Determination of Monosodium Glutamate in Noodles Using a Simple Spectrofluorometric Method based on an Emission Turn-on Approach.

A simple, fast, and ecofriendly spectrofluorometric method was developed and validated for determination of mono sodium glutamate (MSG). The method depended on the reaction between MSG and iron (III) salicylate based on ligand exchange mechanism. Addition of MSG turned-on the fluorescent response of iron (III) salicylate at λem 411 nm. Reaction conditions including reagent concentration, pH, and time were optimized. The method was validated regarding the ICH guidelines. The method determined MSG within the linearity range of 25-250 µM with a coefficient of determination of 0.9967 and a calculated limit of detection of 1.73 µM. Furthermore, the developed method was successfully applied for the determination of MSG in food preparation (instant noodles). The results were compared to those obtained by a published HPLC method using t-test and F-test at 95% confidence interval; no statistically significant difference was found. Based on the analytical Eco-scale and the green analytical procedure index (GAPI), the developed method was assessed to be greener than the published HPLC method. The developed method offered advantages over other spectrophotometric reported methods and was convenient for routine determination of MSG in foodstuffs.

Novel experimental design paradigm for development of eco-friendly gradient chromatographic method for simultaneous determination of metronidazole and spiramycin.

This work describes the innovative experimental design-assisted development of a green gradient chromatographic method for concomitant analysis of metronidazole (MTR) and spiramycin (SPR). Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic conditions involved a mobile phase consisting of ethanol and 20 mM sodium dihydrogen phosphate solution (pH adjusted to 2.5) in the ratio 2:98 (v/v) for 2 min then the ratio changed to 30:70 (v/v). The flow rate was 1.3 mL/minute. Separation and analysis were performed on X-bridge C18 (150 mm × 4.6 mm × 3.5 µm) column with diode array detector set at 230 nm. Column oven temperature was 40°C. A linear response was acquired over the range of 5-125 µg/mL for both drugs. Detection and quantitation limits were 0.86 and 2.62 µg/mL for MTR and 0.92 and 2.83 µg/mL for SPR, respectively. The method was implemented for determination of both drugs in three tablet formulations. The method was proved to be green as evaluated by three assessment tools. The application of experimental designs assists in development of a robust green chromatographic method in gradient elution mode for determination of both drugs within reasonable time.

Green micellar stability-indicating high-performance liquid chromatography method for determination of rupatadine fumarate in the presence of its main impurity desloratadine: Oxidative degradation kinetics study.

A green micellar stability-indicating high-performance liquid chromatography method was developed for rupatadine fumarate determination in existence with its main impurity desloratadine. Separation was attained using Hypersil ODS column (150 × 4.6 mm, 5 µm), the micellar mobile phase consisted of 0.13 M sodium dodecyl sulfate, 0.1 M disodium hydrogen phosphate adjusted by phosphoric acid to pH 2.8 and 10% n-butanol. The column was maintained at 45◦ C and detection was carried out at 267 nm. A linear response was achieved over the range of 2-160 µg/ml for rupatadine and 0.4-8 µg/ml for desloratadine. The method was applied for rupatadine determination in alergoliber tablets and alergoliber syrup without the interference of methyl paraben and propyl paraben present as main excipients. Rupatadine fumarate revealed pronounced susceptibility to oxidation; further study of oxidative degradation kinetics was carried out. Rupatadine was found to follow pseudo-first-order kinetics when exposed to 10% H2 O2 at 60 and 80°C and the activation energy was found to be 15.69 Kcal/mol. At a lower temperature (40°C), degradation kinetics regression was best fitted as a polynomial quadratic relationship, thus rupatadine oxidation at a lower temperature tends to adopt a second-order kinetics rate. Oxidative degradation product structure was revealed using infrared and found to be rupatadine N-oxide at all temperature values.

Stability indicating RP-HPLC method for methylcobalamin determination in different dosage forms: Application to photodegradation kinetics and pH rate profiling.

A stability-indicating RP-HPLC method for methylcobalamin determination was developed. Stress degradation under variable conditions was carried out. Methylcobalamin had pronounced susceptibility to hydrolysis under acidic, alkaline, and photolytic conditions; further study of photolytic degradation kinetics and pH rate profiling over pH range 2-11 was carried out. Photodegradation of methylcobalamin followed zero-order kinetics with half-life 0.99 h equivalent to 1971.53 lux. Methylcobalamin followed pseudo-first-order kinetics upon exposure to acidic and alkaline hydrolysis with highest stability at pH 5 and least stability at pH 2. Optimization of chromatographic conditions was performed using two level full factorial design, and chromatographic analysis was executed using Inertsil column (250 × 4.6 mm, 5 µm) maintained at 25◦ C. Elution was carried out using 25 mM potassium dihydrogen phosphate (pH adjusted with phosphoric acid to 3.8): methanol:acetonitrile (55:35:10, v/v) as mobile phase. The flow rate was 1.0 ml/min. Detection was carried out at 220 nm using diode array detector. The method was validated as per ICH guidelines; the linearity was over concentration range 2-160 µg/ml with coefficient of determination 0.9995. The method was effectively applied for determination of methylcobalamin in Cobalvex ampoule, Cobal tablet, Cobal-F tablet, and Methyltechon oral dissolvable film without interfering from excipients within run time 6 min.

Stability indicating RP-HPLC method for determination of dimethyl fumarate in presence of its main degradation products: Application to degradation kinetics.

A stability-indicating reverse phase-high-perfromance liquid chromatography method for the quantitative determination of dimethyl fumarate in presence of its main degradation products was developed. The chromatographic conditions were optimized using two-level full factorial design, chromatographic analysis was performed using Inertsil® column (250 × 4.6 mm, 5 µm) maintained at 25°C. Mobile phase was a mixture of water (pH 2.6 adjusted with phosphoric acid) and methanol (40:60, v/v) at a flow rate 1.0 mL/min, detection was performed at 210 nm using diode array detector. Stress degradation of dimethyl fumarate under acidic, alkaline, neutral, oxidative, photolytic, and thermal conditions was carried out, it was found to be very susceptible to hydrolysis under acidic and alkaline conditions; further investigation of degradation kinetics over pH range 1-9 was carried out. The degradation rate constant (K), t1/2 and t90 were calculated. Dimethyl fumarate show decreasing in stability in the following pH order: 7 < 5 < 3 < 1 < 9. The method was validated as per ICH guidelines, the method was found to be linear over concentration range 10-150 µg/mL with coefficient of determination (r2 ) 0.9997. The method was successfully applied for dimethyl fumarate determination in Marclerosis® dosage form within run time less than eight minutes without interference from excipients.

Coupling of synchronous fluorescence spectroscopy with derivative amplitude outcomes for simultaneous determination of metoprolol succinate and olmesartan medoxomil in combined pharmaceutical preparation: Application in spiked human plasma.

For the first time a spectrofluorimetric method had been achieved for the concurrent analysis of metoprolol succinate (MET) and olmesartan medoxomil (OLM). The approach depended on assessing the first order derivative (1D) of the synchronous fluorescence intensity of the two drugs in aqueous solution at Δλ of 100 nm. The amplitudes of 1D at 300 nm and 347 nm were measured for MET and OLM, respectively. The linearity ranges were 100-1000 ng/mL and 100-5000 ng/mL for OLM and MET, respectively. This approach is uncomplicated, repetitive, quick, and affordable. The results of analysis had been statistically verified. The validation assessments were carried out following the recommendations of The International Council for Harmonization (ICH). This technique could be employed to assess marketed formulation. The method was sensitive with limits of detection (LOD) of 32 ng/ml and 14 ng/mL for MET and OLM, respectively. Limits of quantitation (LOQ) were 99 ng/ml for MET and 44 ng/mL for OLM. So it can be applied to determine both drugs in spiked human plasma within the linearity ranges of 100-1000 ng/mL for OLM and 100-1500 ng/mL for MET.

Area under the curve and ratio difference spectrophotometric methods with evaluation of the greenness for simultaneous determination of olmesartan medoxomil and metoprolol succinate.

The aim of this study is to develop and validate two simple spectrophotometric methods for simultaneous determination of metoprolol succinate (MET) and olmesartan medoxomil (OLM) in tablet form. Method (I) was area under the curve (AUC) method. This approach involved the measuring of the area over a variety of wavelengths. Two wavelength ranges; 213-230 nm and 244-266 nm were chosen for determination of MET and OLM, respectively. Method (II) was ratio difference spectrophotometricmethod. For determination of MET, the ratio spectra were generated using 15 µg/mL OLM as a divisor then the peak to trough amplitudes between 221 nm and 245 nm were displayed versus the corresponding concentrations of MET. For determination of OLM, the peak-to-peak amplitudes between 247 and 293 nm were chosen and found to be directly proportional to OLM concentrations using 15 µg/mL MET as a divisor. The linearity ranges were 2-30 µg/mL and 2-25 µg/mL for MET and OLM, respectively. The assay results showed good mean %recovery ± SD as well as good agreement with that of the reported method. The developed methods were validated according to ICH guidelines. The developed methods are accurate, precise, eco-friendly and could be applied successfully to estimate OLM and MET in their combined dosage form.

Quality by design paradigm for optimization of green stability indicating HPLC method for concomitant determination of fluorescein and benoxinate.

A green, robust and fast stability indicating chromatographic method has been developed for concomitant analysis of fluorescein sodium and benoxinate hydrochloride in the presence of their degradation products within four minutes. Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic analysis was achieved using a mixture of isopropanol and 20 mM potassium dihydrogen phosphate solution (pH 3.0) in the ratio 27:73 as mobile phase. The flow rate was 1.5 mL/min and column oven temperature was 40 °C. Chromatographic analysis was performed on Eclipse plus C18 (100 mm × 4.6 mm × 3.5 µm) column with DAD detector set at 220 nm. A linear response was acquired over the range of 2.5-60 µg/mL and 1-50 µg/mL for benoxinate and fluorescein respectively. Stress degradation studies were executed under acidic, basic, and oxidative stress conditions. The method was implemented for quantitation of cited drugs in ophthalmic solution with mean percent recovery ± SD of 99.21 ± 0.74 and 99.88 ± 0.58 for benoxinate and fluorescein respectively. The proposed method is more rapid and eco-friendly compared to the reported chromatographic methods for determination of cited drugs.

Design of experiment-oriented development of solvent-free mixed micellar chromatographic method for concomitant determination of metronidazole and ciprofloxacin hydrochloride.

A green, fast and robust solvent-free chromatographic method has been developed for concomitant analysis of ciprofloxacin HCl and metronidazole in bulk powder as well as in dosage form using levofloxacin as internal standard (I.S.). Two different designs including fractional factorial (FFD) and Box-Behnken (BBD) designs were implemented for screening and optimization steps, respectively. The optimum chromatographic separation was accomplished using mobile phase composed of 0.13 M sodium dodecyl sulfate and 0.02 M Birij-35 solution adjusted to pH 2.5 using phosphoric acid at a flow rate of 1.3 mL/min and column oven temperature of 40 °C. Chromatographic analysis was performed on X-Bridge (150 mm × 4.6 mm, 5 µm) column with UV detection at 280 nm. A linear response was acquired over the range of 0.4-50 µg/mL for both drugs. The developed method was applied for quantitation of cited drugs in commercially available tablet with mean percent recovery ± SD of 99.45 ± 0.72 and 100.13 ± 0.81 for metronidazole and ciprofloxacin respectively. The method was proven to be green as evaluated by three greenness assessment tools. The run time was 8 min, thus saving time and reagent.

Using of eosin Y as a facile fluorescence probe in alogliptin estimation: Application to tablet dosage forms and content uniformity testing.

New simple sensitive and reliable spectrofluorimetric approach was established for the determination of the antidiabetic drug; Alogliptin (ALG) in its pure and tablet forms. The developed approach is depended on the suppressive action of ALG on the eosin Y native fluorescence. The quenching action of ALG on the eosin Y native fluorescence was measured at acidic medium pH: 3.5, emission wavelength 541 nm (λex. 260 nm). The relative fluorescence intensity (RFI) was measured, and it was directly proportional to ALG concentration in the concentration range of (15-110) µg/mL. The developed and optimized approach was entirely validated regarding to ICH guidelines. The developed method application was successfully extended for ALG content uniformity test (CU). The distribution fraction (DF), rate constants (K), and free energy changes (ΔG°) were calculated. The results obtained were compared to that of the published spectrophotometric one.

Redox-Based Colorimetric Sensor for the Selective Determination of Ascorbic Acid in Fixed-Dose Combination Tablets.

BACKGROUND: Ascorbic acid (ASC; vitamin C) is a weak chromophore, so its presence cannot be determined directly by spectrophotometry. OBJECTIVE: This work aims to develop and validate a simple and ecofriendly analytical method for the determination of ASC concentration based on the reaction of ASC with the metal complex, ferric salicylate. METHODS: The visible absorbance of ferric salicylate was found to be inversely proportional to the concentration of ASC. The possible mechanism of the reaction between ASC and ferric salicylate was investigated: ferric salicylate was found to be reduced by ASC under the applied experimental conditions. Different parameters that may affect the reaction were also investigated: ferric salicylate concentration, pH, ionic strength, and the time of reaction. The optimum concentration of ferric salicylate was found to be 1000 µM and the optimum pH was 5.5. The developed method was validated according to International Conference on Harmonization (ICH) guidelines. RESULTS: The linearity range of the developed method was 5-70 µg/mL and the correlation coefficient was 0.9994. The limits of detection and quantitation were 0.38 µg/mL and 1.16 µg/mL, respectively. The method was successfully applied to the determination of ASC concentration in commercial Ruta-C60® tablets. The mean recovery ± standard deviation was found to be 101.10 ± 0.70%. The results were statistically compared to those obtained by a reported HPLC method. Good agreement was observed. CONCLUSION: The developed method is simple, fast, cost-effective, and suitable for routine pharmaceutical analysis of ASC. HIGHLIGHTS: The developed method is more sensitive than the other spectrophotometric methods reported for determination of ASC.

Deduction of the operable design space of RP-HPLC technique for the simultaneous estimation of metformin, pioglitazone, and glimepiride.

A reversed-phase RP-HPLC method was developed for the simultaneous determination of metformin hydrochloride (MET), pioglitazone (PIO), and glimepiride (GLM) in their combined dosage forms and spiked human plasma. Quality risk management principles for determining the critical method parameters (CMPs) and fractional factorial design were made to screen CMPs and subsequently, the Box-Behnken design was employed. The analytical Quality by Design (AQbD) paradigm was used to establish the method operable design region (MODR) for the developed method depended on understanding the quality target product profile (QTPP), analytical target profile (ATP), and risk assessment for different factors that affect the method performance to develop an accurate, precise, cost-effective, and environmentally benign method. The separation was carried out using a mobile phase composed of methanol: 0.05 M potassium dihydrogen phosphate buffer pH 3.7 with 0.05% TEA (78:22, v/v). The flow rate was 1.2 mL/min. DAD detector was set at 227 nm. Linagliptin (LIN) was used as an internal standard. The proposed method was validated according to The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). The assay results obtained by using the developed method were statistically compared to those obtained by the reported HPLC method, and a satisfying agreement was observed.

Four chemometric spectrophotometric methods for simultaneous estimation of amlodipine besylate and olmesartan medoxomil in their combined dosage form.

For determination of amlodipine besylate (AML) and olmesartan medoxomil (OLM) at the same time, four UV chemometric spectrophotometric techniques were created and tested in accordance with ICH standards. Method (I) was absorption subtraction method (ASM) using two wavelengths, one of which was of AML at 365 nm and the other was the isoabsorptive point of both drugs at 237 nm. Method (II) was ratio subtraction method (RSM) for determination of OLM at λmax = 254 nm by taking the ratio spectrum and subtracting the constant values using 10 µg/mL of AML as a divisor in combination with extended ratio subtraction method (ERSM) to determine AML at λmax = 239 nm using 10 µg/mL OLM as a divisor. Method (III) was dual wavelength method; the wavelengths used to determine OLM were 221 nm and 235 nm, while those used to determine AML were 246 nm and 259 nm. Method (IV) was the second order derivative (2D) spectrophotometric method at 219 nm for OLM and 227 nm for AML. The proposed approaches were used to achieve linearity in the concentration range of 2-25 µg/mL for both drugs. The approaches were found to be uncomplicated, reproducible, efficient, and cost-effective as well as they were successfully used to determine the cited drugs in both laboratory samples and commercial pharmaceutical formulations.

Validated Spectrophotometric Methods for Simultaneous Determination of Atorvastatin Calcium and Olmesartan Medoxomil in Their Pharmaceutical Formulation.

BACKGROUND: Few spectrophotometric methods have been developed for the simultaneous determination of atorvastatin calcium (ATO) and olmesartan medoxomil (OLM). OBJECTIVE: This work aimed to develop and validate five simple spectrophotometric methods for the simultaneous estimation of ATO and OLM in their tablet form. METHODS: Method I applied the area under curve (AUC) based on the measurement of areas between 241 and 261 nm for ATO, and 248 and 263 nm for OLM. Method II applied second derivative spectrophotometry where the analytical amplitudes at 246.5 and 235 nm were chosen for the estimation of ATO and OLM, respectively. Method III applied the ratio difference (RD) method based on the measurement of amplitude difference (ΔP) within ratio spectra; ΔP (240-260 nm) was directly related to ATO concentration, and ΔP (262-240 nm) was directly related to OLM concentration. Method IV depended on the absorbance ratio method in which the wavelength at the iso-absorptive point (λISP) and the maximum absorbance wavelength (λmax), 277 and 255.5 nm, respectively, were used to calculate OLM concentration, while ATO concentration was determined from the zero-order UV spectra at 300 nm. Method V utilized a dual wavelength (DW) technique where ΔA between 247.5 and 262 nm was directly related to ATO concentration, and ΔA between 216 and 238 nm was directly related to OLM concentration. RESULTS: The results of the assays indicated good mean % recovery ± SD as well as good agreement with the reported method. CONCLUSION: Five simple and rapid spectrophotometric methods were developed, validated, and successfully applied for simultaneous estimation of ATO and OLM tablets. HIGHLIGHTS: None of the developed methods has previously been reported for simultaneous determination of ATO and OLM.

Response surface optimization of a vortex-assisted dispersive liquid-liquid microextraction method for highly sensitive determination of repaglinide in environmental water by HPLC/UV.

A vortex-assisted dispersive liquid-liquid microextraction (DLLME) method, mated to chemometrics and combined with HPLC/UV detection was optimized and validated for enrichment and determination of repaglinide in environmental samples using nateglinide as an internal standard (IS). A phosphate buffer (10 mM, pH 2.5): acetonitrile (45:55, v/v) was used as a mobile phase with a flow rate of 1 mL/min in an isocratic elution mode. Chemometrics-assisted optimization was performed using a quadratic integrated D-optimal design. The developed model assessed the statistical significance of the independent variables and their interactions to attain the optimum conditions revealing that extractant type, extractant volume and pH are the most influential factors. Optimization of the extraction procedures was performed with the aid of Design Expert 8® software, which suggested 58 different experiments. The optimal conditions were 30 µL of 1-octanol as extractant, 100 µL of acetonitrile as a disperser at pH 8. Under the optimized conditions, the method showed linearity over the range of 1-100 ng/mL with a limit of detection of 0.4 ng/mL. The accuracy, the intra- and inter-day precision were assessed, the %recoveries were found to be between 98.48 and 100.81% with %RSD lower than 1.3. Using chemometrics in method optimization helped achieve the maximum possible enrichment with the least effort, time, and reagents while considering all possible interactions between variables.

Analytical quality by design based on design space in reversed-phase-high performance liquid chromatography analysis for simultaneous estimation of metformin, linagliptin and empagliflozin.

Employing the Quality by Design paradigm through this work helped conclude the method operable design region for optimizing the high performance liquid chromatography (HPLC) assay using Design of Experiments and response surface methodology to obtain a good resolution and determination of all analysed compounds and to achieve a suitable analysis time. A deep understanding of the quality target product profile, analytical target profile and risk assessment for parameters that affect the method performance led to developing an accurate, precise and cost-effective method. Quality risk management principles were applied for determining the critical method parameters affecting the simultaneous determination of metformin hydrochloride (MET), linagliptin (LIN) and empagliflozin (EMP) by reversed-phase HPLC . The ternary mixture was successfully resolved in 5 min with a linearity range of (0.1-600) µg ml-1 for MET and (0.05-50) µg ml-1 for LIN and EMP. The newly developed method was validated according to the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. Good agreement was observed with the assay results of the reported UPLC one. To evaluate the greenness of the proposed method, an analytical Eco-Scale method was used.

A highly sensitive switch-on spectrofluorometric method for determination of ascorbic acid using a selective eco-friendly approach.

Ascorbic acid has recently been extensively used due to its role in the management of COVID-19 infections by stimulating the immune system and triggering phagocytosis of the corona virus. The currently used spectrofluorometric methods for determination of ascorbic acid require using derivatizing agents or fluorescent probes and suffer from a number of limitations, including slow reaction rates, low yield, limited sensitivity, long reaction times and high temperatures. In this work, we present a highly sensitive spectrofluorometric method for determination of ascorbic acid by switching-on the fluorescence of salicylate in presence of iron (III) due to a reduction of the cation to iron (II). The addition of ascorbic acid resulted in a corresponding enhancement in the fluorescence intensity of iron (III)-salicylate complex at emission wavelength = 411 nm. The method was found linear in the range of 1-8 µg/mL with a correlation coefficient of 0.9997. The limits of detection and quantitation were 0.035 µg/mL and 0.106 µg/mL, respectively. The developed method was applied for the determination of ascorbic acid in the commercially available dosage form; Ruta C60® tablets. The obtained results were compared with those obtained by a reported liquid chromatographic method at 95% confidence interval, no statistically significant differences were found between the developed and the reported methods. Yet, the developed spectrofluorometric method was found markedly greener than the reference method, based on the analytical Eco-scale and the green analytical procedure index. This work presents a simple, rapid and sensitive method that can possibly be applied for determination of ascorbic acid in pharmaceuticals, biological fluids and food samples.

Chemometric spectrophotometric methods for simultaneous estimation of metoprolol succinate and amlodipine besylate in their tablet formulation.

Two simple chemometric spectrophotometric methods; isosbestic point and dual wavelength methods were developed, validated and applied to the determination of metoprolol succinate in presence of amlodipine besylate in their binary mixtures and in combined tablet formulation while amlodipine besylate was determined by direct spectrophotometry at λ = 365 nm within linearity range of 2-25 µg/mL. The mean percentage recovery ± SD was 99.921 ± 0.089 for amlodipine besylate. Two proposed chemometric spectrophotometric methods were developed for the spectral resolution of metoprolol succinate in presence of amlodipine besylate without preliminary separation with a linearity range of 2-30 µg/mL metoprolol succinate. The first method depended on measuring the absorbance at the isosbestic point at λ = 226 nm. The mean percentage recovery ± SD was 100.110 ± 0.249 for metoprolol succinate. The second method was dual wavelength method, metoprolol could be determined alone using the absorbance difference between 226 nm and 248.7 nm where the absorbance difference was zero for amlodipine at these two wavelengths. The mean percentage recovery ± SD was 99.734 ± 0.438 for metoprolol succinate using dual wavelength method. The developed methods were validated as per ICH guidelines and were successfully applied to analysis of cited drugs in their synthetic tablet formulation. The results obtained by the developed methods were statistically compared to those obtained by a reported one using t-test and F- test. Good agreement was observed.

Experimental design approach for development of spectrofluorimetric method for determination of favipiravir; a potential therapeutic agent against COVID-19 virus: Application to spiked human plasma.

The present work describes development of rapid, robust, sensitive and green spectrofluorimetric method for determination of favipiravir (FAV). Different factors affecting fluorescence were carefully studied and Box Behnken Design was applied to optimize experimental parameters. The proposed method is based on measuring native fluorescence of FAV in 0.2 M borate buffer (pH 8.0) at 432 nm after excitation at 361 nm. There was a linear relationship between FAV concentration and relative fluorescence intensity over the range 40-280 ng/mL with limit of detection of 9.44 ng/mL and quantitation limit of 28.60 ng/mL. The method was successfully implemented for determination of FAV in its pharmaceutical formulation with mean % recovery of 99.26 ± 0.87. Moreover, the high sensitivity of the method allowed determination of FAV in spiked human plasma over a range of 48-192 ng/mL. The proposed spectrofluorimetric method was proved to be eco-friendly according to analytical eco-scale.

Vortex-Assisted Dispersive Liquid-Liquid Microextraction Coupled with Deproteinization for Determination of Nateglinide in Human Plasma Using HPLC/UV.

A validated method for preconcentration and determination of nateglinide in plasma was developed using vortex-assisted dispersive liquid-liquid microextraction. Different variables that affect extraction efficiency were studied and optimized, including type and volume of extractant, type and volume of disperser, pH of diluent, salt addition effect, centrifugation and vortex time. Nateglinide was extracted using 30 µL of 1-octanol as an extractant and 200 µL of methanol as a disperser. The enrichment factor reached 330 under the optimum conditions. High-performance liquid chromatography/ultraviolet was used for detection using phosphate buffer (pH 2.5, 10 mM): acetonitrile (45:55, v/v) as a mobile phase at a flow rate of 1 mL/min. The method was linear over the range of 50-20,000 ng/mL with a limit of detection of 15 ng/mL (signal-to-noise ratio = 3). Intra- and inter-day precision had %relative standard deviation <6% (n = 3) and the %recoveries were found to be between 102.5 and 105.9%. The proposed method is simple, sensitive, eco-friendly, cost-effective and powerful for microextraction of nateglinide from human plasma samples.