Journal of Pharmaceutical Analysis

Highly sensitive chemiluminescence technology for protein detection using aptamer-based rolling circle amplification platform

Zhi-Juan Cao, Qian-Wen Peng, Xue Qiu, Cai-Yun Liu, Jian-Zhong Lu

2011, 01(3): 159-165. doi: 10.1016/j.jpha.2011.06.002

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A robust,selective and highly sensitive chemiluminescent (CL) platform for protein assay was presented in this paper.This novel CL approach utilized rolling circle amplification (RCA) as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier.Typically,the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer-primer sequence.This aptamer-primer sequence was then employed as the primer of RCA.Based on this design,a number of the biotinylated probes and streptavidin-horseradish peroxidase (SA-HRP) were captured on the plate,and the CL signal was amplified.In summary,our results demonstrated a robust biosensor with a detection limit of 10 fM that is easy to be established and utilized,and devoid of light source.Therefore,this new technique will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases,by taking advantages of high sensitivity and selectivity.

Chemiluminescence enzyme immunoassay based on magnetic nanoparticles for detection of hepatocellular carcinoma marker glypican-3

Qian-Yun Zhang, Hui Chen, Zhen Lin, Jin-Ming Lin

2011, 01(3): 166-174. doi: 10.1016/j.jpha.2011.06.004

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Glypican-3 (GPC3) is reported as a great promising tumor marker for hepatocellular carcinoma (HCC) diagnosis.Highly sensitive and accurate analysis of serum GPC3 (sGPC3),in combination with or instead of traditional HCC marker alpha-fetoprotein (AFP),is essential for early diagnosis of HCC.Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay.Here,the magnetic nanoparticles (MnPs) and magnetic microparticles (MmPs) with carboxyl groups were further modified with streptavidin,and applied for the development of chemiluminescence enzyme immunoassay (CLEIA).After comparing between MnPs- and MmPs-based CLEIA,MnPs-based CLEIA was proved to be a better method with less assay time,greater sensitivity,better linearity and longer chemiluminescence platform.MnPs-based CLEIA was applied for detection of sGPC3 in normal liver,hepatoeirrhosis,secondary liver cancer and HCC serum samples.The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

Microfluidics-based assay on the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors

Yang Liu, Jian-Chun Wang, Li Ren, Qin Tu, Wen-Ming Liu, Xue-Qin Wang, Rui Liu, Yan-Rong Zhang, Jin-Yi Wang

2011, 01(3): 175-183. doi: 10.1016/j.jpha.2011.06.001

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A new microfluidic system with four different microchambers (a circle and three equilateral concave polygons) was designed and fabricated using poly(dimethylsiloxane) (PDMS) and the soft lithography method.Using this microfluidic device at six flow rates (5,10,20,30,40,and 50 μL/h),the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors were investigated.Escherichia coli HB101 pGLO,which could produce a green fluorescent protein induced by L-arabinose,was utilized as the model bacteria.The results demonstrated that bacterial adhesion was significantly related to culture time,microenvironment geometry,and aqueous flow rates.Adhered bacterial density increased with the culture time.Initially,the adhesion occurred at the microchamber sides,and then the entire chamber was gradually covered with increased culture time.Adhesion densities in the side zones were larger than those in the center zones because of the lower shearing force in the side zone.Also,the adhesion densities in the complex chambers were larger than those in the simple chambers.At low flow rates,the orientation of adhered bacteria was random and disorderly.At high flow rates,bacterial orientation became close to the streamline and oriented toward the flow direction.All these results implied that bacterial adhesion tended to occur in complicated aqueous flow areas.The present study provided an on-chip flow system for physiological behavior of biological cells,as well as provided a strategic cue for the prevention of bacterial infection and biofilm formation.

Analysis of chiral non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac binding with HSA

Chang-Chuan Guo, Yi-Hong Tang, Hai-Hong Hu, Lu-Shan Yu, Hui-Di Jiang, Su Zeng

2011, 01(3): 184-190. doi: 10.1016/j.jpha.2011.06.005

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The protein binding of non-steroidal anti-inflammatory drugs flurbiprofen,ketoprofen and etodolac with human serum albumin (HSA) was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques.S-(-)-2-(1-naphthyl)-ethylamine (S-NEA) was utilized as chiral derivatization reagent and pre-column derivatization RP-HPLC method was established for the separation and assay of the three pairs of enantiomer.The method had good linear relationship over the investigated concentration range without interference.The average extraction efficiency was higher than 85% in different systems,and the intra-day and inter-day precisions were less than 15%.In serum albumin,the protein binding of etodolac enantiomers showed significant stereoselectivity that the affinity of S-enantiomer was stronger than R-enantiomer,and the stereoselectivity ratio reached 6.06; Flurbiprofen had only weak stereoselectivity in HSA,and ketoprofen had no stereoselectivity at all.Scatchard curves showed that all the three chiral drugs had two types of binding sites in HSA.

Quantitative analysis of a novel antimicrobial peptide in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry

Ruo-Wen Zhang, Wen-Tao Liu, Lu-Lu Geng, Xiao-Hui Chen, Kai-Shun Bi

2011, 01(3): 191-196. doi: 10.1016/j.jpha.2011.04.001

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We described the first results of a quantitative ultra performance liquid chromatographytandem mass spectrometry method for a novel antimicrobial peptide (phylloseptin,PSN-1).Chromatographic separation was accomplished on a Waters bridged ethyl hybrid (BEH) C18 (50 mm × 2.1 mm,1.7 μm) column with acetonitrile-water (25∶75,v/v) as isocratic mobile phase.Mass spectrometry detection was performed in the positive electrospray ionization mode and by monitoring of the transitions at m/z 679.6/120,509.6/120 (PSN-1) and m/z 340.7/165 (Thymopentin,IS).Protein precipitation was investigated and the recovery was satisfactory (above 82%).The method was shown to be reproducible and reliable with intra-day precision below 5.3%,inter-day precision below 14.2%,and linear range from 0.02 to 2 μg/mL with r>0.994.The method was successfully applied to a pharmacokinetic study of PSN-1 in rats after intravenous administration.

Analysis and determination of diterpenoids in unprocessed and processed Euphorbia lathyris seeds by HPLC-ESI-MS

Xiao-Rong Hou, Lei-Lei Wan, Zha-Jun Zhan, Cheng-Ping Li, Wei-Guang Shan

2011, 01(3): 197-202. doi: 10.1016/j.jpha.2011.06.003

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Euphorbia lathyris (Caper spurge) is a toxic and potent Chinese materia medica (T/PCMM).This study sought a method for identifying five diterpenoids (Euphorbia factors L1-L3,L7a and L8) with the spectra of UV and mass,quantifying three diterpenoids L1,L2,and L8 in crude extracts of unprocessed and processed E.lathyris seeds by liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS).The analysis was achieved on an Agilent Eclipse XDB-C18 column (4.6 mm × 150 mm i.d.,5 μm) with an isocratic elution with a mobile phase consisting of water and acetonitrile at a flow rate of 0.25 mL/min at column temperature of 30 ℃ and UV detection was set at 272 nm.An ESI source was used with a positive ionization mode.The calibration curve was linear in the ranges of 9.9-79 μg/mL for Euphorbia factor L1,3.8-30.5 μg/mL for Euphorbia factor L2,and 1.0-20.6 μg/mL for Euphorbia factor L8.The average recoveries (n=6) of three diterpenoids were 98.39%,91.10% and 96.94%,respectively,with RSD of 2.5%,2.4% and 2.1%,respectively.The contents of the three diterpenoids in processed E.lathyris seeds were 3.435,1.367 and 0.286 mg/g,respectively,which decreased more sharply than those in unprocessed E.lathyris seeds which were 4.915,1.944 and 0.425 mg/g,respectively.The method is simple,accurate,reliable and reproducible,and it can be applied to control the quality of unprocessed and processed E.lathyris seeds.

Comparative analysis of essential oils found in Rhizomes Curcumae and Radix Curcumae by gas chromatography-mass spectrometry

Di-Ya Lü, Yan Cao, Ling Li, Zhen-Yu Zhu, Xin Dong, Hai Zhang, Yi-Feng Chai, Zi-Yang Lou

2011, 01(3): 203-207. doi: 10.1016/j.jpha.2011.05.001

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A comparison of the volatile compounds in Rhizomes Curcumae (Ezhu) and Radix Curcumae (Yujin) was undertaken using gas chromatography-mass spectrometry (GC-MS).Ultrasonic extraction and GC-MS methods were developed for the simultaneous determination of five sesquiterpenes,namely,α-pinene,β-elemene,curcumol,germacrone and curdione,in Ezhu and Yunjin.Good linearity (r>0.999) and high inter-day precision were observed over the investigated concentration ranges.The validated method was successfully used for the simultaneous determination of five sesquiterpenes in Ezhu and Yujin.The quantitative method can be effectively used to evaluate and monitor the quality of Chinese curcuma in clinical use.

A rapid method for the determination of dopamine in porcine muscle by pre-column derivatization and HPLC with fluorescence detection

Hong-Xia Zhao, Hui Mu, Yan-Hong Bai, Hu Yu, Ying-Mei Hu

2011, 01(3): 208-212. doi: 10.1016/j.jpha.2011.04.003

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A rapid method has been developed based on the sample preparation procedure named as QuEChERS (Quick,Easy,Cheap,Effective,Rugged and Safe),combined with reversed-phase high performance liquid chromatography with fluorescence detector and C18 column after precolumn derivatization using o-phthalaldehyde and 2-mercaptoethanol to determine dopamine in porcine muscle.Methanol and deionized water (0.1% acetic acid,v/v) with a ratio of 60∶40 was used as mobile phase.The flow rate was 0.8 mL/min and dopamine was eluted within 15 min.The linearity range was 0.003-8 μg/mL with r=0.9992.The detection limit for dopamine was 4 μg/kg and the quantification limit was 9 μg/kg.Recovery studies were carried out at 0.1,0.5 and 1.0 mg/kg fortification levels and the average recoveries obtained ranged from 90.4% to 98.2% with relative standard deviations between 3.5% and 8.1%.The method was found to be suitable for detection of dopamine in animal product tissues at the maximum residue level.

Rapid determination of volatile constituents in safflower from Xinjiang and Henan by ultrasonic-assisted solvent extraction and GC-MS

Ling-Han Jia, Yi Liu, Yu-Zhen Li

2011, 01(3): 213-218. doi: 10.1016/j.jpha.2011.04.002

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The total volatile components were extracted from safflower by ultrasonic-assisted solvent extraction (USE) and their chemical constituents were analyzed by gas chromatographymass spectrometry (GC-MS) to provide scientific basis for the quality control of safflower.Five different solvents (diethyl ether,ethanol,ethyl acetate,dichloromethane and acetone) were used and compared in terms of number of volatile components extracted and the peak areas of these components in TIC.The results showed that USE could be used as an efficient and rapid method for extracting the volatile components from safflower.It also could be found that the number of components in the TIC of ethyl acetate extract was more than that in the TIC of other solvent ones.Meanwhile,the volatile components of safflower from Xinjiang Autonomous Region and Henan Province of China were different in chemical components and relative contents.It could be concluded that both the extraction solvents and geographical origin of safflower are responsible for these differences.The experimental results also indicated that USE/GC-MS is a simple,rapid and effective method to analyze the volatile oil components of safflower.

Simultaneous determination of 12, 13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture by high performance liquid chromatography

Zhi-Sheng Xie, Xin-Jun Xu, Chun-Yan Xie, Jie-Yun Huang, Mei Yang, Rui-Ming Li, Xiao Chen

2011, 01(3): 219-222. doi: 10.1016/j.jpha.2011.06.006

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A reversed phase high performance liquid chromatography (HPLC) method was established for the simultaneous determination of 12,13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture.A Grace Apollo C18 column (250 mm× 4.6 mm,5 μm) was used as the stationary phase and the mobile phase was composed of acetonitrile and aqueous phosphoric acid (0.2%,v/v).Gradient elution was carried out at the flow rate of 1.0 mL/min and the column temperature was 30 ℃.An ultraviolet (UV) detector was used with a selected wavelength of 240 nm.Calibration curves were linear within the concentration range of 4.6-45.75 μg/mL for 12,13-dihydroxyeuparin (r>0.9999) and 106.9-1068.9μg/mL for glycyrrhizic acid (r>0.9999),respectively.Recoveries were 102.18 % for 12,13-dihydroxyeuparin and 101.17% for glycyrrhizic acid.The method developed could be applied to the simultaneous determination of 12,13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture.

INFORMATION

2011, 01(3): 223-234.

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2011 Vol. 1, No. 3