Drug Monitoring Studies Research Articles

Setting the Beta-Lactam Therapeutic Range for Critically Ill Patients: Is There a Floor or Even a Ceiling?

Research articles were sourced from PubMed using search term combinations of "pharmacokinetics," "pharmacodynamics," "toxicity," "neurotoxicity," "therapeutic drug monitoring," "beta-lactam," "cefepime," "meropenem," "piperacillin/tazobactam," "ICU," and "critical illness." Articles were selected if they included preclinical, translational, or clinical data on pharmacokinetic and pharmacodynamic thresholds for effectiveness and safety for beta-lactams in critical illness. Experimental data indicate a beta-lactam concentration above the minimum inhibitory concentration of the organism for greater than or equal to 40-60% of the dosing interval is needed, but clinical data indicate that higher concentrations may be preferrable. In the first 48 hours of critical illness, a free beta-lactam concentration at or above the susceptibility breakpoint of the most likely pathogen for 100% of the dosing interval would be reasonable (typically based on Pseudomonas aeruginosa). After 48 hours, the lowest acceptable concentration could be tailored to 1-2× the observed minimum inhibitory concentration of the organism for 100% of the dosing interval (often a more susceptible organism). Neurotoxicity is the primary dose-dependent adverse effect of beta-lactams, but the evidence remains insufficient to link a specific drug concentration to greater risk. As studies advance the understanding of beta-lactam exposure and response in critically ill patients, it is essential to clearly define the acceptable therapeutic range to guide regimen selection and adjustment.

Development and validation an HPLC - UV method for determination of esomeprazole and pirfenidone simultaneously in rat plasma: application to a drug monitoring study

Background and Aims: It has been observed that the combined treatment of esomeprazole and pirfenidone provides increased efficacy in the treatment of pulmonary fibrosis disease, recently. The aim of this study is to develop a simple, sensitive, and reliable high-performance liquid chromatography method to be used in drug monitoring to increase the effectiveness of esomeprazole and pirfenidone in treatment and to reduce their adverse effects. Methods: Separation was conducted with a C18 reverse-phase column (4.6 mm x 250 mm, 5 µm) used as a mobile phase prepared with the phosphate buffer (10 mM KH2PO4 and 10 mM K2HPO4) and acetonitrile (60:40, v/v) by an isocratic flow (1 mL/min). Mobile phase pH was adjusted to 3.0. Ultraviolet detection was accomplished at 305 nm. The column oven was held at 35°C to ensure an efficient analytical separation. Results: Analytical recovery of esomeprazole was between 92.43 and 105.36% and for pirfenidone it was found between 89.56 and 104.32%. Accuracy values of esomeprazole and pirfenidone were determined between (-2.90) – 4.22 and (-4.45) – 5.78, respectively. Precision (RSD%) was ≤7.89. The quantification limit was determined as 0.58 and 0.36 ng/mL. Plasma esomeprazole and pirfenidone levels were found as 0.87-8296.87 ng/mL (612.99±2212.20, mean ± standard deviation) and 0.45-238.60 ng/mL (61.44±76.35, mean ± standard deviation), respectively. Conclusion: Unexpectedly high RSD values were observed in both plasma (360.88%) and dose-rated results (89.61%) of esomeprazole, and pirfenidone were thought to be related to individual metabolism differences.

An SPR investigation into the therapeutic drug monitoring of the anticancer drug imatinib with selective aptamers operating in human plasma.

The anticancer drug imatinib is often involved in therapeutic drug monitoring (TDM) studies aimed at improving the treatment of several forms of leukemia and gastrointestinal stromal tumors (GIST). To further implement the TDM of imatinib in clinical practice, we developed a detection assay by using an ssDNA aptamer, which demonstrated excellent selectivity and was not affected by interference from the components of human plasma samples. The efficient binding of imatinib to the aptamer was demonstrated by means of surface plasmon resonance (SPR) analysis, which allowed the development of a quantitative assay in the concentration range between 400 and 6000 ng mL-1 (0.7-10 μM), where a lower limit of quantification (LLOQ) of 400 ng mL-1 was achieved. The precision of the assay was found to be within 12.0%, whereas the accuracy was in a range between 97.1 and 101.5%. The sample preparation procedure displayed a recovery in the range of 48.8-52.8%. Solid validation data were collected according to the regulatory guidelines and the method was compared with standard analytical techniques, leading to the development of a feasible aptasensor for the TDM of patients administered with imatinib.

High-Performance Liquid Chromatography for the Simultaneous Estimation of Cefoperazone and Sulbactam in Rat Plasma and its Importance in Therapeutic Drug Monitoring

Therapeutic drug monitoring and pharmacokinetic studies of antibiotics need a sensitive and precise measurement ofplasma drug concentrations. In the present study, we developed a rapid, sensitive and selective chromatographicmethod for simultaneous estimation of cefoperazone (CEF) and sulbactam (SAL) in male Wistar rat plasma. A novelliquid phase extraction method has adopted the preparation of plasma sample preparation. The CEF and SAL wereeluted on a peerless Basic C18 (25 cm; 4.6 mm x 5 μm) column maintained at controlled environmental conditions.The gradient mobile phase comprised of 10 mM ammonium acetate and acetonitrile. A UV detector was set at 250nm and retention times for CEF and SAL were approximately 5.6 and 14.2 min, respectively. The proposed HPLCmethod was validated according to the US FDA guidelines with respect to the linearity, accuracy, precision, detectionand quantitation limits, robustness and specificity. Calibration curves of CEF and SAL were linear across theconcentration range of 600-1000 and 6-10 μg/mL, with correlation coefficients (r2) >0.9977 and (r2) >0.9987,respectively. The limits of detection for CEF and SAL were 70.48 and 0.35 μg/mL, respectively. Additionally, CEFand SAL were stable in plasma for at least 24 h when stored at room temperature and 2-8 C. Furthermore, thedeveloped chromatographic method was effectively utilized to measure the plasma CEF and SAL concentrations in apharmacokinetics study after intravenous injection to the healthy male Wistar rats.

Method Development and Validation for Simultaneous Determination of Six Tyrosine Kinase Inhibitors and Two Active Metabolites in Human Plasma Using UPLC–MS/MS for Therapeutic Drug Monitoring

Over the past decades, therapeutic drug monitoring (TDM) of tyrosine kinase inhibitors (TKIs) and their main active metabolites has shown benefits in improving treatment efficacy and safety. Therefore, a sensitive, simple and economical ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of six oral tyrosine kinase inhibitors (TKIs) and two active metabolites, including imatinib (IMA), N-desmethyl imatinib (NDIMA), sunitinib (SUNI), N-desethyl sunitinib (NDSUNI), regorafenib (REGO), nilotinib (NILO), dasatinib (DASA) and osimertinib (OSI) in human plasma for therapeutic drug monitoring. The plasma samples were deproteinated with acetonitrile after spiking with two deuterated internal standards (ISs, [ 2 H 8 ]-imatinib and [ 2 H 10 ]-sunitinib) and separated on a 40 °C ACQUITY UPLC® BEH C 18 column (1.7 μm, 2.1 mm×50 mm). The mobile phase was composed of acetonitrile (solution A) and water-formic acid-ammonium acetate (1M)(994:1:5, v/v/v, solution B). Gradient elution was applied at a flow rate of 0.4 mL/min. Detection was carried out using a Triple Quad 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The method was validated over the calibration curve (CV) range of 2-400 ng/mL for NDSUNI and DASA, 2.5-500 ng/mL for SUNI, 10-2000 ng/mL NDIMA and OSI, 20-4000 ng/mL for NILO, 30-6000 ng/mL for REGO and 50-10000 ng/mL for IMA using linear regression and 1/x 2 weighting. No difference was observed in the matrix effect (ME) among blank human plasma, hemolytic plasma and lipemic plasma samples in general. And the intra- and inter-batch precision and accuracy of all eight components were acceptable. To provide a clinical reference for the operation, the stability of the whole process from sample collection to drug detection was verified. SUNI and NDSUNI showed obvious photoisomerization under light exposure. Therefore, strict light protection was applied for all sample collection and handling steps of SUNI and NDSUNI. Compared with heparin anticoagulant tubes, the stability of the eight compounds in both whole blood and plasma was better in K3-EDTA and sodium citrate anticoagulant tubes. Given that all the analytes were stable in plasma at 4 °C for 48 h and in whole blood at room temperature for 48 h but OSI and REGO were unstable in whole blood and plasma at room temperature, samples should be centrifuged as soon as possible to be preserved as plasma at 4 °C when OSI or REGO is detected. In conclusion, this validated method can provide support for clinical practice, such as therapeutic drug monitoring (TDM) and pharmacokinetic studies of these six TKIs and two active metabolites.

Determination of phenytoin in human plasma by a validated hplc method: application to therapeutic drug monitoring study

Determination of phenytoin in human plasma by a validated hplc method: application to therapeutic drug monitoring study

1020. Higher efavirenz mid-dose plasma concentration is associated with less weight gain among virologically suppressed people living with HIV

BackgroundRecently, an association between CYP2B6 516 G > T polymorphism and weight change was observed among African people living with HIV (PLWH) who were started on efavirenz(EFV)-based antiretroviral therapy (ART). We aimed to investigate the effect of EFV mid-dose plasma concentration on weight change among Taiwanese PLWH.MethodsThe medical records of adult PLWH who were taking EFV-containing ART and had been enrolled in a EFV therapeutic drug monitoring study between Oct 2009 and May 2014 were accessed. EFV mid-dose plasma concentration (C12) was determined in the previous study and those with serial weight measurements within 48 weeks around the time of EFV C12 measurement were included in the present study. The weight change in the 48 weeks and its associations with mid-dose EFV concentrations and CYP2B6 516 G > T polymorphism were examined by general estimating equations (GEE) after adjusting for age, baseline HIV viremia, baseline weight, and the companion backbone antiretroviral agents.ResultsOne-hundred and fifteen predominantly male (94.8%) PLWH were included in this study (Table 1). The mean CD4 lymphocyte count was 542 cells/μL at the beginning of the observation and 94.8% achieved HIV viral suppression. Forty-four (38.3%) patients had non-wildtype CYP2B6 516 G > T polymorphism. On average, the included PLWH gained 0.8 kilogram at 48 weeks and the weight change did not differ among those with wildtype and non-wildtype CYP2B6 516 G > T(Figure 1). In the GEE models, CYP2B6 516 G > T polymorphism was not associated with weight change (p=0.81), instead, higher EFV C12 was found to be independently associated with less weight gain (p=0.045).Table 1. Baseline characteristics. Figure 1. The absolute weight (A) and weight change from baseline (B) among patients with wildtype and non-wildtype CYP2B6 516G>T polymorphism. ConclusionOur findings support that increased EFV exposure may have a negative effect on weight gain.Disclosures Chien-Ching Hung, MD,PHD, Abbvie (Advisor or Review Panel member, Speaker’s Bureau)Bristol-Myers Squibb (Speaker’s Bureau)Gilead Sciences (Advisor or Review Panel member, Speaker’s Bureau)Janssen (Grant/Research Support, Advisor or Review Panel member)Merck (Grant/Research Support)ViiV (Grant/Research Support, Advisor or Review Panel member, Speaker’s Bureau)

Quantification of pyrazinamide, isoniazid, acetyl-isoniazid, and rifampicin by a high-performance liquid chromatography method in human plasma from patients with tuberculosis.

The aim of this study was to establish and validate an alternative high-performance liquid chromatography method for simultaneous quantification of pyrazinamide, isoniazid, acetyl-isoniazid and rifampicin in plasma of patients under treatment for tuberculosis. The performed method was lineal (r2 >0.99) in the range of 2.00-50.00μg/mL for pyrazinamide, 0.50-20.00μg/mL for both acetyl-isoniazid and isoniazid, and 1.20-25.00μg/mL for rifampicin. Precision and trueness were demonstrated with coefficient of variation<15% and deviations<15%, respectively, for quality controls samples. The lower limits of quantification were 2.00, 0.50, 0.50, and 1.20μg/mL for pyrazinamide, isoniazid, acetyl-isoniazid and rifampicin, respectively. The method was applied for the analysis of plasma from patients with tuberculosis. This method allowed ensuring reliable quantification of the target compounds and their pharmacokinetics parameters. In general, the mean values of maximum concentration of each antituberculosis drug were located within their respective reference therapeutic ranges. However, patients with sub-therapeutic plasma concentrations of isoniazid and rifampicin were detected. This is the first analytical technique that simultaneously quantifies isoniazid, acetyl-isoniazid, rifampicin, and pyrazinamide concentrations from plasma samples by high-performance liquid chromatography with ultraviolet/visible. The proposed method could be applied for therapeutic drug monitoring and pharmacokinetics studies of the four compounds throughout the treatment of tuberculosis patients.

Simultaneous separation and determination of vancomycin and its crystalline degradation products in human serum by ultra high performance liquid chromatography tandem mass spectrometry method and its application in therapeutic drug monitoring.

A rapid and simple ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous separation and determination of vancomycin and its crystalline degradation products in human serum. Vancomycin and two isomers of the degradants were extracted from human serum with a protein precipitation method. The compounds were separated on an Acquity BEH C18 column (2.1 × 50mm, 1.7 μm) eluted with a gradient mixture of acetonitrile and 0.1% formic acid as the mobile phase. Norvancomycin was used as the internal standard. The linear ranges of vancomycin and two degradant isomers were 1.057-105.7, 0.1437-14.37, and 0.2540-25.40μg/mL, respectively. The established methods were validated and successfully applied to a therapeutic drug monitoring study of vancomycin in patients with renal insufficiency.

Toxicity Profile, Pharmacokinetic, and Drug–Drug Interaction Study of Citalopram and Sertraline Following Oral Delivery in Rat: An LC-MS/MS Method for the Simultaneous Determination in Plasma

The burden of depression and other mental disorders is on the rise globally, and successful treatment sometimes requires modifications of drugs and/or dose regimens. The development of novel analytical methods for the determination of antidepressant drugs in biological fluids is thus urgently required. Herein, a sensitive, robust, and rapid liquid chromatographic coupled tandem mass spectrometric method was developed and validated for the determination of citalopram (CIT) and sertraline (SER) in rat plasma after oral administration. The analytes of interest and internal standard (duloxetine (DUL)) were extracted from 100 μL of plasma with solid-phase extraction on an Oasis HLB cartridge followed by the separation with gradient elution with water containing 0.1% formic acid and acetonitrile on an Agilent Eclipse Plus ODS (4.6 × 100 mm, 3.5 μm) column at flow rate 0.2 mL min-1. The triple quadrupole mass spectrometry was applied via electrospray ionization source for detection. The fragmentation pattern of the protonated CIT, SER, and DUL was elucidated using multiple reaction monitoring of the transitions of m/z 325.2 to 109, 306.1 to 158.9, and 298.1 to 154.1 as [M + H]+ for CIT, SER, and DUL, respectively. The proposed method has been validated as per US-FDA bioanalytical guidelines in terms of linearity, accuracy, precision, matrix effects, stability, selectivity, and recovery. The method was linear over the concentration range of 1-2000 and 1-1000 ng mL-1 with the lower limit of detection of 0.12 and 0.19 ng mL-1 for CIT and SER, respectively. The interday and intraday precisions and accuracy expressed by the relative standard deviation and the relative standard error were both lower than 11.1% and 2.1%, respectively. The proposed method was successfully applied for the pharmacokinetics and drug monitoring studies of CIT and SER in rat plasma after a single oral dose of 120 mg kg-1 of CIT and SER. Coadministration of SER with CIT has affected the peak plasma concentrations, maximum plasma concentration time, area under the concentration-time curve, and oral clearance of CIT. Molecular modeling study showed that SER could competitively inhibit CYP2D6, the main enzyme involved in CIT metabolism. A possible drug-drug interaction in psychiatric patients undergoing chronic SER and CIT treatment is therefore worthy of more attention in an effort to avoid side effects and serotonin syndrome.

Electromembrane extraction as a new approach for determination of free concentration of phenytoin in plasma using capillary electrophoresis

Electromembrane extraction is a new membrane-based extraction method in which charged compounds are extracted by an electric field. So far, this method has been used to extract and isolate a variety of acidic and basic drugs from various samples, including blood and plasma. However, in this procedure, it is not yet clear whether only unbound fraction of a drug is extracted or the total drug. The aim of this study is to reveal the nature of drug extraction in the presence of plasma proteins. To determine the nature of the extraction, the electromembrane extraction was performed from plasma solutions of phenytoin with concentrations 0.03 and 1.0μg/mL, then the result was compared with the values obtained from the electromembrane extraction of ultrafiltrate of the same solutions (free concentration) and protein-free ultrafiltrate of plasma with final concentration of 0.03 and 1.0μg/mL (total concentration). For this purpose, EME followed by capillary electrophoresis coupled with diode array detection was optimized and validated. The results showed that the electromembrane extraction method was only able to extract the unbound fraction of phenytoin from plasma samples. The method was validated over a concentration range of 0.03-4μg/mL. The inter and intra-assay precisions were less than 6.7%. The phenytoin protein binding was also determined to be in agreement with the literature data and confirms the validity of this method. This sensitive and quick EME approach for determining the free concentration of a phenytoin, can be a good alternative to classic methods for therapeutic drug monitoring and pharmacokinetic studies.

Validated LC-MS/MS method for the simultaneous determination of enalapril maleate, nitrendipine, hydrochlorothiazide, and their major metabolites in human plasma.

Hypertension is a major risk factor for atherosclerosis and ischemic heart disease. Most hypertensive patients need a combination of antihypertensive agents to achieve therapeutic goals. A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometric method was developed and validated for simultaneous determination of enalapril maleate (ENA) and its major metabolite enalaprilat (ENAT), nitrendipine (NIT) and its major metabolite dehydronitrendipine (DNIT), and hydrochlorothiazide (HCT) in human plasma using felodipine as an internal standard (IS). The drugs were extracted from plasma using one-step protein precipitation. Chromatographic separation was performed on a Symmetry C18 column, with water and acetonitrile (10:90, v/v) as mobile phase. The detection was carried out using multiple reaction monitoring mode and coupled with electrospray ionization source. Multiple reaction monitoring transitions were m/z 377.1 → 234.1 for ENA, m/z 349.2 → 206.1 for ENAT, m/z 361.2 → 315.1 for NIT, m/z 359 → 331 for DNIT, m/z 295.9 → 205.1 for HCT, and m/z 384.1 → 338 for felodipine (IS). The method was linear over concentration ranges of 1-200, 20-500, 5-200, 2-100, and 5-200 ng/mL for ENA, ENAT, NIT, DNIT, and HCT, respectively, with r2 ≥ 0.99. Method validation was performed according to U.S. Food and Drug Administration guidelines. The validated method showed good sensitivity and selectivity and could be applied for therapeutic drug monitoring and bioequivalence studies.

Simultaneous Determination of Docetaxel and Celecoxib in Porous Microparticles and Rat Plasma by Liquid-Liquid Extraction and HPLC with UV Detection: in vitro and in vivo Validation and Application.

A simple, rapid, sensitive, and reliable HPLC method with UV detection was developed and validated for simultaneous quantitation of docetaxel and celecoxib and paclitaxel for dissolution characterization and pharmacokinetic studies. The HPLC assay was performed isocratically on a reversed-phase C18 μ-Bondapack column using a mobile phase of acetonitrile:water (45:55, v/v) at a flow rate of 1.2 mL/min, and the analytes were detected at 230 nm. Paclitaxel was used as an internal standard for analysis of plasma samples following simple liquid-liquid extraction with n-hexane:isoamyl alcohol (97:3). The method was validated for specificity, linearity, sensitivity, precision, accuracy, robustness, and in vitro-in vivo application. The retention times for docetaxel, paclitaxel, and celecoxib were 10.94, 12.4, and 16.81 min, respectively. The standard curves covering 0.1-1 μg/mL and 0.05-4 μg/mL were linear using dissolution medium and rat plasma, respectively. The limit of quantitation of the method was 50 ng/mL using 100 μL of rat plasma sample and injection of 50 μL of the residue. Within- and between-day precision and accuracy did not exceed 16.86% and 12.10%, respectively. This validated method was successfully used to quantify docetaxel and celecoxib simultaneously in the release study of docetaxel- celecoxib -loaded porous microparticles and pharmacokinetics studies. The methods were found to be simple, specific, precise, accurate, and reproducible. In this study, paclitaxel was used as the internal standard while dexamethasone, flutamide, and budesonide proved suitable alternative as an internal standard. Since docetaxel and celecoxib could be co-administered for the treatment of a wide range of cancers such as non-small cell lung carcinoma, the developed method is particularly advantageous for routine therapeutic drug monitoring and pharmacokinetic studies of these drugs.

Simple and Fast Determination of Terbinafine in Human Urine by Dilute and Shoot HPLC-DAD Using a Core-Shell Column.

Terbinafine is an allylamine antifungal that is effective against many fungi, dermatophytes and moulds. Analytical methods are required for the determination of terbinafine in biological fluids to perform therapeutic drug monitoring and pharmacokinetic studies. The aim of this study was to develop and validate a novel and fast method combining dilute and shoot approach and high-performance liquid chromatography coupled with photodiode array detection for the determination of terbinafine in human urine. Chromatographic parameters including mobile phase composition, pH, flow rate and injection volume were assessed and optimized. The separation of terbinafine and naproxen (internal standard) was achieved within 3 min using a C18 core-shell column (Raptor ARC-18, 100 x 4.6 mm, 2.7 μm) under isocratic conditions. Samples were eluted from the column at the flow rate of 1.4 mL/min using a mobile phase containing 0.2% triethylamine in water (pH 3.4 with formic acid): acetonitrile (45:55, v/v). The presented technique was linear in the range of 25-2000 ng/mL. Intra- and inter-day reproducibility at four quality control levels (25, 200, 750 and 1500 ng/mL) were less than 7%, with relative errors ranging from -5.40% to 5.91%. The limit of detection was 12.60 ng/mL. The developed method has three main advantages compared to existing methods: simplicity and greenness of sample preparation, use of core-shell column and short analysis time. The results of this study indicate that the combination of dilute and shoot approach and core-shell column can be regarded as an advantageous application for the fast determination of terbinafine in the urine.

Preparation of magnetic metal organic framework and development of solid phase extraction method for simultaneous determination of fluconazole and voriconazole in rat plasma samples by HPLC

Fluconazole and voriconazole are the two broad-spectrum triazole antifungals. The present work described the fabrication method for the synthesis of the amino-modified magnetic metal–organic framework. This material was applied as a pre-sample treatment sorbent for the selective extraction of fluconazole and voriconazole in rat plasma samples. The material was fabricated by the chemical bonding approach method and was characterized by different parameters. The factors which affect the extraction efficiency of the sorbent material were also optimized in this study. Due to the optimization of solid-phase extraction conditions, the nonspecific interaction was reduced and the extraction recoveries of target drugs were increased in plasma samples. The extraction method was combined with the HPLC-UV method for the analysis. Excellent linearity (0.1–25 µg/mL), detections (0.02, 0.03 µg/mL) and quantification limits (0.04, 0.05 µg/mL) were resulted for fluconazole and voriconazole respectively. The maximum recoveries from spiked plasma samples of fluconazole and voriconazole were 86.8% and 78.6% and relative standard deviation were 0.9–2.8% and 2.2–3.6% respectively. Moreover, this sorbent material was used multiple times which was an improvement over single-use commercial sorbent materials. This validated method has practical potential for the simultaneous determination of these drugs in therapeutic drug monitoring studies as well as for routine pharmacokinetic evaluations.

Preparation of restricted access monolithic tip via unidirectional freezing and atom transfer radical polymerization for directly extracting magnolol and honokiol from rat plasma followed by liquid chromatography analysis

In the present study, a novel strategy based on unidirectional freezing and atom transfer radical polymerization combined with activator regenerated by electron transfer (ARGET-ATRP) was applied to synthesizing orderly macroporous monolithic column with restricted-access (RA) property in a 1000μL pipette tip. The RA column was composed of hydrophobic inner column (poly(styrene-co-ethylene glycol dimethacrylate) and hydrophilic outer layer (poly-hydroxyethyl methacrylate chain) which was grafted on the hydrophobic surface by means of the second ARGET-ATRP reaction. The as-prepared RA monolithic tip was connected to a 2mL syringe for directly extracting magnolol and honokiol from rat plasma just by manually pushing operation. The surface morphology and chemical composition of the column were characterized by scanning electronic microscope, infrared spectroscopy and X-ray photoelectron spectroscopy respectively. The determined results of evaluation experiments based on the optimized solid phase extraction conditions showed that the RA column possessed good protein exclusion power, extraction recovery and reusability. The constructed RA-SPE-HPLC/UV method for simultaneously analyzing magnolol and honokiol in rat plasma was validated with quality control (QC) samples at four concentration levels. Good precision (RSDs, 3.39~11.16%) and acceptable accuracy (relative recoveries, 89.52%~108.42%) were obtained for intra- and inter-day assays. The determined results of real rat plasma as well as the standard-addition samples demonstrated the developed method with good accuracy and precision. It can be extrapolated from the experimental results that this simple and cost-efficient RA-SPE method is also suitable for directly extracting other hydrophobic constituents in biological body fluid for therapeutic drug monitoring or pharmacokinetic study.

Using Kinetic eGFR for Drug Dosing in AKI: Concordance between Kinetic eGFR, Cockroft-Gault Estimated Creatinine Clearance, and MDRD eGFR for Drug Dosing Categories in a Pilot Study Cohort

Introduction: Drug dosing in patients with acute kidney injury (AKI) is based on the Cockroft-Gault (CG) equation-derived estimated Creatinine clearance (CGeCrCl) for historical reasons due to the lack of a validated method for estimating glomerular filtration rate (GFR) when Cr is rapidly changing. The kinetic equation (kinetic estimated GFR [KeGFR]) estimates GFR for the nonsteady-state Cr level. It is being validated in patient cohorts and could potentially be used for drug dosing when the Cr level is in the nonsteady state. Objective: The aim of the study was to measure the concordance and the degree of agreement between KeGFR-, CGeCrCl, and MDRD eGFR-based drug dosing categories in patients with nonsteady Cr levels. Methods: In the pilot study published previously, 80 adult patients with a significant change in Cr level after admission to the acute medical ward were classified as per the Acute Kidney Injury Network (AKIN) criteria and compared to a KeGFR-based criterion. The CG equation and the MDRD equation were applied retrospectively to the same dataset, and the concordance of the eGFR categories between the 3 methods was studied. The 3 eGFR categories (<30, 30–49, and >50 mL/min) were chosen to reflect the frequently used drug dosing categories in patients with renal impairment. Results: The concordance between CGeCRCL and KeGFR for drug dosing categories was only 62%, with 27 (90%) of the 30 discordant subjects falling into a higher eGFR category when KeGFR was used. The agreement between KeGFR and CGeCrCl was also unsatisfactory. There was better concordance (75%), but the agreement was also not satisfactory between MDRD eGFR and KeGFR for the drug dosing categories. Conclusions: In AKI, compared to CGeCrCL, using KeGFR may affect drug dosing significantly by changing the eGFR category. Further studies of KeGFR for drug dosing will need therapeutic drug monitoring and pharmacokinetic studies for validation.

A simple, rapid and sensitive high-performance thin-layer chromatographic method for the simultaneous estimation of berberine and 5-fluorouracil in rabbit plasma

The purpose of the present study was to develop a simple, rapid and sensitive high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of 5-fluorouracil (5FU) and berberine (BER) in rabbit plasma. BER and 5-FU are currently under extensive studies for their synergistic effects in cancer chemotherapy. The HPTLC method was developed using TLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. A mixture of toluene:methanol:ethyl acetate:formic acid (6:2:6:1, v/v) was used as the solvent system for development and detection was carried out at 266 nm. The method was designed to obtain the most optimum retardation factors (Rf) with best resolution. The Rf values of BER and 5-FU were 0.28 ± 0.02 and 0.57 ± 0.02, respectively. The plasma extraction was optimized using ethyl acetate. The method was validated in compliance with the USFDA guideline for bioanalytical method validation. The method was found to be linear in the range of 20 ng/spot to 300 ng/spot with R2 of 0.9964 for 5-FU and 0.9943 for BER. The lower limit for quantification (LLOQ) of BER and 5-FU in rabbit plasma were obtained as 29 ng/spot and 26 ng/spot, respectively. The stability of BER and 5-FU in plasma were confirmed during freeze-thaw cycles (−20 °C), on bench for 5 h and long term for 21 days. The proposed method was validated statistically and was precise, accurate and sensitive for the estimation of BER and 5-FU in rabbit plasma. This method can be extended for therapeutic drug monitoring and pharmacokinetic studies in rabbits.

Development and validation of a new HPLC method for valproic acid determination in human plasma and its application to a therapeutic drug monitoring study

The aim of this study was to develop a new, simple and reliable high performance liquid chromatography (HPLC) method for analysis of valproic acid (VPA) in human plasma and apply to it to a therapeutic drug monitoring study. Also, the relationship between plasma-VPA concentrations and the amount of VPA used by patients was aimed to be evaluated. Plasma samples (0.25 mL) were precipitated with the same volume of acetonitrile and after centrifugation, aliquots were applied to a C18 column (250 mm x 4.6 mm). Mobile phase was prepared with phosphate buffer and acetonitrile (47.5:52.5, v/v). The flow-rate was 1.2 mL/min. Accuracy was between -2.9 and 3.2% and precision was ≤6.6%. Method was specific and sensitive with a detection limit of 2.2 µg/mL and the average recovery was 94.3%. Calibration curve was linear (r2&gt;0.9968) from 10 to 150 µg/mL. Plasma-VPA levels of the epileptic patient population (n=33) treated with VPA between 0.5 and 1.5 g/day were also determined. Patient plasma-VPA concentrations ranged from 2.9 to 166.4 µg/g/mL (56.3±38.8). High RSD% (68.8%) was observed in dose-rated plasma-VPA results. Moreover, VPA plasma levels were found to be outside the recommended treatment range in 30.3% of the patients examined. The procedure described was found to be relatively simple, precise, and applicable for routine therapeutic drug monitoring (TDM) especially in neurology clinics or in toxicology reference laboratories. The high standard deviation (SD) observed in the dose depended plasma-VPA values of the volunteers proved the importance of TDM during the use of this drug. The results showed that for rational drug use, it is important to identify individual polymorphisms in the CYP2C9, CYP2A6 and CYP2B6 subtypes responsible for VPA metabolism, and to rearrange drug doses taking these enzyme activities into account.

DETERMINAÇÃO DE DOCETAXEL E METABÓLITOS EM PLASMA POR UPLC-MS/MS: DESENVOLVIMENTO DE METODOLOGIA ANALÍTICA E APLICAÇÃO CLÍNICA

A highly sensitive ultra-performance liquid chromatography tandem mass spectrometry method for the determination of Docetaxel and metabolites (M1+M3) in human plasma samples was developed and validated. The method employs a liquid-liquid extraction and a reversed phase separation Acquity® C18 column BEH C18 (150 x 2.1 mm, 1.7 µm). Mobile phase was a mixture of formic acid 0.1% (eluent A) and acetonitrile plus 0.1% formic acid (eluent B) in isocratic mode. Total analytical run time was 7 min. The interval of method linearity interval was 50 to 3000 ng mL-1 for docetaxel and 5.0 to 300 ng mL-1, for M1+M3 metabolites. Intra-assay and inter-assay precision was coefficient of variation 2.5-15.7% and 5.9-12.6%, respectively and accuracy was 89.0-114.0%. The method was successfully applied in the measurement of docetaxel plasma levels and area under the curve calculation in 31 cancer patients, demonstrating adequate analytical performance for the clinical application in the therapeutic drug monitoring and pharmacokinetics studies.