RESUMEN
BACKGROUND: Immunoassays and liquid chromatography tandem mass spectrometry (LC-MS/MS) are two major methods for therapeutic drug monitoring (TDM) of immunosuppressant drugs. Compared to the relatively limited analytical performance and cross reactivities of immunoassays, the LC-MS/MS method is considered as a gold standard; however, the lack of systematic evaluation and standardization needs to be addressed. METHODS: A LC-MS/MS method for the determination of cyclosporine A, sirolimus, tacrolimus, and everolimus was developed. One-step protein precipitation was used to prepare blood samples. The newly developed method was systematically evaluated and validated according to the standard guidelines. RESULTS: The quantitative method for four immunosuppressant drugs in human whole blood was validated according to the guidelines. The lower limits of the measuring interval (LLMI) for cyclosporine A, sirolimus, tacrolimus, and everolimus were 5, 0.5, 0.5, and 0.5â¯ng/mL, respectively. Linear correlation coefficients were all >0.999. Internal standard-normalized (IS-normalized) matrix correction factor was within the range 0.88-1.17. The average spiked recoveries of five replicates for the four immunosuppressant drugs were in the range 87.4-109.6%. CONCLUSION: An LC-MS/MS method combined with one-step protein precipitation was developed, providing short sample preparation and chromatographic run time, thus allowing easy clinical diagnosis.
Asunto(s)
Monitoreo de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Inmunosupresores/sangre , Precipitación Química , Cromatografía Liquida/métodos , Ciclosporina/sangre , Everolimus/sangre , Humanos , Límite de Detección , Sirolimus/sangre , Tacrolimus/sangre , Espectrometría de Masas en Tándem/métodosRESUMEN
OBJECTIVE: To explore a feasibility of engraftment of systemically transplanted bone marrow stromal cells (BMSCs) and differentiation into lung epithelial cells in lipopolysaccharides (LPS)-injured lungs. METHODS: BMSCs were isolated from bone marrow of transgenic green fluorescent protein (GFP) C57BL/6J mice and systemically administered to bone marrow-suppressed wild-type C57BL/6J mice. A mouse model of lung injury was prepared by intratracheal instillation of LPS. Recipients were assigned to four groups: intratracheal PBS + BMSCs transplantation (CM), intratracheal LPS + BMSCs transplantation (LM), intratracheal PBS + irradiation + BMSCs transplantation (CIM) and intratracheal LPS+ irradiation + BMSCs transplantation (LIM). BMSCs engraftment in recipient lungs was determined by immunofluorescent staining 14 days after BMSCs administration. Alveolar epithelial type II cells were isolated from recipient lungs and the rate of GFP positive cells was measured by flow cytometry. Expression of surfactant protein (SP)-A, SP-C and aquaporin (AQP)-5 mRNA in the lungs was evaluated by real-time PCR. RESULTS: GFP and cytokeratin positive cells were observed in lung parenchyma of the CIM and the LIM groups, but not in the CM and the LM groups. The LIM group had more positive cells than the CIM group. The rates of GFP positive cells were higher in the CIM (11.10+/- 3.19%) and the LIM groups (14.40+/- 2.40%) than those in the CM and the LM groups (2.82+/- 1.03% and 3.81+/- 0.93%, respectively; P< 0.05). The LIM group had higher mRNA expression of SP-C than the CM group (2.09+/- 0.18 vs 1.38+/- 0.30; P< 0.05). CONCLUSIONS: Donor derived BMSCs can engraft in LPS-injured lungs and differentiate into lung epithelial cells, suggesting BMSCs transplantation might contribute to lung repair.
Asunto(s)
Células de la Médula Ósea/citología , Lipopolisacáridos/toxicidad , Lesión Pulmonar/terapia , Células del Estroma/trasplante , Animales , Acuaporina 5/genética , Diferenciación Celular , Femenino , Péptidos y Proteínas de Señalización Intercelular , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/genética , Proteína A Asociada a Surfactante Pulmonar/genética , Proteína C Asociada a Surfactante Pulmonar , ARN Mensajero/análisis , Células del Estroma/citologíaRESUMEN
The paper is to report the development of a high-performance liquid chromatographic/tandem mass spectrometry (HPLC-MS/MS) method for the determination of icaritin (ICT) in rat plasma. After precipitated with acetonitrile from the plasma, ICT was isolated chromatographically on a Dikma C18 column. The mobile phase consisted of acetonitrile-water-acetic acid (72 : 28 : 1.5, v/v/v). Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 387 --> m/z 313 and m/z 331 --> m/z 315 were used to quantify ICT and the internal standard, respectively. The linear calibration curve was obtained in the concentration range of 2.5-1,000 ng x mL(-1). The lower limit of quantification was 2.5 ng x mL(-1). The inter- and intra-day precision (RSD) were less than 9.63%, and the accuracy (relative error) was within +/-7.42%. The method was proved to be suitable for the pharmacokinetics of ICT, which offers advantages of high sensitivity and selectivity.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Flavonoides/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Administración Oral , Animales , Epimedium/química , Femenino , Flavonoides/administración & dosificación , Flavonoides/aislamiento & purificación , Flavonoides/farmacocinética , Masculino , Plantas Medicinales/química , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To study the effects of conventional mechanical ventilation (CMV) with low tidal volume on developmental porcine lungs by examining the expression of growth factors and inflammatory mediators. METHODS: Twelve preterm piglets born at 99 days of gestational age, 12 term neonatal piglets and 11 young piglets (4-5-weeks old) were randomly placed on CMV or were not ventilated (control group). The ventilator settings were adjusted to provide a tidal volume of 6-8 mL/kg in order to maintain a normal blood-gas value. After 6 hrs (preterm piglets) or 24 hrs (neonatal and young piglets) of mechanical ventilation, the mRNA expression of growth factors PDGF-B, IGF-I, KGF, HGF, VEGF and TGF-beta1 and proinflammatory cytokines IL-1beta, IL-6, IL-8 and TNF-alpha in the lung tissue was measured using RT-PCR. Growth factor protein expression was measured with immunohistochemistry. RESULTS: In preterm piglets, the CMV group had increased mRNA expression of PDGF-B (5.11+/-0.10 vs 4.88+/-0.01), IL-1beta (4.95+/-0.27 vs 4.08+/-0.37), IL-6 (4.76+/-0.27 vs 4.00+/-0.28) and IL-8 (5.31+/-0.57 vs 4.15+/-0.46), but decreased IGF-I mRNA expression (3.54+/-0.13 vs 3.80+/-0.11) compared with those in the control group (P<0.05 or 0.01). In term neonatal piglets and young piglets, there were no significant differences in the mRNA expression of growth factors and proinflammatory cytokines between the CMV and control groups. CONCLUSIONS: CMV caused inflammatory injury in immature lungs by increasing the expression of proinflammatory cytokines and PDGF-B and decreasing IGF-I expression. However, CMV had no effects on pulmonary expression of growth factors and inflammatory mediators in term neonatal piglets and young piglets.