medicines Agency guidelines.Seasonal influenza epidemics are managed through vaccination each winter in the European Union, to prevent infections, complications, and deaths. As circulating virus strains vary unpredictably, vaccines are reformulated annually, and their safety monitored rapidly and continuously at the start of each season, following European Medicines Agency guidelines. This enhanced safety surveillance study assessed pre-specified and other adverse events (AEs) occurring within 7 days of GSK's inactivated quadrivalent seasonal influenza vaccine (IIV4) in children and adults in Spain and Germany. As the study was conducted during the COVID-19 pandemic (2021/2022 season), data were collected electronically, using a web portal or call center. Safety was assessed in 737 participants (median age 49 and 9 years in Germany and Spain, respectively, 19.3% with a chronic medical condition). After Dose 1 and Dose 2, respectively, 332 (45.1%) and 5 (26.3%) participants reported at least one AE, primarily pre-specified AEs. The most common AEs after Dose 1 (adults and children) were injection site pain, swelling or erythema, headache, and fatigue. After Dose 2 (in children), the most common AEs were injection site pain, rhinorrhea, fatigue, and decreased appetite. No new or unexpected safety issues were identified. This study supports and confirms the safety profile of GSK's IIV4 in all age groups with a vaccine indication. The new electronic safety reporting method (with response rates of 75.4% following Dose 1 and 100% following Dose 2) provides an alternative for future studies to reduce the burden on sites or in case site visits are not feasible.

Contraintuitive fallacy in a big-risk-no-gain stroke scenario? Please verify!

Contraintuitive fallacy in a big-risk-no-gain stroke scenario? Please verify!

Human plasma ricinine quantification by LC-HRMS after micro-solid-phase elution.

A rapid, sensitive and specific method for ricinine identification and quantification in plasma has been developed by LC-HRMS. Deuterated ricinine was used as the internal standard. From 100 μL of plasma, ricinine was extracted using micro-solid-phase elution, which allows a reduced extraction time, by eliminating the evaporation step. Eluate is directly injected into the LC-HRMS system. Chromatographic separation was performed using a reverse-phase C18 column with a 4.5 min gradient elution. The method was validated according to European Medicines Agency guidelines. Linearity was verified between 0.25 and 500.0ng/mL; the maximum precision calculated was 19.9% for the lower limit of quantitation and 9.6% for quality control, and accuracy was within ± 5.6% of the nominal concentrations. Selectivity, carryover, matrix effect and stability were also verified according to European Medicines Agency guidelines. The method allows the rapid and reliable identification of ricin-exposed victims in case of terrorist attacks or poisonings: three intoxication cases are reported.

Determination of Carnosic Acid by a Novel HPLC-UV Method in Human Plasma and Application to a Prototype Pharmacokinetic Study.

An HPLC method with UV detection was developed for the determination of carnosic acid in human plasma and applied to a pharmacokinetic study after oral administration of Rosemary extract to a healthy volunteer. Sample preparation depends on liquid-liquid extraction with hexane. Chromatographic separation was achieved with C18 column (150mm × 4.6mm × 5μm), at 25°C with isocratic elution, mobile phase composed of solution A (methanol), and solution B (2% o-phosphoric acid in water) (90:10, v/v) at flow rate of 1.0mL/min. The analyte was detected at 230nm. The retention time is 4.20 ± 0.03min. The method was validated in terms of accuracy, precision, specificity, robustness and detection and quantification limits, in accordance with European Medicines Agency guidelines. LOD and LOQ were found to be 0.075 and 0.25ng/mL, respectively. The method was applied to the analysis of carnosic acid in human plasma with good recovery as 91.7%. The plasma concentration-time profile and pharmacokinetic parameters: AUC0-t, AUC0-∞, Cmax, tmax, t1/2 were calculated according to the assays. The method can certainly be used for routine analysis of carnosic acid in human plasma after oral administration of Rosemary extract, and for phase I clinical studies and bioavailability-bioequivalance studies as well.

Bioanalytical Validated Spectrofluorimetric Method for the Determination of Prucalopride succinate in Human Urine Samples and Its Greenness Evaluation

An economical & eco-friendly spectrofluorometric method has been developed for the determination of prucalopride succinate (PRU) in human urine on the basis of the drug’s native fluorescence. The type of solvent and the wavelengths of excitation and emission have been carefully selected for optimal experimental conditions. In deionized water, the fluorescence intensity was measured at λ emission 362 nm upon excitation at 310 nm. This bio-validated method was carried out using 30uL urine without any preliminary steps. The calibration curve for prucalopride succinate shows a linear relationship in a concentration range of 0.75–5.5 µg/mL. Accuracy and precision were obtained using 4 quality control samples which are: 0.75 μg/ mL (LLOQ), 2.25 μg/mL (QCL), 2.5 μg/mL (QCM) & 4.125 µg/mL (QCH). The validation of this proposed technique obeys European Medicines Agency (EMA) Guidelines for validating bioanalytical methods and the greenness assessment was evaluated according to the Analytical GAPI approach.

Development, validation and application of a selective and sensitive LC-MS/MS method for the quantification of daptomycin in a suspension of Mammaliicoccus sciuri in Mueller-Hinton broth

Reports of therapy failures related to the use of daptomycin (DAP) are steadily increasing. This is mainly due to emerging DAP resistance for which, however, the underlying mechanism is often unknown. To elucidate the mode of action of novel DAP resistance traits it is indispensable to reliably detect and quantify DAP in complex matrices such as bacterial culture media. In this study, we established a selective and sensitive quantification method for DAP upon growth of a DAP resistant Mammaliicoccus sciuri strain in Mueller-Hinton medium. The method combined methanol-induced protein precipitation followed by high performance liquid chromatography with gradient elution coupled to mass spectrometry (LC-MS/MS) using daptomycin-d5 as internal standard. All validation parameters met the acceptance criteria of the European Medicines Agency (EMA) guideline on bioanalytical method validation. We successfully applied this highly selective and sensitive LC-MS/MS method for the quantification of DAP in in vitro studies addressing DAP resistance mechanisms in Gram-positive bacteria.

What is the weight of expectation bias in oncology trials?

What is the weight of expectation bias in oncology trials?

Opportunities of Amlodipine as a Potential Candidate in the Evaluation of Drug Compliance during Antihypertensive Therapy.

Background and Objectives: Blood pressure measurement is essential evidence to establish that the chosen medicine and dosage are appropriate, and also indirectly indicates whether the medicine is being used at all. Therefore, current research compares adherence to the target blood pressure at home and in the hospital between different age groups, using similar combinations of the drugs prescribed by the doctor within ongoing antihypertensive therapy. Moreover, it is very important to develop a method for the determination of amlodipine and its metabolite, which would suitable for clinical applications, when the result is needed as quick as possible. Materials and Methods: This prospective study included patients aged ≥18 years who were diagnosed with hypertension. Subjects were divided into two age groups according to European Society of Cardiology (ESC) hypertension guidelines; older patients (≥65 years) and adult patients (<65 years). Assessment of adherence rate to antihypertensive medications was performed using a measurement of systolic blood pressure and comparing this to ESC hypertension guideline data. A simple liquid chromatography-tandem mass spectrometer (LC-MS/MS) method for determination of amlodipine and dehydroamlodipine was developed and validated according to the European Medicines Agency guideline on bioanalytical method validation at the Latvian Institute of Organic Synthesis. Results: A total of 81 patients with arterial hypertension were enrolled in this study. A significant number of patients were overweight (N = 33, 40.7%) and obese (N = 36, 44.4%). To control arterial hypertension, 70 (86.4%) patients used fixed-dose combinations, where one of the components was amlodipine. Practically, 36 (44.4%) hypertensive subjects were not able to comply with target blood pressure. Nonetheless, 38 (46.9%) patients who received fixed-dose combinations were able to comply with target blood pressure. Conclusions: Adherence to ESC hypertension guideline proposed target blood pressure was relatively low among hypertensive subjects even though a significant number of patients were taking fixed-dose combinations. Therefore, optimizing prevention, recognition, and care of hypertensive young adults require intensive educational interventions. Moreover, survey data suggest that therapeutic drug monitoring using the validated simple, sensitive LC-MS/MS method is pivotal for further understanding factors influencing adherence.

Fast and Sensitive Analysis of Cefiderocol in Human Plasma Microsamples by Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry for Therapeutic Drug Monitoring

Cefiderocol (C) is a parenteral siderophore cephalosporin with relevant inter-individual pharmacokinetic variability among critically ill patients, which may potentially affect effective drug exposure. Therapeutic drug monitoring (TDM) may concur in improving the real-time management of C therapy in clinics. In this study, we developed and validated a fast and sensitive Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry (LC-ITD-MS/MS) method for measuring C in human plasma microsamples, as small as 3 microliters. Analysis was preceded by a user-friendly pre-analytical single-step and was performed by means of a very fast chromatographic run of 4 min, followed by positive electrospray ionization and detection on a high sensitivity triple quadrupole tandem mass spectrometer operated in multiple reaction monitoring mode. The straightforward analytical procedure was successfully validated, based on the European Medicines Agency (EMA) guidelines, in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and stability. The novel method was successfully applied to TDM of C in more than 50 cases of critically carbapenem-resistant Gram-negative bacterial infections and enabled us to optimize antibiotic therapy.