Sodium Dihydrogen Phosphate Buffer Research Articles

Efficient integration of analytical quality-by-design and tri-hued HPLC-DAD approach for synchronized assessment of gemigliptin and rosuvastatin in their fixed-dose pills: Application to stability study

Life-threatening atherosclerotic cardiovascular disorder is a well-recognized risk among diabetics, which is clinically ascribed to their unbalanced plasma lipoproteins. Gemigliptin-Rosuvastatin is a recently introduced polytherapy prescribed for the management of diabetic dyslipidemia. Currently, the main focus of several analysts is to design more ecologically friendly sustainable approaches for pharmaceutical analysis. In this context, an innovative tri-hued (green, blue, and white) stability-indicating HPLC-DAD approach was developed to concurrently quantify Gemigliptin and Rosuvastatin polypills. Consequently, a strategic integration between analytical quality-by-design and green/white analytical chemistry principles was specifically designed to optimize efficiency and practicality while minimizing ecological burden. Two experimental designs, namely two-level fractional factorial and Box-Behnken designs, were employed for screening and optimization procedures, respectively. Optimal separation was attained utilizing an Agilent HC-C18 column in conjunction with a mobile phase consisting of acetonitrile and 10 mM sodium dihydrogen phosphate buffer, pH 3 (60:40 v/v), eluted in an isocratic manner at a 1 mL/min flow rate. Both analytes exhibited good linearities within the concentration range of 0.5–100 μg/mL. Furthermore, stability studies were conducted on both drugs in response to variable stress degradation scenarios. Fortunately, the adopted approach was well-suited to resolve the studied drugs from their respective degradants, thereby verifying the method's stability-indicating power. Tri-hued evaluation for the implemented approach was performed via up-to-date metrics, specifically, the Analytical Eco-scale, AGREE, Blue Applicability Grade Index, and RGB 12. Ultimately, our adopted stability-indicating HPLC approach exhibited great sensitivity, specificity, applicability, and rapidity as well as being ecologically sound. It is worth noting that this is the first stability-indicating HPLC approach for simultaneous assessment of the studied drugs.

Development and validation of stability indicating HPLC-DAD assay of adapalene, methylparaben and propylparaben in a cosmeceutical gel matrix with blueness, greenness and whiteness assessment via relative ranking: An ab initio-sub finem juxtaposed non-traditional approaches

Adapalene (ADA) is primarily used as a cosmeceutical gel in the treatment of mild-moderate acne and is also used off-label to treat keratosis pilaris as well as other skin conditions. A stability-indicating fit-for-purpose (FFP) method has been developed for co-determination of ADA, methylparaben (MPN), and propylparaben (PPN) combined in a cosmeceutical gel dosage form. This method was based on a reversed-phase RP-HPLC separation of the cited analytes. It avoided the use of tetrahydrofuran (THF), which is commonly used by both compendial and published procedures for determination of ADA, thus improving method blueness (applicability), greenness and whiteness. The method development and validation followed non-traditional eco-friendly approaches including chemical simulations and empirical modelling. The HPLC separation was performed on an RP Zorbax Eclipse XDB-C8 analytical column [150 × 4.6 mm, 5 µm (80 Å)] with gradient elution system of HPLC grade acetonitrile and 50 mM aqueous sodium dihydrogen phosphate buffer containing 5 mM ethylenediaminetetraacetic acid (EDTA) adjusted to pH 8.0 as the mobile phase at a flow rate of 2.5 mL min−1 and column oven was set at 25 °C. EDTA played an important role for reproducibility of retention time of the three analytes especially for ADA. It controlled deprotonation of the three acidic analytes under study and hence reproduce their ionization, hydrophobicity, and hence retention time. Quantitation was achieved with the photodiode array detection (DAD) at wavelengths of 256 nm for both MPN and PPN and 312 nm for ADA. Target Measurement Uncertainty (TMU) for the three analytes was determined via two approaches; misclassification rates approach employing 100,000 Monte Carlo simulations (MCS) and in-specification probability (ISP) to ascertain fitness-for-purpose of the proposed method. Microsoft Excel workbooks were developed for both TMU calculation approaches in addition to calculation of procedure capability index (Ppk) and sigma level for each analyte and attached in the Supplementary material. Moreover, linearity of the method underwent additional assessment using the previously mentioned TMU and procedure capability determination techniques. The robustness of the method was checked using Plackett-Burman experimental design (PBD) as an empirical modelling technique which was examined thoroughly to detect some interactions. The calibration graphs for each drug were rectilinear in the range of 0.34–7.08, 0.38–7.00 and 0.08–1.75 µg mL−1 for ADA, MPN, and PPN, respectively. The stress testing revealed photostability of the three components and variable degrees of degradation in the other conditions. The proposed HPLC-DAD method was successfully validated and applied for the determination of the investigated analytes in a gel matrix. In addition, method blueness and greenness were comparatively evaluated via very recent approaches of Blue Applicability Grade Index (BAGI), Green Solvents Selection Tool (GSST), Spider Diagram for Assessment of the Green Analytical Index (SDAGI), Analytical Method Greenness Score (AMGS), Carbon Footprint (CF), Modified Complementary Green Analytical Procedure Index (ComplexMoGAPI), and Analytical GREEnness Metric Approach (AGREE). Furthermore, the method whiteness was assessed via Red Green Blue Twelve (RGB 12) model following a relative ranking approach.

Quality-by-design optimized RP-HPLC approach for the therapeutic drug monitoring of glibenclamide and fluoxetine in human plasma

Glibenclamide (GLB) and fluoxetine (FLX) were reported to have a potential drug-drug interaction when clinically co-administered. FLX is an inhibitor of CYP2C9 (the enzyme responsible for the metabolism of GLB), thus resulting in increased serum levels of GLB followed by severe hypoglycemia. Therefore, a sensitive, simple, and selective HPLC method is urgently needed for their simultaneous determination in plasma to be utilized for monitoring their therapeutic blood concentration levels. The current study introduces the first RP-HPLC approach for the simultaneous estimation of this binary mixture in less than 7 min. Adopting a quality-by-design (QbD) approach allowed for the designed approach to be optimized. Using a two-level full factorial design (25), the effects of various variables on the chromatographic responses were investigated and optimized. Hypersil BDS C18 column was used for the achievement of the optimum chromatographic separation at 19.9 °C using acetonitrile: sodium dihydrogen phosphate buffer (7.5 mM; pH 4, 60:40 % v/v) as a mobile phase, pumped isocratically at a flow rate of 1.036 mL/min. An injection volume of 30.0 μL was used for the binary mixture with UV detection at 230 nm. FLX and GLB calibration curves revealed an excellent linearity over the ranges of 0.025–10.00 and 0.02–10.00 µg/mL with limits of quantitation of 0.018 and 0.02 µg/mL, respectively. Due to the high sensitivity and selectivity, the method was successfully applied to the assay of the two drugs in spiked human plasma samples with reasonable % recoveries (92.70–108.50 %), indicating good bioanalytical applicability. Therefore, this study could be applied for the therapeutic drug monitoring of GLB and FLX in human plasma to control their potential drug-drug interaction. The suggested approach was also applied to estimate the investigated drugs in their dosage forms with reasonable % recoveries (96.80–102.80 %) and small % RSD values (less than 2 %). The method's excellent greenness was evaluated using three metric tools, including Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and Analytical Greenness (AGREE). Validation of the proposed method was performed according to ICH and US FDA guidelines.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION FOR THE QUANTITATIVE ESTIMATION OF SODIUM BUTYRATE IN TABLETS

Objective: To develop a simple, accurate, precise, and linear reverse-phase high-performance liquid chromatographic (RP-HPLC) method and verify the quantitative estimation (assay) of sodium butyrate in tablets. Methods: The optimized RP-HPLC method uses a reverse phase stationary phase which is YMC Triart C18 column, having specifications of 250×4.6 mm; 5 μm, a mobile phase composition of pH 8.0 sodium dihydrogen phosphate buffer and acetonitrile in the proportion of 92:8 v/v, flow rate of 0.5 ml/min, injection volume of 10 μL, and detection wavelength of 210 nm using a UV/PDA detector. Results: The developed method gave sodium butyrate eluting at about 6 min. Sodium butyrate exhibited linearity in the range of 119.56–1195.6 μg/ ml. The precision was exemplified by a relative standard deviation of 0.30%. The percentage of individual recovery was found to be in the range of 97.0 and 103.0 during accuracy studies. Conclusion: A simple, specific, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (assay) of sodium butyrate in tablets and hence this method can be explored for the analysis of sodium butyrate in tablets in various pharmaceutical industries.

STABILITY INDICATING ISOCRATIC HPLC APPROACH TO QUANTIFY MONTELUKAST AND BILASTINE MIX IN BULK AND TABLET FORMULATION

This paper describes an novel, rapid, simple, meticulous and unerring “stability indicating HPLC method” for the assessment of montelukast (MTT) and bilastine (BLE) mix in existence of their degradation products in tablet formulation. Zorbax column XDB-C18 was utilized to separate and analyse MTT and BLE mix. The isocratic mode elution of BLE and MTT mix was made with 0.1M disodium hydrogen phosphate buffer of pH 5.8 (55 % volume ratio) and methanol (45% volume ratio) at 1.0 mL min-1 flow stream. Strong linearity was recorded within the 2.5 - 20 µg mL-1 and 5 - 40 µg mL-1 concentration ranges, with coefficient determination values of 0.9994 and 0.9997, respectively, for MTT and BLE. The recovery percentiles of MTT and BLE were 99.593 % and 99.349 %, respectively. The precision displayed values less than 0.520 % for MTT and less than 0.745 % for BLE. The robustness too was shown in the chromatographic settings by minor variations. The specificity besides stability representing feature was evidenced since the degradation products arisen in stress situations did not interlope in the determination of MTT and BLE mix. Thus, this procedure can be applied for quality regulation analysis of BLE and MTT mix in formulations of tablets.

A long-life aqueous redox flow battery based on a metal–organic framework perovskite [CH3NH3][Cu(HCOO)3] as negative active substance

In this work, one water-soluble metal–organic framework [CH3NH3][Cu(HCOO)3] with a perovskite structure is synthesized as negative active substance, which is used to construct a redox flow battery by combining with the positive active substance 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-OH-TEMPO). The battery voltage of 0.696 V is achieved by utilizing citric acid - disodium hydrogen phosphate (C6H8O7-Na2HPO4) buffer and NaClO4 as supporting electrolyte. Cyclic voltammetry is determined to investigate the electrode reaction kinetics of positive and negative active substance. Moreover, the battery is repeatedly charged and discharged under room temperature at varied current densities, and runs stably for 40 cycles, and the voltage efficiency and energy efficiency at the current density of 7.5 mA cm−2 are up to 74.96 % and 66.52 %, respectively. The highest coulombic efficiency up to 96.87 % is reached at 40 mA cm−2. The infinite ordered structure of [CH3NH3][Cu(HCOO)3] reduces the crossover contamination of electrolyte and the deposition of copper ions, improving the cycle life and stability of the battery.

Analysis of teicoplanin impurities by two-dimensional ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry

A two-dimensional ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry （2D-UPLC-Q/TOF-MS） method was established for the separation and structural analysis of the components in teicoplanin. This method effectively solved the problems associated with chromatographic systems， such as liquid chromatography-mass spectrometry （LC-MS）， which used a non-volatile phosphate buffer as the mobile phase and were not suitable for the rapid identification of impurities. Moreover， this method circumvented the complexities associated with locating and identifying impurities using the original method by re-establishing a chromatographic system suitable for LC-MS. In this study， for one-dimensional （1D） chromatography， the chromatographic separation was performed on an Octadecyl silica （ODS） hypersil column （250 mm×4.6 mm， 5 μm） with gradient elution using 3.0 g/L sodium dihydrogen phosphate buffer （pH 6.0）/acetonitrile=9/1 （v/v） as mobile phase A and 3.0 g/L sodium dihydrogen phosphate buffer （pH 6.0）/acetonitrile=3/7 （v/v） as mobile phase B. The column temperature was maintained at 30 ℃ and an ultraviolet detector was used at 254 nm for analysis. For 2D chromatography， desalting was performed on a Waters ACQUITY UPLC BEH C18 column （50 mm×2.1 mm， 1.7 μm） with gradient elution using ammonium formate buffer （pH 6.0） and acetonitrile as the mobile phases. The column temperature was maintained at 45 ℃. The MS data for the components and impurities were collected by positive ion electrospray ionization （ESI） using the full-information tandem MS mode （MSE）. The cone and nebulizer gas flow rates were set at 50 and 900 L/h， respectively. The ion source and nebulizer gas temperatures were set at 120 ℃ and 500 ℃， respectively. The ESI and cone needle voltages were set at 2500 and 60 V， respectively. The collision energy was set at 20-50 eV. The molecular formulas of the components and impurities were determined using their exact masses and isotope distributions， and the structural components and impurities of teicoplanin were deduced from their fragment ions according to the fragmentation pathway of the TA2-2 component. Moreover， the 10 components reported in the European Pharmacopoeia 10.0 were analyzed and 22 impurities of teicoplanin were identified by 2D-UPLC-Q/TOF-MS. Three new impurities and two characteristic fragment ions of the teicoplanin parent nucleus were detected， and the fragmentation pathway of TA2-2 was deduced. Using this method， 1D-UPLC is applicable for the accurate qualification of components based on relative retention times， and 2D-UPLC-Q/TOF-MS is suitable for the rapid identification of the structure of components based on their fragment ions. The results indicate that 2D-UPLC-Q/TOF-MS may be used to analyze the structure of impurities in teicoplanin based on their exact masses， isotope distributions， and fragment ions. The method is rapid， simple， and sensitive， which provides a novel strategy for the quality control and process optimization of teicoplanin.

Implementation of two sustainable chromatographic methods for the simultaneous micro-quantitation and impurity profiling of metformin and rosuvastatin in recently approved fixed dose pills: Greenness and whiteness studies

Rosuvastatin-Metformin is an emerging fixed dose combination (FDC) for the treatment of diabetes mellitus-associated dyslipidemia. Generally, the beneficial outcomes of FDC in reduction of pills burden, cost and complexity of medications protocols, encouraged analysts and we in turn for the development of rapid sustainable analytical methods for the simultaneous estimation of such important drug combinations. Within this framework, two validated analytical methodologies have been implemented for the neat separation and quantitation of metformin (MET) and rosuvastatin (RSV) in presence of the pharmacopeial impurities cyanoguanidine (CYG) (impurity A of MET) and the rosuvastatin-related substance B (RSV-impurity B). The latter was freshly prepared and then confirmed using Liquid Chromatography-Mass spectrometry (LC-MS). Method I represented the first electro-driven separation method for the assay of this FDC using Micellar Electrokinetic Chromatography (MEKC). The optimized conditions were 50 mM borate buffer containing 100 mM sodium dodecyl sulfate (SDS) at pH 9.2 using a deactivated fused silica capillary (50 cm effective length × 50 μm id). Method II was High Performance Liquid Chromatography (HPLC) method using Microsorb MV-C18 (4.6 × 250 mm, 5 μm particle size) column with 20 mM sodium dihydrogen phosphate buffer (pH 3.5), acetonitrile and methanol as mobile phase constituents. The separation was achieved with a linear gradient program and flow rate 1.5 mL/min all over the run. The Diode Array Detector (DAD) detector was set at wavelengths 243 nm for RSV and 235 nm for MET in both methods. The 4 peaks were separated in about 8 and 5 min for methods I and II, respectively. Linear relationships were obtained between peak areas and concentrations of metformin and rosuvastatin in the ranges 10–100 μg/mL for MEKC and 1–150 μg/mL for HPLC. The 2 techniques fulfilled the International Council for Harmonization (ICH) validation criteria, as well as the green and white analytical chemistry principles which were evaluated using the trendiest AGREE, hexagon and RGB12 tools. The interpretation of scores indicated that the suggested methods outperformed the reported methods in terms of eco-friendliness and sustainability. They offered fast, feasible, stability indicating and accurate analysis of both drugs in bulk and in pills.

A Quality by Design and green LC technique for the determination of mast cell stabilizer and histamine receptor antagonist (Olopatadine HCl) in multiple formulations.

Mast cell stabilizer and histamine receptor antagonist olopatadine hydrochloride (OPT) assay method predicated on LC have been established for the analysis in multiple formulations. The current method dealt with ophthalmic solution, nasal spray, and tablet formulation products. The isocratic chromatography method was optimized and validated with a Boston green C8 column (150 × 4.6 mm, 5μm i.d.). Sodium dihydrogen phosphate buffer (pH3.5) with acetonitrile in the ratio of 75:25 (v/v) was used as a mobile phase at a flow rate of 1.0mL min-1 and at the column temperature of 30°C, and the detection was done at 299 nm. The method was validated as per International Council for Harmonisation (ICH) guidelines and United States Pharmacopoeia (USP). The accuracy results ranged from 99.9 to 100.7%, % relative standard deviation (RSD) from the precision was 0.5, and correlation coefficient from the linearity experiment was > 0.999. Solution stability was established for 24 h at room temperature and refrigerator conditions, and it was found that the solutions were stable. Using quality by design-based experiment designs, critical quality attributes were studied and it was found that the method was robust. In all the forced degradation studies peak purity was passed, and no interference was found at the retention time of the active component. The method validation data demonstrated that the developed method is linear, precise, accurate, specific, robust, and stable for the determination of OPT from multiple formulations. Analytical eco-scale tool, Green Analytical Procedure Index (GAPI) tool, and the National Environmental Method Index (NEMI) were used to evaluate the greenness of the method, and the analytical eco-score of 77 for the presented method was found to be excellent.

Determination of Alpha-Lipoic Acid in a Nutritional Supplement Using High Performance Liquid Chromatography

Alpha lipoic acid has the ability to react and neutralize reactive oxygen species (ROS) such as superoxide radicals, simple oxygen, hydroxyl radicals, hypochlorous acid and peroxyl radicals. A rapid high-performance liquid chromatographic method for determination of lipoic acid in a nutritional supplement was developed. The method involved sample preparation and the mobile phase comprised of 50 mM disodium hydrogen phosphate buffer (pH 2.5 adjusted with 1 M H3PO4): acetonitrile in the ratio of 50:50. The separation was done using a C18 column (150 mm) and detection was carried out using UV detection at 201 nm. The assay was found to be linear in the range of 1.56 - 50 μg/mL with the correlation coefficient of 0.9997. Method precision was determined while LOD was 0.05 μg/mL and LOQ 0.15 μg/mL. The chromatographic peak LA retention time was 6 min.

A simple and isocratic protein-based high performance liquid chromatography method for the enantioseparation of amphetamine derivatives

New amphetamine derivatives, a substance class belonging to the New Psychoactive Substances (NPS), have fundamentally changed the global drug market during the last years. Also known as “legal highs” they are sold under various names like "bath salts", "air fresheners" and so forth. Structures of well-known illicit drugs such as amphetamine, methamphetamine and 3,4-methylenedioxyamphetamine are slightly modified to circumvent law and samples are traded worldwide via various internet shops. Many of these new derivatives possess a chiral centre and the pharmacological differences of the two enantiomers are not yet investigated for many of them. This makes the continuous development of analytical methods for their chiral separation crucial.The aim of this study was to develop a simple, isocratic HPLC-UV method for enantioseparation and to test this method on a set of 26 amphetamine derivatives, including latest products on the drug market. A Daicel Chiralpak® CBH 150×3.0 mm column (5 µm particle size) served for chiral separation. The influence of the organic modifier and type, molarity and pH of the buffer on separation results was investigated. UV detection was performed at 210 and 254 nm. As final mobile phase, a mixture of 5 mM sodium dihydrogen phosphate buffer (pH = 6.9) / methanol (85 : 15 v/v) was chosen. Under these conditions, all 26 tested amphetamines were separated successfully into their enantiomers. Thus, the results showed that all analytes were traded as racemic mixtures.

Quantification of Pyrazinamide in Human Plasma by Validated High Performance Liquid Chromatography Method

Objective: To be able to accurately determine the quantity of Pyrazinamide (PZA) in different tablet preparations and human plasma using an Ultra violet detector equipped high performance liquid chromatography (HPLC). Study Design: Experimental study Place and Duration of Study: Department of Bioequivalence Studies, University of Veterinary and Animal Sciences Lahore and the Department of Pharmacology, University of Health Sciences, Lahore the from 1st April 2017 to 31st March 2018. Methodology: Two mobile phases were used, the first compromised of disodium hydrogen phosphate buffer having a pH of 6.8 and acetonitrile in the proportion of (95:5) and the second was a combination of aforesaid substances in equivalent proportion (50:50 v/v). The gradient for the first 5 min was exclusively Mobile phase “a” after which 5-6 min Mobile phase “b” was raised from 0 to 100% and was kept at 100% till the completion of the cycle. The flow of mobile phase was kept at 1000 µl/min. Determination of PZA was done using a ultraviolet detector at a wavelength of 238 nm. Amount of sample injected was 40 μl. Procedure was done by using Shizmadu Chromatographic System, Japan equipped with a SIL-20AC HT auto-sampler, SPD-M20A, CTO 20 AC, a LC-20AT VP pump, and CBM 20A controller unit. A C18 column was used as well. Results: Retention time of PZA was 6.1±2%. Precision was 0.46 to 2.20% relative standard deviation for intra assay and for inter assay we obtained 0.29 to 34.45% RSD for all quality control levels. The overall recovery of PZA was 96.75%. Conclusion: High selectivity for PZA was seen and no other spikes from drugs present in FDC regimen were observed at the time when PZA is detected in blank plasma samples Key words: Chromatography, High pressure liquid. Pyrazinamide. Tuberculosis

In Vitro Kinetic Hydrolysis Study of Metronidazole Derivatives with Carvacrol and Eugenol Using Validated RP-HPLC Method

Background: Prodrugs principle is widely used to improve the pharmacological and pharmacokinetic properties of some active drugs. Much effort was made to develop metronidazole prodrugs to enhance antibacterial activity and or to improve pharmacokinetic properties of the molecule or to lower the adverse effects of metronidazole. Objective: In this work, the pharmacokinetic properties of some of monoterpenes and eugenol pro metronidazole molecules that were developed earlier were evaluated in-vitro. The kinetic hydrolysis rate constants and half-life time estimation of the new metronidazole derivatives were calculated using the validated RP-HPLC method. Method: Chromatographic analysis was done using Zorbbax Eclipse eXtra Dense Bonding (XDB)-C18 column of dimensions (250 mm, 4.6 mm, 5 μm), at ambient column temperature. The mobile phase was a mixture of sodium dihydrogen phosphate buffer of pH 4.5 and methanol in gradient elution, at 1ml/min flow rate. The method was fully validated according to the International Council for Harmonization (ICH) guidelines. The hydrolysis process carried out in an acidic buffer pH 1.2 and in an alkaline buffer pH 7.4 in a thermostatic bath at 37ºC. Results: The results followed pseudo-first-order kinetics. All metronidazole prodrugs were stable in the acidic pH, while they were hydrolysed in the alkaline buffer within a few hours (6-8 hr). The rate constant and half-life values were calculated, and their values were found to be 0.082- 0.117 hr-1 and 5.9- 8.5 hr., respectively. Conclusion: The developed method was accurate, sensitive, and selective for the prodrugs. For most of the prodrugs, the hydrolysis followed pseudo-first-order kinetics; the method might be utilised to conduct an in-vivo study for the metronidazole derivatives with monoterpenes and eugenol.

Analytical quality by design approach for the control of potentially counterfeit chloroquine with some NSAIDS using HPLC with fluorescence detection in pharmaceutical preparation and breast milk

AbstractChloroquine phosphate (CQ) the antimalarial drug and suggested to treat the pandemic disease coronavirus (COVID-19) is often adulterated with some of the non-steroidal anti-inflammatory drugs (NSAIDs) such as paracetamol, aspirin (ASP), or both. The purpose of this study is to detect such counterfeited drugs, using a reversed phase high pressure liquid chromatography (RP-HPLC) method with fluorescence detection. Analysis was divided into three phases. In the first phase, a Plackett-Burman design (PBD) was used to screen five independent factors, namely, buffer pH, buffer concentration (mM), acetonitrile content (%), flow rate (mL/min) and triethylamine (TEA) content in the buffer preparation (%). The selected dependent variables were (resolution, symmetry of peaks and run time). The objective of the second phase was to optimize the method performance using Box-Behnken design (BBD) and desirability function for multiple response optimization to obtain the best chromatographic performance with the shortest run time. Optimal chromatographic separation was achieved on a YMC-pack pro C18 ODS-A column (15 cm × 4.6 mm, 5 µm) at room temperature The optimum mobile phase consisted of acetonitrile and 5 mM sodium dihydrogen phosphate buffer containing 0.5% triethyamine (30:70, v/v) with the pH adjusted to 3.5 using an orthophosphoric acid solution. The flow rate was maintained at 1 mL/min, and the detection was performed with a fluorescence detector fixed at 380 nm (λemission) after excitation at 335 nm (λexcitation). The third phase was method validation according to ICH guidelines, providing to be specific, precise, accurate, and robust. The method is linear over a range of 0.4–8 µg/mL for chloroquine and ASP, while for paracetamol it is linear over 16–48 µg/mL. The developed RP-HPLC method was used for quantitation of the three drugs in chloroquine dosage form samples. The method shows a great tendency in the classification between the genuine chloroquine and the adulterated ones in pharmaceutical preparations and breast milk.

Bioanalytical Method Development of Atenolol Enantiomers: Stereoselective Behavior in Rabbit Plasma by RP-UFLC Method

Objective: The objective of the method was to develop a new, simple and reliable enantioselective Reverse Phase- Ultra-Fast Liquid Chromatography (RP-UFLC) method for the separation of Atenolol enantiomers. A comprehensive study was performed by extending the work to pharmacokinetic studies using rabbit plasma. Background: Many methods were reported for enantioseparation of Atenolol enantiomers but no attempts were made for chiral separation of Atenolol using rabbit plasma. Moreover, pharmacokinetic data to prove the efficiency of particular enantiomers in rabbit plasma was not studied. Methods: In the present examination, the binary RP-UFLC technique was developed on Phenomenex® Lux cellulose i5 segment (150×4.6 mm, 5μ) using di-sodium hydrogen phosphate buffer (pH 6.8): acetonitrile (35:65 v/v) as the mobile phase. Results: The elution of Atenolol was observed at 225 nm with a stream rate of 1 mL.min-1. The described technique offered a linear relationship with a regression coefficient of r2 = 0.997 and 0.996 for (R) and (S)-enantiomer respectively, between the concentration range of 2-10 ng.mL-1. Atenolol enantiomers were detected at a retention time (tR) of 2.7 min and 3.10 min for R and S-enantiomer respectively. The rate of recovery of both Atenolol enantiomers was observed to be (R) 98.18% and (S) 100.45% individually. USFDA guidelines May 2018 were systematically followed for the development and validation of the bioanalytical method. Conclusion: The developed technique was applied for the separation of Atenolol enantiomers and for the pharmacokinetic determination of Atenolol enantiomers in rabbit plasma.

Determination of A2 β-casein and total β-casein in cow milk and milk powder by capillary zone electrophoresis

β-casein (β-CN) is one of the major casein proteins in cow milk. There are 13 different variants documented for β-CN in cow milk, among which A1 and A2 are the major variants. The separation and quantitation of A2 β-CN are imperative for A2 dairy products. A new capillary zone electrophoresis (CZE) method with UV detection at 214 nm was established for the separation and quantification of the A2 variant and total β-CN content in cow milk and milk powders. The separation of β-CN variants was achieved on bare fused silica capillaries (50 μm×30/40 cm (effective/total length)). The separation buffer was a mixture of 4 mol/L urea, 0.2% (mass fraction) hydroxypropyl methylcellulose, 140 mmol/L citric acid, and 50 mmol/L disodium hydrogen phosphate buffer (pH 2.7). The corrected peak areas and the concentrations of total β-CN and the A2 variant showed good linearity, with correlation coefficients (r2) ranging from 0.9968 to 0.9997. The intra-day precisions for A2 β-CN and total β-CN determination in four samples (two pasteurized milk samples and two milk powder samples) were in the ranges of 2.4%-4.7% and 2.6%-4.8%, respectively. The inter-day precisions for A2 β-CN and total β-CN determination in four samples were in the ranges of 4.0%-6.3% and 3.9%-6.7%, respectively. The recoveries of A2 and total β-CN ranged from 85.5% to 106.4%. With the established CZE method, the A2 β-CN variant and total β-CN protein in liquid and powder bovine milk products could be separated and accurately quantified. By calculating the A2 β-CN content in the total β-CN, the quality of A2 dairy products can be evaluated, and this in turn would aid in the protection of consumer rights.

Application of green methodology to pharmaceutical analysis using eco-friendly ethanol-water mobile phases

Green analysis techniques based on solvent consumption and substitution are two major applications in greening chromatographic analysis. Decreasing the toxicity of solvents used in mobile phases by substituting them with less or non-hazardous ones or revealing the consumed amounts by using lower diameter columns or miniaturizing instruments are some examples that can be implemented to an analysis. Environmentally friendly water and ethanol based mobile phases reduce the use of toxic solvents such as methanol and acetonitrile, and consequently the necessary cleaning of waste is reduced. In the presented study, ethanol having less toxic and hazardous effects has been selected as organic modifier. Famotidin, Paracetamol and Thiocolchicoside were selected molecules for demonstration of the applicability of green HPLC in pharmaceutical analysis. Experiments were carried out using an LC system connected to a Diode Array Detection (DAD) at 254 nm. C8 (150 × 4.6 mm, 5 µm) analytical column was tested as stationary phases. In the mobile phase optimization, ethanol and sodium dihydrogen phosphate buffer contents were adjusted to the desired concentrations for the elution of selected analytes both in isocratic and gradient elution modes. Initial flow rate of the mobile phase was set to 1.0 mL/min and the injection volume was 20 µL. Developed mobile phase consisted of sodium dihydrogen phosphate (pH 4.6; 50 mM), and ethanol in gradient elution mode. All three active pharmaceutical ingredients were well separated both form baseline and each other with the capacity factors of 2.14, 2.53 and 4.26, respectively. Caffeine was selected as internal standard.The developed EtOH based mobile phase method in RP-HPLC was validated in terms of ICH requirements and found to be selective, linear, sensitive, accurate, precise, repeatable, rugged and robust. Developed method was also successful in pharmaceutical analysis of famotidin, paracetamol and thiocolchicoside from Turkish drug market. The findings of the presented study suggested that environmentally friendly ethanol and water based mobile phases could successfully apply in the pharmaceutical analysis.

Optimized and Validated Stability Indicating RP-HPLC Method for Estimation of Nadolol

A stability indicating RP-HPLC method for the determination of nadolol was developed and validated using Enable C18 (250 mm × 4.6 mm, 5 μm) column with mobile phase consisting of methanol, acetonitrile and 0.01 M sodium dihydrogen phosphate buffer pH 7.0 (60:15:25 v/v). The spectrophotometric detection was carried out at 220 nm and the retention time of nadolol was found to be 4.6 min. Forced degradation studies were performed as per ICH guidelines under acid/alkaline hydrolytic, oxidative, thermal, and photolytic conditions, and the degradation products were separated chromatographically. Nadolol was found to be susceptible towards acidic and alkaline hydrolysis and oxidation, while it was stable to thermal and photolytic degradation. The acid hydrolytic degradation study was optimized using 33 factorial design to achieve 5 – 25% desired drug degradation. The proposed method was validated as per ICH guidelines. Linearity was observed in the range of 100 to 500 μg/mL (R2 = 0.999). The drug recovery was within 98.0 – 99.8%. The LOD and LOQ values for the drug were found to be 0.21 and 0.66 μg/mL, respectively.

Extraction and determination of pyridinium ionic liquid cations in environmental water samples by using strong cation exchange solid phase extraction and ion chromatography

ABSTRACTThis paper presents a novel analytical method for the determination of pyridinium ionic liquid cations in environmental water samples by ion chromatography coupled with solid phase extraction. The water samples were purified and enriched by a sulphonic acid extraction column, and then the impurities were washed with 10 mL water – 20% (v/v) acetonitrile solution and the analytes eluted with 0.5 mol L−1 phosphoric acid – sodium dihydrogen phosphate buffer solution – 55% methanol. The eluted analytes were determined by ion chromatography with 3.0 mmol L−1 methane sulphonic acid – 8% acetonitrile as the mobile phase and direct conductivity detection. Three pyridinium cations were completely separated in 20 min and the retention mechanism basically conforms to the ion exchange mode. The newly developed method has been successfully applied to the analysis of pyridinium cations in spiked environmental water samples, which provided satisfactory recoveries in the range of 98.8% to 100.0% with the relative standard deviations less than 2.8%. The concentration of 0.004 to 0.01 mg L−1 in water samples can be preconcentrated by this method. The method is accurate, reliable, simple and practical. This research provides a new reference for detecting ionic liquid cations in environmental water samples and studying the environmental risk assessment of ionic liquids.

Green micellar HPLC-fluorescence method for simultaneous determination of metoprolol and amlodipine in their combined dosage form: Application on metoprolol in spiked human plasma

A fast, sensitive and reliable green micellar liquid chromatographic method has been developed and validated for simultaneous determination of metoprolol (MET) and amlodipine (AMD). Separation was performed on X-Bridge ODS column (150 × 4.6 mm, 5 μm particle size) using a micellar mobile phase consisting of 100 mM sodium dodecyl sulfate, 20 mM sodium dihydrogen phosphate buffer pH 3.0 and 10% v/v n-butanol. The fluorescence detector was set at 275/ 303 nm (excitation/ emission) for MET and 364/ 455 nm (excitation/emission) for AMD. A linear response was obtained over the ranges of 0.1–10 μg/mL and 0.2–2 μg/mL for MET and AMD, respectively. The method reached limits of detection of 20 ng/mL for MET and 50 ng/mL for AMD. The proposed method was successfully employed for the determination of the two drugs in their combined pharmaceutical dosage form and in addition, it was extended for the determination of MET in spiked human plasma. Furthermore, the green character of the developed method was evaluated using the new tool “green analytical procedure index (GAPI)”. The developed method was verified to be an excellent green method.