Journal of Pharmaceutical Analysis

2012, 02(3): 后插1-后插2,封3-封4.

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Simultaneous determination of pioglitazone and candesartan in human plasma by LC-MS/MS and its application to a human pharmacokinetic study

Vijaya Kumari Karra, Nageswara Rao Pilli, Jaswanth Kumar Inamadugu, J.V.L.N. Seshagiri Rao

2012, 02(3): 167-173. doi: 10.1016/j.jpha.2012.01.002

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A simple and rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS)assay method has been developed and fully validated for simultaneous quantification of pioglitazone and candesartan in human plasma.Irbesartan was used as an internal standard.The analytes were extracted from human plasma samples by solid-phase extraction technique using a Strata-X 33 μm polymeric sorbent.The reconstituted samples were chromatographed on a C18column by using a 80∶20 (v/v) mixture of acetonitrile and 0.1% formic acid as the mobile phase at a flow rate of 0.8 mL/min.The calibration curves obtained were linear (r(≥)0.99) over the concentration range of 15-3000 ng/mL for pioglitazone and 5-608 ng/mL for candesartan.The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits.A run time of 2.7 min for each sample made it possible to analyze more than 300 plasma samples per day.The proposed method was found to be applicable to clinical studies.

Capture of cervical exfoliative cells on a glass slide coated by 3-glycidyloxypropyl trimethoxysilane and poly-L-lysine

Gao-Wa Xing, Sen Xiang, Wei Xue, Gao-Wa Aodeng, Yan Liu, Jing-Hua Zhang, Jin-Ming Lin

2012, 02(3): 174-179. doi: 10.1016/j.jpha.2012.02.008

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A new modification method for glass slides was developed and applied to make ThinPrep Pap smears,in order to increase the adhesion ability of cervical exfoliative cells.3-glycidyloxypropyl trimethoxysilane (GOPS) was coated on the glass slides firstly on the slides,then poly-L-lysine (PLL)was covalently modified onto the above epoxy-terminated slides to form GOPS-PLL double decorated slides.The modified slides were characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM).The cell adhesion ability effect was tested and compared with traditional PLL coated slides by fixing the cervical exfoliative cells on the double adorned slides.The control test was conducted by the bare glass slides unmodified.The cell morphology of cervical exfoliative cells adhered on different slides was observed under the microscope after Papanicolaou staining.The number of cervical exfoliative cells on the unmodified slides,PLL coated slides and GOPS-PLL coated slides was 1030±300,3283±226 and 4119±280 (n=12),respectively.The data among the three different modification methods showed significant differences (one-way analysis of variance,ANOVA test,P< 0.05).The cell capturing effect of the GOPS-PLL slide was the best among the three different modified slides.In addition,the GOPS-PLL slide could enhance the uniformity of the adhered cells and be widely applied to the ThinPrep system for cervical carcinoma screening to increase the accuracy rate of diagnosis.

Quantification of desloratadine in human plasma by LC-ESI-MS/MS and application to a pharmacokinetic study

Venkata Suresh Ponnuru, B.R. Challa, Ramarao Nadendla

2012, 02(3): 180-187. doi: 10.1016/j.jpha.2012.01.008

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A simple,sensitive,and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of desloratadine (DL) in human plasma using desloratadine-d5 (DLD5) as an internal standard (IS).Chromatographic separation was performed using an Xbridge C18 column (50 mm × 4.6 mm,5 μm) with an isocratic mobile phase composed of 10 mM ammonium formate:methanol (20∶80,v/v),at a flow rate of 0.7 mL/min.DL and DLD5 were detected with proton adducts at m/z 311.2→259.2 and 316.2→264.3 in multiple reaction monitoring (MRM)positive modes,respectively.Liquid-liquid extraction (LLE) method was used to extract the drug and the IS.The method was validated over a linear concentration range of 5.0-5000.0 pg/mL with a correlation coefficient of (r2)(≥)0.9994.This method demonstrated intra- and inter-day precision within 0.7-2.0% and 0.7-2.7%,and an accuracy within 101.4-102.4%,and 99.5-104.8%.DL was found to be stable throughout the freeze-thaw cycles,bench-top,and postoperative stability studies.This method was successfully applied in the analysis of plasma samples following oral administration of DL (5 mg) in 35healthy Indian male human volunteers under fasting conditions.

Preparation of gastro-resistant pellets containing chitosan microspheres for improvement of oral didanosine bioavailability

Patrícia Severino, George G.G. de Oliveira, Humberto G. Ferraz, Eliana B. Souto, Maria H.A. Santana

2012, 02(3): 188-192. doi: 10.1016/j.jpha.2012.02.005

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The purpose of this work was to introduce a new concept of coated pellets containing chitosan microspheres loaded with didadosine for oral administration,aiming at reducing the frequency of administration and improving the bioavailability by a suitable release profile.Chitosan microspheres were produced under fluidized bed,followed by extrusion and spheronization to obtain pellets with a mean diameter of about 1 mm.The pellets were then coated with Kollidon(R) VA64 and Kollicoat(R) MAE100P in water dispersion to depict a sustained release profile.Conventional hard gelatine capsules were loaded with these pellets and tested in vitro for their release profile of didadosine.Dissolution testing confirmed that chitosan microsphere pellets provides appropriate sustained release up to 2 h behavior for didanosine.

Adsorptive stripping voltammetric methods for determination of aripiprazole

Derya A(s)angil, (I)brahim Hüdai Ta(s)demir, Esma K(i)l(i)(c)

2012, 02(3): 193-199. doi: 10.1016/j.jpha.2012.01.009

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Anodic behavior of aripiprazole (ARP) was studied using electrochemical methods.Charge transfer,diffusion and surface coverage coefficients of adsorbed molecules and the number of electrons transferred in electrode mechanisms were calculated for quasi-reversible and adsorption-controlled electrochemical oxidation of ARP at 1.15 V versus Ag/AgCl at pH 4.0 in Britton-Robinson buffer (BR) on glassy carbon electrode.Voltammetric methods for direct determination of ARP in pharmaceutical dosage forms and biological samples were developed.Linearity range is found as from 11.4 μM (5.11 mg/L) to 157 μM (70.41 mg/L) without stripping mode and it is found as from 0.221 μM (0.10 mg/L) to 13.6 μM (6.10 mg/L) with stripping mode.Limit of detection (LOD) was found to be 0.11 μM (0.05 mg/L) in stripping voltammetry.Methods were successfully applied to assay the drug in tablets,human serum and human urine with good recoveries between 95.0% and 104.6% with relative standard deviation less than 10%.

Validated spectrofluorimetric method for the determination of atorvastatin in pharmaceutical preparations

Mohie M.K. Sharaf El-Din, Fathy M.M. Salama, Mohamed W.I. Nassar, Khalid A.M. Attia, Mohamed M.Y. Kaddah

2012, 02(3): 200-205. doi: 10.1016/j.jpha.2012.01.005

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A rapid,sensitive and simple spectrofluorimetric method was developed for the estimation of atorvastatin.In this method,the native fluorescence characteristics of atorvastatin have been studied in both acidic and basic media.High sensitivity was obtained with 5% acetic acid at 389 nm using 276 nm for excitation.Regression analysis showed a good correlation coefficient (r=0.9995) between fluorescence intensity and concentration over the range of 1.5-4 μg/mL with detection limit of 0.012 μg/mL.The proposed method was successfully applied to the analysis of atorvastatin in pure and pharmaceutical dosage forms with average recovery of 100.29±0.47%.The results were compared favorably with those of the reported method.

High performance liquid chromatography mass spectrometric method for the simultaneous quantification of pravastatin and aspirin in human plasma:Pharmacokinetic application

Srinivasa Rao Polagani, Nageswara Rao Pilli, Venkateswarlu Gandu

2012, 02(3): 206-213. doi: 10.1016/j.jpha.2012.01.001

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A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS)assay method has been developed and fully validated for the simultaneous quantification of pravastatin and aspirin in human plasma.Furosemide was used as an internal standard.Analytes and the internal standard were extracted from human plasma by liquid-liquid extraction technique using methyl tertiary butyl ether.The reconstituted samples were chromatographed on a Zorbax SB-C18 column by using a mixture of 5 mM ammonium acetate buffer and acetonitrile (20∶80,v/v) as the mobile phase at a flow rate of 0.8 mL/min.The calibration curve obtained was linear (r(≥)0.99)over the concentration range of 0.50-600.29 ng/mL for pravastatin and 20.07-2012.00 ng/mL for aspirin.Method validation was performed as per FDA guidelines and the results met the acceptance criteria.A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day.The proposed method was found to be applicable to clinical studies.

Simple and sensitive determination of sparfloxacin in pharmaceuticals and biological samples by immunoassay

Hua-Jin Zeng, Ran Yang, Bing Liu, Li-Fang Lei, Jian-Jun Li, Ling-Bo Qu

2012, 02(3): 214-219. doi: 10.1016/j.jpha.2012.02.001

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Plasma quinolone concentrations are not routinely measured in clinical practice.However,in order to optimize quinolone treatment,monitoring of plasma concentrations could sometimes be useful particularly in critically ill patients.In this study,anti-sparfloxacin antibody was obtained by immunizing rabbits with sparfloxacin conjugated with bovine serum albumin using isobutyl chloroformate method.After the assay procedure was optimized,the standard curve of sparfloxacin was established.The practical measuring range of the competitive ELISA extended from 5 ng/mL to 2 μtg/mL.The recovery rates and coefficients of variation for rat plasma,urine and tissues were 87.7-106.2% and 4.8-15.3%,respectively.To demonstrate the potential of the ELISA,a preliminary pharmacokinetics and tissue distribution study of sparfloxacin in rats and quantitative analysis of sparfloxacin in several pharmaceuticals were performed and compared with high-performance liquid chromatography (HPLC).The experimental data indicated that the proposed method would be a valuable tool in therapeutic drug monitoring (TDM) for sparfloxacin.

Analysis of species-dependent hydrolysis and protein binding of esmolol enantiomers

Yi-Hong Tang, Jun-Yan Wang, Hai-Hong Hu, Tong-Wei Yao, Su Zeng

2012, 02(3): 220-225. doi: 10.1016/j.jpha.2012.01.007

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The stereoselective hydrolysis of esmolol in whole blood and in its separated components from rat,rabbit and human was investigated.Blood esterase activities were variable in different species in the order of rat > rabbit > human.Rat plasma showed the high esterase activity and had no stereoselectivity to enantiomers.Rabbit red blood cell (RBC) membrane,RBC cytosol and plasma all hydrolyzed esmolol but with different esterase activity,whereas the hydrolysis in RBC membrane and cytosol showed significant stereoselectivity towards R-(+)-esmolol.Esterase in RBC cytosol from human blood mainly contributed to the esmolol hydrolysis,which was demonstrated with no stereoselctivity.Esterase in human plasma showed a low activity,but a remarkable stereoselectivity with R-(+)-esmolol.In addition,the protein concentration affected the hydrolysis behavior of esmolol in RBC suspension.Protein binding of esmolol enantiomers in human plasma,human serum albumin (HSA) and α1-acid glycoprotein (AGP) revealed that there was a significant difference in bound fractions between two enantiomers,especially for AGP.Our results indicated that the stereoselective protein binding might play a role in the different hydrolysis rates of esmolol enantiomers in human plasma.

Validated gradient stability indicating HPLC method for determining Diltiazem Hydrochloride and related substances in bulk drug and novel tablet formulation

Vivekanand A. Chatpalliwara, Pawan K. Porwal, Neeraj Upmanyu

2012, 02(3): 226-237. doi: 10.1016/j.jpha.2012.01.003

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A stability-indicating liquid chromatographic method has been developed and validated for the determination of Diltiazem Hydrochloride (DTZ) together with its six related substances (Diltiazem sulphoxide,Imp-A,Imp-B,Imp-D,Imp-E,and Imp-F) in a laboratory mixture as well as in a novel tablet formulation developed in-house.Efficient chromatographic separation was achieved on a Hypersil BDS C18 (150mm ×4.6mm,5.0μm) with mobile phase containing 0.2% Triethylamine (TEA) in gradient combination with acetonitrile (ACN) at a flow rate of 1.0 mL/min and the eluent was monitored at 240 nm.In the developed method,the resolution of DTZ from any pair of impurities was found to be greater than 2.0.The test solution and related substances were found to be stable in the diluent for 24 h.The developed method resolved the drug from its known impurities,stated above,and also from additional impurities generated when the formulation was subjected to forced degradation; the mass balance was found close to 99.9%.Regression analyses indicate correlation coefficient value greater than 0.997 for DTZ and its six known impurities.The LOD for DTZ and the known impurities was at a level below 0.02%.The method has shown good,consistent recoveries for DTZ (99.8-101.2%) and also for its six known impurities (97.2-101.3%).The method was found to be accurate,precise,linear,specific,sensitive,rugged,robust,and stability-indicating.

Simultaneous determination of oleanolic acid and ursolic acid by RP-HPLC in the leaves of Eriobotrya japonica Lindl.

Xiao-Hong Xu, Qing Su, Zhi-He Zang

2012, 02(3): 238-240. doi: 10.1016/j.jpha.2012.01.006

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Oleanolic acid (OA) and ursolic acid (UA) are isomeric triterpenic acids and only one methyl's position is different between them.OA and UA always exist in the same plant,so it is difficult to separate them when determining contents by RP-HPLC.In this study,a very simple mobile phase for HPLC was developed to simultaneously determine UA and OA,and the factors affecting separation were also discussed.The mobile phase is methanol:water (95∶5) with flow rate 0.4mL/min.The retention time for OA and UA was 20.58 and 21.57 min,respectively,the resolution was 1.61.The average contents of OA and UA of three Loquat leaves sets were 1.4 mg/g and 5.6 mg/g,respectively.Regarding the HPLC,we found that changing mobile phase,adjusting the pH value or adding ion-pairing agent could not affect the separation between UA and OA greatly.While adjustment of the flow rate and column temperature could improve the resolution greatly.

2012 Vol. 2, No. 3