Journal of Pharmaceutical Analysis

2012, 02(2): 后插1-后插3,封3-封4,前插1.

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Immobilized enzyme reactors in HPLC and its application in inhibitor screening: A review

Si-Meng Fang, Hai-Na Wang, Zhong-Xi Zhao, Wei-Hong Wang

2012, 02(2): 83-89. doi: 10.1016/j.jpha.2011.12.002

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This paper sets out to summarize the literatures based on immobilized enzyme biochromatography and its application in inhibitors screening in the last decade.In order to screen enzyme inhibitors from a mass of compounds in preliminary screening,multi-pore materials with good biocompatibility are used for the supports of immobilizing enzymes,and then the immobilized enzyme reactor applied as the inmobilized enzyme stationary phase in HPLC.Therefore,a technology platform of high throughput screening is gradually established to screen the enzyme inhibitors as new anti-tumor drugs.Here,we briefly summarize the selective methods of supports,immobilization techniques,co-immobilized enzymes system and the screening model.

Determination of drug, excipients and coating distribution in pharmaceutical tablets using NIR-CI

Anna Palou, Jordi Cruz, Marcelo Blanco, Jaume Tomàis, Joaquin de los Rios, Manel Alcalà

2012, 02(2): 90-97. doi: 10.1016/j.jpha.2011.11.003

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The growing interest of the pharmaceutical industry in Near Infrared-Chemical Imaging (NIR-CI) is a result of its high usefulness for quality control analyses of drugs throughout their production process (particularly of its non-destructive nature and expeditious data acquisition).In this work,the concentration and distribution of the major and minor components of pharmaceutical tablets are determined and the spatial distribution from the internal and external sides has been obtained.In addition,the same NIR-CI allowed the coating thickness and its surface distribution to be quantified.Images were processed to extract the target data and calibration models constructed using the Partial Least Squares (PLS) algorithms.The concentrations of Active Pharmaceutical Ingredient (API) and excipients obtained for uncoated cores were essentially identical to the nominal values of the pharmaceutical formulation.But the predictive ability of the calibration models applied to the coated tablets decreased as the coating thickness increased.

Voltammetric quantification of anti-hepatitis drug Adefovir in biological matrix and pharmaceutical formulation

Rajeev Jain, Ramkishor Sharma

2012, 02(2): 98-104. doi: 10.1016/j.jpha.2011.10.002

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Electrochemical reduction behavior of Adefovir was studied using Hanging Mercury Drop Electrode (HMDE) in Britton-Robinson (BR) buffer solution.Voltammetric study showed one well-defined reduction peak in the potential range -1.2 to -1.4 V (vs.Ag/AgCl) due to reduction of C=N bond of the imidazole ring.Solid-phase extraction and protein-precipitation techniques were employed for extraction of Adefovir from human plasma.The proposed method allows quantification of Adefovir in human plasma over the concentration range 0.50-5.00 μg/mL with the detection limit 0.17 μg/mL,whereas in pharmaceutical formulation 0.25-2.25 μg/mL with the detection limit 0.08 μg/mL.

Validated stability indicating methods for determination of nitazoxanide in presence of its degradation products

Nouruddin W.Ali, Samah Sayed Abbas, Hala El-Sayed Zaazaa, Maha Mohamed Abdeirahman, Mohamed Abdelkawy

2012, 02(2): 105-116. doi: 10.1016/j.jpha.2011.11.004

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Three sensitive,selective and reproducible stability-indicating methods are presented for determination of nitazoxanide (NTZ),a new anti-protozoal drug,in presence of its degradation products.Method A utilizes the first derivative of ratio spectra spectrophotometry by measurement of the amplitude at 364.4 nm using one of the degradation products as a divisor.Method B is a chemometric-assisted spectrophotometry,where principal component regression (PCR) and partial least squares (PLS) were applied.These two approaches were successfully applied to quantify NTZ in presence of degradation products using the information included in the absorption spectra in the range 260-360 nm.Method C is based on the separation of NTZ from its degradation products followed by densitometric measurement of the bands at 254 nm.The separation was carried out on silica gel 60F254,using chloroform-methanol-ammonia solution-glacial acetic acid (95∶5∶1∶1 by volume,pH=5.80) as a developing system.These methods are suitable as stability-indicating methods for the determination of NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations.Statistical analysis of the results has been carried out revealing high accuracy and good precision.

The expert system of genotype discrimination for D5S818 locus based on near-infrared spectroscopy-principal discriminant variate

Zai-Zhen Wu, Jian-Hua Tang, Bin Zhang, Li-Ping Guo, Hong-Ping Xie, Bing-Ren Gu

2012, 02(2): 117-122. doi: 10.1016/j.jpha.2011.10.007

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This paper studied the expert system of genotype discrimination for the STR locus D5S818 based on near-infrared spectroscopy-principal discriminant variate (PDV).Six genotypes,i.e.genotypes 10-10,10-11,11-11,11-12,11-13 and 13-13,were selected as research subjects.Based on the optimum polymerase chain reaction (PCR) conditions,about 54 measuring samples for each genotype were obtained; these samples were tested by near-infrared spectroscopy directly.With differences between homozygote genotypes and heterozygote ones,and differences of the total number of core repeat units between the six genotypes,two types of genotyping-tree structure were constructed and their respective PDV models were studied using the near-infrared spectra of the samples as recognition variables.Finally,based on the classification ability of these two genotyping-tree structures,an optimum expert system of genotype discrimination was built using the PDV models.The result demonstrated that the built expert system had good discriminbility and robustness; without any preprocessing for PCR products,the six genotypes studied could be discriminated rapidly and correctly.It provided a methodological support for establishing an expert system of genotype discrimination for all genotypes of locus D5S818 and other STR loci.

Voltammetric behavior of sedative drug midazolam at glassy carbon electrode in solubilized systems

Rajeev Jain, Rajeev Kumar Yadav

2012, 02(2): 123-129. doi: 10.1016/j.jpha.2011.11.008

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Redox behavior of midazolam was studied at a glassy carbon electrode in various buffer systems,supporting electrolytes and pH using differential paise,square-wave and cyclic voltammetry.Based on its reduction behavior,a direct differential pulse voltammetric method has been developed and validated for the determination of midazolam in parenteral dosage.Three welldefined peaks were observed in 0.1% SLS,Britton-Robinson (BR) buffer of pH 2.5.The effect of surfaetants like sodium lauryl sulfate (SLS),cetyl trimethyl ammonium bromide (CTAB) and Tween 20 was studied.Among these surfactants SLS showed significant enhancement in reduction peak.The cathodic peak currents were directly proportional to the concentration of midazolam with correlation coetfficient of 0.99.

Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

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

2012, 02(2): 130-135. doi: 10.1016/j.jpha.2011.10.001

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A chemiluminescence enzyme immunoassay based on magnetic microparicles (MmPsCLEIA) was developed to evaluate serum α-fetoprotein (AFP) in parallel with tramional colorimetric enzyme-linked immunsorbrnt assay (ELISA).A sestematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer of immunoreagents,less total assay time,and better linearity,recovery,precision,senitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results werecompared with commercial electrochemilunminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R2=0.6703; however,the correlation between MPs-CLEIA and ECLIA (R2=0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R2=0.6866).

Development and validation of RP-HPLC and RP-UPLC methods for quantification of parathyroid hormones (1-34) in medicinal product formulated with meta-cresol

Shaligram S.Rane, Alkesh Ajameri, Rustom Mody, P.Padmaja

2012, 02(2): 136-142. doi: 10.1016/j.jpha.2011.12.001

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Rapid and sensitive reversed phase high performance liquid chromatography (RP-HPLC) and ultra performance liquid chromatography (RP-UPLC) method with UV detection has been developed and validated for of parathyroid hormone (PTH)in presence of meta-cresol as a stabilizer in a pharmaceutical formulation.Chromatography was performed with phase containing 0.1% Trifluoroacetic acid (TFA) in MilliQ water and 0.1% TFA in acetonitrile with gradient and flow rate at 0.3 mL/min for HPLC and 0.4 mL/min for UPLC.Quantification was accomplished with internal reference standard (qualified against innovator product and National for for Biological Standards and Control (NIBSC) standard).The methods were validated for linearity (correlation coefficient=0.99),range,accuracy,precision and robustness.Robustness was confirmed by considering three factors; mobile phase composition,column temperature and flow rate/age of mobile phase.Intermediate precision was confirmed on different equipments,different columns and on different days.The relative standard deviation (RSD) (<2% for RP-HPLC and <1% for UPLC n=30) indicated a good precision.Retention time was found about 17min and 2min by HPLC and UPLC methods,respectively.Both methods are simple,highly sensitive,precise and accurate and have the potential of being useful for routine quality control.

Separation, identification, and quantification of active constituents in Fructus Psoraleae by high-performance liquid chromatography with UV, ion trap mass spectrometry, and electrochemical detection

Qinhua Chen, Yuan Li, Zilin Chen

2012, 02(2): 143-151. doi: 10.1016/j.jpha.2011.11.005

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The qualitative and quantitative analysis of active constituents in Fructus Psoraleae is presented by high-performance liquid chromatography (HPLC) coupled with different detections.Extracts of Fructus Psoraleae were examined by HPLC with ion trap mass spectrometry (IT-MS) and 18 major compounds of coumarins,benzofuran glycosides,flavonoids,and meroterpene were identified.The determination of four major constituents including bavachin,isobavachalcone,bavachinin,and bakuchiol was accomplished by HPLC with UV,MS,and electrochemical detection (ECD).These methods were evaluated for a number of validation characteristics (repeatability,LOD,calibration range,and recovery).ECD obtained a high sensitivity for analysis of the four components; MS provided a high selectivity and sensitivity for determination of bavachin,isobavachalcone,and bavachinin in negative-ion mode.After optimization of the methods,separation,identification.and quantification of the four components in Fructus Psoraleae were comprehensively tested by HPLC with UV,MS,and ECD.

Development and validation of a normal-phase HPTLC method for the simultaneous analysis of Lamivudine and Zidovudine in fixed-dose combination tablets

Palani Venkatesh, Mahesh Daggumati

2012, 02(2): 152-155. doi: 10.1016/j.jpha.2011.11.002

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Simultaneous quantification of Lamivudine and Zidovudine in tablets by HPTLC method was developed and validated.The chromatograms were developed using a mobile phase of toluene:ethyl acetate:methanol (4∶4∶2,v/v/v) on pre-coated plate of silica gel GF aluminum TLC plate and quantified by densitometric absorbance mode at 276 nm.The Rf values were 0.41±0.03and 0.60±0.04 for Lamivudine and Zidovudine,respectively.The linearity of the method was found to be within the concentration range of 50-250 ng/spot for Lamivudine and for Zidovudine,it was 100-500 ng/spot.The lower limits of detection and quantification were 2.23 ng/spot and 7.90 ng/spot for Lamivudine and 2.90 ng/spot and 8.85 ng/spot for Zidovudine.The method was also validated for precision,specificity and recovery.This developed method was used to analyze fixed-dose tablets (Duovir,Cipla Ltd) samples of Lamivudine and Zidovudine.

New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA

Hidetoshi Arakawa, Shigeo Nakabayashi, Ken-ichi Ohno, Masako Maeda

2012, 02(2): 156-159. doi: 10.1016/j.jpha.2012.01.004

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Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA).The procedure used for HRP detection in EIA is critical for sensitivity and precision.This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate.The principle of the assay is as follow:sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol.The dimer is fluorescent and can be detected sensitively at ex.347 nm,em.427 nm.The measurable range of HRP was 1.0 × 10-18 to 1.0 × 10-15 mol/assay,with a detection limit of 1.0 × 10-18 tmol/assay.The coefficient of variation (CV,n=8) was examined at each point on the standard curve,with a mean CV percentage of 3.8%.This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

2012 Vol. 2, No. 2