Beta-galactosidase of Aspergillus sp. AF crude homogenate was immobilized in Ca-alginate gel beads and used for the production of galactooligosaccharides (GOS) from lactose. Optimum pH and temperature, thermal and storag...Beta-galactosidase of Aspergillus sp. AF crude homogenate was immobilized in Ca-alginate gel beads and used for the production of galactooligosaccharides (GOS) from lactose. Optimum pH and temperature, thermal and storage stability of the enzyme activity were investigated and compared with those of the free enzyme. The study on the improvement of mechanical strength of the alginate beads was carried out through various methods, which demonstrates that the hardening process, where the alginate beads were treated with 0.225 M CaCl(2) solution after three batches to compensate the lost of calcium in the beads, provided a high mechanical stability for repeated use in large-scale production. The experiment results show that GOS yield increased with the increase of lactose concentration, and also increased with excessive addition of lactose (exceeding its solubility) at the beginning of the reaction. The immobilization of beta-galactosidase of Aspergillus sp. AF crude homogenate is cheap in processing cost and easy to carry out, and the immobilized enzyme possesses high performance for industrial application.
Abstract: Nerve regeneration and re-innervation are usually difficult after peripheral nerve injury. Epineurium neurorrhaphy to recover the nerve continuity was the traditional choice of peripheral nerve mutilation witho...Abstract: Nerve regeneration and re-innervation are usually difficult after peripheral nerve injury. Epineurium neurorrhaphy to recover the nerve continuity was the traditional choice of peripheral nerve mutilation without nerve defects, whereas the functional recovery was not quite satisfactory. In this article, the authors review the literature focused on peripheral nerve injury research and possible clinical application, including introducing peripheral nerve selective regeneration theory, small gap sleeve bridging nerve methodepineurium neurorrhaphy, kinds of biological conduit, and microenvironment research between nerve stumps.
Our previous study showed that Leukotriene B4 can directly stimulate osteoclast differentiation independent of RANKL. In order to determine whether Leukotriene B4 could indirectly stimulate human osteoclast differentiati...Our previous study showed that Leukotriene B4 can directly stimulate osteoclast differentiation independent of RANKL. In order to determine whether Leukotriene B4 could indirectly stimulate human osteoclast differentiation through increasing RANKL expression of rheumatoid arthritis fibroblast-like synoviocytes, we utilize the coculture model of rheumatoid arthritis fibroblast-like synoviocytes and monocyte, which were stimulated in the presence of 2.5 ng/ml M-CSF in the control group, 2.5 ng/ml M-CSF+10(-8)M LTB4 in the experimental group a, and 2.5 ng/ml M-CSF+10(-8)M LTB4+100 ng/ml OPG in the experimental group b. After culture for 3 weeks, the number of multinucleated TRAP staining positive osteoclast-like cells stained with TRAP was counted to evaluate the differentiation effect in each group. There was almost no osteoclast-like cell in the control group and the experimental group b. There were many osteoclast-like cells in the experimental group a. These results indicated that Leukotriene B4 is capable of inducing osteoclast differentiation by a RANKL-dependent mechanism.
OBJECTIVE: To study the effect of programmed cell death 5 (PDCD5) on apoptosis of rheumatoid arthritis fibroblast-like synoviocyte (RAFLS) induced by triptolide. METHOD: Cultured synovial cells in vitro from RA patients...OBJECTIVE: To study the effect of programmed cell death 5 (PDCD5) on apoptosis of rheumatoid arthritis fibroblast-like synoviocyte (RAFLS) induced by triptolide. METHOD: Cultured synovial cells in vitro from RA patients were transfected with Ad-PDCD5. In protein level, expression of PDCD5 protein in Ad-PDCD5 transfected RAFLS was detected by Western blot. RAFLS transfected with Ad-PDCD5 were cultured in presence or absence of triptolide and RAFLs apoptosis was determined by flow cytometry. RESULT: Transfection of RAFLS with increasing concentration of Ad-PDCD5 (50-300 MOI) resulted in dose-dependent increase of PDCD5 production. Apoptotic cells percentage of no transfection group, Ad-null group and Ad-PDCD5 group were, respectively, (22.41 +/- 3.87)%, (28.77 +/- 12.97)%, and (48.87 +/- 12.69)%. Alternatively, transfection without triptolide stimuli had no effect. The data showed that gene transfection of PDCD5 alone without triptolide was not sufficient to activate RAFLS apoptosis; PDCD5 acted as an enhancer rather than inductor of apoptosis. CONCLUSION: Overexpression of PDCD5 could enhance apoptosis of RA FLS induced by triptolide; PDCD5 may be a potential therapeutic target to RA.
The purpose of this study was to evaluate the feasibility of using tissue-engineered nerve grafts for delayed repair of peripheral nerve defects. A 1-month delayed, 10-mm long sciatic nerve defect was created for rats, w...The purpose of this study was to evaluate the feasibility of using tissue-engineered nerve grafts for delayed repair of peripheral nerve defects. A 1-month delayed, 10-mm long sciatic nerve defect was created for rats, which were divided into three grafted groups and a non-grafted group. For bridging the nerve defects, the rats in three grafted groups were subjected to surgical repair with tissue-engineered nerve grafts made of a chitosan/polyglycolic acid (PGA) conduit filled with neural stem cells (NSCs), chitosan/PGA conduits, and autologous nerve grafts, respectively. At 3 months after nerve grafting, the data from electrophysiology, retrograde tracing and histological investigation revealed that the better outcomes in sciatic nerve regeneration and target muscle re-innervation were achieved in three grafted groups as compared to those in non-grafted group without major differences between three grafted groups. Our results suggest that grafting of chitosan/PGA conduits might be a promising technique for repairing peripheral nerve injuries after a 1-month delay, while introduction of NSCs seem to show no significant additional benefits to regenerative outcomes.
To describe the phenotypic changes of Schwann cells during nerve regeneration, we made two different neurorrhaphy models after rat sciatic nerves injury, the epineurium neurorrhaphy and small gap bridging suture. Then, a...To describe the phenotypic changes of Schwann cells during nerve regeneration, we made two different neurorrhaphy models after rat sciatic nerves injury, the epineurium neurorrhaphy and small gap bridging suture. Then, at selected time points after surgery (1d, 3d, 5d, 7d, 2 weeks, 3 weeks), the materials were drawn for detecting the expression of GFAP, Sox2, Krox20 by immunofluorescence. GFAP is expressed in non-myelin-forming Schwann cells and Krox20 is a marker for myelin-forming cells in the adult nerve. Sox2 is a marker for neural stem and progenitor cells. Our findings showed the rule of phenotypic changes of Schwann cells during nerve regeneration. Furthermore, the difference in the phenotypic changes of Schwann cells between two operation methods indicated that the small gap changes the regenerated microenvironment and may be one of the reasons that small gap bridging suture is superior to the epineurium suture.
To investigate the possible association between V249I and T280M polymorphisms and carotid atherosclerosis (CAS) in a Han population in northern China we studied 328 patients with increased carotid artery intima-media thi...To investigate the possible association between V249I and T280M polymorphisms and carotid atherosclerosis (CAS) in a Han population in northern China we studied 328 patients with increased carotid artery intima-media thickness (C-IMT) and 292 healthy controls with normal C-IMT. T280M and V249I polymorphic genotypes of CX3CR1 were determined by polymerase chain reaction restriction fragment length polymorphism. Results showed that the M280 allele frequency in CAS group was significantly lower than that in the controls. Logistic regression analysis revealed that the decreased frequency of the M280 allele was an independent risk factor for CAS.
The nitrilase produced from a new isolate is evaluated for its activity in presence of a number of different ions and compounds at optimal conditions. It was found that the activity of nitrilase increased up to 10-20% in...The nitrilase produced from a new isolate is evaluated for its activity in presence of a number of different ions and compounds at optimal conditions. It was found that the activity of nitrilase increased up to 10-20% in presence of most of the divalent ions at a concentration of 5 mM relative to the control. Silver, mercury, tin, DTT, ascorbic acid and thiourea, respectively, were observed as potential inhibitors of the enzyme catalysis. The investigation on storage stability of whole cells in presence of a number of stabilizers showed that the enzyme is stable (relative activity 50%) for more than 120 days at various temperatures.
In addition to heme-irons, reactive (beta-globin thiols (betaCys93s) of hemoglobin (Hb) also have been shown to interact with endogenous nitric oxide (NO) thereby contributing to vascular tone regulation. What relative r...In addition to heme-irons, reactive (beta-globin thiols (betaCys93s) of hemoglobin (Hb) also have been shown to interact with endogenous nitric oxide (NO) thereby contributing to vascular tone regulation. What relative roles do these NO binding sites contribute to the overall Hb-mediated vasoactivity? Several test Hbs with either or both the NO binding sites preliganded or blocked were prepared and tested in a rat thoracic aortic ring model. Hbs tested were: NEM-Hb (ferrous Hb with masked thiols), HbNO (ferrous Hb preliganded with NO), Hb(+)CN (ferric Hb liganded with CN(-)), NEM-HbNO and NEM-Hb(+)CN (Hbs with both heme-iron and cysteine sites preliganded or blocked). Typically, >0.2 microM control Hb significantly increased isometric tension in agonist stimulated vessel rings (58.1 +/-7.0% over baseline). At comparable concentrations, NEM-Hb also caused a significant contraction (50.7+/-9.5%) while HbNO and Hb(+)CN did not (-5.5+/-6.0% and -3.7+/-4.6%, respectively). For these Hbs, masking thiols as well did not significantly alter respective vascular effects. Ferrous sperm whale myoglobin (Mb), which has no reactive thiol, elicited a significant contraction (55.1+/-13.2%) while metMb did not (-0.8+/-3.2%), suggesting the relative importance of heme-iron ligand and oxidation state in Hb vasoactivity. Additionally, ferrous or ferric equine heart cytochrome-C, a heme protein with no readily available heme-iron and cysteine binding sites, did not elicit notable contraction. Human Hb variants in which (betaCys93s are deleted or substituted with non-cysteine residues did not reveal any documented significant hemodynamic abnormalities. These results indicate that reactive globin-thiols do not appear to play a prominent role relative to heme-irons in Hb-mediated vasoconstriction.
A new amperometric cholesterol biosensor was prepared by immobilizing cholesterol oxidase by a glutaraldehyde crosslinking procedure on polypyrrole-polyaniline (ppy-pani) composite film on the surface of a platinum elect...A new amperometric cholesterol biosensor was prepared by immobilizing cholesterol oxidase by a glutaraldehyde crosslinking procedure on polypyrrole-polyaniline (ppy-pani) composite film on the surface of a platinum electrode. In order to prepare a biosensor for the determination of cholesterol, electropolymerization of pyrrole and aniline on Pt surface was performed with an electrochemical cell containing pyrrole and aniline in sulphuric acid by cyclic voltammetry between 0.0 and 0,7 V (vs.Ag/AgCl) at a scan rate of 50 mV upon Pt electrode. The amperometric determination is based on the electrochemical detection of H(2)O(2), which is generated in enzymatic reaction of cholesterol. The cholesterol determined by the oxidation of enzymatically generated H(2)O(2) at 0.7 V vs. Ag/AgCl. The optimized cholesterol oxidase biosensor displayed linear working range and a response time of 300 s. The effects of pH and temperature were investigated and optimum parameters were found to be 7.0, 25 degrees C, respectively. In addition to this, the stability and reproducibility of biosensor were tried. Operational stability of the proposed cholesterol biosensor was obtained by periodical measurements of the biosensor response. Biosensor at optimum activity conditions was used in 30 activity assays in one day to determine the operational stability. The results show that 82% of the response current was retained after 30 activity assays. The electrode was stored in a refrigerator at 4 degrees C after the measurements. The storage stability of the biosensor was determined by performing activity assays within 23 days. The results demonstrate that 60% of the response current was retained after 23 days. Preparing biosensor is used for the analysis of cholesterol in serum.
Platelets are enriched with Transforming Growth Factor-beta (TGF-beta). However, information is limited concerning TGF-beta's effects at the molecular level. Nevertheless, it has been demonstrated that TGF-beta activates...Platelets are enriched with Transforming Growth Factor-beta (TGF-beta). However, information is limited concerning TGF-beta's effects at the molecular level. Nevertheless, it has been demonstrated that TGF-beta activates cell proliferation and its positive influence on cartilage formation has been proven within the field of Tissue Engineering (TE). As Platelet Rich Plasma (PRP) contains TGF-beta, it was the purpose of this study to optimize PRP-isolation for further TGF-beta extraction. Red blood cell count (RBC) was separated from whole blood by centrifugation. From the supernatant PRP and platelet poor plasma (PPP) layer, the latter supernatant was re-centrifuged to extract PRP. Various experimental series were run to investigate influences concerning anticoagulating alternatives, different amounts of buffer, various centrifugal forces, or substituting centrifugation for sedimentation. TGF-beta levels were determined using ELISA. The technique of platelet-/ TGF-beta-extraction described here proves to be more effective than other methods, is easily repeatable and not time-consuming, which predisposes it for TE requirements.
Plasma expander-like properties of albumin induced on hexa as well as dodecacPEGylation using Extension Arm Facilitated PEGylation platform make it an excellent resuscitation fluid. PEGylation induced changes in the stru...Plasma expander-like properties of albumin induced on hexa as well as dodecacPEGylation using Extension Arm Facilitated PEGylation platform make it an excellent resuscitation fluid. PEGylation induced changes in the structure, drug binding, and plasma expander-like properties of bovine serum albumin has been now investigated as a function of PEGylation. The molecular volume of albumin increases on PEGylation nearly linearly; in the beginning up to about six PEG chains are conjugated, then plateau off, while the viscosity and colloidal osmotic pressure change very little initially and then increase exponentially as a function of PEG chains conjugated. PEGylation has essentially no influence on the secondary structure or drug properties of albumin. Tryphtophyl fluorescence of albumin is quenched on PEGylation as a direct correlate of the changes in molecular radius of PEG-albumin. It is concluded that hexaPEGylated and dodecaPEGylated albumin belong to two different configurational states of PEG-albumin in terms of packing of PEG-chains on the molecular surface of the protein. The results suggest a transition of PEGylated albumin from the initial mushroom-like conformation to brush conformation as the PEGylation increases. The therapeutic efficacy of the two PEGylated species is needed to establish the optimum level of PEGylation to function as resuscitation fluids.
MDCK cells were engineered to express luciferase driven by cytomegalovirus (CMV) or hybrid ubiquitin B (UbB) promoter and encapsulated in alginate-poly-L-lysine-alginate microcapsules. In vitro experiments showed capsule...MDCK cells were engineered to express luciferase driven by cytomegalovirus (CMV) or hybrid ubiquitin B (UbB) promoter and encapsulated in alginate-poly-L-lysine-alginate microcapsules. In vitro experiments showed capsules could be monitored individually or in multi-layers quantitatively. When luciferase-expressing and non-luciferase expressing MDCK cells were mixed at different ratios and encapsulated, the signals increased linearly according to the number of capsules, in vitro and in vivo. For CMV-driven luciferase expression, the strongest signal was seen at 4 hours post-implantation, with a subsequent 50% decrease by 24 hours and then declined gradually to 10-20% until day 20. However, retrieved capsules showed good cell viability. When capsules contained plasmid driven by UbB promoter, there was no decline in signal. Our results indicate that luciferase could be used as a marker for microencapsulated cells to monitor the viability and gene expression of the implanted cells.
Kidney failure is a common disease with high frequency. Food-grade recombinant bacteria that can effectively remove urea has great potential for treatment of renal failure. A nonpathogenic strain, L. lactis MG1363, was t...Kidney failure is a common disease with high frequency. Food-grade recombinant bacteria that can effectively remove urea has great potential for treatment of renal failure. A nonpathogenic strain, L. lactis MG1363, was transformed with plasmid pMG36eure, which carries urease gene. The expression of transgene urease in genetically modified L. lactis MG1363 and the urease activity in removal of urea were investigated. It was found that the removal of urea by recombinant L. lactis MG1363 was pH- and nickel-dependent. At pH 6.5 and in the presence of 250 microM of NiSO4, 50 approximately 60% of urea could be removed in 24 hours. The urea removal activity was also evaluated in imitative gastroenteric environment. After being exposed to acidic solution (pH2.5-4.0) for 2 hours, the cells were then grown in a medium containing 0.1 cfu/ml bile acid salt, 30 mg/dl urea, and 250 microM NiSO4 at pH 6.8. The concentration of urea decreased over time, and the removal was about 30% at 10 hours and 65% at 24 hours, respectively. The safety tests were performed by feeding normal rats with either L. lactis MG1363 or recombinant L. lactis MG1363. The two materials did not cause any changes in blood cells and blood biochemical indexes. There were no differences in terms of body weight and water/food consumption between the two materials. These results indicate the safety, feasibility, and capacity of urease gene modified Lactococcus Lactis in removal of urea under the gastroenteric circumstances. Further investigation may generate a food-grade strain for treatment of chronic renal failure.
To prepare timozolomide liposomes for administration through nasal mucous membrane, we studied the factors of the preparation of the liposomes. The timozolomide liposomes were prepared by the ammonium sulphate gradient m...To prepare timozolomide liposomes for administration through nasal mucous membrane, we studied the factors of the preparation of the liposomes. The timozolomide liposomes were prepared by the ammonium sulphate gradient method; electroscopy and laser particle analyzer were utilized to determine the conformation, size and distribution of timozolomide liposomes; high performance liquid chromatography (HPLC) was applied to determine the entrapping efficiency of timozolomide liposomes; then we studied the influences of the concentration of ammonium sulphate solution, temperature, and the drug-to-lipid ratio on the entrapping efficiency. The average size of timozolomide liposomes was 185 nm; the entrapping efficiency was 90.3%. The entrapping efficiency was enhanced with the increasing of the concentration of ammonium sulphate solution and the rising of temperature, and decreased with the increasing of the drug-to-lipid ratio. The timozolomide liposomes with high entrapping efficiency, small and even particle sizes could be prepared by the simple and convenient ammonium sulphate gradient method. The primary influencing factors on the entrapping efficiency of timozolomide liposomes were the concentration of ammonium sulphate solution, the temperature, and the drug-to-lipid ratio.
After the nerve selective regeneration theory was put forth [1], many researchers had focused on it and its possible application. According to our previous study, the results of small gap sleeve bridging peripheral nerve...After the nerve selective regeneration theory was put forth [1], many researchers had focused on it and its possible application. According to our previous study, the results of small gap sleeve bridging peripheral nerve mutilation had been confirmed [2]. But the imminent causes of those results haven't been unveiled completely. This study tries to explore these reasons in some aspects. The results indicate the advantages of this new surgical method not only because of nerve selective regeneration but also because the small gap can create a suitable microenvironment for peripheral nerve regeneration.
OBJECTIVE: To investigate whether it is feasible to use the chondrogenic microenvironment provided by cartilage cells to construct cartilage tissues in vitro with bone marrow stromal cells (BMSC). MATERIALS AND METHODS:...OBJECTIVE: To investigate whether it is feasible to use the chondrogenic microenvironment provided by cartilage cells to construct cartilage tissues in vitro with bone marrow stromal cells (BMSC). MATERIALS AND METHODS: We isolated and cultured BMSC and cartilage cells from Sprague Dawley rats (SD rats). The supernatant of cartilage culture was used as inducing solution to cause differentiation of BMSC from the second generation of cells cultured in vitro. Cells were examined seven days later, using immunohistochemistry to determine the expression of collagen specific to type II cartilage. RT-PCR was used to detect the expression of type II collagen and aggrecan mRNA. BMSC and cartilage cells were isolated from SD rats and cultured in vitro. The BMSC and cartilage cells in culture were mixed evenly in an 8:2 ratio and inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold to a final concentration of 5.0x10(7) cells/ml. PGA/PLA preparations with pure cartilage cells or pure BMSC served as the positive and negative controls, respectively. The control group of low-concentration cartilage cells consisted of PGA/PLA preparations containing cartilage cells at 20% of the above mentioned concentration (1.0x10(7) cells/ml). Samples were collected eight weeks later, at which time general observations, wet weight, and glycosaminoglycan (GAG) levels were determined, and histological and immunohistochemical examinations were performed. RESULTS: Immunohistochemistry showed the induction of BMSC type II collagen, and RT-PCR indicated the expression of type II collagen and aggrecan mRNA. In the mixed-cell group and the positive control group, pure mature cartilage cells were produced after eight weeks of culture in vitro, and the size and shape of the scaffold were maintained throughout the culture period. The two groups gave rise to newly generated cartilage cells essentially identical in appearance and histological properties. The immunohistochemical results showed that the cartilage cells of both groups expressed abundant cartilage-specific type II collagen. The average wet weight and GAG content were more than 70% of the values in the positive control group. Only an extremely small amount of immature cartilage tissue formed in local regions in the BMSC-only sample, and the scaffold was obviously shrunken and deformed. Although the wet weight of newly generated cartilage tissue in the low-concentration cartilage cell sample reached 30% of the value of the positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly generated cartilage was obviously less than in the co-culture and positive control groups. CONCLUSIONS: Cartilage cells can provide a microenvironment for cartilage formation to some extent, and also effectively induce BMSC to differentiate into cartilage cells and form tissue-engineered cartilage in vitro.
Living cells encapsulated in polymeric shells are receiving increasing attention because of their possible biotechnological and biomedical applications. The aim of this work is to evaluate how different polyelectrolyte c...Living cells encapsulated in polymeric shells are receiving increasing attention because of their possible biotechnological and biomedical applications. The aim of this work is to evaluate how different polyelectrolyte coatings, characterized by different numbers of polyelectrolyte layers and by different polyelectrolyte conformations, affect the viability of encapsulated biological material. We demonstrate the ability to individually encapsulate HL-60 cells as well as rat pancreatic islets within polymeric shells consisting of different PE layers using the layer-by-layer process. Coating of HL-60 cells allows for surviving and functioning of cells for all applied PE as well as for different numbers of layers. The islets encapsulated in applied polyelectrolytes exhibited the lower level of mitochondrial activity as compared to non-encapsulated islets. Nevertheless, encapsulated islets exhibited comparable absorbance values during the whole period of culture. Polyelectrolyte coating seems to be a promising way of allowing capsule void volume minimization in a model of encapsulated biological material for local production of biologically active substances.
To find new biomarkers and establish histopathology protein fingerprint models for early detection of oral squamous cell carcinoma (OSCC), laser capture microdissection (LCM) technology was utilized in 21 OSCC tissues an...To find new biomarkers and establish histopathology protein fingerprint models for early detection of oral squamous cell carcinoma (OSCC), laser capture microdissection (LCM) technology was utilized in 21 OSCC tissues and 7 oral leukoplaque (OLK) tissues as well as their adjacent normal tissues. Each sample was then detected by SELDI-TOF-MS technology and CM10 protein chip as well as bioinformatics tools. Three proteomic biomarker patterns were identified. Pattern 1 distinguishes patients with OLK from healthy individuals. Pattern 2 distinguishes patients with OSCC from healthy individuals. Pattern 3 distinguishes patients with OSCC from patients with OLK. The analysis yielded both a specificity and a sensitivity of 90.48% for pattern 1, a specificity of 100.00% and a sensitivity of 85.71% for pattern 2, and a specificity of 100.00% and a sensitivity of 85.71% for pattern 3. Proteome mass/charge 3714, 3515, and 4944 built the distinguished protein peaks between the OSCC tumor and adjacent normal tissues. The accuracy of the blind prediction was 90.48% (38/42). M/Z 15122 and 7569 built the distinguished protein peaks between the OLK and adjacent normal tissues. M/Z 3738 and 11366 built the distinguished protein peaks between the OSCC and the OLK. By employing LCM technology combined with SELDI-TOF-MS technology and bioinformatics approaches, histopathology would not only facilitate the discovery of better biomarkers for OSCC and OLK, but also provide a useful tool for molecular diagnosis by potential biomarker.
A rapid, highly sensitive and specific high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS-MS) quantitation method was developed and validated for the determination of hydrocodone in hum...A rapid, highly sensitive and specific high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS-MS) quantitation method was developed and validated for the determination of hydrocodone in human plasma. Sample was extracted from 0.5mL heparinized plasma by a simple liquid-liquid extraction method and analyzed on a C18 column with a mobile phase of acetonitrile-water (78:22,v/v,0.1% acetic acid). Detection was carried out by positive elevtrospray ionization (ESI) in multiple reactions monitoring (MRM) mode of 300.3-->199.2 (m/z) for hydrocodone and 341.2-->107.2 (m/z) for canrenone (I.S.), respectively. A good linearity was obtained from 0.5 to 60 ng x mL(-1) and the lower limit of quantification (LLOQ) was 0.1 ng x mL(-1). Compared to an existing method, the extraction method, internal standard and chromatographic conditions were modified and the cost of a large amount of samples determination was decreased obviously. The method was successfully applied to the pharmacokinetic and bioequivalence studies in healthy Chinese volunteers.