Method Validation and Rapid Determination of Monosodium Glutamate in Various Food Products by HPLC-Fluorescence Detection and Method Optimization of HPLC-Evaporative Light Scattering Detection Approach without Derivatization.

In this study, an effective, simple and rapid high-performance liquid chromatography (HPLC) using fluorescence (FLD) method was developed and validated for the determination of monosodium glutamate (MSG) in 57 various food samples. Besides, HPLC-Evaporate Light Scattering Detection (ELSD) method was carried out for determination of MSG without derivatization. MSG analysis was performed by derivatization with dansyl chloride at excitation 328, emission 530nm with fluorescence detector. HPLC-FLD method was carried out by using C18 (150 mm, 4.6 mm, 2.7 µm) column with the mobile phase consisting of (Water: Methanol:Glacial Acetic Acid)/(54:45:1,v/v/v). The column temperature was set at 25°C and the flow rate was set at 0.5 mL min-1 with an injection volume 20 µL. The results were linear (R2 = 0.9999) with very low quantification limits. The applied method was optimized and the validated parameters such as LOD, LOQ, accuracy, precision, linearity and robustness were calculated. The obtained results were statistically compared with each other. The validated HPLC-FLD method was successfully applied for the analysis of MSG in all of the food samples. Moreover, HPLC-ELSD method was optimized and successfully demonstrated for detect the MSG without derivatization.

ltrafiltration-based sample preparation and HPLC-UV determination of diclofenac in human plasma samples.

The sample preparation step is the initial step in pharmaceutical analysis. While ultrafiltration is a well-known technique used in the food and pharmaceutical industries, it has rarely been used to measure the plasma concentration of active pharmaceutical ingredients. This study aimed to analyze diclofenac sodium (DS) in human plasma samples using ultrafiltration-based sample preparation before high-performance liquid chromatography (HPLC) analysis. The advantages and limitations of ultrafiltration-based sample preparation in bioanalysis were evaluated by comparing the results with conventional methods. The precipitating agent was used before ultrafiltration. The analysis was carried on an HPLC-UV system with a C18 column (250 ×4.6 mm, 5 µm) and acetonitrile : phosphate buffer (pH 3.0, 10 mM) (70 : 30 v/v) was used as the mobile phase. The bioanalytical method was validated according to FDA guidelines and applied to spiked samples of DS in commercial human plasma samples. The LOD and LOQ values were 0.006 µg mL-1 and 0.020 µg mL-1, respectively. The method was linear in the range of 0.025-0.50 µgmL-1 with excellent determination coefficients (R2 > 0.9991). The findings of this analysis with low LOD and LOQ values and high recovery values with high trueness and precision proved the matrix minimizing the effect of the presented sample preparation technique.

Determination of Potential Genotoxic Impurity, 5-Amino-2-Chloropyridine, in Active Pharmaceutical Ingredient Using the HPLC-UV System.

A novel analytical method, based on high-performance liquid chromatography with a UV (HPLC-UV) detection system for the sensitive detection of a genotoxic impurity (GTI) 5-amino-2-chloropyridine (5A2Cl) in a model active pharmaceutical ingredient (API) tenoxicam (TNX), has been developed and validated. The HPLC-UV method was used for the determination of GTI 5A2Cl in API TNX. The compounds were separated using a mobile phase composed of water (pH 3 adjusted with orthophosphoric acid): MeOH, (50:50: v/v) on a C18 column (150 × 4.6 mm i.d., 2.7 µm) at a flow rate of 0.7 mL min-1. Detection was carried out in the 254 nm wavelength. Column temperature was maintained at 40°C during the analyses and 10 µL volume was injected into the HPLC-UV system. The method was validated in the range of 1-40 µg mL-1. The obtained calibration curves for the GTI compound was found linear with equation, y = 40766x - 1125,6 (R2 = 0.999). The developed analytical method toward the target compounds was accurate, and the achieved limit of detection and limit of quantification values for the target compound 5A2Cl were 0.015 and 0.048 µg mL-1, respectively. The recovery values were calculated and found to be between 98.80 and 100.03%. The developed RP-HPLC-UV analytical method in this research is accurate, precise, rapid, simple and appropriate for the sensitive analysis of target GTI 5A2Cl in model API TNX.

Capillary Electrophoresis Method for Determination of Escitalopram Oxalate in Urine Samples and Different Dosage Forms.

Application of capillary electrophoresis (CE) has become a rapidly growing analytical technique for the estimation of drugs in pharmaceutical dosage forms and biological fluids. In this study, an effective and sensitive method was developed for the determination of escitalopram oxalate (ESC-OX) by CE with diode-array detection at 200 nm. The separation was achieved by a fused silica capillary with 40 cm effective length (48.5 cm total, 75 µm i.d.). The background electrolyte was composed of 15 mM phosphate buffer (pH 2.5). The applied potential was 22.5 kV, and the samples were injected at 50 mbar pressure for 10 s. Metoprolol was used as an internal standard (IS). The migration time under these optimum conditions was 6.51 ± 0.07 and 6.73 ± 0.08 min for ESC-OX and IS, respectively, with total run time 7 min. The method was validated for linearity, precision, accuracy, specificity and sensitivity. The limit of detection was calculated as 3.85 and 5.07 ng mL-1 for standard and urine samples, respectively. The developed method was employed successfully for the determination of ESC-OX in different pharmaceutical dosage forms and spiked human urine after simple liquid-liquid extraction with good recovery.

Development of a RP-HPLC method for simultaneous determination of reference markers used for in-situ rat intestinal permeability studies.

One of the most common techniques for assessing the intestinal absorption characteristics of drugs is single-pass intestinal perfusion (SPIP) method. Metoprolol tartrate (MT, reference standard) and phenol red (PR, zero permeability marker) are the compounds that are normally used in SPIP studies. The aim of this study was to develop a reverse phase high-performance liquid chromatography (RP-HPLC) method combined with UV-detection for the simultaneous determination of MT and PR in the perfusion medium used in SPIP experiments. Elution was performed using a Restek Raptor C18 column (5 µm, 4.6 mm × 250) at a temperature of 25 °C. The mixture of the mobile phase consisted of (MeOH):(Phosphate buffer solution, PBS), (20 mM, pH 3.0 adjusted with ortho-phosphoric acid),(55:45, v/v). Flow rate and column temperature were set at 1.2 mL min-1 and 25 °C, respectively. MT and PR were injected as 20 µL into the HPLC system. UV detection was performed at 227 nm. The obtained retention times were reported as 2.89 and 3.80 min for MT and PR, respectively. The developed RP-HPLC method was validated according to Q2(R1) guideline of The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The method was linear within the range of 2-50 µg mL-1 for PR and 10-75 µg mL-1 for MT. The developed RP-HPLC method was successfully applied on determination of MT and PR in perfusion medium. The developed method could be helpful for researchers working on in-situ rat intestinal permeability studies and it could be easily modified on further studies.

Application of LC-ESI-MS/MS Method for Analysis of Escitalopram Oxalate in Human Urine and Pharmaceutical Dosage Forms.

An effective and sensitive liquid chromatographic-electrospray ionization tandem mass-spectrometric (LC-ESI-MS/MS) method was developed and validated for quantification of escitalopram oxalate (ESC-OX), antidepressant drug in spiked human urine and pharmaceutical formulations. In this work, simple liquid-liquid extraction was optimized and used for extraction of cited drug from urine samples. The chromatographic separation was attained within 6 min including re-equilibration time by using gradient elution with 0.1% formic acid in acetonitrile and 0.1% formic acid in water as mobile phase, Zorbax Eclipse RP C18 (50 × 2.1 mm) column was used with a particle size of 1.8 µm; the flow-rate was 0.35 mL min-1. Ion signal m/z 262.0 and 109.0 for ESC-OX product ions were monitored at positive ESI mode. Validation of the method was carried out according to the ICH Q2 (R1) guidelines and EMEA criteria. The method was linear over 79-196,450 pg mL-1 with a regression of 0.9999 and 0.9993 for both standard and urine samples. The LOD was 3.88 and 10.66 pg mL-1 for standard and urine samples, respectively, while lower limit of quantification was 79 pg mL-1.

A novel dilute and shoot HPLC assay method for quantification of irbesartan and hydrochlorothiazide in combination tablets and urine using second generation C18-bonded monolithic silica column with double gradient elution.

Irbesartan (IRB) and hydrochlorothiazide (HCT) are angiotensin-II receptor antagonist and thiazide-class diuretic compounds, respectively, which are in use in the treatment of hypertension. A novel dilute-and-shoot HPLC assay method for simultaneous quantification of IRB and HCT in fixed-dose combination tablets and urine samples was described. The separation of IRB, HCT and agomelatine (internal standard) was carried out using a second generation C18-bonded monolithic silica column (Chromolith(®) High Resolution RP-18e, 100×4.6mm, Merck KGaA), utilizing both mobile phase and flow rate gradient elution programs. The analytes were detected at 230 nm wavelength using photodiode array detector within 24 minutes with high resolution, observing about 50 percent more peak capacity when using second generation C18-bonded monolithic silica column. Urine samples were introduced into the system effortlessly, with only filtration and subsequent dilution. Validation studies were performed according to the official recommendations of USP and ICH, and the developed method was successfully applied to pharmaceutical tablets and urine samples.

A novel RP-HPLC method for simultaneous determination of potassium sorbate and sodium benzoate in soft drinks using C18-bonded monolithic silica column.

Potassium sorbate and sodium benzoate are food additives that are generally employed for prevention of food spoilage originating from bacteria, molds or yeasts. Although these compounds were generally recognized as safe due to their low risk of acute and chronic toxicity, they have limitations of usage to protect human health. Development and validation of a novel RP-HPLC method, in which a C18-bonded monolithic silica column was used as stationary phase to assay these compounds, is described for the first time. Aliquots of 10 µL of samples were injected into chromatograph and eluted using phosphate buffer (0.025 M, pH 2.0)-water-acetonitrile (50:45:5, v/v/v) solution, which was pumped at the rate of 3.0 mL/min. To sharpen the peaks, 10 mM octylamine was added to the mobile phase. Potassium sorbate and sodium benzoate were detected at about 12(th) and 14(th) min, respectively, and quantified at 230 nm using photodiode array detector. A total of 41 samples were prepared by simply filtering through 0.45 µm filters after sonication, and injected into the system without any pre-treatment steps. Applicability of the method was demonstrated by performing total procedure on samples of different brands and types, and their compliance to official regulations was assessed.

Reversed-phase HPLC analysis of levetiracetam in tablets using monolithic and conventional C18 silica columns.

Development and validation of an RP-HPLC method for determination of levetiracetam in pharmaceutical tablets is described. The separation and quantification of levetiracetam and caffeine (internal standard) were performed using a single analytical procedure with two different types of stationary phases, conventional Phenomenex Gemini C18 (100 x 4.6 mm, 5 microm) and Merck Chromolith Performance RP18e (100 x 4.6 mm, macropore size 2 mm, micropore size 13 nm) monolithic silica. Five-microliter aliquots of samples were injected into the system and eluted using water-acetonitrile (90 + 10, v/v) mobile phase pumped at the rate of 1 mL/min. The analyte peaks were detected at 200 nm using a diode array detector with adequate resolution. Validation studies were performed using the method recommended by the International Conference on Harmonization, the U.S. Pharmacopeia, and AOAC INTERNATIONAL, which includes accuracy, precision, range, limits, robustness, and system suitability parameters. Levetiracetam and caffeine were detected in about 7 min using the conventional column, whereas less than 5 min was required when the monolithic column was used. Calibration plots had r values close to unity in the range of 0.8-8.0 microg/mL. Assay of levetiracetam in a tablet formulation was demonstrated as an application to real samples.