Objective To establish an ion chromatography method to determine the content of galactosamine in heparin sodium sample. Methods The content of galactosamine was determined by the ratio of response value of galactosamine and glucosamine.The determination was performed on an Dionex ICS, and the separation was carried out on a Amino acids capture column (30 mm ×3 mm), Series protect column (30 mm × 3 mm)and analytical column CarboPac PA20 (150 mm ×3 mm).The mobile phase was 14 mM potassium hydroxide solution at a flow rate of 0.4 mL/min; the column tempertature was at 30℃; the injection volume was 10μL.Results Glucosamine hydrochloride had good linearity within the range of 1.013 -16.211μg/mL(Y=2.303 4X+0.824 2,r=0.998 3), the average accuracy was 92.7%, and RSD was 3.2%(n=9), the limit of detection was 0.101 3μg/mL, and the limit of quantitation was 0.337 7μg/mL.D-Galactosamine hydrochloride had good linearity within the range of 0.010 2 -0.162 5 g/mL, (Y=31.157X-0.114 4,r=0.999 3).The accuracy was 102.1%, RSD was 2.4%(n=9).The limit of detection was 0.001 0μg/mL, and the limit of quantitation was 0.003 4μg/mL.The determination of galactosamine in 3 batches of heparin sodium raw material was not detected, (0.02 ±2.1)%, (0.03 ±1.5)%, respectively, which were all lower than the limit value (1%) of United States Pharmacopeia regulation.Conclusion The method for the determination of galactosamine in total hexose amine is successfully developed , which could be used as reference for improvement of the quality standard of heparin sodium.

Objective:To investigate the mechanism of learning and memory impairment of apolipoprotein E -/- ( ApoE-/-) mice after transient global cerebral ischemia by diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS). Methods:Ten-week-old C57BL/6(WT) mice and ApoE-/- mice were randomly divided into WT sham-operated group ( n=8), WT 7 d group ( n=12) and WT 30 d group ( n=12), and ApoE-/- sham-operated ( n=8), ApoE-/- 7 d group ( n=12) and ApoE-/- 30 d group ( n=12). The mice in the WT 7 d group, WT 30 d group, ApoE-/- 7 d group, and ApoE-/- 30 d group received bilateral common carotid artery ischemia-reperfusion injury, while mice in the WT sham-operated group and ApoE sham-operated group only stripped the blood vessels without ligation. On the 7 th and 30 th d of modeling, Morrris water maze test was employed to detect the learning and memory abilities of these mice; DTI was used to detect the fractional anisotropy (FA) values in the bilateral hippocampus of mice, and MRS was used to detect the contents of choline complex (Cho) and N-acetylaspartate (NAA) in the bilateral hippocampus of mice. Results:(1) On 2 nd, 3 rd, and 4 th d of water maze experiment, the escape latency of mice in ApoE-/- 7 d group was significantly prolonged as compared with that in the ApoE-/- sham-operated group and WT 7 d group ( P<0.05); since the 3 rd d of water maze experiment, the escape latency of mice in ApoE-/- 30 d group was significantly prolonged as compared with that in WT 30 d group ( P<0.05). The number of times crossing platform in ApoE-/- 7 d group was significantly smaller than that in WT 7 d group, and the residence time in the third quadrant was significantly shorter ( P<0.05); the number of times crossing platform in ApoE-/- 30 d group were significantly smaller as compared with that in the WT 30 d group, and the residence time in the third quadrant was significantly shortened ( P<0.05). (2) DTI results showed that there was no significant difference in bilateral hippocampal FA values between ApoE-/- 7 d group and WT 7 d group ( P>0.05); the bilateral hippocampal FA values of mice in the ApoE-/- 30 d group were statistically lower than those in WT 30 d group ( P<0.05). (3) MRS results showed that the relative contents of hippocampal Cho and NAA in the ApoE-/- 7 d group were significantly lower than those in the WT 7 d group, and the relative contents of hippocampal Cho and NAA in the ApoE-/- 30 d group were significantly lower than those in the WT 30 d group ( P<0.05). Conclusion:ApoE-/- mice have poor learning and memory abilities after transient global cerebral ischemic injury, whose mechanism is closely related to the damage of hippocampal white matter fibers and abnormal metabolism of nerve cells.

This study aimed to isolate and prepare highly purified impurity C from doxycycline hyclate by a preparative HPLC method and to inspect the toxicity and in vitro antimicrobial activity of the impurity C of doxycycline hyclate. The solution of doxycycline hyclate treated with heat produced a solution containing 10% of impurity C which was firstly separated by the Sapphire C18(21. 2 mm×250 mm, 5 μm)column with 0. 1% acetic acid-acetonitrile(83 ∶17)as the mobile phase at 20 mL/min. Secondly, rotary evaporation of the eluted solution at the time of 8. 4 min was performed at 50 °C to remove organic solvent. Then the target product was prepared after freeze drying of evaporated solution adjusting pH to 1. 8 with formic acid. The target product was identified with ultraviolet absorbance(UV), infrared(IR), mass spectrometry(MS)and nuclear magnetic resonance(NMR), and its purity was be determined by HPLC. Meanwhile, cytotoxicity and genotoxicity in the Chinese hamster lung cells, toxicity on the development of zebrafish embryos and in vitro antimicrobial activity were compared among impurity C of doxycycline hyclate, doxycycline, metacycline and β-doxycycline. Results showed that prepared product was confirmed to be the impurity C of doxycycline hyclate. Its purity was 90. 1%, which had been the highest so far. In the cellular toxic tests and genetic toxic tests of Chinese hamster lung cells, impurity C of doxycycline hyclate, doxycycline, metacycline and β-doxycycline were somewhat toxic to Chinese hamster lung cells. Toxicity gradually decreased from doxycycline, impurity C of doxycycline hyclate, β-doxycycline to metacycline from -S9mix test results; toxicity gradually decreased from doxycycline, β-doxycycline, impurity C of doxycycline hyclate to metacycline from +S9mix test results; the aberration rate of all the tested related substances was less than 5%, and no obvious genotoxicity was found. According to test results of the development of zebrafish embryos, impurity C of doxycycline hyclate showed the strongest teratogenicity and lethality. Invitro antimicrobial tests revealed that impurity C of doxycycline hyclate had a weaker antimicrobial activity, and invitro antimicrobial activity potential of the tested compounds followed the order: metacycline, doxycycline, impurity C of doxycycline hyclate, β-doxycycline. Studies on safety and effectiveness indicated that impurity C of doxycycline hyclate belonged to toxic and ineffective impurity and need to be controlled individually in quality standard. A useful suggestion was given to revise the quality standard of doxycycline hyclate and its preparation in the current Pharmacopoeia of the People′s Republic of China.