Engy Shokry

Abstracts

4 Spectral Analysis Approaches for Simultaneous Determination of Binary Mixtures with Overlapping Spectra: An Application on Pseudoephedrine Sulphate and Loratadine

Authors: Engy Shokry, Sara El-Hanboushy, Hayam Lotfy, Yasmin Fayez, Mohammed Abdelkawy

Abstract:

Simple, specific, accurate and precise spectrophotometric methods are developed and validated for simultaneous determination of pseudoephedrine sulphate (PSE) and loratadine (LOR) in combined dosage form based on spectral analysis technique. Pseudoephedrine (PSE) in binary mixture could be analyzed either by using its resolved zero order absorption spectrum at its λ max 256.8 nm after subtraction of LOR spectrum or in presence of LOR spectrum by absorption correction method at 256.8 nm, dual wavelength (DWL) method at 254nm and 273nm, induced dual wavelength (IDWL) method at 256nm and 272nm and ratio difference (RD) method at 256nm and 262 nm. Loratadine (LOR) in the mixture could be analyzed directly at 280nm without any interference of PSE spectrum or at 250 nm using its recovered zero order absorption spectrum using constant multiplication(CM).In addition, simultaneous determination for PSE and LOR in their mixture could be applied by induced amplitude modulation method (IAM) coupled with amplitude multiplication (PM).

Keywords: dual wavelength (DW), induced amplitude modulation method (IAM) coupled with amplitude multiplication (PM), loratadine, pseudoephedrine sulphate, ratio difference (RD)

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3 Development and Validation of Selective Methods for Estimation of Valaciclovir in Pharmaceutical Dosage Form

Authors: Engy Shokry, Eman M. Morgan, Hayam M. Lotfy, Yasmin M. Fayez, Mohamed Abdelkawy

Abstract:

Two simple, selective, economic, safe, accurate, precise and environmentally friendly methods were developed and validated for the quantitative determination of valaciclovir (VAL) in the presence of its related substances R1 (acyclovir), R2 (guanine) in bulk powder and in the commercial pharmaceutical product containing the drug. Method A is a colorimetric method where VAL selectively reacts with ferric hydroxamate and the developed color was measured at 490 nm over a concentration range of 0.4-2 mg/mL with percentage recovery 100.05 ± 0.58 and correlation coefficient 0.9999. Method B is a reversed phase ultra performance liquid chromatographic technique (UPLC) which is considered superior in technology to the high-performance liquid chromatography with respect to speed, resolution, solvent consumption, time, and cost of analysis. Efficient separation was achieved on Agilent Zorbax CN column using ammonium acetate (0.1%) and acetonitrile as a mobile phase in a linear gradient program. Elution time for the separation was less than 5 min and ultraviolet detection was carried out at 256 nm over a concentration range of 2-50 μg/mL with mean percentage recovery 100.11±0.55 and correlation coefficient 0.9999. The proposed methods were fully validated as per International Conference on Harmonization specifications and effectively applied for the analysis of valaciclovir in pure form and tablets dosage form. Statistical comparison of the results obtained by the proposed and official or reported methods revealed no significant difference in the performance of these methods regarding the accuracy and precision respectively.

Keywords: hydroxamic acid, related substances, UPLC, valaciclovir

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2 A New Second Tier Screening for Congenital Adrenal Hyperplasia Utilizing One Dried Blood Spot

Authors: Giancarlo La Marca, Engy Shokry, Maria Luisa Della Bona

Abstract:

Newborn screening for Congenital Adrenal Hyperplasia (CAH) relies on quantification of 17α-hydroxyprogesterone using enzyme immunoassays. These assays, in spite of being rapid, readily available and easy to perform, its reliability was found questionable due to lack of selectivity and specificity resulting in large number of false-positives, consequently family anxiety and associated hospitalization costs. To improve specificity of conventional 17α-hydroxyprogesterone screening which may experience false transient elevation in preterm, low birth weight or acutely ill neonates, steroid profiling by LC-MS/MS as a second-tier test was implemented. Unlike the previously applied LC-MS/MS methods, with the disadvantage of requiring a relatively high number of blood drops. Since newborn screening tests are increasing, it is necessary to minimize the sample volume requirement to make the maximum use of blood samples collected on filter paper. The proposed new method requires just one 3.2 mm dried blood spot (DBS) punch. Extraction was done using methanol: water: formic acid (90:10:0.1, v/v/v) containing deuterium labelled internal standards. Extracts were evaporated and reconstituted in 10 % acetone in water. Column switching strategy for on-line sample clean-up was applied to improve the chromatographic run. The first separative step retained the investigated steroids and passed through the majority of high molecular weight impurities. After the valve switching, the investigated steroids are back flushed from the POROS® column onto the analytical column and separated using gradient elution. Found quantitation limits were 5, 10 and 50 nmol/L for 17α-hydroxyprogesterone, androstenedione and cortisol respectively with mean recoveries of between 98.31-103.24 % and intra-/ inter-assay CV% < 10 % except at LLOQ. The method was validated using standard addition calibration and isotope dilution strategies. Reference ranges were determined by analysing samples from 896 infants of various ages at the time of sample collection. The method was also applied on patients with confirmed CAH. Our method represents an attractive combination of low sample volume requirement, minimal sample preparation time without derivatization and quick chromatography (5 min). The three steroid profile and the concentration ratios (17OHP + androstenedione/cortisol) allowed better screening outcomes of CAH reducing false positives, associated costs and anxiety.

Keywords: cortisol, LC-MS/MS, congenital adrenal hyperplasia (CAH), androstenedione

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1 Therapeutic Drug Monitoring by Dried Blood Spot and LC-MS/MS: Novel Application to Carbamazepine and Its Metabolite in Paediatric Population

Authors: Giancarlo La Marca, Engy Shokry, Fabio Villanelli

Abstract:

Epilepsy is one of the most common neurological disorders, with an estimated prevalence of 50 million people worldwide. Twenty five percent of the epilepsy population is represented in children under the age of 15 years. For antiepileptic drugs (AED), there is a poor correlation between plasma concentration and dose especially in children. This was attributed to greater pharmacokinetic variability than adults. Hence, therapeutic drug monitoring (TDM) is recommended in controlling toxicity while drug exposure is maintained. Carbamazepine (CBZ) is a first-line AED and the drug of first choice in trigeminal neuralgia. CBZ is metabolised in the liver into carbamazepine-10,11-epoxide (CBZE), its major metabolite which is equipotent. This develops the need for an assay able to monitor the levels of both CBZ and CBZE. The aim of the present study was to develop and validate a LC-MS/MS method for simultaneous quantification of CBZ and CBZE in dried blood spots (DBS). DBS technique overcomes many logistical problems, ethical issues and technical challenges faced by classical plasma sampling. LC-MS/MS has been regarded as superior technique over immunoassays and HPLC/UV methods owing to its better specificity and sensitivity, lack of interference or matrix effects. Our method combines advantages of DBS technique and LC-MS/MS in clinical practice. The extraction process was done using methanol-water-formic acid (80:20:0.1, v/v/v). The chromatographic elution was achieved by using a linear gradient with a mobile phase consisting of acetonitrile-water-0.1% formic acid at a flow rate of 0.50 mL/min. The method was linear over the range 1-40 mg/L and 0.25-20 mg/L for CBZ and CBZE respectively. The limit of quantification was 1.00 mg/L and 0.25 mg/L for CBZ and CBZE, respectively. Intra-day and inter-day assay precisions were found to be less than 6.5% and 11.8%. An evaluation of DBS technique was performed, including effect of extraction solvent, spot homogeneity and stability in DBS. Results from a comparison with the plasma assay are also presented. The novelty of the present work lies in being the first to quantify CBZ and its metabolite from only one 3.2 mm DBS disc finger-prick sample (3.3-3.4 µl blood) by LC-MS/MS in a 10 min. chromatographic run.

Keywords: Therapeutic Drug Monitoring, LC-MS/MS, carbamazepine, carbamazepine-10, dried blood spots

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