RESUMEN
Equisetum arvense, known as common horsetail, is used for the treatment of inflammatory diseases and is the plant with the highest concentration of silica. Yet it is unknown if the medicinal properties are mediated by its silica content. In the current study, optimal conditions for silica-rich horsetail preparations were identified. Bioactivity of the preparations was analyzed in vitro using flow cytometry-based activity and functionality profiling of primary human lymphocytes as well as cytokine measurement using a classical ELISA technique. Experiments revealed that horsetail preparations suppress activation and proliferation of lymphocytes by an interleukin-2-dependent mechanism. The effect increased with the silica concentration in the decoctions. Lymphocytes' polyfunctionality was also influenced, shown by a downregulation of IFN-γ. Analytical profiling by HPLC-UV-MS and bioactivity testing revealed relevant immunosuppressive concentrations of a component that has been identified as isoquercitrin. Our results show that both silica and isoquercitrin are active compounds of horsetail preparations.
Asunto(s)
Antiinflamatorios/farmacología , Equisetum/química , Preparaciones de Plantas/farmacología , Quercetina/análogos & derivados , Dióxido de Silicio/farmacología , Antiinflamatorios/química , Cromatografía Líquida de Alta Presión , Humanos , Linfocitos/efectos de los fármacos , Preparaciones de Plantas/química , Quercetina/química , Quercetina/aislamiento & purificación , Quercetina/farmacología , Dióxido de Silicio/químicaRESUMEN
Background: In an interdepartmental cooperation, we investigated the feasibility and benefits of implementing dose banding of chemotherapy at our medical center. Based on this concept, chemotherapy doses are clustered into bands of similar dosage levels, thereby allowing the preproduction of frequently used standard doses of drugs, with sufficient physicochemical stability. Although established practice in the United Kingdom, there is little published evidence of its introduction elsewhere. Methods: We performed an analysis of local prescribing practice (22,310 chemotherapies) and identified gemcitabine, 5-fluorouracil, and carboplatin, among various others, as cytotoxic drugs suitable for dose banding. Results: First, we determined the physicochemical stability of the selected chemotherapy drugs during 12-weeks' storage by performing pH analysis and visual examination for color change or particles. No relevant changes were identified. Gemcitabine was selected for quantitative high-performance liquid chromatography analysis and we were able to show that ≥95% remained after 12 weeks' storage, in accordance with international guidelines. To simulate a worst case scenario, we performed microbiological stability testing of simulated cytotoxic compounding by replacing the cytotoxic drug with liquid media. Samples were incubated over defined storage time points (3, 6, and 12 weeks) and evaluated using the direct inoculation method. For the container integrity test, we deposited the samples into highly contaminated broth for 1 hour. Microbiological stability was demonstrated in both tests for the full storage period. Conclusions: Our data show that 12-weeks' storage of selected cytotoxic products is feasible from a microbiological perspective. Sterility of prepared products was maintained under extreme storage conditions. Gemcitabine content was in accordance with international guidelines after 12-weeks' storage. These results support the introduction of dose-banded gemcitabine products with the predicted advantages of optimized pharmacy workflow and reduced patient waiting times. We highlight the need for further research and consensus on the performance of purity analyses in dose-banded drug products.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/epidemiología , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/normas , Contaminación de Medicamentos , Prescripciones de Medicamentos/estadística & datos numéricos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Humanos , Pautas de la Práctica en Medicina , Factores de TiempoRESUMEN
Personalized antibiotherapy ensures that the antibiotic concentration remains in the optimal therapeutic window to maximize efficacy, minimize side effects, and avoid the emergence of drug resistance due to insufficient dosing. However, such individualized schemes need frequent sampling to tailor the blood antibiotic concentrations. To optimally integrate therapeutic drug monitoring (TDM) into the clinical workflow, antibiotic levels can either be measured in blood using point-of-care testing (POCT), or can rely on noninvasive sampling. Here, a versatile biosensor with an antibody-free assay for on-site TDM is presented. The platform is evaluated with an animal study, where antibiotic concentrations are quantified in different matrices including whole blood, plasma, urine, saliva, and exhaled breath condensate (EBC). The clearance and the temporal evaluation of antibiotic levels in EBC and plasma are demonstrated. Influence of matrix effects on measured drug concentrations is determined by comparing the plasma levels with those in noninvasive samples. The system's potential for blood-based POCT is further illustrated by tracking ß-lactam concentrations in untreated blood samples. Finally, multiplexing capabilities are explored successfully for multianalyte/sample analysis. By enabling a rapid, low-cost, sample-independent, and multiplexed on-site TDM, this system can shift the paradigm of "one-size-fits-all" strategy.
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Antibacterianos , Técnicas Biosensibles , Animales , Monitoreo de Drogas , Pruebas en el Punto de AtenciónRESUMEN
The aim of this study was to evaluate the concentration of penicillin G in bone affected by antiresorptive agent-related osteonecrosis of the jaw (ARONJ) following a single preoperative dose of 10 million international units (6000 mg). ARONJ is a major concern in patients administered antiresorptive agents for conditions associated with pathologically increased bone resorption. Antibiotic therapy is a key component of most treatment approaches for ARONJ and penicillin based regimens, providing a cost effective therapy option with a favorable side effect profile, are administered most frequently. In this study, high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) was applied to evaluate penicillin G concentration in serum and bone samples of 19 patients suffering from ARONJ and undergoing surgical treatment under perioperative intravenous (IV) antibiotic therapy. Penicillin G bone concentrations were above the limit of detection (0.1 µg/g bone tissue) in 16 out of 19 samples, with a median concentration of 2.7 µg/g (range 0.1-8.8 µg/g). Penicillin G concentrations in intraoperative serum samples were above the limit of detection in all serum samples, with a median concentration of 116 µg/mL (range 1-232 µg/mL). Thus, considering bacteria frequently found in ARONJ lesions, penicillin G at levels providing adequate antimicrobial activity was detected in the serum and 16 out of 19 osteonecrotic lesions of patients suffering from ARONJ.
RESUMEN
This work presents two cases of codeine intoxication in 3-year-old monozygotic twin brothers while treated with a codeine slow-release formulation. One child had to be admitted to the hospital, whereas the other one died at home after aspiration of gastric content. The concentrations of codeine and major metabolites including morphine and corresponding glucuronide conjugates were measured by liquid chromatography-tandem mass spectrometry in serum, urine, cerebrospinal fluid, and brain tissue, respectively. A genetic polymorphism study was carried out in order to determine the ability of the children to metabolize codeine by O-demethylation. A pharmacokinetic calculation was also performed to estimate the administered dose of codeine in question. High concentrations of all substances were found in samples of both children. The pharmacokinetic estimate suggests an overdose of codeine, and the possible reasons for the high opiate concentrations are discussed. Furthermore, the postmortem distribution--during and after resuscitation--might play a major role in the interpretation of postmortem concentration levels.
Asunto(s)
Antitusígenos/farmacocinética , Antitusígenos/envenenamiento , Codeína/farmacocinética , Codeína/envenenamiento , Errores de Medicación , Antitusígenos/análisis , Química Encefálica , Edema Encefálico/patología , Preescolar , Cromatografía Liquida , Codeína/análogos & derivados , Codeína/análisis , Citocromo P-450 CYP2D6/genética , Preparaciones de Acción Retardada , Sobredosis de Droga , Resultado Fatal , Toxicología Forense , Genotipo , Glucurónidos/análisis , Humanos , Morfina/análisis , Derivados de la Morfina/análisis , Polimorfismo Genético , Aspiración Respiratoria/patología , Espectrometría de Masas en Tándem , Distribución Tisular , Gemelos MonocigóticosRESUMEN
UNLABELLED: In spite of the lack of evidence for its efficacy, and of sporadic reports of severe adverse events, codeine is still widely used as an antitussive agent in children. A 3-year-old boy (twin 1) was found lying in vomit and apnoeic at night; he was resuscitated and immediately transferred to our paediatric intensive care unit (PICU). Two and a half hours later, his twin brother (twin 2) was found dead in his bed at home. Twin 1 required mechanical ventilation for 3 days, but he eventually made a full recovery; autopsy in twin 2 showed massive aspiration of gastric content. History revealed that the monozygotic twins had an upper respiratory tract infection for several days and had both been given codeine at a dose of "10 drops per day" by their mother. The blood of both twins was found to contain high levels of codeine and its metabolites. The weight of "10 drops" was determined experimentally and was found to range from 494 to 940 mg. Thus, the highest possible dose given by mother was 23.5 mg of codeine instead of the recommended 10 mg. The twins had identical CYP2D6 gene polymorphisms corresponding to the "extensive metaboliser" type. CONCLUSIONS: Because of the variability of drop size drug dosage, dosage "by drops" is unprecise and may result in accidental overdose. The combination of repeated overdosing and extensive metabolism to morphine is likely to have caused apnoea in these twins. These cases illustrate the danger of codeine as an antitussive in young children.
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Antitusígenos/administración & dosificación , Antitusígenos/efectos adversos , Codeína/administración & dosificación , Codeína/efectos adversos , Aspiración Respiratoria/terapia , Gemelos , Vómitos/complicaciones , Administración Oral , Preescolar , Citocromo P-450 CYP2D6/genética , Sobredosis de Droga/complicaciones , Resultado Fatal , Genotipo , Humanos , Masculino , Polimorfismo Genético , Respiración Artificial/métodos , Aspiración Respiratoria/etiología , Aspiración Respiratoria/fisiopatología , Resultado del Tratamiento , Vómitos/inducido químicamenteRESUMEN
OBJECTIVE: In Europe extracts of Rosmarinus officinalis were traditionally used for the treatment of rheumatic diseases. We investigated the capacity of standardized aqueous extracts of Rosmarinus officinalis on human primary lymphocyte function in vitro, as activated lymphocytes are an important mediator of rheumatic diseases. METHODS: Lymphocyte proliferation was measured using membrane-permeable dye carboxyfluorescein diacetate succinimidyl ester (CFSE). Apoptosis was analysed by surface staining of phosphatidylserine (annexin V-assay) and necrosis was analysed by staining with propidium iodide. Modification of cell activity was detected by surface staining of CD69 and CD25. The activity of STAT3 in T-lymphocytes was determined by intracellular staining of STAT3 molecules. All endpoints were analyzed by using flow cytometry. The Rosmarinus officinalis extract was investigated at concentrations of 0.05-25â¯mg/mL. Analysis of the extract was performed using HPLC methods. RESULTS: Rosmarinus officinalis inhibited proliferation of human lymphocytes and CD4+ T-cells in a dose-dependent manner (3.1-25â¯mg/mL) through induction of apoptosis. The intracellular signalling pathway STAT3 in T-cells, but not NF-kappaB and ERK1/2 in T- and B-cells was inhibited in a dose-dependent manner by Rosmarinus officinalis (0.2-6.2â¯mg/mL). Rosmanol, carnosolic acid, carnosol and trans-caffeic acid were tested in the same cellular models as the crude extract. From these, only trans-caffeic acid inhibited lymphocyte proliferation and STAT3 (30-100⯵g/mL). Trans-caffeic acid was found in the extract in a concentration of 14.7⯵g/mL. CONCLUSIONS: We conclude that an immunosuppressive effect of Rosmarinus officinalis is mostly due to the effect of trans-caffeic acid. It results in inhibition of the activity of STAT3 causing induction of apoptosis and inhibition of proliferation of T-lymphocytes.
Asunto(s)
Inmunosupresores/farmacología , Extractos Vegetales/farmacología , Rosmarinus/química , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ácidos Cafeicos/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: The aim of this study was to determine the elution of monomers of two conventional resin composite materials after different polymerization and storage times. METHODS: Two resin composites (a hybrid and a flowable) were used. Four groups (n=10, diameter: 4.5mm, thickness: 2mm) of each material were fabricated, one for each polymerization time of: 0s, 20s, 40s, and 80s. The samples were stored in 1ml of 75% ethanol at room temperature, and the storage medium was renewed after 24h, 7 days, and 28 days. From the storage medium that was removed, samples were prepared and evaluated, with LC-MS/MS. RESULTS: Bisphenol A and UDMA were not detected in the samples. Regardless of the polymerization time, the material or the storage time, a higher amount of BisGMA was eluted compared to TEGDMA. The amount of monomer that was released from the polymerized samples of the hybrid resin composite (Tetric Ceram) was significantly higher (p<0.0001) compared to the flowable (Tetric Flow). No significant difference was found between samples polymerized for 20s compared to 40s concerning the elution of monomers. Only a polymerization time of 80s resulted in a decreased release of monomers. The release of TEGDMA decreased after 28 days; however, the elution of BisGMA remained at high levels. SIGNIFICANCE: The release of monomers remains at a high level for a long time (7-28 days) after polymerization. The 40s that are usually used for the polymerization of resin composites seems insufficient in order to prevent a high release of monomers.
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Resinas Compuestas/química , Análisis de Varianza , Bisfenol A Glicidil Metacrilato/análisis , Cromatografía Líquida de Alta Presión , Restauración Dental Permanente , Almacenaje de Medicamentos , Etanol , Ensayo de Materiales , Transición de Fase , Polietilenglicoles/análisis , Ácidos Polimetacrílicos/análisis , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
Voriconazole is a novel broad-spectrum antifungal agent. We developed an on-line LC-LC-MS-MS method for fully automated and direct analysis of voriconazole in raw human serum. After injection of human serum size-selective sample fractionation and analyte extraction was achieved using an extraction column (25 mm x 4 mm) packed with a restricted access material (RAM, LiChrospher ADS C(8), 25 microm). On-line transfer of voriconazole from the extraction column was followed by chromatography separation on a C(18) column. Detection was done by ESI-MS-MS. The total analysis time was 13 min, managed by parallel extraction and chromatographic separation. This LC-MS assay was fully validated. The lower limit of quantification was 0.05 microg/ml. The automated inline extraction of voriconazole described here eliminates the need for difficult and time-consuming sample pre-treatment. Other advantages of the new method are that only a small quantity (5 microl) of serum is needed and that the method is very specific.
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Antifúngicos/sangre , Cromatografía Liquida/métodos , Pirimidinas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Triazoles/sangre , Automatización , Calibración , Cromatografía Liquida/instrumentación , Estándares de Referencia , VoriconazolAsunto(s)
Acetamidas/efectos adversos , Interacciones Farmacológicas/genética , Oxazolidinonas/efectos adversos , Rifampin/efectos adversos , Acetamidas/administración & dosificación , Acetamidas/sangre , Adulto , Quimioterapia Combinada , Femenino , Humanos , Inyecciones Intravenosas , Linezolid , Masculino , Oxazolidinonas/administración & dosificación , Oxazolidinonas/sangre , Rifampin/administración & dosificación , Rifampin/uso terapéutico , Factores de TiempoRESUMEN
Posaconazole is a new broad-spectrum antifungal agent that is currently only available as an oral suspension and shows high intra- and inter-individual differences in oral bioavailibility. Pre-existing methods for the determination of the substance involve the use of internal standards or require a quite complicated and time-consuming sample pre-treatment. Our HPLC method is fast and fully-automated and there is no need for any manual sample pre-treatment. On-line transfer of posaconazole from the extraction column was followed by chromatographic separation on a C18 column and fluorescence detection (lambda(ex): 261 nm, lambda(em): 357 nm). Retention time of posaconazole was about 11.7 min, the lower limit of quantification was found to be 0.1 mg/l. A linear calibration curve was obtained over the concentration range of 0.1-5 mg/l using a 50 microl sample (r(2)=0.999). The relative standard deviations of intra-day variations ranged from 2.3% to 9.4%, intra-day accuracy from 88.8% to 114.8%.
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Antifúngicos/sangre , Cromatografía Líquida de Alta Presión/métodos , Triazoles/sangre , Humanos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Tormentil extracts (TE) have antioxidative properties and are used as a complementary therapy for chronic inflammatory bowel disease. In individual patients with ulcerative colitis (UC) positive effects have been observed. GOALS: To assess the safety, pharmacology, and clinical effects of different doses of TE in patients with active UC. STUDY: Sixteen patients with active UC [clinical activity index (CAI) >/=5] received TE in escalating doses of 1200, 1800, 2400 and 3000 mg/d for 3 weeks each. Each treatment phase was followed by a 4-week washout phase. The outcome parameters were side effects, CAI, C-reactive protein, and tannin levels in patient sera. RESULTS: Mild upper abdominal discomfort was experienced by 6 patients (38%), but did not require discontinuation of the medication. During therapy with 2400 mg TE per day, median CAI and C-reactive protein improved from 8 (6 to 10.75) and 8 (3 to 17.75) mg/L at baseline to 4.5 (1.75 to 6) and 3 (3 to 6) mg/L, respectively. During therapy, the CAI decreased in all patients, whereas it increased during the washout phase. Neither undegraded nor metabolized tannins could be detected by liquid-mass spectrometry (LC-MS) in patient sera. CONCLUSIONS: TE appeared safe up to 3000 mg/d. Tannins from TE are not systemically absorbed. The efficacy in patients with UC should be further evaluated.
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Colitis Ulcerosa/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/administración & dosificación , Potentilla/química , Adolescente , Adulto , Anciano , Proteína C-Reactiva/efectos de los fármacos , Proteína C-Reactiva/metabolismo , Cromatografía Liquida , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Extractos Vegetales/efectos adversos , Extractos Vegetales/farmacología , Plantas Medicinales , Rizoma , Índice de Severidad de la Enfermedad , Taninos/sangreRESUMEN
OBJECTIVES: The oxazolidinone class of antibiotics such as linezolid have a narrow spectrum of activity that targets Gram-positive bacteria. We hypothesized that the poor activity of linezolid in Gram-negative bacteria is in part caused by relatively low intracellular concentration due to efflux. METHODS: Using whole cell accumulation assays we estimated the intracellular concentration of linezolid in Escherichia coli and other Enterobacteriaceae. We included test strains with enhanced RND-type multidrug efflux pump activity and with genetic inactivation of the pump or functional inhibition by carbonyl cyanide m-chlorophenylhydrazone as inhibitor of the proton motive force or 1-(1-naphthylmethyl)-piperazine (NMP), an efflux pump inhibitor. RESULTS: Consistent with susceptibility studies, enhanced pump activity caused decreased accumulation, and pump inactivation and inhibition caused increased accumulation, of linezolid. The accumulation levels in test strains of E. coli, Citrobacter freundii and Enterobacter aerogenes with functional pumps were lower than in control strains of Staphylococcus aureus and Enterococcus faecium, but were higher after pump inactivation and correlated with ethidium bromide and pyronin Y accumulation. CONCLUSIONS: The intracellular concentration of linezolid is comparatively low owing to efficient efflux of the drug and could be increased substantially by inhibition of RND-type efflux pumps.
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Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Acetamidas/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacología , Citrobacter freundii/metabolismo , Enterobacter aerogenes/metabolismo , Escherichia coli/metabolismo , Oxazolidinonas/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Cromatografía Líquida de Alta Presión , Colorantes , Etidio , Fluorometría , Linezolid , Pruebas de Sensibilidad Microbiana , PironinaRESUMEN
OBJECTIVES: YhiV (MdtF) is an resistance nodulation division (RND) type efflux pump in Escherichia coli with significant homology to AcrB but usually expressed at a low level in clinical isolates. When overexpressed the pump confers decreased susceptibility to a variety of substances including erythromycin and ethidium bromide (EtBr). We characterized two mutants of E. coli E12 (DeltaacrB DeltaacrF) overexpressing yhiV that showed surprising differences in their spectrum of multidrug resistance (MDR). METHODS: The two mutants obtained after repeated exposure of E. coli E12 to levofloxacin were tested for antimicrobial susceptibility to a variety of agents and for intracellular accumulation of selected pump substrates. Gene expression was studied by quantitative RT-PCR, and yhiV was sequenced. Gene inactivation and replacement were done by phage lambda-based homologous recombination. RESULTS: Mutant DKO20/1 overexpressed yhiV, showed a wild-type yhiV sequence and had >2-fold increased MICs of fluoroquinolones, novobiocin, macrolides/ketolides, EtBr, oxacillin and Phe-Arg-beta-naphthylamide (PAbetaN, a putative efflux pump inhibitor) compared with the E12 parent. A second mutant, strain DKO1/17 that had the Val-610-->Phe point mutation in YhiV differed from DKO20/1 by faster growth, >2-fold increased MICs of linezolid and tetracycline, but >2-fold decreased MICs of PAbetaN, azithromycin and telithromycin. Inactivation of yhiV in DKO1/17 and reintroduction of the wild-type and mutant yhiV sequence confirmed that the differing MICs of most of the drugs were associated with the observed single point mutation. Intracellular drug accumulation studies with linezolid and PAbetaN were consistent with the MIC results. CONCLUSIONS: The region around amino acid Val-610 in YhiV appears to be involved in determining recognition and efficiency of export of a number of MDR efflux pump substrates. This single point mutation in the periplasmic loop of the pump can increase resistance to a given drug such as a fluoroquinolone while decreasing resistance to another one.
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Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , Mutación Puntual/genética , Acetamidas/metabolismo , Alelos , Antibacterianos/farmacología , ADN Bacteriano/biosíntesis , ADN Bacteriano/genética , ADN Complementario/biosíntesis , ADN Complementario/genética , Levofloxacino , Linezolid , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Ofloxacino/farmacología , Oxazolidinonas/metabolismo , Fenotipo , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Caspofungin (MK-0991; L-743,872) is the first representative of a new important class of antifungal agents, the glucan synthesis inhibitors. To the authors' best knowledge, to date only one high-performance liquid chromatography (HPLC) method has been published for the determination of caspofungin in serum. Severe difficulties with sorption were described. We developed a new method which addresses these difficulties using an advanced column-switching technique for fully automated analysis of caspofungin in serum without any pre-treatment. Extraction was performed automatically inline, using a diol column, followed by chromatography on a CN column. Detection was performed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with isolation and fragmentation in the positive ion mode. Total analysis time was 30 min. Detection of caspofungin was achieved by retention time, isolation and fragmentation of the double positively charged caspofungin ion. This LC/MS assay was validated for between-run accuracy (max. 110%) and precision (max. CV 16.1%). The lower limit of quantification was 0.2 microg/mL. The analytical method with fully automated inline extraction of caspofungin described here removes the need for difficult and time-consuming sample pre-treatment. Sorption of caspofungin is not of importance. Additional advantages of the new method are that only a small quantity of serum (5 microL) is needed and that the method is very specific.
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Antifúngicos/sangre , Cromatografía Líquida de Alta Presión/métodos , Péptidos Cíclicos/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Caspofungina , Equinocandinas , Lipopéptidos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
A sensitive and rapid HPLC assay for the determination of fluconazole in serum is described. HPLC-integrated sample preparation allows direct injection of serum samples without any pretreatment. The in-line extraction technique is carried out by automatically switching from the extraction column (Lichrospher ADS C8) to the analytic column (Nucleosil C18). After 6 minutes the matrix passes the extraction column, and the retained analyte is quantitatively transferred to the analytic column, where separation by isocratic HPLC is performed. The extraction eluent is sodium dihydrogen phosphate buffer, pH 5.0 (50 mM), and the analytic eluent is acetonitrile/sodium dihydrogen phosphate buffer, pH 5.0 (50 mM) (26.8/73.2, vol/vol). Fluconazole is detected according to its absorption maximum at 210 nm. The lower limit of quantification (LLOQ) is 0.65 microg/mL, the limit of detection (LOD) is 0.2 microg/mL, and the quantification range is 0.65-23.3 microg/mL. The assay was precise with a between-run coefficient of variation of < or = 5.59%. The within-run accuracy was 99.8% and 103.4%, and the between-run accuracy was 99.2% and 99.7%, respectively, for the concentrations 23.3 microg/mL and 1.3 microg/mL. The recovery was 78%. The described procedure allows sample cleanup and determination within 20 minutes, thereby facilitating drug monitoring in clinical routine. The method was applied successfully.