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ABSTRACT OBJECTIVE：To establish a method for the determination of 5-hydroxyindole acetic acid（5-HIAA），glutamine， hippurate，pimelate，proline，tryptophan，tyramine，tyrosine and valine in human urine. METHODS：Morning urine samples were collected from depression patients. The sample was extracted with acetonitrile after addition of internal standard cortisone. LC-MS/ MS method was adopted. The determination was performed on XTerra RP18 column with mobile phase consisted of 0.1％ acetic acid-water as mobile phase A and 0.1％ acetic acid-acetonitrile as mobile phase B（gradient elution）. The column temperature was set at 40 ℃，and flow rate was 0.45 mL/min. Electrospray ion source（ESI）was used for quantitative analysis by multiple reaction monitoring （MRM）. The precursor/product ion transitions （m/z） were monitored at m/z 192.2→146.1，m/z 147.2→130.0，m/z 180.1→105.1，m/z 161.1→125.2，m/z 116.1→70.2，m/z 205.2→188.2，m/z 138.2→121.1，m/z 182.0→123.0，m/z 118.2→72.1 and m/z 361.2→163.0（+ion-mode）for 5-HIAA，glutamine，hippurate，pimelate，proline，tryptophan，tyramine，tyrosine，valine and cortisone，respectively. RESULTS：The linear range of 5-HIAA，glutamine，pimelate，proline，tyramine and valine were 10.00-3 200 ng/mL（r＝0.993 8-0.998 9，n＝6）. The lower limit of quantification was 10 ng/mL；the linear range of hippurate，tryptophan and tyrosine were 1 600-51 200 ng/mL（r＝0.999 2-0.999 7，n＝6）. The lower limit of quantitation was 1 600 ng/mL. The results of accuracy tests were 86.29％-98.65％（n＝6）. RSDs of intra-day and inter-day precision tests were no more than 14.65％（n＝6）CV of matrix effect were 6.18％-14.37％（n＝6）. Extraction recovery rates were 86.21％-98.14％（n＝6）. RE of stability tests were no more than 14.71％（n＝3-6）. CONCLUSIONS：The method is sensitive，accurate and suitable for the determination of 9 substances in human.
ABSTRACT
Objective To study the effect of salidroside (Sal) on learning and memory abilities and expression of p75NTR signal transducers and Aβ in AD rat hippocampus.Methods Totally,ninety-six male SD rats were randomly divided into sham-operation group,model control group,Sal low,medium and high dose group,Hup-A group (16 rats in each group).Except for sham-operation group,AD model in the other group was established by slowly injecting 1μL(10 μg)Aβ1-40 in tobilateral hippocampal CA1 area.Sham-operation group was given the same volume of 0.9％ sodium chloride solution.Sal low,medium and high dose group took orally as early as 24 h postoperatively with salidroside 25,50,100 mg· kg-1 for 21days,while Hup-A group took 50 mg kg-1 of Hup-A orally.The same volume of 0.9％ sodium chloride solution was administrated in shanmoperation group and model control group.After administration,the learning and memory function of rats were tested by Morris water maze.The contents of Aβ in serum and hippacampus were determined by ELISA,and protein expression of p75NTR and p-JNK in the hippocampus of the rats were measured by ELISA.Results Compared with model control group,the latent time in water maze training of Sal low,medium and high dose groups were significantly shortened (P ＜ 0.05 or P ＜ 0.01),the number of times of crossing platform of Sal low,medium and high dose group was significantly increased.The contents of Aβ in the hippocampus and the protein expression levels of p75NTR and p-JNK in the hippocampus of Sal low,medium and high dose group were decreased significantly (P ＜ 0.05,P ＜ 0.01).Conclusion Salidroside has protective effects on learning and memory consolidation of dysmnesia rats caused by Aβ.Its mechanism might be related to inhibition of p75NTR regulation pathways for Aβ,reducing the neurotoxic effect of Aβ,so as to reduce cell apoptosis of hippocampus neuron.