Detection of Phosphatidylethanol in Whole Blood by UPLC-MS/MS / 法医学杂志

OBJECTIVES@#To establish the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to detect ethanol metabolites phosphatidylethanol (PEth) in whole blood.@*METHODS@#An appropriate amount of aqueous solution including 1% formic acid was added to 100 μL whole blood, the protein was precipitated with acetone, centrifuged and the supernatant was purified and enriched by using Bond Elut Certify column. The eluent was redissolved with 1/1 isopropanol/acetonitrile (v/v) solution after nitrogen blowing and then tested by UPLC-MS/MS. Selective reaction monitoring scanning was carried out in negative ionization mode, and quantitative analysis was performed by external standard method.@*RESULTS@#PEth showed a linear relationship over the concentration range of 1-160 ng/mL in whole blood (r=0.999 9) with peak area. The detection limit was 0.2 ng/mL, the quantification limit was 1 ng/mL, the recovery rate was 97.43%-103.61%, the accuracy was 0.99%-1.77%, the intra-day precision was 0.4%-2.4%, and the inter-day precision was 1.1%-3.3%, and the matrix effect was 91.00%-99.55%. PEth was not detected in the in vitro blood samples supplemented with ethanol. PEth was detected positive in three drunk driving cases, and the concentration were 195.49, 83.67 and 876.12 ng/mL, respectively.@*CONCLUSIONS@#The established method has high sensitivity and specificity and the analysis results are accurate. It is suitable for the qualitative and quantitative analysis of PEth in whole blood.

\Quantitative determination of 10 illicit drugs in wastewater by liquid-liquid extraction-ultra performance liquid chromatography-tandem mass spectrometry / 中国药科大学学报

@#A deuterated internal standard quantitative analysis method based on liquid-liquid extraction-ultra performance liquid chromatography-tandem mass spectrometry (LLE-UPLC-MS/MS) for simultaneous determination of 10 illicit drugs in wastewater was established.Wastewater samples were concentrated by liquid-liquid extraction with dichloromethane: ethyl acetate (1∶1), and separated by a linear gradient of 0.1% formic acid-5 mmol/L ammonium formate aqueous solution and acetonitrile on a C18 column. The samples were then detected by ESI positive ion mode and multiple reaction monitoring mode (MRM) for quantitative analysis.All analytes had a good linear relationship (r ≥ 0.995 7) within the range of their respective standard curves; the limit of quantification was 1 ng/L (except amphetamine at 2.5 ng/L); the relative recovery rate ranged from 96.36% to 106.43%, and the intra- and inter-day precisions were less than 4.70%.This method is accurate, reliable and reproducible, and is suitable for the quantitative determination of 10 illicit drugs in wastewater.It is also suitable for wastewater with complex matrixes that affect solid phase extraction and enrichment.It provides a new analytical method for real-time monitoring of drug abuse.

Regulatory Effect of Polygala tenuifolia and Licorice-simmered P. tenuifolia on Learning and Memory， HPA Axis Function and Neurotransmitters in Rats with Heart-kidney Imbalance Insomnia / 中国实验方剂学杂志

Objective:To compare the regulatory effects of <italic>Polygala tenuifolia</italic> and licorice-simmered <italic>P. tenuifolia</italic> on learning and memory， hypothalamus-pituitary-adrenal axis （HPA axis） function and neurotransmitters in rats with heart-kidney imbalance insomnia. Method:The rat model of insomnia induced by multi-factor stimulation was established. After the model being made， the administration groups were given the extracts of <italic>P. tenuifolia</italic> and licorice-simmered <italic>P. tenuifolia</italic> by gavage （dose of 8 g·kg^-1·d^-1）， while the normal group and model group were given the same volume of normal saline for 7 days. Morris water maze was used to detect the changes of learning and memory ability of rats in each group. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of corticotropin-releasing hormone （CRH）， adrenocorticotropic hormone （ACTH） and cortisol （CORT） in serum of rats from each group. The contents of 5-hydroxytryptamine （5-HT）， dopamine （DA）， <italic>γ</italic>-aminobutyric acid （GABA） and glutamic acid （Glu） in the hypothalamus of rats were determined simultaneously by ultra performance liquid chromatography tandem mass spectrometry （UPLC-MS/MS）. Result:Compared with the normal group， the spatial learning and memory ability of rats in the model group<italic> </italic>was decreased， the times and time of staying in target quadrant were significantly reduced （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， the levels of CORT， CRH and ACTH in serum were significantly increased （<italic>P<</italic>0.01）， the contents of GABA， DA， 5-HT in hypothalamus tissue were significantly decreased （<italic>P<</italic>0.01）， and the content of Glu was significantly increased （<italic>P<</italic>0.01）. Compared with the model group， the spatial learning and memory ability of rats in the <italic>P. tenuifolia</italic> group and licorice-simmered <italic>P. tenuifolia </italic>group<italic> </italic>were increased， the times and time of staying in the target quadrant were significantly increased （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， the levels of CORT， CRH and ACTH in serum were significantly decreased （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， the contents of GABA， DA and 5-HT in hypothalamus tissue were significantly increased （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， and the content of Glu was significantly decreased （<italic>P<</italic>0.05）. The recovery degree of each index in the licorice-simmered <italic>P. tenuifolia</italic> group was better than that in the <italic>P. tenuifolia</italic> group. Conclusion:Both <italic>P. tenuifolia</italic> and licorice-simmered <italic>P. tenuifolia</italic> can improve the learning and memory ability， improve the function of HPA axis， regulate the level of central neurotransmitters， and have the effect of calming the mind and improving the intelligence of rats with heart-kidney imbalance insomnia. The effect of licorice-simmered <italic>P. tenuifolia</italic> is better than that of <italic>P. tenuifolia.</italic>

Analysis on Content Differences of Artemisinin and Arteannuin B in Different Provenances of Artemisia annua Under Indoor Hydroponic Conditions / 中国实验方剂学杂志

Objective:To establish a method for the determination of artemisinin and arteannuin B in different <italic>Artemisia annua</italic> germplasms， compare the differences of the two compounds among different <italic>A. annua</italic> germplasm under the condition of hydroponic homogenization and explore the key factors affecting contents of principal compounds in different<italic> A. annua</italic> germplasms. Method:Seedlings from different <italic>A. annua</italic> germplasms were arranged randomly and fed in a hydroponic cultivation system. Contents of artemisinin and arteannuin B were detected by ultra performance liquid chromatography tandem mass spectrometry （UPLC-MS/MS） with multi-reaction monitoring mode and ACQUITY UPLC^® BEH C₁₈ column （2.1 mm×100 mm， 1.7 μm）， mobile phase was water-acetonitrile （95∶5， containing 0.1% formic acid， A） and acetonitrile-water （95∶5， containing 0.1% formic acid， B） for gradient elution （0-3.5 min， 25%-1%A； 3.5-3.6 min， 1%-25%A； 3.6-5.0 min， 25%A）， the flow rate was set at 0.4 mL·min^-1. The content differences of artemisinin and arteannuin B in different provenances of <italic>A. annua</italic> were detected and analyzed statistically. Result:The established method had high sensitivity and good separation. A significant difference of artemisinin and arteannuin B contents was observed in different germplasms under the same culture conditions， that is， under the constant temperature of 25 ℃ in hydroponics. The provenance with higher artemisinin content was Yunnan， and the content was 3 810.597 μg·g^-1. The highest strain of arteannuin B was Shanxi provenance germplasm with the content of 1 691.747 μg·g^-1. According to the content of artemisinin， the provenances were arranged as follows：Yunnan， Hainan， Hubei， Guangxi， Zhejiang， Shanxi， Beijing， Shandong， Heilongjiang， and Gansu province germplasms. Correlation analysis showed that there was a significant negative correlation between artemisinin content and latitude of <italic>A. annua</italic>， but there was no significant correlation between contents of artemisinin and arteannuin B and longitude. Conclusion:The contents of artemisinin and arteannuin B among different <italic>A. annua</italic> germplasms were significantly different under the same culture environment， and the dominant factors affecting biosynthesis and accumulation of artemisinin and arteannuin B in <italic>A. annua</italic> may be the genetic background， suggesting that germplasm improvement is the key factor to improve the medicinal quality of <italic>A. annua</italic> in subsequent cultivation.

Difference Analysis of Pharmacokinetic Behavior of Active Ingredients in Wujiwan Between Normal Rats and Rats with Irritable Bowel Syndrome / 中国实验方剂学杂志

Objective:To establish an ultra-performance liquid chromatography-mass spectrometry （UPLC-MS/MS） for determining the plasma concentrations of 10 active ingredients in Wujiwan at different time points after oral administration， and to compare the pharmacokinetic characteristics between normal rats and rats with chronic visceral hypersensitive irritable bowel syndrome （CVH-IBS）. Method:CVH-IBS rat model was prepared by the neonatal rat colon percutaneous transluminal coronary angioplasty （PTCA） balloon stimulation method. After intragastric administration of Wujiwan （0.245 g·kg^-1）， blood was collected from the jugular vein at different time points， and the plasma concentrations of 10 active ingredients （berberine hydrochloride， palmatine hydrochloride， coptisine hydrochloride， jatrorrhizine hydrochloride， epiberberine， dihydroberberine， evodiamine， evodine， paeoniflorin， albiflorin） in Wujiwan was detected simultaneously by UPLC-MS/MS， the pharmacokinetic parameters of each component in normal rats and CVH-IBS rats were calculated. Result:The established UPLC-MS/MS could sensitively and accurately detect the plasma concentrations of 10 active ingredients of Wujiwan in rats. Compared with the normal group， the absorption rates of these 10 active ingredients of Wujiwan in the blood of CVH-IBS rats all decreased to a certain extent， and the peak time （<italic>t</italic>_max） was prolonged. Among them， the <italic>t</italic>_max of berberine hydrochloride and jatrorrhizine hydrochloride were significantly prolonged from 54 minute and 39 minute to 90 minute， respectively （<italic>P</italic><0.05， <italic>P</italic><0.01）. Area under the plasma concentration-time curve （AUC_0-<italic>_t</italic>） of each component increased， and evodiamine and paeoniflorin were significantly different （<italic>P</italic><0.05，<italic> P</italic><0.01）. The clearance rates （CL/<italic>F</italic>） of these 10 active ingredients were all decreased， among which berberine hydrochloride， palmatine hydrochloride and evodiamine had significant differences （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:There are significant differences in the pharmacokinetic behavior of the active ingredients in Wujiwan between normal rats and CVH-IBS rats， which may be related to the destruction of microstructure of intestinal epithelial cells and the change of activity of liver enzymes under the pathological state of IBS.

Determination and Risk Assessment of Six Hepatotoxic Pyrrolizidine Alkaloids in Verbenae Herba / 中国实验方剂学杂志

Objective:To establish ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） for simultaneous determination of six hepatotoxic pyrrolizidine alkaloids in Verbenae Herba， and to carry out preliminary risk assessment according to the research results. Method:An ACQUITY UPLC HSS T3 column （2.1 mm×100 mm， 1.8 μm） was used for analysis with 0.05% formic acid and 2.5 mmol·L^-1 ammonium formate in water （A）-0.05% formic acid and 2.5 mmol·L^-1 ammonium formate in acetonitrile （B） as mobile phase for gradient elution （0-12 min， 3%-8%B； 12-25 min， 8%-15%B； 25-26 min， 15%-3%B； 26-30 min， 3%B）， the flow rate was 0.3 mL·min^-1， the column temperature was 40 ℃， and the injection volume was 1 μL. MS system was operated by electrospray ionization （ESI） in the positive ion mode with multiple reaction monitoring mode. MS parameters of triple quadrupole and six analytes were optimized for qualitative and quantitative analysis. According to the determination results， the risk assessment was carried out by using margin of exposure （MOE） combined with transfer rate of hot water extraction. Result:Based on the instrument precision， linear range， repeatability， stability， recovery and other methodological validations， the results were in conformity with relevant standards of quantitative analysis. The linear ranges of intermedine， lycopsamine， intermedine <italic>N</italic>-oxide， lycopsamine<italic> N</italic>-oxide， echimidine<italic> N</italic>-oxide and echimidine were good （<italic>r</italic>≥0.999 0） between peak area and mass concentration in the ranges of 0.984-49.20， 0.994-49.70， 1.012-50.60， 1.032-51.60， 1.004-50.20， 1.016-50.80 µg·L^-1， respectively. The average recoveries of these six analytes were 87.2%-94.2% with relative standard deviation （RSD）<4.0%. Their MOE values were >10 000. Conclusion:The UPLC-MS/MS established in this study is stable and feasible， which can provide scientific basis for the quality control and safety evaluation of hepatotoxic pyrrolizidine alkaloids in Verbenae Herba.

Effect of Lidan Ruanjian Prescription on Lipid-lowering and Antiobesity andMetabolic Profile of Bile Acid / 中国实验方剂学杂志

Objective:To investigate the pharmacodynamics effects of antiobesity, lipid-lowering and the regulations of serum bile acid profiles of Lidan Ruanjian prescription (LDRJ) in obesity rats induced by high-fat diet. Method:The 42 rats were fed high-fat diet for 9 weeks to establish model of obese rats，24 rats were randomly divided into model group, high and low-dose LDRJ group (30,15 g·kg-1). Another 8 normal rats were selected as the normal group.The model group and normal group were given normal saline, and drug group was given the corresponding dose of drug for 4 weeks. Body weight, liver weight, white adipose tissue (WAT) weight were determined after administration medicine for 4 weeks. The bile flow of the rats was measured by bile duct intubation and fasting serum lipid levels of total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) were detected by automatic biochemical analyzer. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay was used to test serum bile acid profile of each group rats. Result:Compared with the control group, the average body weight, liver weight, WAT weight of the model group were significantly increased (P<0.01), while the fasting serum TC, TG and LDL-C levels were elevated (P<0.05，P<0.01). The total bile secretion and bile flow at each test point within 2 h were decreased and the proportion of primary bile acids was decreased (P<0.05).The serum total bile acid content decreased significantly（P<0.01），levels of cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA), taurohyodeoxycholic acid (THDCA) and glycodeoxycholic acid (GDCA) were significantly reduced (P<0.05，P<0.01). Compare with model group, body weight, liver weight in high and low-dose LDRJ groups reduced significantly(P<0.05，P<0.01). Fasting serum TC, TG and LDL-C levels were decreased in high-dose group(P<0.05，P<0.01), so did as TG levels in low-dose group(P<0.05). The bile flow rate increased significantly in high-dose group 1~1.5 h after administration (P<0.05). All dose treatment groups increased the proportion of primary bile acids (P<0.05) and changed the bile acid profile, especially elevated the bile acid levels of TCA, DCA, glycocholic acid (GCA), GDCA in high-dose LDRJ group (P<0.05，P<0.01), while TCA and TCDCA in low-dose group (P<0.05). Conclusion:LDRJ has significant lipid-lowering and antiobesity effects and the mechanism might involve the increase of bile secretion, the stimulation of primary bile acid synthesis and the regulation of bile acid profile.

Simultaneous determination of illicit drugs and their metabolites in wastewater by SPE-UPLC-MS/MS / 中国药科大学学报

@#A quantitative analysis method based on solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) for simultaneous determination of illicit drugs and their metabolites in wastewater was established. Samples filtered at pH of 2 and spiked with internal standard were loaded to Oasis Prime MCX cartridges for solid-phase extraction. The samples were washed with 4 mL of methanol and eluted with 4 mL of 5% ammonia in acetonitrile before reconstituting with 0.1% formic acid/water solution. ZORBAX Eclipse Plus C18 column was used for chromatography, and gradient elution was performed with 0.1% formic acid/water solution and acetonitrile as mobile phase. The samples were then detected by electrospray ionization (ESI) in positive ion mode, and multiple reaction monitoring mode (MRM) was adopted for quantitative analysis. All analytes had a good linear relationship (r ≥ 0.993 2) within the range of their respective standard curve; the limit of quantification was 1 ng/L (except amphetamine at 2.5 ng/L); the extraction recovery ranged from 82.13% to 99.96%; and the intra- and inter-day precisions were less than 9.43%. The method is accurate, reliable and reproducible, and is suitable for the quantitative determination of illicit drugs and their metabolites in wastewater and can provide an analytical method for real-time monitoring of drug abuse.