Ahmed M. Abdel-Megied

Development a validated highly sensitive LC–MS/MS method for simultaneous quantification of Ledipasvir, sofosbuvir and its major metabolite GS-331007 in human plasma: Application to a human pharmacokinetic study

&NA; A highly sensitive and rapid LC–MS/MS method was developed, fully optimized and validated for the simultaneous determination of Ledipasvir (LED) and Sofosbuvir (SOF) in the presence of its major metabolite GS‐331007 in human plasma using Daclatasvir as internal standard (IS). The extraction of analytes and IS from plasma was performed using liquid‐liquid extraction with ethyl acetate. The chromatographic separation of these prepared samples was achieved on Xterra MS C8 column (4.6 × 50 mm,5 &mgr;m) using gradient elution with a mobile phase of ammonium formate buffer (pH 3.5; 10 mM), acetonitrile and methanol pumped at a flow rate 0.7 mL min−1.The detection was performed on API4000 triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM) positive electrospray ionization interface. The method was validated according to FDA guidelines for bio‐analytical methods with respect to linearity, accuracy, precision, selectivity, carry‐over, stability and dilution integrity. Linearity was obtained over a concentration range of 0.1–1000, 0.3–3000 and 3.0–3000 ng mL−1 for LED, SOF and GS‐331007; respectively by applying weighted least‐squares linear regression method (1/x2). The wider range of quantification in a shorter period of separation time less than 5.0 min allowed monitoring the serum concentration of analytes up to 144 h. The proposed method can be successfully applied for pharmacokinetic and bioequivalence studies in healthy human volunteers. Graphical abstract Figure. No caption available. HighlightsA highly sensitive LC‐MS/MS method was developed for analysis of Ledipasvir, sofosbuvir in the presence of its metabolite GS‐331007 in human plasma.The method was fully validated according to FDA guidelines for bio‐analytical methods with respect to linearity, accuracy, precision, recovery, selectivity, carry over, stability and dilution integrity.The proposed method can be applied to pharmacokinetic and bioequivalence studies in human volunteers.Our study proved that there was no significant difference in pharmacokinetic parameters with other reported papers.

Development and validation of LC-MS/MS method for simultaneous determination of sofosbuvir and daclatasvir in human Plasma: Application to pharmacokinetic study

A simple and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method was developed and fully validated for the first time for the simultaneous determination of newly discovered antiviral drugs, namely sofosbuvir (SOF) and daclatasvir (DAC) in human plasma. Tadalafil (TAD) was used as internal standard (IS). SOF, DAC and TAD (IS) were extracted from plasma using liquid-liquid extraction technique with methyl tert-butyl ether. The chromatographic separation was carried out using ZorbaxSB-C18 column (4.6 × 50 mm,5 μm) and 5 mm ammonium formate buffer (pH 3.5)-acetonitrile (50:50, v/v) as mobile phase in an isocratic elution mode pumped at a flow rate 0.7 mL min-1 . The quantitation was performed on API4500 triple quadrupole tandem mass spectrometer with positive electrospray ionization interface in multiple reaction monitoring mode. Validation was applied according to US Food and Drug Administration guidelines for bio-analytical methodswith respect to linearity, precision, accuracy, selectivity, carry-over, stability and dilution integrity. Linearity was obtained over concentration ranges of 0.3-3000 and 3-3000 ng mL-1 for SOF and DAC, respectively, by applying a weighted least-squares linear regression method (1/x2 ). The proposed method could be applied successfully in bioequivalence and/or clinical studies for therapeutic drug monitoring of patients undergoing dual combination therapy as the latter combination proved more efficacious and powerful tool for the complete treatment of hepatitis C genotype 3 within 16 weeks. The suggested method has been applied successfully to pharmacokinetic studies with excellent assay ruggedness and reproducibility.

Chiral separation of perindopril erbumine enantiomers using high performance liquid chromatography and capillary electrophoresis

Two separation methods were developed for the determination of S- and R-perindopril tert-butylamine (erbumine salt) (PER): high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The HPLC method uses a chiral stationary phase (CSP), ChiraDex column constituting β-cyclodextrin chemically bonded to spherical silica gel particles. The mobile phase consisted of phosphate buffer (50 mM, pH 3.0) and acetonitrile (45:55 v/v). The flow rate was 1.0 mL min−1 and the detection wavelength was 210 nm. In CE, 2-hydroxylpropyl-β-cyclodextrin (10 mM) was used as a chiral selector. It was added to the background buffer composed of phosphate buffer (100 mM, pH 7.0) and methanol (15% v/v). The applied voltage was 15 kV and the detection was carried out using a diode array detector. All factors affecting the chromatographic or electrophoretic separations were studied and optimized. The linear concentrations ranged from 5–150 and 25–800 μg mL−1 with detection limits of 2.3 and 14.7 μg mL−1 for HPLC and CE methods, respectively. The methods were validated according to ICH and USP guidelines. The suggested methods were applied for the determination of S-PER in bulk powder and commercial tablets containing PER erbumine racemate.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, TLC-DENSITOMETRY, AND FIRST-DERIVATIVE SPECTROPHOTOMETRY FOR SIMULTANEOUS DETERMINATION OF AMLODIPINE AND PERINDOPRIL IN BULK POWDER AND ITS TABLETS

Three simple, sensitive, and specific methods were developed for simultaneous determination of amlodipine besylate (AML) and Perindopril Erbumine (PER) without previous separation. The first method was dependent on the first derivative of the ratio spectra by measuring the amplitudes at 348 nm for amlodipine using 50 µg mL−1 of perindopril as a divisor and at 227 nm for perindopril using 30 µg mL−1 of amlodipine as a divisor. The second method was based on ion-pair RP-HPLC. Satisfactory resolution was achieved using RP-C18 chromatographic column Zorbax Extend column and a mobile phase consists of potassium dihydrogen phosphate buffer (0.05 M, pH 3.0 ± 0.02 adjusted by orthophosphoric acid): acetonitrile 30:70 v/v at a flow rate 1 mL/min using 0.002 M sodium heptanesulfonate in the aqueous phase. UV detection was performed at 215 nm. The third method was based on TLC; the separation was carried out on Fluka TLC aluminum sheets silica gel 60 F254, using n-butanol:water:glacial acetic acid (4:5:1, v/v/v) as the mobile phase. The validation of the proposed methods was applied according to ICH guidelines and LOD and LOQ were calculated. The suggested methods were successfully applied for the determination of the cited drugs in bulk powder and commercial tablets.

Simultaneous spectrophotometric determination of compounds having relatively disparate absorbance and concentration ranges; application to antidiabetic formulation of linagliptin and metformin

The limited linear range of UV-Visible spectrophotometry may be insufficient to occupy multiple components with wide variations in their concentrations or absorptivities that will hinder the simultaneous spectrophotometric determination and may require spiking or measurements in subsequent dilution steps. The current work introduces the absorptivity target concentration (ATC) values, a simple way for the proper choice of the working spectral region to execute accurate and linear spectrophotometric measurements. Simultaneous spectrophotometric determination of linagliptin (LNG) and metformin (MET) that are present in a ratio of 1:400 was carried out using traditional spectrophotometric techniques such as third derivative and derivative ratio as well as recently developed techniques such as ratio difference and factorized dual wavelength. The proposed methods were able to determine MET in the concentration range of 50-1200 μg mL-1. On the other hand, LNG was successfully determined from its zero-order absorption UV-spectrum at λmax (296 nm) in the concentration range 2.5-25 μg mL-1. The mentioned methods were successfully applied for the determination of the LNG and MET in their combined dosage form. The methods were validated according to the ICH guidelines. The proposed ATC value can be employed as a novel concept for the proper choice of the working spectral region where spectrophotometric measurements can be deployed accurately and precisely.

Development and Validation of a Versatile UPLC-PDA Method for Simultaneous Determination of Paracetamol, Tizanidine, Aceclofenac, and Nimesulide in Their New Combinations

A simple, rapid, and validated UPLC method was developed for the simultaneous quantitation of paracetamol (PAR), tizanidine (TIZ), aceclofenac (ACF), and nimesulide (NIM) either in pure forms or in their different tablet dosage forms. Chromatographic separation was attained on an ACQUITY UPLC™ BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of 20 mM phosphate buffer (pH 7.0) : acetonitrile in the proportion (60 : 40 v/v) isocratically pumped at a flow rate of 1.25 mL·min−1, and detection was monitored at 305 nm. All analytes were separated simultaneously at a retention time (tr) of 1.42, 2.31, 3.63, and 5.62 min for PAR, TIZ, ACF, and NIM, respectively, with a total run time less than 6.0 min. The proposed method was validated according to ICH guidelines with respect to accuracy, precision, linearity, limit of detection, limit of quantitation, and robustness. Linearity was obtained over a concentration range of 81.25–487.5, 0.5–3.5, 25–150, and 25–150 µg·mL−1 for PAR, TIZ, ACF, and NIM, respectively. The development method can be successfully employed in QC laboratories for the routine analysis of the investigated drugs in their new combination.

A Validated Enantioselective HPLC Method for Assay of S-Amlodipine Using Crown Ether as a Chiral Stationary Phase

Address: 1Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, October 6 University, 6th October City, Cairo, Egypt, 2Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt, 3Pharmaceutical Chemistry & Phytochemistry. Department, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia, 4Analytical Chemistry Department, Faculty of Pharmacy, Minia University, Minia, Egypt, 5Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Center, Dokki, Giza 12622, Egypt