Development of a highly sensitive ELISA for determination of darunavir in plasma samples using a polyclonal antibody with high affinity and specificity

Bioanalysis ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 355-366 ◽  
Author(s):  
Abdulrahman A Almehizia ◽  
Rashed N Herqash ◽  
Ibrahim A Darwish
Keyword(s):  
High Throughput ◽  
Polyclonal Antibody ◽  
Routine Analysis ◽  
Therapeutic Monitoring ◽  
Pharmacokinetic Studies ◽  
Plasma Samples ◽  
High Affinity ◽  
Spiked Plasma ◽  
Highly Sensitive

Aim: To support pharmacokinetic studies and therapeutic monitoring of darunavir (DRV), a highly sensitive ELISA was developed for the determination of DRV in plasma samples at picogram levels. Results: The assay LOD and LOQ were 15 and 30 pg ml-1, respectively. The working range of the assay was 20–2000 pg ml-1. Analytical recoveries of DRV from spiked plasma were in the ranges of 98.4–113.0 and 86.0–99.1% for intra-assay and inter-assay runs, respectively. The precision of the assay was satisfactory. Conclusion: The ELISA is characterized by high throughput and it is expected to significantly contribute to routine analysis of DRV in its pharmacokinetic studies and therapeutic monitoring.

scholarly journals Spectrofluorimetric Determination of Vigabatrin and Gabapentin in Dosage Forms and Spiked Plasma Samples Through Derivatization with 4-Chloro-7-Nitrobenzo-2-Oxa-1,3-Diazole

2001 ◽  
Vol 84 (4) ◽  
pp. 1017-1024 ◽  
Author(s):  
Ekram M Hassan ◽  
Fathalla Belal ◽  
Omar A Al-Deeb ◽  
Nasr Y Khalil
Keyword(s):  
Reaction Pathway ◽  
Dosage Forms ◽  
Specific Method ◽  
Plasma Samples ◽  
Spiked Plasma ◽  
Highly Sensitive ◽  
Percent Recovery ◽  
Spectrofluorimetric Determination ◽  
Label Claim

Abstract A highly sensitive and specific method is proposed for the determination of vigabatrin (I) and gabapentin (II) in their dosage forms and spiked human plasma. The method is based on coupling the drugs with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole in borate buffer at pH 7.1 and measuring the resulting fluorescence at 532 nm after excitation at 465 nm. The fluorescence intensity was a linear function of the concentration of the drugs over the ranges of 1.3–6.5 and 1.7–8.5 μg/mL for I and II, respectively. Minimum detectability values were 0.54 μg/mL (4.2 × 10−6M) and 0.97 μg/mL (5.7 × 10−6M) for I and II, respectively, under the described conditions. The proposed method was successfully applied to the determination of the 2 drugs in their dosage forms, and the percent recoveries ± standard deviation (SD) were 104.53 ± 1.2 and 100.00 ± 1.32 of the label claim for I and II, respectively. The method was further applied to the determination of vigabatrin in spiked plasma samples. The percent recovery ± SD was 101.58 ± 2.68. Interference from endogenous α-amino acids was overcome through selective complexation with freshly prepared Cu(OH)2. The interference likely to be encountered from co-administered drugs, such as carbamazepine, cimetidine, clonazepam, clopazam, phenobarbital, valproic acid, and lamotrigine, was also studied. A reaction pathway is suggested.

scholarly journals Highly Sensitive LC–MS-MS Method for the Determination of Tacrine in Rat Plasma: Application to Pharmacokinetic Studies in Rats

2015 ◽  
pp. bmv155
Author(s):  
Suresh Ponnayyan Sulochana ◽  
Vishnuvardh Ravichandiran ◽  
Ramesh Mullangi ◽  
Sathesh Kumar Sukumaran
Keyword(s):  
Rat Plasma ◽  
Pharmacokinetic Studies ◽  
Highly Sensitive

A New, Rapid And Sensitive Determination Of Fibrinopeptide- A (FPA) By A High Affinity Antibody To FPA Antiserum

1981 ◽  
Author(s):  
G Stehle ◽  
J Harenberg ◽  
H Schmidtgayk ◽  
R Zimmermann
Keyword(s):  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Standard Curve ◽  
High Affinity ◽  
Microtiter Plates ◽  
Highly Sensitive ◽  
Ethanolic Extraction ◽  
Sensitive Determination ◽  
Radioimmunological Determination

The clinical relevance of the determination of FPA is not yet fully recognized because of the still time consuming radioimmunological techniques. Shortening of the incubation times allways lead to a critical loss of the sensitivity of the assay. We present now a modification which provides highly sensitive and reproducable results within 2hrs.The ethanolic extraction of FPA from plasma was shortened to 20 min including centrifugation. Samples were analysed in triplicates and evaporated at 50°C on microtiter plates. The addition of 0.2μl normal rabbit serum (NRS) lead to a 20% increase of the reaction rate of the FPA antiserum to FPA. A second antibody with high affinity to rabbit immunglobulin i.e. the FPA antiserum improved the maximal binding to 35% after an incubation period of only 10 min. 35-40000 cpm tracer FPA were added to each sample. FPA antiserum, second antibody and NRS were preincubated for 1-24 hrs and then added together with the tracer to the samples. Thus a sensitive standard curve was obtained between 0.16 and 160 ng/ml (12000-600 cpm). The correlation coefficient of this modification to our previously described method was r=0.96 (n=60) The variation coefficient could be improved substantially to 3.1 for low, 4.0 for medium and 4.5% for high FPA. Normal FPA were measured between 0.16 and 2.5 ng/ml (mean 1.4 ng/ml, n=32).The presented modification of the radioimmunological determination of FPA overcomes the difficulties of previously described methods and provides acurate results within 2 hrs.

scholarly journals Determination of nimodipine in plasma by HPLC-MS/MS and pharmacokinetic application

2010 ◽  
Vol 46 (4) ◽  
pp. 665-677 ◽  
Author(s):  
Demétrius Fernandes do Nascimento ◽  
Manoel Odorico de Moraes ◽  
Fernando Antônio Frota Bezerra ◽  
Andréa Vieira Pontes ◽  
Célia Regina Amaral Uchoa ◽  
...  
Keyword(s):  
Human Plasma ◽  
Internal Standard ◽  
Pharmacokinetic Profile ◽  
Plasma Samples ◽  
Linear Calibration ◽  
Limit Of Quantification ◽  
Standard Curve ◽  
Liquid Liquid Extraction ◽  
Highly Sensitive

To develop and validate a rapid, specific and highly sensitive method to quantify nimodipine in human plasma using dibucaine as the internal standard (IS). The analyte and IS were extracted from plasma samples by liquid-liquid extraction using hexane-ethyl acetate (1:1 v/v). The chromatographic separation was performed on a Varian® Polaris C18 analytical column (3 μm, 50 x 2.0 mm) and pre-column SecurityguardTM C18 (4.0 x 3.0 mm) with a mobile phase of Acetonitrile-Ammonium acetate 0.02 ml/L (80:20v/v). The method had a chromatographic run time of 4.5 min and linear calibration curve over the range of 0.1- 40 ng/mL (r > 0.9938). The limit of quantification was 100 pg/mL. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. This validated method was successfully applied in determining the pharmacokinetic profile of nimodipine tablets of 30 mg administered to 24 healthy volunteers. The proposed method of analysis provided a sensitive and specific assay for nimodipine determination in human plasma. The time for the determination of one plasma sample was 4.5 min. This method is suitable for the analysis of nimodipine in human plasma samples collected for pharmacokinetic, bioavailability or bioequivalence studies in humans.

Highly sensitive LC-MS/MS method for determination of galantamine in rat plasma: application to pharmacokinetic studies in rats

2014 ◽  
Vol 28 (12) ◽  
pp. 1633-1640 ◽  
Author(s):  
P. S. Suresh ◽  
Ramesh Mullangi ◽  
Sathesh Kumar Sukumaran
Keyword(s):  
Rat Plasma ◽  
Pharmacokinetic Studies ◽  
Highly Sensitive
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