Reversed-phase High-performance Liquid Chromatography System Research Articles

Nanoscale Pluronic® micellar templates with varying %EO content for controlled drug release and cytotoxicity

This study investigates the self-assembly in ethylene oxide (EO)-propylene oxide (PO)-based block copolymers (BCPs) with various molecular features like molecular weight, EO/PO ratio, HLB (Hydrophobic lipophilic balance) in aqueous solution environment. Our BCP solution systems exhibit significant changes in solution behaviour that showed micellization, micelle growth/ transition, and progressively phase separation (2ϕ), all of which are well explained by clear solution, blue point (BP) and cloud Point (CP) respectively. The solubilization of the highly hydrophobic anticancer drug-Quercetin (QCT) in such BCPs is examined using UV–Visible spectroscopy. The spectral findings inferred the dissolution capability of QCT in the examined copolymeric micellar systems in terms of drug loading efficiency (DL%), encapsulation efficiency (EE%), partition coefficient (P), and standard free energy of solubilization (ΔGo). Amongst the varied tested BCPs, it was observed that Pluronics® P123 and F127 exhibited an enhanced QCT solubilization capability than others and is explained in terms of hydrophobicity and hydrophilicity. The micellar size distribution profile expressed as hydrodynamic diameter (Dh) was determined for QCT-loaded and QCT-unloaded BCP micelles employing dynamic light scattering (DLS). The drug release profile was fitted employing various kinetic models, allowing this study to serve as an excellent foundation for QCT delivery. Reversed-phase High-performance liquid chromatography (HPLC) system determined the retention period in the QCT-loaded micelle while the structural alterations involved in Pluronics®-QCT system is inferred using small-angle neutron scattering (SANS). Fourier transform infrared spectroscopy (FT-IR) depicted the compatibility between Pluronics® and QCT which was validated further from the evaluated optimum descriptors using Gaussian 09 computational simulation framework. It was discovered that the QCT-loaded micelles exhibited a greater anticancer effect than free drug when tested in vivo on cancer cells. The anticancer activity of QCT-loaded F127 micelles was determined to be the strongest. Thus, the current study on QCT solubilization in Pluronics® will benefit considerably from its investigated outcomes.

P076 The promising antimycotic activities of a novel cyclic antifungal lipopeptide against Human Dermatophyte Isolates

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTo determine the minimum inhibitory concentrations (MICs) of a novel antifungal lipopeptide against clinical isolates of dermatophytes of human origin.MethodsTo perform antifungal susceptibility testing (AFST) by CLSI microbroth dilution method, a reversed-phase high-performance liquid chromatography (RP-HPLC) purified lipopeptide of 1071.4 Da from a wild-type soil isolate Bacillus subtilis was used and compared with the standard allylamine terbinafine MICs. Briefly, 1200 ml of cell-free culture supernatant was extracted using a solvent mixture and silica gel (230-400 mesh size)-based adsorption chromatography. The semi-preparative RP-HPLC system consisted of an Agilent quaternary pump and a variable wavelength detector equipped with a Phenomenex Luna C18 column (10 mm × 250 mm, 5 μm). The solvent system for RP- HPLC was (A) water with 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile containing 0.1% TFA. The gradient of solvent B used for purification was as follows: 0%-54% for 0-20 min at the flow rate of 1 ml/min, 54%-60% from 20-48 min at 0.5 ml/min, 60%-100% from 48-58 min at 0.5 ml/min, 100%-0% from 58-65 min at 1 ml/min and monitored at 210 nm.ResultsSuperficial skin infections are caused by dermatophytes including Trichophyton spp. Nowadays, resistance to terbinafine in Trichophyton spp. isolates with higher MICs have been documented in India. We report here the antifungal prowess of a novel small antifungal lipopeptide against 20 clinical isolates of Trichophyton spp. of human origins (from human skin scrapings and nails) with the clinical diagnosis of tinea corporis/cruris and tinea unguium. The representative photographs of T. tonsurans, T. rubrum, and T. mentagrophytes complex are provided below. A total of 6 isolates of T. mentagrophytes and T. rubrum carry point mutations at F397L of squalene epoxidase (SQLE) protein. The in vitro antifungal efficacies determined by AFST revealed that the lipopeptide showed less or equivalent MICs (100% inhibition) in the case of five dermatophytes. The lipopeptide drug has exhibited improved MICs against two T. mentagrophytes complex and two T. rubrum isolates with amino acid substitution F397L in SQLE protein. Trichophyton mentagrophytes complex and T. rubrum with F397L mutations were inhibited at MICs 4-32 μg/mL of terbinafine. In comparison, the lipopeptide showed 4-16 μg/ml (100% inhibition). In the case of all four Trichophyton tonsurans, the MICs ranged between 2-4 μg/ml for the lipopeptide.ConclusionsThe broad-spectrum lipopeptide showed promising antifungal activity against dermatophytes and may be considered for nano-emulsion formulation and tested for topical application in a mice model.

Determination of Glufosinate-P-Ammonium in Soil Using Precolumn Derivation and Reversed-Phase High-Performance Liquid Chromatography

This study developed an analytical method to quantify glufosinate-P-ammonium (GLUF-P) in farmland soil using a reversed-phase high-performance liquid chromatography (HPLC) system with a fluorescence detector after derivatization. GLUF-P in farmland soil was extracted with a mixed alkaline solution and was further derivatized with 9-fluorenyl methyl chloroformate (FMOC) at 25 °C for 1 h. The derivatives were separated with an ACE-C18 column, gradient eluted with a mobile phase A of acetonitrile and a mobile phase B of 0.2% phosphoric acid solution, and finally determined by high-performance liquid chromatography (HPLC) with fluorescence detection at an excitation wavelength of 254 nm and an emission wavelength of 279.8 nm. The limits of detection (LODs) in the four types of soil ranged from 0.004 to 0.015 mg/kg, and the limits of quantification (LOQs) ranged from 0.0125 to 0.05 mg/kg. The mean recoveries of GLUF-P ranged from 94% to 119.8%, and the relative standard deviations (RSDs) varied between 2.8% and 9.0% when the spiked concentrations of GLUF-P were 0.1 mg/kg and 1.0 mg/kg, respectively. The coefficients of regression for the linearity equation were more than 0.99. The proposed method had high sensitivity and could be used for the determination of GLUF-P residues in farmland soil.

Modified aqueous mobile phases: A way to improve retention behavior of active pharmaceutical compounds and their impurities in liquid chromatography

Most commonly used analytical technique for determination of active pharmaceutical ingredients and their impurities in quality control throughout all phases of drug research, development and manufacture is definitely reversed-phase high performance liquid chromatography (RP-HPLC). However, pharmaceutical industry professionals are often faced with various challenges in RP mode, which cannot be resolved with common variations in the composition of the mobile phase. These challenges often occur when analyzing compounds that contain basic ionizable groups, possess large differences in polarities and require consumption of high amounts of toxic organic solvents. Among available strategies for addressing the aforementioned issues, the most convenient one includes RP-HPLC mobile phase modifications by an addition of the proper chemical compounds. In that respect, RP-HPLC method can be easily adapted to the needs of the analysis without time-consuming and expensive equipment procurement. In this review the chaotropic chromatography, micellar liquid chromatography, and cyclodextrin modified RP-HPLC systems are presented and discussed in details. Special attention is devoted to the theoretical background, the possibility of retention modeling and applications in various fields of pharmacy, as well as their prospective in further research.

SIMULTANEOUS ESTIMATION OF CLONAZEPAM AND METRONIDAZOLE IN PHARMACEUTICAL TABLETS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY MODE WITH UV DETECTION

Clonazepam (CLO) plays a significant role in treating seizures, myoclonic seizures, and other clinical infections, while metronidazole (MTZ) is an antiprotozoal, antibacterial drug. The reversed-phase high performance liquid chromatographic (RP-HPLC) technique has provided greater precision and sensitivity over other methods, especially the colorimetric and spectrophotometric. This study aimed to implement a simple method in pure and pharmaceutical tablets for the simultaneous determination of CLO and MTZ. The RPHPLC method's development and optimization for validating a separation method for the simultaneous estimation of both drugs in pharmaceutical formulation involved studying the optimum mobile phase, buffer concentration, and the pH value. Under the chromatographic conditions, RP-HPLC system achieved excellent separation on a Phenomenex HyperClone BDS (250 x 4.60 mm, 130A, and 5μ) at a temperature of 45°C and the following conditions: 65:35% acetonitrile:acetic acid and sodium acetate buffer solution (NaOAc/HAc) as mobile phase (pH 3.5); 20 μL injection volume at a flow rate of 0.75 mL/min and detection wavelength of 310 nm. The proposed RP-HPLC procedure demonstrated high precision (RSD% 1%), as well as the good linear relationship of the calibration graph at concentration ranges of 50-160, 35-100 ppb with a coefficient of determination (r2) of the regression line of 0.9996 and 0.9997 for metronidazole and clonazepam respectively. The proposed method offered excellent validated values for both LOD (4.24 and 3.06 ppb) and LOQ (14.15– 10.21 ppb) for both metronidazole and clonazepam drugs, respectively. The RP-HPLC method has been successfully applied to estimate metronidazole and clonazepam in the well-known commercial pharmaceutical tablets and gave an excellent recovery> 98% for both drugs. The results of the method was compared with the standard pharmacopeial method for both drugs using statistical tests, which indicated no difference in accuracy between the methods

Deep eutectic solvent-based liquid-phase microextraction coupled with reversed-phase high-performance liquid chromatography for determination of α-, β-, γ-, and δ-tocopherol in edible oils.

For simultaneous analysis of four fat-soluble tocopherols (α-, β-, γ-, and δ-) in edible oils, an efficient and green method using deep eutectic solvent-based liquid-phase microextraction (DES-LPME) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) was developed. The DESs formed by different quaternary ammonium salts and ethanol were used as the extractants. Tetrabutylammonium chloride (TBAC)-ethanol DES at a molar ratio of 1:2 achieved the best extraction efficiency. Under the optimized conditions, the detection limits were in the range of 2.1-3.0ngmL-1. The intra-day and inter-day repeatability were in the ranges of 3.9-5.3% and 4.8-7.1%, respectively, and the recoveries for the real samples varied from 80.7% to 105.4%. The developed method was successfully employed for the determination of all four tocopherol homologues with an RP-HPLC system containing a COSMOSIL π-NAP column in five edible oils collected locally. Graphical abstract.

VALIDATION OF A BIOANALYTICAL REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE QUANTITATION OF NYSTATIN IN AN ANIMAL MODEL AFTER INTRANASAL IN SITU GEL ADMINISTRATION

Objective: The aim of the study was to develop and validate a bioanalytical reverse-phase high-performance liquid chromatographic (HPLC) method for the estimation of nystatin in rat plasma after intranasal administration.
 Methods: The reversed-phase HPLC system was equipped with a Luna C18 column, the mobile system comprised of methanol, water, and dimethylformamide (55:30:15) and the flow rate was set at 0.9 ml/min.
 Results: The elution time for nystatin was 4.096±0.025 min. The calibration curves constructed in rat plasma were linear from 0.25 to 50 μg/ml. The lower limit of quantification (LOQ) was found to be 0.25 μg/ml. The standards for accuracy and precision of the intra- and inter-day variation studies were in the acceptable ranges as per the FDA guidelines.
 Conclusion: The LOQ value determined by the proposed method was noted to be satisfactory for inspecting the plasma pharmacokinetics of nystatin in rats’ post-administration of a nasal in situ gelling liquid crystalline precursor formulation in an in vivo study.

PH‐adjusted solvent extraction and reversed‐phase HPLC quantification of isoflavones from soybean (Glycine max (L.) Merr.)

Soybeans, Glycine max (L.) Merr., are among the most important food crops worldwide. Isoflavones are major bioactive phytochemicals in soybeans, and have a variety of health benefits, including antioxidative, antiatherosclerotic, antiinflammatory, and weak estrogen-like effects. The isoflavone content and composition of soybeans vary according to the cultivar and the extraction solvent conditions. Therefore, we investigated the effects of three different solvent pHs (1.0, 5.5, and 10.0) on the isoflavone, total phenolic, and total flavonoid contents and antioxidant capacities of eight soybean cultivars developed in Korea. Twelve isoflavones in soybeans were efficiently separated and identified on a reversed-phase high-performance liquid chromatography (HPLC) system. The percentage distribution of isoflavones measured by HPLC in the eight soybean cultivars at various extraction pHs decreased as follows: malonyl isoflavones (67.2% to 81.3%) > isoflavone glucosides (16.2% to 29.0%; as nonacylated form) > acetyl isoflavones (1.6% to 5.9%). The highest contents of isoflavone glucosides, malonyl derivatives, and acetyl derivatives were extracted at solvent pHs of 10.0, 1.0, and 5.5, respectively. The solvent extraction at pH 1.0 yielded a lower total isoflavone content than those at pHs 5.5 and 10.0. However, the highest total phenolic and flavonoid contents were extracted from soybeans at pH 1.0. Soybeans extracted at pH 10.0 displayed the highest antioxidant capacities in the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical assay. Taken together, these results suggest that proper solvent pH adjustment is needed to maximize the extraction of targeted forms of isoflavones from soybeans. PRACTICAL APPLICATION: Soybeans contain a variety of bioactive compounds, including isoflavones, which function as antioxidants and weak phytoestrogens. Chemical and instrumental analyses can facilitate the selection of soybean cultivars with high amounts of isoflavones for soybean breeding and isoflavone-enriched product development. Proper solvent pH adjustment allows for the efficient extraction of high amounts of targeted isoflavone subgroups (acetyl and malonyl forms) from soybeans for functional food products.

SIMPLE AND RAPID HPLC METHOD FOR SIMULTANEOUS QUANTIFICATION OF LEVODOPA AND CARBIDOPA IN A FAST DISINTEGRATING TABLET FORMULATION

Objective: Fast disintegrating tablets (FDTs) are found helpful in dysphagia (difficulty in swallowing) especially in Parkinson patients. Levodopa is still the first choice in Parkinson disease treatment and is co-administered by carbidopa for better efficacy.
 Methods: In the present study, a rapid and simple isocratic Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for simultaneous quantification of levodopa and carbidopa in optimized Fast Disintegrating Tablets (FDTs). The linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) of the method were determined. FDTs were prepared using direct compression, dry and wet granulation and were optimized for faster disintegration time. Tablets thickness, weight, hardness, friability, drug content and dissolution profile were also evaluated.
 Results: A RP-HPLC system with C18 column and mobile phase 90:10 (v/v) phosphate buffer: methanol was used. The method linearity was found to be within the concentration range of 3.125-50 μg/ml for levodopa, and 3.125-25 μg/ml for carbidopa. The intra and inter-day precision and accuracy were acceptable. LOD and LOQ of levodopa-carbidopa were 0.2-0.8 μg/ml and 0.5-2.4 μg/ml, respectively. The total chromatographic run time was 5 min. The optimized FDTs hardness was 3.81±0.4 and tablets were disintegrated within 30 sec. Levodopa and carbidopa were dissolved in dissolution media within 5 min.
 Conclusion: Results indicated that this method was suitable for simultaneous quantification of levodopa and carbidopa in the presence of different ingredients of a pharmaceutical solid dosage form. Therefore, this method could be applied in pharmaceutical quality control for rapid quantification of structurally similar substances with different physicochemical properties.

Reverse phase high-performance liquid chromatography (RP-HPLC) system for analysis of vitamin D3 soft capsules with soybean oil matrix

ObjectiveTo develop a method for the determination and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in reverse phase high performance liquid chromatography (RP-HPLC) system. MethodsRP-HPLC system had Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) with detection wavelength of 265 nm, column temperature of 25 ℃ and flow rate of 1 mL/min. Retention behaviors of vitamin D3 and its 3 isomers were studied by altering the mobile phase. Firstly, acetonitrile was mixed with different proportions of methanol, water and ethanol as the mobile phase to investigate the effects of these 4 mobile phase components on the retention behavior of vitamin D3 and its 3 main related substances (isomers) on a C18 column. Then, a suitable mobile phase was selected for content determination and impurity analysis according to the retention behavior study. ResultsThe recovery was only 80.55%-84.37% with 100% acetonitrile as the mobile phase.The addition of ethanol in acetonitrile was found to make remarkably significant improvement. Recovery rate was achieved between 98.07% and 103.23% with V (acetonitrile):V (ethanol)=90:10 as the mobile phase, while improving peak shape. The method showed good linearity [(0.52-5.2)×10-4 μmol/L, R2>0.999] and fine density (RSD < 2.32%) which can be used for determination. For impurities profile, it could be achieved using V (acetonitrile):V (water)=95:5 as the mobile phase which can obviate interference from soybean oil matrix. ConclusionsThe method established in this experiment can easily and accurately determine the content and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in a RP-HPLC system.

Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most popular chromatographic mode, accounting for more than 90% of all separations. HPLC itself owes its immense popularity to it being relatively simple and inexpensive, with the equipment being reliable and easy to operate. Due to extensive automation, it can be run virtually unattended with multiple samples at various separation conditions, even by relatively low-skilled personnel. Currently, there are >600 RP-HPLC columns available to end users for purchase, some of which exhibit very large differences in selectivity and production quality. Often, two similar RP-HPLC columns are not equally suitable for the requisite separation, and to date, there is no universal RP-HPLC column covering a variety of analytes. This forces analytical laboratories to keep a multitude of diverse columns. Therefore, column selection is a crucial segment of RP-HPLC method development, especially since sample complexity is constantly increasing. Rationally choosing an appropriate column is complicated. In addition to the differences in the primary intermolecular interactions with analytes of the dispersive (London) type, individual columns can also exhibit a unique character owing to specific polar, hydrogen bond, and electron pair donor-acceptor interactions. They can also vary depending on the type of packing, amount and type of residual silanols, "end-capping", bonding density of ligands, and pore size, among others. Consequently, the chromatographic performance of RP-HPLC systems is often considerably altered depending on the selected column. Although a wide spectrum of knowledge is available on this important subject, there is still a lack of a comprehensive review for an objective comparison and/or selection of chromatographic columns. We aim for this review to be a comprehensive, authoritative, critical, and easily readable monograph of the most relevant publications regarding column selection and characterization in RP-HPLC covering the past four decades. Future perspectives, which involve the integration of state-of-the-art molecular simulations (molecular dynamics or Monte Carlo) with minimal experiments, aimed at nearly "experiment-free" column selection methodology, are proposed.

A two-dimensional non-comprehensive reversed/normal phase high-performance liquid chromatography/tandem mass spectrometry system for determination of limonene and linalool hydroperoxides

A two-dimensional non-comprehensive high-performance liquid chromatographic (HPLC) system coupled to electrospray ionization tandem mass spectrometry was developed for the determination of skin allergenic hydroperoxides of limonene and linalool. These compounds are some major components behind skin sensitization and contact (skin) allergy to fragrances.Fragrance hydroperoxides usually occur in complex compositions, often as constituents of the natural essential oils added to a large number of commercial products. Their similarities to interfering compounds, many with identical elemental composition, make the determination difficult even when using selective detection methods like mass spectrometry. In this work, a first-dimension chromatographic heart-cut isolation of the hydroperoxides on a reversed-phase HPLC system was combined with a second-dimension normal-phase HPLC system for separation of the hydroperoxides. The intersystem transfer was made by trapping the heart-cut fraction on a short graphitized carbon column, exchanging the mobile phase and back-flushing the hydroperoxides into the second dimension.Each analysis was performed within 60 min without any pretreatment, except dilution, prior to injection. The obtained instrumental limits of detection (LODs) at a signal-to-noise ratio of 3 were lower than 1.2 ng injected on column and method LODs were below 0.3 ppm. An after-shave product was shown to contain the highest concentrations of the measured hydroperoxides, with 445 ± 23 ppm of total linalool hydroperoxides. This level is likely able to elicit skin reactions in already sensitized individuals.

Lepiota cristata does not contain amatoxins or phallotoxins

It is believed that Lepiota cristata is inedible and slightly poisonous, so it should always be avoided. This study analyzed the alpha, beta and gamma amanitin, phalloidin (PHN) and phallacidin (PCN) content of L. cristata collected from different regions of Turkey. Toxin levels have been assessed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system with ultraviolet detectors. In this study, the L. cristata mushroom was not found to contain any amatoxins or phallotoxins. However, this finding does not mean that this mushroom is edible. New studies regarding the toxin content of L. cristata may be useful to understand the toxicity of this species.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD VALIDATION OF α-MANGOSTIN ASSAY IN MANGOSTEEN (Garcinia mangostana L.) FRUIT RIND EXTRACT FORMULATED IN ORAL SOLUTION

&lt;p&gt;Mangosteen fruit rind extract contain a lot of antioxidants. α-Mangostin is a component in mangosteen fruit rind that has highest antioxidant effect. The oral solution containing mangosteen fruit rind extract is required an assay method for quality assessment. Determination of a very low concentration of analyte in sample with very complex matrix, such as α-mangostin in oral solution, needs a selective and sensitive method, such as high performance liquid chromatography (HPLC). In this study, α-mangostin assay was performed by reverse phase HPLC system using octadecylsilane (C18) as stationary phase, methanol-water (90:10) as mobile phase, the flow rate is 1.0 mL/min, and the UV detector at 316 nm. The retention time of α-mangostin was 9.622 minutes. Peak of α-mangostin was well separated with resolution of 1.725. Linearity was in the range of 1.67-5.01 ppm with correlation coefficient of 0.9986. The relative standard deviation (RSD) was 1.30 %, the recovery was in the range of 95.80-100.76 &lt;/p&gt;

Influence of carboxylic ion-pairing reagents on retention of peptides in thin-layer chromatography systems with C18 silica-based adsorbents

One of the main problems related to chromatography of peptides concerns adverse interactions of their strong basic groups with free silanol groups of the silica based stationary phase. Influence of type and concentration of ion-pairing regents on peptide retention in reversed-phase high-performance liquid chromatography (RP-HPLC) systems has been discussed before. Here we present influence of these mobile phase additives on retention of some peptide standards in high-performance thin-layer chromatography (HPTLC) systems with C18 silica-based adsorbents. We prove, that due to different characteristic of adsorbents used in both techniques (RP HPLC and HPTLC), influence of ion-pairing reagents on retention of basic and/or amphoteric compounds also may be quite different. C18 silica-based HPTLC adsorbents provide more complex mechanism of retention and should be rather considered as mixed-mode adsorbents.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD VALIDATION OF α-MANGOSTIN ASSAY IN MANGOSTEEN (Garcinia mangostana L.) FRUIT RIND EXTRACT FORMULATED IN ORAL SOLUTION

&lt;p&gt;Mangosteen fruit rind extract contain a lot of antioxidants. α-Mangostin is a component in mangosteen fruit rind that has highest antioxidant effect. The oral solution containing mangosteen fruit rind extract is required an assay method for quality assessment. Determination of a very low concentration of analyte in sample with very complex matrix, such as α-mangostin in oral solution, needs a selective and sensitive method, such as high performance liquid chromatography (HPLC). In this study, α-mangostin assay was performed by reverse phase HPLC system using octadecylsilane (C18) as stationary phase, methanol-water (90:10) as mobile phase, the flow rate is 1.0 mL/min, and the UV detector at 316 nm. The retention time of α-mangostin was 9.622 minutes. Peak of α-mangostin was well separated with resolution of 1.725. Linearity was in the range of 1.67-5.01 ppm with correlation coefficient of 0.9986. The relative standard deviation (RSD) was 1.30 %, the recovery was in the range of 95.80-100.76 &lt;/p&gt;

Development of simple and sensitive HPLC method for determination of glyphosate residues in soybean

The purpose of this study was to develop a simple and sensitive high performance liquid chromatography (HPLC) method for determination of glyphosate (GP) residues in soybean grains. From soybean matrix, glyphosate was extracted with a mixture of water and methanol (4:1, v/v) from soybean samples followed by protein precipitation with equal volume of methanol. No preconcentration and further clean up of the sample were required. Pre-column derivatization was carried out with excess amount of 9- fluorenylmethyl chloroformate (FMOC-Cl) in the presence of borate buffer. The gradient program developed in this method was successfully applied to a reverse phase HPLC system with a C18 column (ACE 5 μm 4.6 x 250 mm), and eluted with a mobile phase consisting of 50 mM phosphate buffer, pH 2.5, and acetonitrile at the flow rate of 0.8 ml/min and fluorescence detection. Parameters and conditions affecting extraction, derivatization reaction and chromatographic separation were systematically examined. Linearity of the method ranged from 0.005 - 1.0 μg/ml. The correlation coefficient (r2) of calibration curve for glyphosate in soybean sample was found to be 0.99929. The limit of detection (LOD) and limit of quantitation (LOQ) values were determined to be 0.125 mg/kg and 0.25 mg/kg, respectively. Average recovery was 95.2%. Repeatability and intermediate precision calculated on the basis of peak area were excellent and showed relative standard deviation ranged from 0.15 - 1.29% and 1.15 - 3.87%, respectively. The developed method has been successfully applied for determination of glyphosate residues in soybean grains obtained from Thailand and Nepal. Soybean samples (53) from two different lots were analyzed and glyphosate residues ranged from 0.23 mg/kg to 5.06 mg/kg. Almost 50% soybean samples contained nearly consistent residue levels in both lots but in remaining samples there was a significant variation of glyphosate levels between two lots. Relatively higher residues were detected in samples from Thailand (0.27-5.06 mg/kg) compared to Nepal (0.23-0.99 mg/kg). The results suggest that the proposed method can be used to determine glyphosate residues in foods derived from soybean and other crops such as corn, cotton, wheat, etc. where glyphosate is widely applied to these crops.

Theoretical Models and QSRR in Retention Modeling of Eight Aminopyridines.

In this article, retention modeling of eight aminopyridines (synthesized and characterized at the Faculty of Pharmacy) in reversed-phase high performance liquid chromatography (RP-HPLC) was performed. No data related to their retention in the RP-HPLC system were found. Knowing that, it was recognized as very important to describe their retention behavior. The influences of pH of the mobile phase and the organic modifier content on the retention factors were investigated. Two theoretical models for the dependence of retention factor of organic modifier content were tested. Then, the most reliable and accurate prediction of log k was created, testing multiple linear regression model-quantitative structure-retention relationships (MLR-QSRR) and support vector regression machine-quantitative structure-retention relationships (SVM-QSRR). Initially, 400 descriptors were calculated, but four of them (POM, log D, M-SZX/RZX and m-RPCG) were included in the models. SVM-QSRR performed significantly better than the MLR model. Apart from aminopyridines, four structurally similar substances (indapamide, gliclazide, sulfamethoxazole and furosemide) were followed in the same chromatographic system. They were used as external validation set for the QSRR model (it performed well within its applicability domain, which was defined using a bounding box approach). After having described retention of eight aminopyridines with both theoretical and QSRR models, further investigations in this field can be conducted.

Abstract 4627: Fasting reduces the systemic exposure to irinotecan and its active metabolite SN-38

Abstract Limitations to the use of chemotherapeutic agents are the often severe adverse side-effects which may lead to early discontinuation of treatment. Most anticancer agents have a narrow therapeutic index. In previous work we have shown that 72 hours of fasting prior to treatment with a high dose of irinotecan prevents the occurrence of adverse side effects in C26 colon carcinoma bearing mice, while the antitumor activity is not abrogated. To elucidate the mechanism of fasting induced resistance against adverse side effects, we have examined the pharmacokinetics of irinotecan in 72 hours fasted mice in plasma and liver. Male BALB/c mice were divided into four groups (n=18/group). Two groups were fasted for 72 hours and two groups were fed ad libitum. After the fasting period, mice were fed ad libitum again. One group of ad libitum and fasted animals was treated with 50 mg/kg and the other with 100 mg/kg irinotecan intraperitoneally. Plasma and liver were collected at 1,4,8,12,24 and 48 hours after irinotecan injection. Tissues were homogenized in lithium heparinized plasma and concentrations of irinotecan (CPT-11) and the active metabolite SN-38 were determined using a validated reversed-phase high-performance liquid chromatography (HPLC) system with fluorescence detection. Pharmacokinetic parameters, including peak concentration, area under the plasma concentration-time curve (AUC), clearance (CL), and the half-life of the terminal disposition phase were calculated. In the fasted group, plasma levels of SN-38 in the 50 mg/kg and 100 mg/kg group showed a 57% and 53% reduction, respectively, compared with ad libitum fed controls (AUC0-inf 5,149 vs. 2,242 ng*h/mL and 7,507 vs. 3,562 ng*h/mL). For CPT-11 the effects of fasting were smaller. An increase of 26% in the 50 mg/kg group and a reduction of 15% in the 100 mg/kg group were observed (AUC0-inf 9,715 vs. 12,235 and 37,360 vs. 31,924 ng*h/mL). In the liver, SN-38 levels in the 50 mg/kg and 100 mg/kg group showed a reduction of 51% and 30%, respectively, in the fasted animals (AUC0-inf 85.4 vs. 41.5 µg*h/g and 126 vs. 87.6 µg*h/g), while CPT-11 levels were reduced with 19% in the 50 mg/kg group and 28% in the 100 mg/kg group (AUC0-inf 218 vs. 177 µg*h/g and 712 vs. 512 µg*h/mg). Our data demonstrate that 72 hours of fasting prior to irinotecan administration induces an important change in its metabolism. Plasma and liver levels of the pro-drug CPT-11 were only slightly reduced in fasted animals. Levels of the active metabolite SN-38 were considerably lower in fasted animals compared to ad libitum fed animals. These data suggest that the reduction of side effects by fasting is due to the lower systemic exposure to SN-38, and may have important clinical implications if also found in humans. Citation Format: Sander A. Huisman, P de Bruijn, I.M. Ghobadi Moghaddam-Helmantel, J.N.M. IJzermans, E Wiemer, A.H.J. Mathijssen, R.W.F. de Bruin. Fasting reduces the systemic exposure to irinotecan and its active metabolite SN-38. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4627. doi:10.1158/1538-7445.AM2014-4627

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid-liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30-1000ng for nicotine and 0.06-1000ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8-15ng/mL and 156-372ng/mL, respectively.