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61.
ObjectiveLevetiracetam and its acid metabolite have almost identical MRMs. They therefore need to be separated chromatographically prior to quantitation.Research design and methodsThe sample is deproteinized with acetonitrile containing Ritonavir as internal standard, centrifuged and the supernatant diluted with water (1:2 v/v). Sixty microliters of the supernatant is injected into the LC–MS/MS and Levetiracetam (LEV) and LEV metabolite separated chromatographically at room temperature employing a Supelco C18 column and a 0.1% formic acid methanol gradient at pH of 2.5.ResultsThe retention times for LEV metabolite, LEV and Ritonavir were 4.50, 5.38 and 9.18 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 0–50 μg/mL for LEV and 0.0–5.0 µg/mL for LEV metabolite. Intra- and inter-run imprecision (n = 10) gave CVs of 2.3–4.7%, 3.4–8.9% for LEV and 2.9–3.9%, 3.3–7.4% for LEV metabolite. Recoveries of both LEV and LEV metabolite were close to 100%. Results for LEV were compared with those obtained by a commercial reference laboratory (r = 0.974).ConclusionThe procedure is reliable, quick, and inexpensive. LEV and LEV metabolite co-elute using C-18 columns at pHs > 3.0 and previously published methods employing these conditions could therefore be subject to metabolite interference. In this method LEV and LEV metabolite are separated at pH 2.5. The total run time including the washing step is 10 min/sample, making this method suitable when moderate throughput is needed such as in clinical or commercial reference laboratories. 相似文献
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S J Soldin N Rifai E W Palaszynski A Morales R N Lipnick 《Therapeutic drug monitoring》1992,14(2):164-168
We have developed a radioreceptor assay to measure glucocorticoids. The assay employs the partially purified 95-kDa receptor isolated from human liver and purified by size fractionation on high-performance liquid chromatography (HPLC). In the assay [3H]prednisolone competes with steroids (endogenous and exogenous) for binding to the receptor. Bound and free are separated by treatment with charcoal. The between-day precision [% coefficient of variation (CV)] at concentrations of 9.4, 18.7, and 69.9 micrograms/L prednisolone is 16.6, 9.3 and 4.5%, respectively. Specificity studies revealed that hydrocortisone, deoxycorticosterone, 4-pregnene-17 alpha,21-diol-3,20-dione, 17 alpha-hydroxyprogesterone, corticosterone and beta-hydroxyprogesterone all compete with [3H]prednisolone for binding to the receptor. Prednisone and 6 alpha-methyl prednisolone displace [3H]prednisolone to only a minor degree. The assay has been used to assess "glucocorticoid activity" in children with rheumatic diseases treated with prednisolone. 相似文献
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Natasa Kahric-Janicic Steven J Soldin Offie P Soldin Threvia West Jianghong Gu Jacqueline Jonklaas 《Thyroid》2007,17(4):303-311
OBJECTIVE: Pregnancy is a time of rapidly changing demands on the thyroid axis, and knowledge of thyroid hormone levels, especially during the first trimester, is important for ensuring maternal and fetal health. The thyroid hormone assays currently in use become more inaccurate at extremes of binding protein concentrations and when heterophilic antibodies are present. Pregnancy is characterized by both these conditions, making accurate determination of free thyroid hormone levels by conventional direct analog immunoassay methods difficult. The objective of this study was to characterize the performance of a novel tandem mass spectrometric assay for free thyroxine during the physiologic conditions of pregnancy. DESIGN: Healthy women without a history of thyroid abnormalities were recruited from the obstetrics and gynecology and endocrinology clinics of a university medical center and their thyroid status was monitored. Free thyroxine levels were assessed by both immunoassay and tandem mass spectrometry during the course of their pregnancy. Serum thyrotropin levels were also measured. The distributions of free thyroid concentrations obtained by the two assays were compared. MAIN OUTCOME: The tandem mass spectrometry and immunoassay values did not correlate well with each other. However, tandem mass spectrometry values correlated well with the current gold standard equilibrium dialysis values. Moreover, the good agreement between equilibrium dialysis and tandem mass spectrometry was maintained across all weeks of gestation. CONCLUSIONS: We conclude that tandem mass spectrometry has a superior performance to immunoassay for the measurement of free thyroxine during pregnancy. Furthermore, it is ideally suited to generating trimester-specific reference intervals for free thyroxine levels. Future studies will determine if it is a better assay to use in most clinical circumstances. 相似文献
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WILLEM P. VERMEULEN JACOB J. BRIEDE GERTRUDE BUNT JOS A. F. OP DEN KAMP ROB J. KRAAIJENHAGEN BEN ROELOFSEN 《British journal of haematology》1995,90(1):56-64
Hereditary spherocytosis (HS) is a congenital haemolytic anaemia which is characterized by a great variety of structural defects in the red cell's membrane skeleton and/or deficiencies in particular membrane (skeletal) proteins. Enhanced (Mg2+ )-dependent adenosine triphosphatase (Mg2+ -ATPase) activities, varying from 115% to 160%, were invariably found in erythrocyte ghosts derived from 13 HS patients. Similarly, an enhancement of Mg2+ -ATPase activity by 30% is observed in normal red cell ghosts that have been stripped of the greater part of their membrane skeletal proteins by treatment with a low ionic strength buffer. Reassociation of those stripped ghosts with spectrin reduces the enhanced Mg2+ -ATPase activity to its original level. Since in both cases, HS ghosts and stripped normal ghosts, the stabilizing effects that the membrane skeleton exerts on the maintenance of an endofacial localization of the aminophospholipids are impaired, the enhanced Mg2+ -ATPase activity is interpreted to reflect an increased activity of the aminophospholipid translocase. The present observations therefore support a role of the membrane skeleton in the stabilization of phospholipid asymmetry in the red cell membrane and consequently in reducing the energy consumption of the translocase. 相似文献
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OBJECTIVES: This study was designed to improve our previously developed tandem mass spectrometry (MS/MS) method for free thyroxine (FT4) by enhancing sensitivity and permitting simultaneous measurements of both free triidothyronine (FT3) and FT4 using a smaller plasma/serum sample. DESIGN AND METHODS: An API-5,000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standard, T4-d(5). Four hundred microliters of human plasma/serum was filtered through a Centrifree YM-30 ultrafiltration device by centrifugation, and 450 microL of internal standard in methanol was then added to 150 microL of ultrafiltrate for deproteinization. After centrifugation, 500 microL of supernatant was diluted with 400 microL of distilled de-ionized water and a 650 microL aliquot was injected onto a C-18 column. After washing, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode. RESULTS: The within-day and between-day coefficients of variation (CVs) were 相似文献
70.