HPLC Conditions Research Articles

Study of chromatographic behavior of antibiotic drugs and their metabolites based on quantitative structure-retention relationships with the use of HPLC-DAD

The separation of eleven antibiotics and ten metabolites were studied using high performance liquid chromatography. The C18-PFP octadecyl with integral PFP, C18-AR octadecyl with integral phenyl, C18-HL octadecyl and phenyl phase were used as stationary phases. Mixtures of acetontrile–0.1 % formic acid in water were investigated as mobile phases. The elution order of the target compounds was similar for all four HPLC columns applied. The best separation was obtained using the column with the pentafluorophenylpropyl chain. In addition, in order to optimize the parameters of retention elution for the column and to predict the conditions for the best separation of the active compounds studied biologically the ChromSword software was used. To obtain reliable information of the physicochemical properties and to estimate the relative biological activity of a group of the studied analytes, the QSRR approach was applied. Molecular descriptors were calculated using the HyperChem software. The study was based on multiple linear regression and the results were presented as quantitative structure–retention relationships equations. The QSRR models were determined using 16 molecular descriptors mainly related to the dipole moment (μ), the solvent accessible surface area (SAS), the van der Waals surface area (VWS), the minimum charge (δmin) as well as the polar surface area (PSA). Moreover, structural descriptors of the target compounds were used to describe their chromatographic retention behavior under the optimized HPLC conditions.

Oxaliplatin and [Pt(R,R-DACH)(panobinostat-2H)] show nanomolar cytotoxicity towards diffuse intrinsic pontine glioma (DIPG).

We report the synthesis of two novel platinum(ii) complexes which incorporate histone deacetylase (HDAC) inhibitors: [PtII(R,R-DACH)(Sub-H)] (1), [PtII(R,R-DACH)(panobinostat-2H)] (2), where SubH = suberoyl-bis-hydroxamic acid; DACH = (1R,2R)-(-)-1,2-diaminocyclohexane and panobinostat = (E)-N-hydroxy-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enamide. Complexes 1 and 2 were characterised by 1H, 13C, 195Pt NMR spectroscopy and ESI-MS. Whilst oxaliplatin demonstrated considerable cytotoxicity in two patient-derived low-passage paediatric glioma DIPG cell lines (IC50 values of 0.333 μM in SU-DIPG-IV, and 0.135 μM in SU-DIPG-XXI), complex 2 showed even greater cytotoxicities, with IC50 values of 0.021 μM (SU-DIPG-IV), 0.067 μM (BIOMEDE 194) and 0.009 μM (SU-DIPG-XXI). Complex 2 also demonstrated superior aqueous solubility in comparison to panobinostat. Complex 2 released free intact panobinostat under HPLC conditions, as determined by ESI-MS. Incubation of solutions of oxaliplatin (H2O) and panobinostat (DMF) resulted in instantaneous reactivity and precipitation of a panobinostat derivative which was not a platinum complex; the same reactivity was not observed between carboplatin and panobinostat.

Stability Indicating Forced Degradation Studies to Assess Degradation behaviour of Chlordiazepoxide and Amitriptyline Hydrochloride in Pharmaceutical Dosage Form by RP-HPLC

Stability Indicating Forced Degradation Studies was carried out to Assess Degradation Behaviour of Chlordiazepoxide and Amitriptyline Hydrochloride in Pharmaceutical Dosage Form by RP-HPLC. The HPLC conditions consists of methanol Buffer: Acetonitrile: THF (50: 20: 30 v/v/v) mobile Phase, μBondapak C18 column (300 x 3.9 mm, 10 μm particle size), wavelength (254nm). The method was validated according to ICH Guidelines. The validation parameters, linearity range, recovery, robustness and sensitivity was studied. Linearity for Chlordiazepoxide in the range of 50–200μg/mL and R2 Value 0.9999 and Amitriptyline hydrochloride 125–500μg/mL R2 value 0.9998. The percentage recovery of Chlordiazepoxide was found to be 98.75–98.99% and for Amitriptyline Hydrochloride it was found to be 99.66–99.49% and the limit of detections (LOD) are 0.258μg/ml and 0.283μg/ml for Chlordiazepoxide and Amitriptyline hydrochloride, respectively. The developed method is simple, specific, precise and cost effective for estimation of Chlordiazepoxide and Amitriptyline Hydrochloride. Hence developed method was stability indicating and can be used for routine analysis of production samples and also check the stability of bulk and pharmaceutical formulation. The developed method was superior when compared to the reported method with less retention time and composition of the mobile phase with good separation.

Confirmation of organic compounds hydrates formation under the reversed-phase HPLC conditions

The confirmation of the correspondence of the detected forms of analytes to their molecular structures seems to be one of the principal problems of analytical chemistry. The previous analysis of the series of drugs by reversed-phase HPLC allowed revealing that the retention regularities of the complex polyfunctional organic compounds containing sulfonamide groups -SO2-N< differ from those of other compounds. It was explained by reversible formation of their hydrate forms. To verify this proposition, three model N-substituted arylsulfon­ami­des were synthesized, namely N,N-diethyl- (I), N-allyl- (II), and N-phenyl (III). The consideration of the dependencies of these analytes net retention times (tR) in reverse-phase HPLC on the content of orga­nic solvent (C, methanol) in an eluent did not permit us to reveal any regularities or anomalies due to the non-linearity of such depen­den­cies. However, to achieve this, the recurrent appro­ximation was re­com­mended, tR(C + DC) = atR(C ) + b (*), where DC = const – con­stant varia­tions of concentration of methanol (5% in our case), а and b – coefficients calculated by Least Squares Method. If the chemical origin of the analytes at the outlet of the chromatographic column remained the same within concentration range Сmin < С < Сmax, dependencies (*) were linear with correlation coefficients R > 0.999. Yet, if additional chemical transformations of analytes took place, na­me­ly rever­sible formation of hydrates (or variations of their composition) due to the presence of water in an eluent, it led to the deviations of recurrent dependencies (*) from linea­rity. Three sulfonamides under characterization corresponded to different kinds of such deviations: non-linearity within the whole range Сmin < С < Сmax (amide I), presence of two linear dependencies instead of one (amide II), and linearity within the whole range Сmin < С < Сmax (amide III). First two cases corres­ponded to the interconver­si­on of anhydrous forms and hydrates, or (less probable) different hydrates, while the latter meant the existence of analyte in the single form (pro­ba­b­ly hydrate). Thus, the ana­ly­sis of mo­del compounds confirmed that the presence of the polar functionality -SO2-N< was just the reason of hydrate formation in water soluti­ons.

Determining the Levels of Four Phenylethanoid Glycosides and Five Triterpene Acids in Liuwei Dihuang Capsule Using Solid Phase Extraction with HPLC-UV.

In this study, we used quantitative high-performance liquid chromatography equipped with an ultraviolet detector (HPLC-UV) and solid phase extraction (SPE) to determine the levels of four phenylethanoid glycosides and five triterpene acids in Liuwei Dihuang capsules (LDCs). LDCs were methanol-extracted and purified using a 500 mg/6 mL silica-based C18 SPE cartridge. Two elutions were analyzed on a ChromCore C18 column under two HPLC conditions. To improve the pretreatment clean-up, an array of silica- and polymer-based SPE cartridges were compared. Both wash and elution steps were also optimized to achieve the highest removal of impurities. Under optimal chromatographic conditions, good linearity was achieved for all compounds (correlation coefficient of r ≥ 0.999), with a quantification limit ranging from 0.0076 to 0.418 μg/mL. The method had satisfactory efficiency and reproducibility with recovery rates ranging from 91.6 to 99.3% with a relative standard deviation below 1.5%. Taken together, this demonstrated SPE as a suitable extension of HPLC-UV for the determination of phenylethanoid glycosides and triterpene acids in complex LDCs.

Development of a method for preparing octreotide derivative for diagnosis of neuroendocrine tumors

Currently the development of technologies for labeling somatostatin with technetium-99m for diagnosing radionuclide neuroendocrine tumors is under way. Somatostatin analogues are binded with technetium99m only by the preliminary addition of a chelating agent. Therefore, it is important to develop a method for preparation of an octreotide derivative by modifying octreotide with precursors: ligands with high chelating ability for its tight binding with technetium-99m. ω-Bis(pyridin-2-ylmethyl)amino)aliphatic acids can be used successfully as such precursors.The purpose of the study was to develop a method for obtaining a new octreotide derivative for diagnosing neuroendocrine tumors.Materials and methods. The somatostatin octreotide analogue was used as the object of the study; succinimid-1-yl 6-(bis(pyridin-2-ylmethyl)amino)hexanoate was used as a chelating agent. Methods of high performance liquid chromatography and mass spectrometry were used to separate and analyze the synthesized compounds.Results. A method to produce an original octreotide derivative using a succinimid-1-yl 6-(bis(pyridin2-ylmethyl)amino)hexanoate as a chelating agent was proposed. The conditions of analytical and semipreparative HPLC for the analysis and purification of the active octreotide derivative (a monosubstituted derivative of the amino acid residue of D-phenylalanine) were suggested.Conclusion. The synthesized derivative of octreotide has a chelating center for strong binding to technetium-99m in its structure, which can be useful for diagnosing neuroendocrine tumors.

Preparation, optimization and in vitro\u2013in vivo evaluation of Shunxin sustained release granules

BackgroundShunxinzufang decoction is tutors, empirical formula and has been used in Chinese patients of HFpEF for several years. The aim of this study was to make into sustained release granules and select the best formula for the preparation of Shunxin sustained release granules and to evaluate its in vivo and in vitro drug release behavior.MethodsResponse surface methodology and Center composite design were applied to screen the optimal formula of Shunxin sustained release granules. HPLC was used to detect indicative ingredients—paeoniflorin, calycosin-7-glucoside and ferulic acid in Shunxin sustained release granules. The in vitro sustained release character of indicative ingredients was investigated in simulated digestive fluids. In-vivo process of active components was studied through pharmacokinetics.ResultsThe optimal formula of Shunxin sustained release granules consisted of 35% shunxinzufang extract and 65% HPMC/starch (HPMC/starch ratio = 2:1). Three indicative components can be separated well under selected HPLC conditions. Compared with Shunxinzufang extract, the active components of Shunxin sustained release granules have obvious sustained-release character and improved bioavailability.ConclusionShunxin sustained release granules has obvious sustained-release character and improved bioavailability.

HPLC and SFC enantioseparation of (±)-Corey lactone diol: Impact of the amylose tris-(3,5-dimethylphenylcarbamate) coating amount on chiral preparation.

As an important intermediate of prostaglandins and entecavir, optically pure Corey lactone diol (CLD) has great value in the pharmaceutical industry. In this work, the enantioseparation of (±)-CLD was evaluated using high-performance liquid (HPLC) and supercritical fluid chromatography (SFC). In HPLC, the separations of CLD enantiomers on polysaccharide-based chiral stationary phases with both normal phase and polar organic phase were screened. And the conditions for the enantioseparation were optimized in HPLC and SFC, including the selection of mobile phase, temperature, back-pressure, and other conditions. More important, it was found that the chiral resolutions were greatly enhanced by the increase of the coating amount of ADMPC (amylose tris-(3,5-dimethylphenylcarbamate)) under both HPLC and SFC conditions, which can lead to the increase of the productivity and the decrease of the solvent consumption. The preparations of optically pure CLD were evaluated on a semi-preparative (2×25cm) column packed with 30% ADMPC-coated CSP under HPLC and SFC conditions. Preparative performances in terms of kkd are 1.536kg racemate/kg CSP/day and 1.248kg racemate/kg CSP/day in HPLC and SFC, respectively.

Development and Validation of an HPLC Method Using a Less Hazardous Mobile Phase for the Determination of some β‐Lactams

A simple, cost‐effective, and greener HPLC method was investigated to determine 14 β‐lactams. To achieve this, various strategies were implemented such as the use of an aqueous mobile phase, ionic liquids, or an alternative organic solvent. Preliminary experiments were conducted using “one‐factor‐at‐a‐time” to identify the significant factors affecting chromatographic performance. The experimental design (central composite design) was then applied to thoroughly examine the effects of the key factors and exploit them to achieve optimal HPLC condition. The developed method was less hazardous and improved the retention and peak shape, compared with other conventional methods. The optimized assay condition was validated according to the ICH guidelines and effectively applied for the quantification of some β‐lactams in various formulations. These results imply that this novel HPLC method can be employed for routine analyses of some β‐lactams in pure drug and commercial formulations.

Selectivity of the Separation of Isomeric Chlorogenic Acids under the Conditions of Reversed-Phase HPLC

The selectivity of the separation of isomeric chlorogenic acids often found in natural samples (3, 4, and 5-caffeoylquinic acids, 3CQA, 4CQA, and 5CQA, respectively) under the conditions of reversed-phase chromatography on stationary phases of different brands, Kromasil (C18, C8, and C4), Symmetry C18, Reprosil-Pur C18-AQ, Nucleosil RPC18, and Diasfer C18, is studied. The results obtained favor the retention mechanism with predominant surface adsorption. The change in the selectivity of separation of a pair of 5CQA and 4CQA correlates with a change in the activity of residual silanol groups. At their low activity, the order of elution 3CQA < 5CQA < 4CQA does not depend on the mode (isocratic or gradient), the type of organic modifier, and the addition of an acid (formic, orthophosphoric) to the mobile phase. With an increase in the activity of silanol groups, the separation of the pair of 5CQA and 4CQA deteriorates up to the inversion of retention times. A Kromasil 100-5C18 column (250 × 4.6 mm) was chosen for a reliable separation of three acids in plant samples (instant coffee, juice, and “Mate” tea).

Влияние строения производных 1,2,4-триазола на их сорбцию гиперкарбом в условиях ОФ ВЭЖХ

The results of 1,2,4-triazole derivatives studies by high-performance liquid chromatography using water-acetonitrile mixture in various volume ratios as eluent are presented. A porous graphitic carbon – hypercarb was used as a sorbent. It has been shown that the main physicochemical parameters determining the sorption of 1,2,4-triazole derivatives under the conditions of RP HPLC are the lipophilicity, polarizability and the volume of analyte molecules. At the same time, almost all the investigated substances are relatively polar, which contributes to their interaction with the components of the polar mobile phase. In general, among the derivatives of 1,2,4-triazole selected for the study, the values of the molecular volume, polarizability, lipophilicity and dipole moment vary within relatively wide limits, which leads to significant differences in the sorption characteristics of these compounds, which are also determined by the nature of the sorbent. At the same time, a symbiotic relationship was established in changing the values of the retention factor of the studied substances, their polarizability and lipophilicity. The observed deviations from the dependences obtained are due to specific interactions of sorbate molecules with the components of the mobile phase. The effect of temperature on the chromatographic retention of azoles was investigated. The sorption isotherm of 2-(1H-triazol-1-ylmethyl) phenol is obtained, which is almost linear in the initial part, which corresponds to the Henry region. The thermodynamic sorption characteristics of triazole derivatives have been experimentally determined. It is established that the values of changes in standard molar enthalpy and Gibbs energy of sorption during the transition of the molecules of the analyzed analytes from the bulk solution to the surface layer of the hypercarb are predominantly negative, which indicates the exothermic process and its spontaneous flow. At the same time, these values lie in a relatively narrow range and are determined mainly by the nature of the substituent and its position in the heterocycle.

A New Compound (8,9) -Furanyl-Pterocarpan-3-Ol Used for Standardization of Bengkuang (Pachyrhizus erosus) Extract as Sunscreen and Skin Whitening Agent

Bengkuang (Pachyrhizus erosus) has been traditionally used as sun screening and skin whitening. The active compounds in bengkuang extract already published included their activities in antioxidant and skin whitening. However, standardization of bengkuang extract has not been studied. This research aims to find out the analysis procedure by High Performance Liquid Chromatography to make standardization bengkuang extract.The first step of this research was collecting bengkuang from Prembun, Central Java, Indonesia in dry season. After cleaning and peeling, bengkuang root was sliced, dried and ground to make powder. Then followed by extraction using Soxhlet in petroleum ether and subsequently in methanol. Methanol extract was evaporated and then partitioned with ethyl acetate-water. Ethyl acetate fraction was evaporated and then separated in open column chromatography using silica gel as stationary phase and a gradient mixture of chloroform-ethyl acetate-methanol as mobile phase. Bio guided fraction method was used for separation and purification to get isolated compounds. The isolated compounds obtained from this fractionation were then elucidated and analyzed their activities.A new compound (8,9-furanyl-pterocarpan-ol) has been selected as a biomarker for extract standardization. The optimum of HPLC condition for standardization consisted of a column (Zorbax SB-C18; i.d. 0.46 cm; 5 μm particle size), mobile phase (gradient elution of MeOH-water) with flow rate of 1 ml/min and detector (UV-detector at 293 nm). The obtained LOD value was 0.51 ± 0.02 µg. The potentials of this compound to absorb UV ray, antioxidant and anti-tyrosinase were 4.018 mAU*S/mml; 2.113±0.001mM (SC50); 7.19±0.11 mM (IC50), respectively.Keywords : bengkuang (Pachyrhizus erosus) extract, (8,9)-furanyl-pterocarpan-3-ol, standardization, sunscreen, skin whitening

Multi-fingerprint profiling analysis for screening and quantification of illegal adulterated antidiabetics in a functional food using HPLC coupled to diode array detection/fluorescence detection

A method for screening illegal adulterated antidiabetics in bitter melon and ginseng soft gels (BMGSG) was developed by multi-fingerprinting profiling analysis. HPLC fingerprints of BMGSG samples were investigated for chemical feature and similarity evaluation. Based on chemical structure and spectroscopic properties, chromatographic behaviors of common antidiabetics were further studied under different HPLC conditions combining with multi-wavelength monitoring with diode array detector (DAD) and fluorescence detector (FLD) to highlight and discriminate illegal adulterants in BMGSG products. Thereby, a hierarchical strategy for screening illegal adulterated antidiabetics was designed by multi-fingerprinting profiling analysis coupled to spectral purity examination. Detection limit of identification (ILOD) and detection limit of spectra (SLOD) were defined in the range of 0.115–2.65 μg/mL and 0.382–2.72 μg/mL, respectively. The method also demonstrated good quantification performance through validation tests. It had been successfully applied to screening real samples, and the results were confirmed by ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometer (UPLC-Q-Orbitrap/MS). Thus, the proposed method was an efficient and reliable approach for detection of illegal adulterated antidiabetics in the dietary supplement.

An improved automated one-pot synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) based on a purification by cartridges

IntroductionO-(2-[18F]Fluoroethyl)-L-tyrosine ([18F]FET) is an established radiotracer used for oncology investigations by Positron Emission Tomography (PET). Main limitations to its widespread use are the synthesis itself (time; cost; radiochemical yield; complexity) and a troublesome and time-consuming HPLC purification. Aim of this work was to improve the preparation overall efficiency and, most important, to achieve an efficient and reliable purification by means of disposable cartridges. Methods[18F]FET was synthesized by direct nucleophilic radiofluorination of O-(2-tosyloxy-ethyl)-N-trityl-L-tyrosine t-butylester (TET) followed by acid hydrolysis with HCl. Several conditions and materials were tested for the synthesis and purification step. For the latter, a number of different commercial cartridges, varying in amount, particulate size and adsorbent, were examined. Best results were obtained by a combination of STRATA-X, tC18 and QMA cartridges. ResultsStarting from only 5 mg of TET, up to 11 GBq of injectable solutions of [18F]FET were produced within 36 min with 54–65% radiochemical yields and radiochemical purities >99%. No D-form was observed by chiral HPLC. Chemical purity was 1–2 order of magnitude below the limits imposed by the European Pharmacopoeia's monograph on [18F]FET.A radiochemical purity decrease by radiolysis, observed only on relatively large batches of [18F]FET, was efficiently suppressed by preloading in the receiving final vial a small amount of ethanol (<2% v/v). ConclusionsBy combining improvements to a known synthetic route with a novel cartridge-based purification, [18F]FET was obtained in a very efficient and reproducible way. The whole process was easily implemented on a commercial automated module presently used for [18F]FDG production. Advances in knowledge and implications for patient careA few drawbacks regarding the HPLC conditions recommended in the European Pharmacopoeia were highlighted. An alternative method able to cope with them is herein proposedThe simplified preparation herein described is expected to encourage a more widespread clinical use of [18F]FET.

Rapid determination of theaflavins by HPLC with a new monolithic column

The quantitative determination of four theaflavin monomers by a rapid reversed-phase high performance liquid chromatographic method was developed. A new RP-18 end-capped column with particle size 2 µm and equilibrated to 35°C in a Shimadzu temperature controller module was used. Four theaflavin monomers were successfully separated in 8 min by the new strategy, comparing to 20–85 min by HPLC in the peer literature reports. Linear gradient elution: from 92% mobile phase A (v) to 76% mobile phase A (v) during early 3 min and then 92% mobile phase A (v) till 8 min at elution flow rate 1.5 ml/min. The limits of detection and quantification were in the range of 0.1–0.3 and 0.4–1.1 mg/l. Satisfactory recoveries of theaflavin, theaflavin-3-gallate, theaflavin-3’-gallate and theaflavin-3,3’-gallate were 97.5–102.6, 98.6–102.4, 99.6–105.4, and 95.5–105.4%, respectively. The new method was applied to quantitative analysis theaflavins of tea samples, including 10 black teas, 5 oolong teas, and 5 green teas. This method is suitable for the rapid, accurate and inexpensive quantitative analysis of theaflavins under the basic detection conditions of HPLC.

Development of simple isocratic HPLC methods for siRNA quantitation in lipid-based nanoparticles

This paper describes the development of simple and user-friendly HPLC methods that can quantitate the amount of small interfering RNA (siRNA) in lipid-based nanoparticle (LNP) formulations. The methods have been used as alternative chromatographic approaches to the size exclusion chromatography in order to perform “fit for purpose” analysis such as determining the amount of released siRNA from LNP formulations as a part of in-vitro release testing. Two HPLC conditions were optimized using reversed phase (a 250 × 4.6 mm Waters XSelect CSH column) and cation exchange columns (a 250 × 4.6 mm Zorbax SCX-300 column) maintained at 30 °C with a mobile phase of 0.1 M ammonium bicarbonate aqueous solution containing 20–30% acetonitrile. All the siRNA variants were excluded from pores in stationary phase and led to a single peak eluted earlier than the solvent front. The impact of chromatographic conditions and column configurations on method specificity, accuracy, and sensitivity have been investigated. Validation data for both methods in terms of precision, linearity, accuracy and sensitivity are also presented.

VALIDATION AND QUANTITATIVE ANALYSIS OF CARMINE AND RHODAMINE B IN LIPSTICK FORMULATION

Objective: The objective of this study was to validate reversed phase-high performance liquid chromatography (RP-HPLC) method using photo-diode array detector for the simultaneous determination of carmine (CAR) and rhodamine B (RHO B) in lipstick products.&#x0D; Methods: CAR and RHO B were analysed using Cosmosil C18 column (250 mm x 4.6 mm i.d., 5 µm) using Shimadzu LC 20AD chromatograph equipped with photo-diode array (PDA) detector at 245-600 nm. The mobile phase used was acetonitrile-phosphate buffer pH 3.4 (5.5:4.5 v/v) delivered isocratically at flow rate of 1.0 ml/min, using column temperature at 35 °C. The optimized HPLC condition was validated by determining several characteristics including selectivity, linearity, sensitivity expressed by the limit of detection and limit of quantification, precision and accuracy.&#x0D; Results: The optimized HPLC condition could separate CAR and RHO B successfully with resolution value&gt;2.0. The method was linear over studied concentration with a coefficient of determination (R2) 0.9999. The relative standard deviation (RSD) values were lower than those required RSD Horwitz and the recovery values were also acceptable, therefore, the developed method was precise and accurate.&#x0D; Conclusion: HPLC with PDA detector has been accurately and successfully used for quantitative analysis of CAR and RHO B. The developed method is simple and can be used for routine analysis of CAR and RHO B for quality assurance purposes.

DETERMINATION OF SHORT CHAIN FATTY ACIDS IN RAT FECES TREATED WITH RESISTANT STARCH BY HPLC

Background: Short-chain fatty acids (SCFAs), mostly found in colon feces, is an important group of gut microbial metabolites from anaerobic fermentation of indigestible carbohydrates. Objectives: To develope an HPLC method to determined SCFAs in rat feces treated with resistant starch. Materials and methods: Sample is the rat feces fed with acetate wheat starch and normal starch; fatty acid hydrazides are derived from SCFAs in feces and measured by HPLC. After validated as guidance of US-FDA, method is applied to identify SCFAs in rat treated two types of starch. Results: the HPLC condition was optimized as follow: Eclipse XDB–C8 (4.6mmx150mm, 5µm) column; mobile phase: methanol, acetonitrile and 0,057 mM acid triflouroacetic (pH 4.5) (0:13:87 – 10:20:70 – 0:13:87, v/v/v) in 40 mins;, examinized wavelength: 396 nm. Method was validated with parameters: system suitability, specificity, linearity, precision, accuracy and stability. The result showed amount ratio of SCFAs in feces of mice group treated with acetate wheat starch containing resistant starch higher than from the diet containing normal starch significantly. Conclusion: This method can be used to investigate SCFAs in the gastrointestinal segments of the living organism. Key words: SCFAs, HPLC, resistant starch, feces

High throughput residue analysis of paraquat and diquat involving hydrophilic interaction liquid chromatographic separation and mass spectrometric determination

ABSTRACTA selective, sensitive and robust LC-MS/MS method is reported for the determination of the residues of paraquat and diquat in various fruit matrices, including grape, apple and pomegranate. The extraction with acidified water (0.1 M HCl) at 80°C (15 min) offered superior recoveries for both analytes with a significantly lower matrix effects as compared to the extraction with acidified methanol by the methods reported in the existing literature. The optimised HPLC conditions on hydrophilic interaction liquid chromatography (HILIC) columns, when coupled with electrospray ionisation-tandem mass spectrometry, offered their limit of quantification at 0.01 mg kg−1. The analysis on an XBridge HILIC column required a thorough optimisation of the gradient programme to induce chromatographic separation and minimise matrix effects. This was not necessary when a CORTECS HILIC column was used, which provided selective and sensitive analysis within 5 min runtime using isocratic flow. Isotopically labelled internal standards corrected the recoveries of both analytes within 70–120% (RSD < 20%). For the first time, the applications of high resolution accurate mass analysis in the ‘time of flight – multiple reaction monitoring’ mode have been demonstrated as a complementary means of targeted screening of these compounds at 0.01 mg kg−1 level. The method has a strong potential for applications in both official control and by those involved in food production for checking compliance with the EU MRLs.

Effect of the Nature of the Sorbent on the Retention of 1,2,4-Triazole and 1,2,4-Triazine Derivatives under the Conditions of Reversed-Phase High-Performance Liquid Chromatography

The chromatographic retention of 1,2,4-triazine and 1,2,4-triazole derivatives on octadecyl silica gel, super–cross linked polystyrene, and porous graphitized carbon is studied using water-acetonitrile mobile phase. The effect the structure of sorbate molecules and the nature of the sorbent have on the retention characteristics under conditions of RP HPLC is analyzed. The influence of the acetonitrile concentration in the eluent on the retention of the investigated compounds by sorbents of various nature is illustrated.