Biological sample pretreatment is an important step in biological sample analysis. Due to the diversity of biological matrices, the analysis of target substances in these samples presents significant challenges to sample processing. To meet these emerging demands on biopharmaceutical analysis, this paper summarizes several new techniques of on-line biological sample processing: solid phase extraction, solid phase micro-extraction, column switching, limited intake filler, molecularly imprinted solid phase extraction, tubular column, and micro-dialysis. We describe new developments, principles, and characteristics of these techniques, and the application of liquid chromatography-mass spectrometry (LC-MS) in biopharmaceutical analysis with these new techniques in on-line biological sample processing.

Zidovudine (AZT), the first drug approved by the US Food and Drug Administration for the treatment of human immunodeficiency virus (HIV) infection, is metabolized in the host cells to 5'-AZT triphosphate (AZT-TP) which inhibits HIV reverse transcriptase. As the pharmacokinetics of AZT and its phosphorylated metabolites in human peripheral blood mononuclear cells (hPBMCs) is limited, the aim of this study was to determine the pharmacokinetic parameters of AZT and its phosphorylated metabolites in hPBMCs from 12 healthy Chinese male subjects after a single oral dose of 600 mg of AZT. Blood samples were collected prior to drug administration, then at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8 and 10 h after drug administration. Mononuclear cells collected by Ficoll-Hypaque density gradient centrifugation were used for determination of AZT and metabolites [AZT monophosphate (AZT-MP), AZT diphosphate (AZT-DP) and AZT-TP] and the plasma was used to evaluate the pharmacokinetics of AZT. Plasma concentration of AZT peaked within 0.583 h and intracellular concentrations of AZT, AZT-MP, AZT-DP and AZT-TP peaked within 1.083, 1.500, 1.417 and 1.583 h, respectively. AZT in plasma was eliminated rapidly with t 1/2 of 2.022 h, and AZT-MP, AZT-DP and AZT-TP were eliminated with t 1/2 of 13.428, 8.285 and 4.240 h, respectively. The plasma concentration of the phosphorylated metabolites was not quantifiable.

Objective: To observe the effects of nootkatone on depression-like behavior, neurogenesis and protein kinase A(PKA)/cyclic adenosine monophosphate response element-binding protein(CREB)/brain-derived neurotrophic factor(BDNF) signaling pathway in hippocampus of chronic unpredictable stress(CUS) treated mice, and to explore the role and molecular mechanism of nootkatone′s antidepressant effect. Methods: Male C57 BL/6 mice were randomly divided into 3 groups: the control group(saline), the CUS group(CUS+saline), the CUS+nootkatone group(CUS+nootkatone). The sucrose preference test and forced swim test were used to evaluate the depression-like behaviors. The mRNA expression of BDNF in hippocampus was measured by RTPCR. The expression levels of BDNF, PKA and phosphorylated cyclic adenosine monophosphate response element-binding protein(p-CREB) in hippocampus were determined by Western blot. The level of neurogenesis was measured by immunofluorescence. Results: Compared with the control group, the sucrose preference decreased(P<0.05) and the latency decreased(P<0.05), and immobility time increased in forced swim test(P<0.05) in CUS group, the expression levels of BDNF mRNA(P<0.05) and protein(P<0.05), PKA(P<0.05) and p-CREB(P<0.05) decreased. The sucrose preference of the CUS+nootkatone group increased(P<0.05) and the latency increased(P<0.05), and immobility time decreased in forced swim test(P<0.05), the expression levels of BDNF mRNA(P<0.05) and protein(P<0.05), PKA(P<0.05) and p-CREB(P<0.05) increased in comparison with the CUS group. Compared with control group, the number of hippocampal doublecortin(DCX) labeled neurons decreased(P<0.05) in CUS group. Compared with the CUS group, the number of DCX labeled neurons increased in the CUS+nootkatone group(P<0.05). Conclusion The improvement of depressive symptoms in CUS mice by nootkatone may be related to the neurogenesis in dentate gyrus of hippocampus and the activation of PKA/CREB/BDNF signaling pathway.

The aim of this study was to apply the reference-scaled average bioequivalence (RSABE) approach to evaluate the bioequivalence of 2 formulations of agomelatine, and to investigate the pharmacokinetic properties of agomelatine in Chinese healthy male subjects. This was performed in a single-dose, randomized-sequence, open-label, four-way crossover study with a one-day washout period between doses. Healthy Chinese males were randomly assigned to receive 25 mg of either the test or reference formulation. The formulations were considered bioequivalent if 90% confidence intervals (CIs) for the log-transformed ratios and ratio of geometric means (GMR) of AUC and C max of agomelatine were within the predetermined bioequivalence range based on RSABE method. Results showed that both of the 90% CIs for the log-transformed ratios of AUC and C max of 7-desmethyl-agomelatine and 3-hydroxy-agomelatine were within the predetermined bioequivalence range. The 90% CIs for natural log-transformed ratios of C max, AUC0-t and AUC0-∞ of agomelatine (104.42-139.86, 101.33-123.83 and 97.90-117.94) were within the RSABE acceptance limits, and 3-hydroxy-agomelatine (105.55-123.03, 101.95-109.10 and 101.72-108.70) and 7-desmethyl-agomelatine (104.50-125.23, 102.36-111.50 and 101.62-110.64) were within the FDA bioequivalence definition intervals (0.80-1.25 for AUC and 0.75-1.33 for C max). The RSABE approach was successful in evaluating the bioequivalence of these two formulations.