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Hilal M.A.,Sohag University | Mohamed K.M.,Assiut Chemical Laboratory
Journal of chromatographic science | Year: 2014

A sensitive and accurate method for the extraction and quantification of tramadol (T) and its active metabolite, O-desmethyltramadol (ODT) in human plasma with high-performance liquid chromatography-diode array detection was developed and validated. The analytes were extracted from plasma samples by tert-butylmethyl ether in the presence of ammonium hydroxide as alkaline medium and back extraction with 1.0 M hydrochloric acid. Propranolol was used as internal standard. The extraction efficiencies of T and ODT were 83.51 and 78.72%, respectively. The calibration curves were linear (r(2) > 0.99) in the concentration range of 250-2000 ng/mL for T and ODT. Limits of detection and quantification were 125 and 250 ng/mL for both analytes. Intra- and interassay precision for T and ODT were ranged from 1.89 to 10.91% and 2.16 to 5.85%, respectively. Intra- and interassay accuracy for T and ODT were ranged from -13.07 to 4.99% and -2.03 to -6.98%, respectively. The method was successfully applied to quantify T and ODT from authentic plasma samples received from Hospital Sohag University. The method was completely validated and can be of interest to clinical and forensic laboratories. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

El-Sayed A.A.Y.,Al - Azhar University of Egypt | Mohamed K.M.,Assiut Chemical Laboratory | Nasser A.Y.,Assiut University | Button J.,St Georges, University of London | Holt D.W.,St Georges, University of London
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2013

Analytical procedures for the determination of tramadol (T), O-desmethyltramadol (ODT), and N-desmethyltramadol (NDT) in human urine have been developed and validated using gas chromatography-mass spectrometry (GC/MS). Sample preparation involved liquid-liquid extraction with methyl-tert-butyl ether (MTBE) and followed by back extraction with 0.1M hydrochloric acid. Proadifen (SKF525A) was selected as internal standard (IS). Extraction efficiencies of T, ODT and NDT were 102.12, 101.30, and 98.21%, respectively. The calibration curves were linear (r2>0.99) in the concentration range 10-1000ng/mL for all compounds. Limits of quantification (LOQ) were 10, 10 and 20ng/mL for T, ODT and NDT, respectively. Intra-assay precision was within 1.29-6.48% and inter-assay precision was within 1.28-6.84% for T, ODT and NDT. Intra-assay accuracy was within 91.79-106.89% for all analytes. This method detected urine concentrations of T, ODT and NDT in six healthy volunteers for 7 days after administration of 50mg oral doses of tramadol. © 2013 Elsevier B.V.

Mohamed K.M.,Assiut Chemical Laboratory | Cromarty D.,University of Pretoria | Steenkamp V.,University of Pretoria
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2015

A sensitive LC-MS/MS method was developed and validated for the simultaneous determination of p-phenylenediamine (PPD) and its metabolites N-acetyl-. p-phenylenediamine (MAPPD) and N,. N-diacetyl-. p-phenylenediamine (DAPPD) in human blood. Acetanilide was used as an internal standard (IS). The LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray positive ionization technique. The transition ions m/z 109. →. 92, m/z 151. →. 92, m/z 193. →. 92, and m/z 136. →. 77 were selected for the quantification of PPD, MAPPD, DAPPD, and IS, respectively. The linear range was 10-2000. ng/mL for all the compounds. The absolute recoveries were 51.94, 56.20 and 54.88% for PPD, MAPPD and DAPPD, respectively. Intra- and inter-assay imprecision were lower than 14% (RSD), and the bias of the assay was lower than 15% for all the compounds. The stability studies demonstrated that critical degradation for PPD in blood samples and autosampler occurred after 6. h, while MAPPD and DAPPD were stable in blood samples and the autosampler up to 48. h and 24. h, respectively. This newly developed method allows for the detection of PPD and its metabolites in blood samples in the clinical and forensic setting. © 2015 Elsevier B.V..

Omran A.A.,Assiut University | Omran A.A.,King Khalid University | El-Sayed A.-A.,Assiut University | Shehata A.,Assiut Chemical Laboratory
Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy | Year: 2011

The binding constants (K values) of three benzodiazepine drugs to bovine serum albumin were determined by a second derivative spectrophotometric method. Despite the sample and reference samples were prepared in the same way to maintain the same albumin content in each sample and reference pair, the absorption spectra show that the baseline compensation was incomplete because of the strong background signals caused by bovine serum albumin. Accordingly, further quantitative spectral information could not be obtained from these absorption spectra. On the other hand, the calculated second derivative spectra clearly show isosbestic points indicating the complete removal of the residual background signal effects. Using the derivative intensity differences (ΔD values) of the studied benzodiazepine drugs before and after the addition of albumthe binding constants were calculated and obtained with R.S.D. of less than 8%. The interactions of drugs with bovine serum albumin were investigated using Scatchard's plot. In addition, the consistency between the fractions of bound benzodiazepine calculated from the obtained K values and the experimental values were established. The results indicate that the second derivative method can be advantageously applicable to the determination of binding constants of drugs to serum albumin without prior separation. Moreover, the validity of the proposed method was confirmed. © 2011 Elsevier B.V.

Omran A.,Assiut University | Omran A.,King Khalid University | El-Sayed A.-A.,Assiut University | Shehata A.,Assiut Chemical Laboratory
Journal of Solution Chemistry | Year: 2012

Second derivative spectrophotometry was applied to determine the binding constant (K) between codeine phosphate (COD) and bovine serum albumin (BSA) at simulated physiological conditions (37.00 °C and pH = 7.4). The second derivative spectra of COD in buffer solutions containing various amounts of BSA showed derivative isosbestic points. The residual background signals derived from incomplete suppression of BSA signals can be entirely eliminated in the second derivative spectra indicating that BSA has spectrophotometrically one kind of binding site for COD. The fractions of COD bound to BSA were calculated from the derivative intensity differences (ΔD values) of COD before and after the addition of BSA. Scatchard plot calculation suggested that the binding of COD to BSA can be explained by a partition-like non-specific binding model. The binding constant (K) was calculated from ΔD values according to the non-specific binding model by a nonlinear least-squares method. K values were almost constant for all of the COD concentrations studied with good reproducibility. The fractions predicted by the K values were in good agreement with the observed values. The results indicate the usefulness of the derivative method in drug-albumin binding studies without the need for prior separation procedures which may disturb the equilibrium states of the samples solutions. © Springer Science+Business Media, LLC 2012.

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