Revisiting the dependence of retention factor on the content of organic component in the mobile phase in reversed-phase HPLC.

In high-performance liquid chromatography, the dependence of retention factor k on volumetric fraction ϕ of organic phase is expressed by log k = F(ϕ) with F(ϕ) obtained by measuring log k at different ϕ values. From F(ϕ), a value kw is calculated by taking ϕ = 0. The equation log k = F(ϕ) is applied for predicting k, and kw is a descriptor of hydrophobic character of solutes and stationary phases. Calculated kw should not depend on the nature of organic component of mobile phase but extrapolation procedure leads to different kw for different organic components. The present study shows that the expression of F(ϕ) changes depending on the range of ϕ and the same function F(ϕ) cannot be used for the full range of ϕ from 0 to 1. Consequently, kw obtained by extrapolation of ϕ to zero is not correct because the expression of F(ϕ) was generated by fitting the data using ϕ with higher values. The present study shows the proper way to obtain the value of kw .

Occurrence and fate of Adsorbable Organic Halogens (AOX) in two WWTPs from Romania.

Absorbable organic halogens (AOX) are a global parameter which refers to a group of chemical compounds that contain one or more chlorine, bromine or iodine atoms in their molecule and can easily adsorb on activated carbon. The global concern related to the occurrence of the AOX compounds in the environment is due to their toxic and mutagenic effects on aquatic organisms and their potential role as inhibitors of microorganism growth, even at AOX low concentrations. The purpose of this study was to analyze the presence, occurrence and composition of absorbable organic halogens in wastewater and sewage sludge. In addition, their genotoxicity effect on the environment was tested on a bacterial biological model. Daily mass loading, mass emission and fate of AOX parameter were investigated in two wastewater treatment plants (wastewater and sewage sludge samples) from Romania, Galati and Iasi. Their AOX daily mass loadings (151 and 55.4 g/day/1000people) and mass emissions into the environment (47.8 and 23.5 g/day/1000 people) for both locations were correlated with the concentration level of volatile organic compounds, chlorophenols, organochlorine pesticides and polychlorinated biphenyls from both wastewater and sewage sludge, respectively. Concentration levels of detected halogenated organic compounds (regulated by current standards) accounted only for a small percentage (3.70-14.5%) from the total AOX amount. An exception was observed in the case of dehydrated sludge samples where the identified compounds accounted for 80% of the AOX content from Iasi WWTP and 53% for Galati. Evaluating the genotoxic activity of AOX in sludge samples showed that genotoxicity was not induced up to 100 µg/mL dehydrated sludge.

Derivatization procedures and their analytical performances for HPLC determination in bioanalysis.

Derivatization, or chemical structure modification, is often used in bioanalysis performed by liquid chromatography technique in order to enhance detectability or to improve the chromatographic performance for the target analytes. The derivatization process is discussed according to the analytical procedure used to achieve the reaction between the reagent and the target compounds (containing hydroxyl, thiol, amino, carbonyl and carboxyl as the main functional groups involved in derivatization). Important procedures for derivatization used in bioanalysis are in situ or based on extraction processes (liquid-liquid, solid-phase and related techniques) applied to the biomatrix. In the review, chiral, isotope-labeling, hydrophobicity-tailored and post-column derivatizations are also included, based on representative publications in the literature during the last two decades. Examples of derivatization reagents and brief reaction conditions are included, together with some bioanalytical applications and performances (chromatographic conditions, detection limit, stability and sample biomatrix).

Occurrence and Fate of Bisphenol A and its Congeners in Two Wastewater Treatment Plants and Receiving Surface Waters in Romania.

The present study investigated the distribution and environmental fate of Bisphenol A (BPA), the 4-hydroxyacetophenone (4-HAP) metabolite, and 5 other bisphenol congeners in 2 municipal wastewater treatment plants (WWTPs) and their receiving rivers in Romania. Accordingly, a new, highly sensitive and accurate solid-phase extraction-liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed and validated. This technique generated low limit of quantitation values: below 2.3 ng/L for surface water and less than 2.4 and 2.7 ng/L for WWTP effluent and influent water. The sum of detected analytes in wastewater was between 1337 and 16 118 ng/L for influent samples and between 15 and 96 ng/L for effluent samples. In surface water, the total of all compounds was somewhere between 34 and 240 ng/L. The highest concentration observed was for BPA in all 3 types of analyzed water (up to 9140 ng/L for influent, as high as 75 ng/L for effluent, and a maximum of 135 ng/L in surface waters). All analyzed samples were free of bisphenols B, C, and F. For all analytes detected in surface water, the concentration values were higher than those determined in the effluent samples, which may be caused by intrinsic contamination of the 2 rivers (Danube and Jiu Rivers). Values of environmental risk coefficients, calculated for both effluents and surface waters, indicated a low ecological risk or no ecological risk for 3 types of organisms (algae, daphnia, and fish). Human risk assessment calculation suggests no risk to human health as a result of the presence of BPA in either of the 2 rivers. Environ Toxicol Chem 2021;40:435-446. © 2020 SETAC.

Fast and sensitive detection of acrolein in environmental water samples without derivatization using liquid chromatography tandem mass spectrometry.

A fast and sensitive SPE-LC-MS/MS method for the determination of acrolein in environmental water samples using activated charcoal as SPE adsorbent was developed. The novelty of this study consists in acrolein extraction, separation and detection without the need of a derivatization process. Physicochemical properties of acrolein, such as low molecular weight and high polarity represent real challenges for extraction, separation, and detection of this pollutant using SPE-LC-MS/MS. These were addressed by choosing a suitable chromatographic column which ensures a good peak symmetry and retention for the analyte, as well as the choice of SPE adsorbent suitable for retaining very polar compounds like acrolein from the aqueous matrix. The chromatographic column was a Synergi Fusion RP (150 × 2.0 mm, 4.0 µm) with a C18 stationary phase modified with polar embedded amide groups. Activated charcoal adsorbent used as SPE extraction media was able to extract efficiently highly polar molecules such as acrolein and 13C3-acrylamide (internal standard) from water samples. Using this method, the obtained extraction recovery for acrolein was 88% at a 50 ng/L concentration level. Overall method quantitation limit (LOQ) for acrolein in water was established at 3.8 ng/L. The newly developed SPE-LC-MS/MS method was successfully applied to detect acrolein occurrence in wastewater and drinking water samples. Acrolein level in these samples ranged from LOQ to 122 ng/L.

Simultaneous ESI-APCI+ ionization and fragmentation pathways for nine benzodiazepines and zolpidem using single quadrupole LC-MS.

Nine important 1,4-benzodiazepines and zolpidem were characterized by liquid chromatography-mass spectrometry using a multimode ionization source able to generate ions using both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), and a single quadrupole mass analyzer. An optimum chromatographic separation was applied for all target compounds in less than 8 minutes using a Zorbax Eclipse Plus column (100 × 4.6 mm, 3.5 µm) kept at 35°C and a 0.3% HCOOH/ACN/IPA (61:34:5) mobile phase pumped at 1 ml/min. Optimization of LC-MS method generated low limit of quantitation (LOQ) values situated in the range 0.3-20.5 ng/ml. Comparison between differences in method sensitivity, under specified chromatographic conditions, when using ESI-only, APCI-only, and simultaneous ESI-APCI ionization with such a multimode source was discussed. Mixed ESI-APCI(+) mode proved to be the most sensitive ionization generating an average 35% detector response increase compared to ESI-only ionization and 350% detector response increase with respect to APCI-only ionization. Characterization of the nine benzodiazepines and zolpidem concerning their MS fragmentation pathway following 'in-source' collision-induced dissociation is discussed in detail and some general trends regarding these fragmentations are set.

Effects of large volume injection of aliphatic alcohols as sample diluents on the retention of low hydrophobic solutes in reversed-phase liquid chromatography.

Recent studies showed that injection of large volume of hydrophobic solvents used as sample diluents could be applied in reversed-phase liquid chromatography (RP-LC). This study reports a systematic research focused on the influence of a series of aliphatic alcohols (from methanol to 1-octanol) on the retention process in RP-LC, when large volumes of sample are injected on the column. Several model analytes with low hydrophobic character were studied by RP-LC process, for mobile phases containing methanol or acetonitrile as organic modifiers in different proportions with aqueous component. It was found that starting with 1-butanol, the aliphatic alcohols can be used as sample solvents and they can be injected in high volumes, but they may influence the retention factor and peak shape of the dissolved solutes. The dependence of the retention factor of the studied analytes on the injection volume of these alcohols is linear, with a decrease of its value as the sample volume is increased. The retention process in case of injecting up to 200µL of upper alcohols is dependent also on the content of the organic modifier (methanol or acetonitrile) in mobile phase.

Retention studies for large volume injection of aromatic solvents on phenyl-silica based stationary phase in RP-LC.

The use of a large volume injection of hydrophobic solvents as diluents for less hydrophobic solutes has already been proven for C18 and C8 stationary phases in reversed-phase liquid chromatography. The same possibility is investigated for a phenyl-hexyl stationary phase using aromatic solvents (benzene, toluene, ethylbenzene and propylbenzene) as diluents for several model analytes also containing aromatic rings. Both hydrophobic interaction and π-π stacking account for the competitive interaction of both the diluent and model analytes with the phenyl-hexyl phase. A linear decrease in analyte retention factor was observed with an increase of injection volume in the range of 1-100 µL. A moderate peak efficiency decrease was also observed, but peaks of model analytes remained undistorted with minimum band broadening up to 100 µL injection volume. A very small retention decrease was observed when changing the sample diluent in the homologous series: benzene, toluene, ethylbenzene and propylbenzene. The critical conditions for a successful large volume injection of analytes dissolved in studied hydrophobic solvents are for the analyte to have lower hydrophobicity and for the specified solutes to have proper solubility.

Deviation from van't Hoff dependence in RP-LC induced by tautomeric interconversion observed for four compounds.

The most encountered situations in reversed-phase liquid chromatography for temperature dependence of retention are those obeying the linear equation known as the van't Hoff plot. When studying compounds that are involved in structural modifications, it is likely that the temperature dependences of their retention factors do not follow this rule. It is the aim of this paper to report some particular cases when compounds involved in tautomeric interconversion have a different retention behavior with temperature: a deviation from the linearity of ln k on 1/T, or, in certain temperature ranges, temperature increase leading to a retention increase. Examples of compounds exhibiting deviation from the van't Hoff temperature dependence are piroxicam, drotaverine, vincamine, and epivincamine. A simple thermodynamic model relying on tautomeric equilibria in mobile phase is proposed for these compounds, which gives a polynomial dependence between ln k and 1/T.

Competitional hydrophobicity driven separations under RP-LC mechanism: application to sulfonylurea congeners.

Retention of a model set of sulfonylurea compounds has been studied under RP-LC conditions, considering competitional effects brought by different alcohols (ethanol, 1-propanol, 2-propanol, 1-butanol, 1-pentanol, and 1-octanol) used as additives in the organic component of the mobile phase (methanol). The capacity factors determined for the model compounds decreased with the increase of the hydrophobic character of the organic additive in the mobile phase. The amount of the additive within the organic component of the mobile phase was kept constant (1% as volumetric ratio). Retention was studied at different mobile phase compositions (aqueous to organic component ratios). Different functional fitting models were used to correlate retention to the content of the organic component in the mobile phase. Extrapolation of retention expressed as capacity factor to a mobile phase composition free of organic component is well correlated to the hydrophobic characteristics of the organic additives. The adsorption model was used for tuning the experimental find-outs. The possibility of controlling retention through the competitive effects induced by hydrophobic additives in the mobile phase is highlighted.

Assay of free captopril in human plasma as monobromobimane derivative, using RPLC/(+)ESI/MS/MS: validation aspects and bioequivalence evaluation.

A sensitive method for determination of free captopril as monobromobimane derivative in plasma samples is discussed. The internal standard (IS) was 5-methoxy-1H-benzimidazole-2-thiol. Derivatization with monobromobimane immediately after blood collection and plasma preparation prevents oxidation of captopril to the corresponding disulfide compound and enhances the ionization yield. Consequently, derivatization enhances sample stability and detection sensitivity. Addition of the internal standard was made immediately after plasma preparation. The internal standard was also derivatized by monobromobimane, as it contains a thiol functional group. Preparation of plasma samples containing captopril and IS derivatives was based upon protein precipitation through addition of acetonitrile, in a volumetric ratio 1:2. The reversed-phase liquid chromatographic separation was achieved on a rapid resolution cartridge Zorbax SB-C(18), monitored through positive electrospray ionization and tandem MS detection using the multiple-reaction monitoring mode. Transitions were 408-362 amu for the captopril derivative and 371-260 amu for the internal standard derivative. The kinetics of captopril oxidation to the corresponding disulfide compound in plasma matrix was also studied using the proposed method. A linear log-log calibration was obtained over the concentration interval 2.5-750 ng/mL. A low limit of quantitation in the 2.5 ng/mL range was obtained. The analytical method was fully validated and successfully applied in a three-way, three-period, single-dose (50 mg), block-randomized bioequivalence study for two pharmaceutical formulations (captopril LPH 25 and 50 mg) against the comparator Capoten 50 mg.

High-throughput liquid-chromatography method with fluorescence detection for reciprocal determination of furosemide or norfloxacin in human plasma.

A simple, high-throughput, highly selective and sensitive HPLC-FLD method for isolation and determination of furosemide and/or norfloxacin in human plasma samples following a simple organic solvent deproteinization step with acetonitrile as sample 'clean-up' procedure is reported. One of the two drug substances plays the internal standard role for the determination of the other. Separation of analyte and internal standard was achieved in less than 5.3 min (injection to injection) on a Chromolith Performance RP-18e column, using an aqueous component containing 0.015 mol/L sodium heptane-sulfonate and 0.2% triethylamine brought to pH = 2.5 with H(3)PO(4). The composition of the mobile phase was: acetonitrile-methanol-aqueous component = 70:15:15 (v/v/v) and the flow-rate was set up to 3 mL/min. The chromatographic method applied to the determination of furosemide relies on fluorescent detection parameters of 235 nm for the excitation wavelength, and 402 nm for the emission wavelength. In case of norfloxacin, the excitation wavelength is set up to 268 nm and the emission wavelength is set up to 445 nm. The overall method leads to quantitation limits of about 27 ng/mL for furosemide, and 19.5 ng/mL for norfloxacin, using an injection volume of 250 microL. The method was applied to the bioequivalence study of two furosemide-containing formulations.

Achiral-chiral LC/LC-FLD coupling for determination of carvedilol in plasma samples for bioequivalence purposes.

Bioequivalence data for two pharmaceutical formulations (solid oral dosage forms) containing carvedilol is presented for both racemic and enantiomers of the active substance. This was achieved by on-line coupling of two liquid chromatographic separations followed by fluorescence detection. The first LC dimension was used for a fast separation of racemic carvedilol from propranolol (IS) and the endogenous matrix, by means of a reversed phase mechanism. The peak of racemic carvedilol was on-line transferred to the second enantioselective LC dimension, based on a reversed phase separation on cellulose tris(3,5-dimethyl-phenylcarbamate) stationary phase. Both stages were monitored over a single run by means of a fluorescence detector operated at an excitation wavelength of 285 nm and an emission wavelength of 355 nm. Automated shortcutting of the racemic carvedilol peak to the chiral column and simultaneous detection over the two LC dimensions have been obtained by using an experimental set-up based on two six-port rotative switching valves. Linearity was demonstrated on the interval 2-150 ng/mL for racemic carvedilol and on 1-75 ng/mL intervals for enantiomers. LLOQ fits between 0.7 and 1.4 ng/mL. Recoveries of the target compounds are 87+/-4 and 81+/-4% for the IS. Precision ranged from 0.6 to 2.5% and the mean accuracy obtained on quality control samples (measured as % bias) over the whole study falls between -0.8 and 6.3%.

Fast RPLC-UV method on short sub-two microns particles packed column for the assay of tenoxicam in plasma samples.

An extraction-less sample preparation technique followed by a RPLC-UV method on sub-two microns particles packed short column were used for the assay of tenoxicam in plasma samples. Protein precipitation was made by means of trichloroacetic acid addition. Supernatant was injected to the chromatographic column without any further pH adjustment. The mobile phase consisted in a mixture of acetonitrile and aqueous 0.1% phosphoric acid, at 2 mL/min flow rate and gradient elution. The Zorbax SB-C18 column (50 mm length, 4.6 mm internal diameter and 1.8 microm particle size) was thermostated at 60 degrees C. The mobile phase gradient composition program allowed separation of tenoxicam and piroxicam (internal standard), column clean-up and re-equilibration within 4 min. UV detection was achieved at 368+/-10 nm. The method is characterized by a low limit of quantitation of 25 ng/mL for tenoxicam, with a linearity interval up to 5500 ng/mL. The use of a low volume detection cell and detector high frequency data acquisition rate produced high precision and accuracy through a whole bioequivalence study of tenoxicam in two commercially available tablet formulations, after a single oral administration dose. Full method validation is presented. The high throughput characteristic of the proposed method allowed full validation and bioanalytical study completion within a 96 h period.