Therapeutic Drug Monitoring Purposes Research Articles

Hapten Synthesis, Antibody Generation, and Immunoassay Development for Linezolid Therapeutic Monitoring.

Linezolid (LNZ) is a broad-spectrum antibiotic used for the treatment of severe Gram+ infections. Significant pharmacokinetic variability in different groups of patients requires therapeutic drug monitoring (TDM) to ensure optimal therapy. This article presents the first description of the development of immunoanalytical TDM systems for LNZ. Four LNZ derivatives (H1-H4) with spacer arms of a different length (0-6.2-9.4-11.8 Å) and chemistry (aliphatic and heterocyclic) were synthesized to prepare H1-H3-immunogens and H1/H4-coating antigens or H1/H2-enzyme tracers. Three distinct antibody types against H1-H3 haptens were generated in rabbits and served as the basis for the development of ELISAs in heterologous coated antigen and coated antibody formats. The length of the spacer in the immunogen had an insignificant effect on the quality of the antibody generated or the performance of the designed coated antibody-ELISA formats (IC50 = 0.48-0.75 ng/mL) and coated antigen ELISAs (IC50 = 11.5-28.3 ng/mL). The coated antigen-based assays were considered more appropriate for monitoring therapeutic levels of LNZ due to their more convenient reduced sensitivity but wider dynamic range than the coated antibody-based assays (2.0-160 vs 0.16-2.1 ng/mL). The best recovery of LNZ (82.1-99.6%) from spiked human serum among the sample pretreatments examined was provided by the trichloroacetic acid deproteinization procedure. Serum samples derived from surgical patients with superimposed Gram-positive infections treated with LNZ were evaluated by parallel ELISAs using different immunoreagents. The results obtained in the various assays were found to be concordant, thereby confirming the reliability of the measurements and the developed assay systems suitable for TDM purposes.

Recent Trends in Therapeutic Drug Monitoring of Peptide Antibiotics.

Antimicrobial peptides take a specific position in the field of antibiotics (ATBs), however, from a large number of available molecules only a few of them were approved and are used in clinics. These therapeutic modalities play a crucial role in the management of diseases caused by multidrug-resistant bacterial pathogens and represent the last-line therapy for bacterial infections. Therefore, there is a demand for a rationale use of such ATBs based on optimization of the dosing strategy to minimize the risk of resistance and ensure the sustainable efficacy of the drug in real clinical practice. Therapeutic drug monitoring, as a measurement of drug concentration in the body fluids or tissues, results in the optimization of the patient´s medication and therapy outcome. This strategy is beneficial and could result in tailored therapy for different types of infection and the prolongation of the use and efficacy of ATBs in hospitals. This review paper provides an actual overview of approved antimicrobial peptides used in clinical practice and covers current trends in their analysis by convenient and advanced methodologies used for their identification and/or quantitation in biological matrices for therapeutic drug monitoring purposes. Special emphasis is given to the methods with perspective clinical outcomes.

Blueness, Whiteness, and greenness Appraisal of sensitive chromatographic approach for determination of semaglutide concurrently with Pioglitazone and metformin using green mobile Phase; alongside with pharmacokinetic study

The replacement of hazardous solvents with greener alternatives was under consideration to achieve a sustainable and green approach. The assessment of reliable white analytical method for the determination of semaglutide as a novel member in glucagon-like peptide agonists class is still of a great importance. That is because pharmacological, clinical, and therapeutic drug monitoring purposes. This research proposes and fully validates a simple and sensitive RP-HPLC method for the determination of semaglutide simultaneously with pioglitazone and metformin. The suggested procedures utilizing a green mobile phase of 8 % glycerol in 0.01 M phosphate buffer (pH 6). Separation was achieved using a cyano (5.0 μm, 150 × 4.6 mm) column maintained at a temperature of 40 °C. The mixture was isocratically eluted under a flow rate of 1 mL/min with a total run time of less than 8 min. The UV-detector was adjusted at 215 nm. The method provides low detection (ranging from 0.003 to 0.63 µg/mL) and quantification (ranging from 0.008 to 1.9 µg/mL) limits, with good precision (%RSD ranging from 0.66 % to 0.89 %). The suggested technique has been effectively used to determine each drug in its pharmaceutical preparation and spiked human plasma. Beside the remarkable sensitivity and sustainability of the evolved approach, the relevance of therapeutic drug monitoring motivated us to perform a pharmacokinetics study for both semaglutide and metformin in real human plasma samples. Greenness, whiteness and blueness assessment of the developed method have been carried out using different tools (Green Analytical Procedure Index (GAPI), the analytical GREEnness metric approach (AGREE), RGB-12 model and BAGI). The proven green and white profile of the evolved method could open future prospective for the clinical research and regular quality control examination of the investigated mixture with the least negative environmental impacts.

Design of point-of-care electrochemical sensor for therapeutic drug monitoring of ofloxacin in biological fluids

A disposable screen-printed sensor has been crafted specifically for therapeutic drug monitoring purposes, particularly for detecting ofloxacin in biological fluids. To enhance selectivity toward ofloxacin, a supramolecular calix [6] arene serves as the ionophore of choice. The sensor incorporates a graphene nanocomposite as an ion-to-electron transducer layer, which not only boosts potential stability but also mitigates potential drift. The developed ofloxacin sensor underwent rigorous characterization following IUPAC guidelines. The linearity range spans from 1 × 10–6 to 1 × 10–2 M, with a measured slope of 59.0 mV/decade. Impressively, it boasts a percentage recovery of 100.18 ± 1.60 and a low detection limit (LOD) of 6 × 10–7 M. Stability assessments indicate reliable performance over an extended period of 8 weeks. The versatility of this sensor extends to various applications, including the determination of ofloxacin in pharmaceutical formulations, bulk powder, and biological fluids. Notably, it has demonstrated efficacy post-bioanalysis validation, adhering to Food and Drug Administration regulations. This advancement holds promise for personalized therapeutic drug monitoring in clinical pharmacy studies and quality control laboratories, thereby optimizing patient care at the point-of-care.Graphical abstract

Influence of ultrafiltration conditions on the measurement of unbound drug concentrations: flucloxacillin as an example.

When performing therapeutic drug monitoring (TDM) for flucloxacillin, it is advised to measure the unbound, not the total, flucloxacillin concentration. To be able to accurately quantify unbound flucloxacillin concentrations, a reliable analytical method is indispensable. To determine the influence of temperature and pH of the sample during ultrafiltration on the measured unbound fraction of flucloxacillin. We performed three different experiments. In a single laboratory experiment, we investigated the influence of ultrafiltration temperature (10°C, room temperature and 37°C) on the measured unbound fraction of flucloxacillin for three concentration levels. In a multiple laboratory experiment, the results of eight laboratories participating in an international quality control programme measuring unbound flucloxacillin concentrations were analysed. In the third experiment, patient samples were ultrafiltrated using four different conditions: (i) physiological pH and room temperature; (ii) unadjusted pH (pH 9 after freezing) and room temperature; (iii) physiological pH and 37°C and (iv) unadjusted pH and 37°C. For all experiments, measurement of samples that were ultrafiltrated at room temperature resulted in a substantially lower unbound fraction compared to samples that were ultrafiltrated at 37°C. Adjusting the pH to physiological pH only had a minimal impact on the measured unbound fraction. On the basis of these findings and considering the need for fast, simple and reproducible sample pretreatment for TDM purposes, we conclude that ultrafiltration of flucloxacillin should be performed at physiological temperature (37°C), but adjustment of pH does not seem to be necessary.

Hollow-fiber liquid phase microextraction for determination of fluoxetine in human serum by nano-liquid chromatography coupled to high resolution mass spectrometry

Therapeutic drug monitoring (TDM) is a personalized care tool based on the determination of a target drug concentration in human serum. An antidepressant drug of interest for such investigations is fluoxetine (FXT), due to a severe impact of genetic polymorphisms on its metabolism. A bioanalytical method employed for TDM purposes must exhibit satisfactory selectivity and detectability, which becomes more difficult due to highly complex biological matrices. In this study, a highly selective bioanalytical method for the determination of FXT in human serum is proposed, which provides excellent clean-up efficiency based on a low cost hollow fiber liquid-phase microextraction (HF-LPME) sample preparation step and nano-liquid chromatography coupled to high-resolution mass spectrometry (nano-LC-HRMS). HF-LPME was performed using a two-phase “U” configuration, with 6 cm fiber, 20 µL of 1-octanol acting as supported liquid membrane, and ammonium hydroxide (pH 10) as the donor phase with NaCl (10 % m/v) and methanol (5 % v/v) as additives, requiring only 250 µL of the sample. The procedure was conducted for 30 min under a 750 rpm stirring rate. Gradient elution was carried out employing an acetonitrile–water as mobile phase, the composition of 30:70 to 100:00 (v/v) for 15 min, using formic acid 0.1 % (v/v) as an additive. MS1 was acquired in an Orbitrap mass analyzer, while MS2 was acquired in a linear trap quadrupole. Satisfactory linearity (Pearson’s r = 0.99709) was obtained for a concentration range of 0.02 to 2.5 µg mL−1, which is compatible with the therapeutic and toxic range for FXT. The developed method presents adequate precision (1.61 to 7.45 %) and accuracy (95 to 114 %) and allows the dilution of high concentration samples in a 1:4 ratio (v/v), enabling its application for forensic serum samples. To our knowledge, this is the first study reporting a method based on HF-LPME and nano-LC-HRMS with any analytical purpose, especially with a TDM focus.

Meropenem Model-Informed Precision Dosing in the Treatment of Critically Ill Patients: Can We Use It?

The number of pharmacokinetic (PK) models of meropenem is increasing. However, the daily role of these PK models in the clinic remains unclear, especially for critically ill patients. Therefore, we evaluated the published meropenem models on real-world ICU data to assess their suitability for use in clinical practice. All models were built in NONMEM and evaluated using prediction and simulation-based diagnostics for the ability to predict the subsequent meropenem concentrations without plasma concentrations (a priori), and with plasma concentrations (a posteriori), for use in therapeutic drug monitoring (TDM). Eighteen PopPK models were included for evaluation. The a priori fit of the models, without the use of plasma concentrations, was poor, with a prediction error (PE)% of the interquartile range (IQR) exceeding the ±30% threshold. The fit improved when one to three concentrations were used to improve model predictions for TDM purposes. Two models were in the acceptable range with an IQR PE% within ±30%, when two or three concentrations were used. The role of PK models to determine the starting dose of meropenem in this population seems limited. However, certain models might be suitable for TDM-based dose adjustment using two to three plasma concentrations.

Capsule Phase Microextraction Combined with Chemometrics for the HPLC Determination of Amphotericin B in Human Serum

This article discusses the use of a sorbent-based microextraction technique employing a capsule device to isolate amphotericin B (AMB) from human serum before analysis by high performance liquid chromatography (HPLC). AMB is a macrocyclic compound used for the treatment of invasive fungal infections. Before determining AMB in human serum by HPLC, a sample preparation step is required. Capsule phase microextraction (CPME) integrates the stirring and filtration mechanisms in a single unit, simplifying the sample preparation procedure. Moreover, it results in fast extraction kinetics and high extraction efficiency, while it has proved to be a powerful tool for bioanalysis. Different sol–gel sorbent encapsulated microextraction capsules were investigated, and sol–gel Carbowax 20 M was finally chosen as the basis for the microextraction device. Accordingly, the sample preparation protocol was investigated using a face-centered central composite design to achieve good extraction performance. The optimum protocol was validated in terms of linearity, selectivity, limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy. The linear range of the developed approach was 0.10–10.0 μg mL−1. The LOD value was 0.03 μg mL−1, and the LOQ value was 0.10 μg mL−1. Method accuracy (expressed as relative recovery) was 87–113%, while the relative standard deviation of the repeatability (sr) and within-laboratory reproducibility (sR) were <12.4%. The sol–gel sorbent encapsulated microextraction capsules were reusable for at least 10 extraction cycles. All things considered, the proposed method exhibited good overall performance, and it could be used in bioanalysis for quality control, therapeutic drug monitoring and research purposes.

A simple and easy non-derivatization gas chromatography-mass spectrometry method for simultaneous quantification of valproic acid, gabapentin, pregabalin, and vigabatrin in human plasma.

Immunoassays are currently not available in commercial kits for the quantification of valproic acid, vigabatrin, pregabalin, and gabapentin, which also cannot suffer the limitations of interferences of substances with similar structures. Chromatography is a good alternative to immunoassay. In this study, a simple and robust non-derivatization gas chromatography-mass spectrometry method for simultaneous determination of the above four drugs in human plasma was developed and validated for therapeutic drug monitoring purposes. This method employed benzoic acid as the internal standard with hydrochloric acid for plasma acidification and ACN for precipitate protein. The supernatant was directly injected into gas chromatography-mass spectrometry for analysis. Good linearity was obtained with linear correlation coefficients of the four analytes of 0.9988-0.9996. Extraction recoveries of valproic acid, vigabatrin, pregabalin, and gabapentin were respectively in the ranges of 91.3%-94.5%, 90.0%-90.9%, 90.0%-92.1%, and 88.0%-92.2% with the relative standard deviation values less than 12.6%. Intra- and inter-batch precision and accuracy, and stability assays were all acceptable. Taken together, the novel method developed in this study provided easy plasma pretreatment, good extraction yield, and high chromatographic resolution, which has been successfully validated through the quantification of valproic acid in the plasma of 46 patients with epilepsy.

Fabric phase sorptive extraction combined with high performance liquid chromatography for the determination of favipiravir in human plasma and breast milk

A fast procedure obtained by the combination of fabric phase extraction (FPSE) with high performance liquid chromatography (HPLC) has been developed and validated for the quantification of favipiravir (FVP) in human plasma and breast milk. A sol-gel polycaprolactone-block-polydimethylsiloxane-block-polycaprolactone (sol-gel PCAP-PDMS-PCAP) coated on 100% cellose cotton fabric was selected as the most efficient membrane for FPSE in human plasma and breast milk samples. HPLC-UV analysis were performed using a RP C18 column under isocratic conditions. Under these optimezed settings, the overall chromatographic analysis time was limited to only 5 min without encountering any observable matrix interferences. Following the method validation procedure, the herein assay shows a linear calibration curve over the range of 0.2–50 µg/mL and 0.5–25 µg/mL for plasma and breast milk, respectively. The method sensitivities in terms of limit of detection (LOD) and limit of quantification (LOQ), validated in both the matrices, have been found to be 0.06 and 0.2 µg/mL for plasma and 0.15 and 0.5 µg/mL for milk, respectively. Intraday and interday precision and trueness, accordingly to the International Guidelines, were validated and were below 3.61% for both the matrices. The herein method was further tested on real samples in order to highlight the applicability and the advantage for therapeutic drug monitoring (TDM) applications. To the best of our knowledge, this is the first validated FPSE-HPLC-UV method in human plasma and breast milk for TDM purposes applied on real samples. The validated method provides fast, simple, cost reduced, and sensitive assay for the direct quantification of favipiravir in real biological matrices, also appliyng a well-known rugged and cheap instrument configuration.

Clinical Effectiveness and Pharmacokinetics of Dalbavancin in Treatment-Experienced Patients with Skin, Osteoarticular, or Vascular Infections.

Dalbavancin (DBV) is a lipoglycopeptide approved for the treatment of Gram-positive infections of the skin and skin-associated structures (ABSSSIs). Currently, its off-label use at different dosages for other infections deserves attention. This work aimed to study the clinical effectiveness and tolerability of DBV in outpatients with ABSSSIs, osteoarticular (OA), or other infections, treated with either one or two 1500 mg doses of dalbavancin, for different scheduled periods. A liquid chromatography–tandem mass spectrometry method was used to measure total DBV concentrations. PK/PD parameters and the clinical and microbiological features of this cohort were evaluated in order to investigate the best predictors of treatment success in real-life settings. Of the 76 screened patients, 41 completed the PK study. Long-term PK was comparable to previous studies and showed significant differences between genders and dosing schedules. Few adverse events were observed, and treatment success was achieved in the vast majority of patients. Failure was associated with lower PK parameters, particularly Cmax. Concluding, we were able to describe DBV PK and predictors of treatment success in selected infections in this cohort, finding DBV Cmax as a possible candidate for therapeutic drug-monitoring purposes, as well as highlighting the dual-dose one-week-apart treatment as the optimal choice for OA infections.

Pharmacokinetic assessment of vancomycin in critically ill patients and nephrotoxicity prediction using individualized pharmacokinetic parameters.

Introduction: Therapeutic drug monitoring (TDM) and pharmacokinetic assessments of vancomycin would be essential to avoid vancomycin-associated nephrotoxicity and obtain optimal therapeutic and clinical responses. Different pharmacokinetic parameters, including trough concentration and area under the curve (AUC), have been proposed to assess the safety and efficacy of vancomycin administration. Methods: Critically ill patients receiving vancomycin at Nemazee Hospital were included in this prospective study. Four blood samples at various time intervals were taken from each participated patient. Vancomycin was extracted from plasma samples and analyzed using a validated HPLC method. Results: Fifty-three critically ill patients with a total of 212 blood samples from June 2019 to June 2021 were included in this study. There was a significant correlation between baseline GFR, baseline serum creatinine, trough and peak concentrations, AUCτ, AUC24h, Cl, and Vd values with vancomycin-induced AKI. Based on trough concentration values, 66% of patients were under-dosed (trough concentration <15 μg/ml) and 18.9% were over-dosed (trough concentration ≥20 μg/ml). Also, based on AUC24h values, about 52.2% were under-dosed (AUC24h < 400 μg h/ml), and 21.7% were over-dosed (AUC24h > 600 μg h/ml) that emphasizes on the superiority of AUC-based monitoring approach for TDM purposes to avoid nephrotoxicity occurrence. Conclusion: The AUC-based monitoring approach would be superior in terms of nephrotoxicity prediction. Also, to avoid vancomycin-induced AKI, trough concentration and AUCτ values should be maintained below the cut-off points.

Dried Volumetric Microsampling Approaches for the Therapeutic Drug Monitoring of Psychiatric Patients Undergoing Clozapine Treatment.

Clozapine is one of the most widely used second-generation antipsychotic drugs (SGAs) for the treatment of schizophrenia. Despite advantages over first-generation drugs, clozapine still shows significant side effects and interindividual variations in efficacy. In order to ensure frequent therapeutic drug monitoring (TDM) and improve the compliance of psychiatric patients undergoing clozapine treatment, two novel dried microsampling approaches based on whole blood and plasma volumetric absorptive microsampling (b-VAMS and p-VAMS) and microfluidic generated-dried blood spot technology (mfDBS) were developed and coupled to HPLC with electrochemical detection (ED). The proposed miniaturized strategies by means of VAMS and microfluidic channel-based devices provide several advantages in terms of collection, storage, and handling compared to classical blood and plasma processing. Satisfactory validation results were obtained for all microsampling platforms, with mean extraction yields >85.1%, precision as relative standard deviation (RSD) < 5.1%, and stability < 4.5% analyte loss after 30 days for p-VAMS; mean extraction yields > 83.4%, precision RSD < 5.4%, and stability < 4.6% analyte loss after 30 days for b-VAMS, and mean extraction yields > 74.0%, precision RSD < 5.6%, and stability < 4.9% analyte loss after 30 days for mfDBS. The original microsampling methodologies have been successfully applied to the blood and plasma collected from five psychiatric patients for the monitoring of the levels of clozapine and its main metabolites, providing robust and reliable quali-quantitative results. Comparisons between results of the two dried microsampling technologies with those obtained by classic fluid plasma analysis were in good agreement and have demonstrated that the proposed miniaturized approaches could be suitable for TDM purposes.

Disease activity and therapeutic drug monitoring of polyglutamates of methotrexate after daily or weekly administration of low-dose methotrexate in patients recently diagnosed with rheumatoid arthritis.

Low-dose methotrexate can be challenging to treat rheumatoid arthritis due to side effects, lack of adherence and risk of medication errors. The aim of this study was to explore the safety and efficacy of low-dose methotrexate administered daily or weekly in patients with rheumatoid arthritis. Patients were randomized according to a total oral dose of 12.5 mg of methotrexate administered: (A) divided in 5 days/week and (B) once per week. Patients were assessed along 24 weeks after starting treatment. Polyglutamates of methotrexate were quantified by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometer. Patients from groups A and B showed a good response to methotrexate treatment in 29% and 25.5%, respectively, and a global frequency of adverse events of 37%. Methotrexate polyglutamate 3 concentrations were higher in normal weight (body mass index 18.5-24.9 kg/m2 ) than in obese (body mass index 30 kg/m2 ) patients with a median (interquartile range) of 28 (17.95-45.15) and 10.35 (5.22-30.88) nM without differences between dosage groups. Daily dosage regimen represents a therapeutic alternative without compromising the efficacy and safety of methotrexate treatment and with similar adherence patterns than weekly dosage regimen; further, methotrexate polyglutamate 3 concentrations could be a useful tool for therapeutic drug monitoring purposes.

Isotope dilution LC-MS/MS quantification of the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their major metabolites in human serum.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105-115 and 96.5-103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.

An Overview of Different Methods for Aminoglycoside Residue Determination

Aminoglycosides (AGs) are chemical substances that exist in the bacteriologic category of traditional antibiotic (AB) therapy. The importance of the determination of AG as has been described in many situations by researchers. Because of the narrow therapeutic ranges of AGs, considerable efforts have been attributed to the analysis of AGs in pharmaceutical preparations, serum, and urine specimens for therapeutic drug monitoring purposes. Residues of ABs in many different cases like environment and human food, causes a major concern, as prolonged exposure to ABs is a serious health hazard, related to both side effects of prolonged use and the risk of developing bacterial resistance to various ABs. The major challenge is finding a sensitive and reliable method to determine AGs in a complex matrix. The microbiological assay was a simple and old method for the determination of AGs. Chromatography and spectroscopy methods are the main instrumental methods for analysis that have been employed for these purposes. Biosensor based instrumental systems have been recently used to determine the AG residues in many cases. Each of these methods has its advantages and disadvantages. This review summarizes different ways (microbiological methods, instrumental methods, and biosensor) for the determination of AGs in all cases. Different databases including PubMed, Scopus, and Web of Science with the words of AGs determination and related words for antimicrobial keywords searched without time limitation.

Determination of Voriconazole Plasma Concentration by HPLC Technique and Evaluating Its Association with Clinical Outcome and Adverse Effects in Patients with Invasive Aspergillosis.

Purpose Invasive aspergillosis is a prevalent fungal disease, especially in Asian countries with a high mortality rate. Voriconazole (VRZ) is the first choice for invasive aspergillosis treatment. Plasma concentration of this drug is unpredictable and varies among individuals. This variability is influenced by many factors leading to clinical implication. Therapeutic drug monitoring (TDM) may have a crucial role in the patients' treatment process. The HPLC method provides sufficient specificity and sensitivity for plasma VRZ concentration determination for TDM purposes of this drug. Methods Patients who initiated oral or intravenous VRZ for invasive aspergillosis were enrolled in this study. Demographic characteristics and clinical data, outcome, and adverse effects were documented. For each patient, the plasma sample was collected under steady-state condition and analyzed using a validated HPLC method. Results A total of 22 measurements were performed. Fifty percent of patients were out of the therapeutic range. From them, 27.27% and 22.73% were in subtherapeutic and supratherapeutic ranges (<1 μg/mL and >5.5 μg/mL), respectively. There was a significant correlation between VRZ plasma concentration and treatment outcomes (P=0.022). Treatment failure was five times higher than treatment success in those in the subtherapeutic range. Adverse effects were observed more frequently in patients with supratherapeutic concentrations compared to those with non-supratherapeutic levels. Furthermore, the mortality rate in patients experiencing treatment failure was 2.17 times higher than those with treatment success. Conclusions TDM of VRZ plays an important role in better evaluation of efficacy and toxicity during treatment. Therefore, determination of the drug level may be of clinical significance.

Liquid Chromatography-Tandem Mass Spectrometry to Monitor Unbound and Total Ceftriaxone in Serum of Critically Ill Patients.

Ceftriaxone is recommended for empiric antimicrobial therapy in patients with sepsis. Therapeutic Drug Monitoring (TDM) guided dose optimisation could elucidate pharmacokinetic variabilities, improving treatment efficacy. However, detailed data on Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) for unbound ceftriaxone quantification in serum are scarce. The authors aimed to develop a reliable UPLC-MS/MS method for serum ceftriaxone quantification and exhibit its application potential in routine hospital settings. In this observational, single centre study, UPLC-MS/MS method validation included specificity, carry-over, linearity, repeatability, intermediate precision, accuracy, the limit of quantification, and plasma protein binding. Unbound and total ceftriaxone were quantified in the serum of 5 critically ill patients. Pharmacokinetic/Pharmacodynamic (PK/PD) target attainment calculations were performed for both unbound and total ceftriaxone. The PK/PD target for unbound ceftriaxone in serum was set at 4 times the non-species related minimum inhibitory concentration breakpoint of 1 mg/L for at least 60% of the dosing interval. The UPLC-MS/MS method revealed acceptable limits for clinical samples, with coefficients of variation < 15.0% for concentrations between 0.2 - 400.0 mg/L. Ceftriaxone eluted at 1.94 min and ceftazidime, as internal standard, eluted at 1.42 min. Patients demonstrated a median unbound ceftriaxone fraction of 29.1% (IQR: 15.2 - 52.2). Day 1 of ceftriaxone treatment presented a median PK/PD target attainment of 100.0% (IQR: 81.1 - 100.0) for unbound ceftriaxone in serum, while for calculations based on total concentrations, this figure was 23.9% (IQR: 10.5 - 80.6). The described UPLC-MS/MS method enables reliable and rapid ceftriaxone quantification in the serum of critically ill patients. Method feasibility was exhibited for TDM purposes in routine clinical practice.

Method Validation for the Determination of Carbamazepine in Spiked-saliva Using HPLC-UV for Therapeutic Drug Monitoring Application

Background:: Carbamazepine has been used in the treatment of bipolar disorder, both in acute mania and maintenance therapy, particularly in developing countries. Not only its interaction with various drugs and auto-inducer nature, but the narrow therapeutic range of carbamazepine also makes monitoring necessary to guarantee the adequacy of its safety and therapeutic concentration. To date, the most common biological specimen used for therapeutic drug monitoring (TDM) purposes is still plasma, but saliva can become an alternative biological matrix since its level in saliva strongly correlates with carbamazepine plasma concentration. Objective:: This study validated the bioanalytical method parameters used for carbamazepine in spiked-saliva in accordance with the Food and Drug Administration (FDA) criteria in the Guidance for Industry Bioanalytical Method Validation. Methods: HPLC-UV detector was employed at 285 nm λ with methanol: water: glacial acetic acid (65:34:1) as the mobile phase and C8 as the stationary phase (4.6x150 mm; 5 μm). Results:: The linearity test in a range of 0.0 - 5 μg/mL carbamazepine concentration resulted in a correlation coefficient of 0.999 with 0.20 μg/mL LoD, 0.30 μg/mL LLoQ, and 0.61 μg/mL LoQ. The coefficient of variation and 0iff in the selectivity, accuracy, and precision parameters remained below 20%, indicating fulfillment of the criteria for a bioanalytical method, while the average % recovery was more than 90%. Conclusion:: The currently-developed bioanalytical method has fulfilled the stipulated validation criteria to be used for determining carbamazepine concentration in spiked-saliva as an alternative method for relative bioequivalence studies or TDM application in a clinical setting.

Measurement of Free Plasma Concentrations of Beta-Lactam Antibiotics: An Applicability Study in Intensive Care Unit Patients.

The antibacterial effect of antibiotics is linked to the free drug concentration. This study investigated the applicability of an ultrafiltration method to determine free plasma concentrations of beta-lactam antibiotics in ICU patients. Eligible patients included adult ICU patients treated with ceftazidime (CAZ), meropenem (MEM), piperacillin (PIP)/tazobactam (TAZ), or flucloxacillin (FXN) by continuous infusion. Up to 2 arterial blood samples were drawn at steady state. Patients could be included more than once if they received another antibiotic. Free drug concentrations were determined by high-performance liquid chromatography with ultraviolet detection after ultrafiltration, using a method that maintained physiological conditions (pH 7.4/37°C). Total drug concentrations were determined to calculate the unbound fraction. In a post-hoc analysis, free concentrations were compared with the target value of 4× the epidemiological cut-off value (ECOFF) for Pseudomonas aeruginosa as a worst-case scenario for empirical therapy with CAZ, MEM or PIP/tazobactam and against methicillin-sensitive Staphylococcus aureus for targeted therapy with FXN. Fifty different antibiotic treatment periods in 38 patients were evaluated. The concentrations of the antibiotics showed a wide range because of the fixed dosing regimen in a mixed population with variable kidney function. The mean unbound fractions (fu) of CAZ, MEM, and PIP were 102.5%, 98.4%, and 95.7%, with interpatient variability of <6%. The mean fu of FXN was 11.6%, with interpatient variability of 39%. It was observed that 2 of 12 free concentrations of CAZ, 1 of 40 concentrations of MEM, and 11 of 23 concentrations of PIP were below the applied target concentration of 4 × ECOFF for P. aeruginosa. All concentrations of FXN (9 samples from 6 patients) were >8 × ECOFF for methicillin-sensitive Staphylococcus aureus. For therapeutic drug monitoring purposes, measuring total or free concentrations of CAZ, MEM, or PIP is seemingly adequate. For highly protein-bound beta-lactams such as FXN, free concentrations should be favored in ICU patients with prevalent hypoalbuminemia.