Aqueous Mobile Phase Research Articles

Innovative preparation of ureido/dodecyl dual-functionalized silica as a versatile mixed-mode stationary phase for high-resolution chromatographic separations

Developing a versatile stationary phase to achieve the purpose of efficiently separating various target analytes is of great significance. In this work, ureido/dodecyl dual-functionalized silica (Sil-Ur-DD) was synthesized and employed as a new mixed-mode stationary phase for high-resolution chromatographic analysis. It was observed that multiple interactions, including hydrophobic, hydrophilic, hydrogen bonding, electrostatic and π-π interactions, contributed to the retention. Nucleosides and nucleobases could be effectively determined in hydrophilic interaction chromatography mode, and the separation performance of the Sil-Ur-DD was found to be even superior to that of the commercial XAmide column. Environmental endocrine disruptors, including alkylphenols, bisphenols, steroid hormones and phthalates, were more favorably separated in reversed-phase chromatography mode with highly aqueous mobile phases compared with the commercial C18-silica column. The experimental results indicated that the combined effect of ureido and long-chain alkyl groups obviously enhanced separation efficiency and selectivity. More significantly, the proposed method concerning the preparation of an efficient separation material with powerful separation ability is economical and environmentally friendly, which provides a proven and effective way for the recycling and utilization of the discarded chromatographic packing materials, thereby showing a good application prospect.

A Rapid HPLC Method for the Simultaneous Determination of Organic Acids and Furans: Food Applications

Concerns over the potential adulteration of commercially produced foods give rise to a requirement for a simple and fast analytical method capable of quantifying potential adulterants. This work demonstrates a simple HPLC method tailored to detect major organic acids and furans within ingredients in commercial food products, for example, pomegranate molasses, balsamic vinegar, and apple cider vinegar. The relative importance of this method is in its simplicity and its use of an environmentally friendly aqueous mobile phase under isocratic conditions, providing results in a less than 20 min runtime. The chromatographic separation was achieved using an Acclaim® OA, 5 µm, 120 Å (4.0 × 250 mm) column; a UV-DAD detector set at 210 nm; and a 200 mM Na2SO4 mobile phase with 0.55 mL/L methanosulfonic acid as a pH modifier. The method was then validated by quantifying the concentration of acetic acid, formic acid, citric acid, and hydroxymethyl furfural (HMF) in pomegranate molasses, balsamic vinegar, and apple cider vinegar commercial products. The concentration of acetic acid and HMF in balsamic vinegar was 80.380 mg/mL (±1.272 mg/mL) and 2.153 mg/mL (±0.021 mg/mL), respectively. The apple cider vinegar was composed only of acetic acid with a concentration of 44.139 mg/mL (±0.053 mg/mL). The concentrations of citric acid and HMF were 123.425 mg/mL (±2.502 mg/mL) and 11.382 mg/mL (±0.582 mg/mL), respectively, in pomegranate molasses. Furthermore, this method is also capable of determining various organic acids and furans in biomass: levulinic acid, formic acid, furfurals, diformylfuran, and gamma-valerolactone.

Per-Aqueous Liquid Chromatography for the Separation of Polar Compounds-Preparation and Characterization of Poly(ethylene oxide-co-dimethylsiloxane) Based Stationary Phase

Poly(ethylene oxide-co-dimethylsiloxane) was thermally immobilized on silica particles- Si(PEO)-to separate small polar compounds with water-rich mobile phases. Poly(ethylene oxideco- dimethylsiloxane) content on Si(PEO) stationary phase was optimized using a central composite design. Infrared spectroscopy, scanning electron microscopy, and thermogravimetric analysis morphologically and structurally characterized the optimized material. Separation of standard test mixtures showed that the Si(PEO) phase had a typical reversed-phase elution order. However, the Si(PEO) phase retained polar compounds better than C18 or aqueous C18 phases under waterrich mobile phases. Under this condition, small changes in the acetonitrile fraction resulted in a marked increase in the retention of some polar drugs on the Si(PEO) phase. A typical condition observed in per aqueous liquid chromatography separations, a more environmentally friendly liquid chromatography approach. On the other hand, hydrophobic compounds showed lower mass transfer rates due to their low solubility in the aqueous mobile phase. Thus, the Si(PEO) phase was more suitable and efficient for separating polar or hydrophilic compounds.

Validated quantitative analysis method for routine residue monitoring of oxytetracycline and its 4-epimer in milk: Pipetting sample preparation and isocratic mobile phase HPLC-diode array under organic solvent-free conditions

The author introduces an organic solvent-free, small-scale, time shortening, economical procedure for quantitation of oxytetracycline (OTC) and its 4-epimer, 4-epi-oxytetracycline (4eOTC), in cow’s milk. Sample preparation was performed by homogenization using a handheld ultrasonic-homogenizer with water followed by Mono Tip® C18 pipette tip contains silica monolith bonded with octadecyl group with water eluent. The HPLC separation was achieved a C4 column with an isocratic aqueous mobile phase and a photodiode array detector (PDAD). The total time required for the analysis of one sample was less than 6 min. The method validation data were well within the international analytical method acceptance criteria. The limits of quantitation for OTC and 4eOTC were 0.035 and 0.048 μg/mL, respectively. The present technique may be proposed as an international standardized analytical method for routine residue monitoring for OTC and 4eOTC in milk.

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.

Uranium partitioning from contaminated wetland soil to aqueous and suspended iron-floc phases: Implications of dynamic hydrologic conditions on contaminant release

Environmental mobility of anthropogenic heavy metal releases is governed by site-specific hydrobiogeochemical factors. Uranium (U) releases from 1965 to 1988 resulted in contamination of the Tims Branch wetland at the United States Department of Energy Savannah River Site (SRS) near Aiken, South Carolina. Today, 80% of the 43,000 kg released remain sequestered in soils of two former ponds, Beaver Pond and Steed Pond. The objective of this study was to understand U partitioning from contaminated wetland sediments to aqueous and particulate iron-floc phases. The approach was to correlate U, iron (Fe), and organic matter (OM) in soil cores at key depositional areas; determine U and Fe concentrations in aqueous- and solid-phases in the stream; and compare these stream water data following two storm events. Uranium was released into an outfall canal leading away from the SRS M-Area and first deposited in a topographically low area where a beaver pond is frequently in place. Uranium soil concentrations in Beaver Pond exceed 14,000 µg/g and a second deposition area 1 km downgradient in a former farm pond (Steed Pond) has U soil concentrations near 300 µg/g. Uranium releases to the stream are minimal at Beaver Pond, but U incorporation into Fe-OM colloids, or flocs, at Steed Pond results in appreciable fluxes of U from the wetland. This work yields four key conclusions. At the kilometer and meter scales, geochemistry cannot be the sole predictor of U mobility, as the anthropogenic U deposition is geologically young relative to Fe and OM. Storm intensity and duration do not appear to be directly proportional to U releases from the wetland, as flooding decreases U inclusion in mobile flocs. The mass of suspended solids present in a stream reach often governs how much U moves. The solid-bound U inventory at Beaver Pond does not enter the mobile aqueous phase as easily as Steed Pond U, resulting in U sequestration amid wetland metal cycling in Tims Branch. This work expands upon studies conducted in the 1990′s and early 2000′s, improving our understanding of the geochemical controls of uranium migration through Tims Branch, particularly with respect to the changes over the last 30+ years. Current wetland conditions do not favor significant U movement, but long-term changes in local and regional hydrology may alter this delicate biogeochemical balance.

Temperature responsive chromatography for therapeutic drug monitoring with an aqueous mobile phase

Therapeutic drug monitoring is a key technology for effective pharmacological treatment. In the present study, a temperature-responsive chromatography column was developed for safe and simple therapeutic drug monitoring without the use of organic solvents. Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel-modified silica beads were prepared via a condensation reaction and radical polymerization. The temperature-dependent elution behavior of the drugs was observed using a PNIPAAm-modified silica-bead packed column and an all-aqueous mobile phase. Sharp peaks with reproducible retention times were observed at temperatures of 30 °C or 40 °C because the PNIPAAm hydrogel on the silica beads shrinks at these temperatures, limiting drug diffusion into the PNIPAAm hydrogel layer. The elution behavior of the sample from the prepared column was examined using a mixture of serum and model drugs. The serum and drugs were separated on the column at 30 °C or 40 °C, and the concentration of the eluted drug was obtained using the calibration curve. The results show that the prepared chromatography column would be useful for therapeutic drug monitoring because the drug concentration in serum can be measured without using organic solvents in the mobile phase and without any need for sample preparation.

A carbonylative coupling approach to alkyl stationary phases with variable embedded carbamate groups for high-performance liquid chromatography

A highly efficient carbonylative coupling method for the preparation of alkyl stationary phases with variable numbers of carbamate groups was established. The effectiveness of such method was verified through elemental analysis, X-ray photoelectron spectroscopy and solid-state 13C nuclear magnetic resonance spectroscopy of three as-synthesized stationary phases bearing different alkyl chains and different numbers of carbamate groups (octadecyl/one carbamate group, C18C; docosyl/three carbamate groups, C22C3; triacontyl/two carbamate groups, C30C2). The comparative evaluation of these stationary phases using a great variety of analytes, including three sets of isomers of alkylbenzenes, two sets of standard mixtures of polycyclic aromatic hydrocarbons (SRM 1647e and 869b), nine polychlorinated biphenyls, fiveteen N-substituted ureas, ten sulfonylureas, five xanthines and some other phytonutrients, revealed their remarkable applicability in reversed-phase liquid chromatography. Notably, the intercalated carbamate groups rendered the resultant stationary phases compatible with 100% aqueous mobile phase. The suppression of silanol activity was positively related to the number of polar groups embedded in the bonded selector, and the smallest peak tailing factor (1.14) for amitriptyline was obtained by C22C3. The molecular shape-related selectivity was found to be more closely related to the length of the selector's aliphatic chain, as supported by the lowest αTBN/BaP value (0.31) by C30C2. These carbamate-embedded alkyl stationary phases constituted another class of polar-embedded stationary phases possessing a single type of functional ligand.

Chromatographic preparation of food-grade prebiotic oligosaccharides with defined degree of polymerization

Prebiotic oligosaccharides are of widespread interest in the food industry due to their potential health benefits. This has triggered a need for research into their sensory properties. Such research is currently limited due to the lack of available food-grade oligosaccharide preparations with specific degree of polymerization (DP). The aim of this study was to develop economical approaches for the preparation and characterization of prebiotic oligosaccharides differing with respect to composition and DP. Such preparations were prepared by chromatographic fractionation of commercially available prebiotic mixtures using microcrystalline cellulose stationary phases and aqueous ethanol mobile phases. This approach is shown to work for the preparation of food-grade fructooligosaccharides of DP 3 and 4, galactooligosaccharides of DP 3 and 4, and xylooligosaccharides of DP 2–4. Methods for the characterization of the different classes of oligosaccharides are also presented including those addressing purity, identity, total carbohydrate content, moles per unit mass, and DP.

POTENSI KEBERADAAN SENYAWA AKRILAMIDA DALAM MAKANAN

This of the study was to examine the potential presence of acrylamide compounds in foods containing the amino acid asparagine and reducing sugars that are processed at high temperatures. The initial research was carried out to determine the levels of precursors in the raw material and then analyzed the amount of acrylamide using HPLC-RP LC-6A Shimadzu with a C18 column and sterile aqueous mobile phase. The optimization condition of the instrument was achieved with a column temperature of 35oC, a wavelength of 254 nm and a flow rate of 0.8 mL/min. The retention time of acrylamide was observed at 3.8 min. The food sample tested was banana chips. The results showed that banana chips with a moisture content of 7.74% (w/w) ± 0.01 contained acrylamide 115.5 to 565 g/kg. Acrylamide content is influenced by the amount of precursor, temperature and duration of food processing.

An investigation of aspects of radiochemical purity of 99mTc-labelled human serum albumin nanocolloid

BackgroundNanocolloidal human serum albumin radiolabelled with 99mTc provides a diagnostic radiopharmaceutical for sentinel node lymphoscintigraphy. NanoHSA (Nanotop), a commercially available kit, enables the simple preparation of this radiopharmaceutical via reconstitution with pertechnetate eluted from a generator. Thin-layer chromatography is widely used for determining radiochemical purity in clinical nuclear medicine. Quality control methods recommended by the manufacturer were sometimes reported to yield variable results. Therefore, we proposed and evaluated three alternative thin-layer chromatography methods for the quality control of [99mTc]Tc-NanoHSA from a commercially available kit.ResultsThe radiochemical purity of [99mTc]Tc-NanoHSA determined with all methods was reproducible and met the requirements of the SPC and the European Pharmacopoeia (≥ 95%). Our quality control using iTLC-SG chromatographic paper in methyl ethyl ketone mobile phase identified only free pertechnetate as impurity, resulting in > 99% RCP. The quality control using iTLC-SG in 85% methanol or iTLC-SA in 0.9% NaCl identified an additional small fraction of a hydrophilic impurity, resulting in 95–97% RCP. Glucose was identified as a potential 99mTc-carrying hydrophilic species contributing to hydrophilic impurities.ConclusionOur quality control of [99mTc]Tc-NanoHSA with non-polar mobile phase tended to underestimate the amount of hydrophilic impurities, although without compromising the final quality of the radiopharmaceutical. Alternative TLC methods using aqueous mobile phases enabled a more accurate determination of hydrophilic impurities.

Rapid and sensitive analytical method for the determination of amoxicillin and related compounds in water meeting the requirements of the European union watch list

The presence of antibiotics in the aquatic environment is becoming one of the main research focus of scientists and policy makers. Proof of that is the inclusion of four antibiotics, amongst which is amoxicillin, in the EU Watch List (WL) (Decision 2020/1161/EU)) of substances for water monitoring. The accurate quantification of amoxicillin in water at the sub-ppb levels required by the WL is troublesome due to its physicochemical properties. In this work, the analytical challenges related to the determination of amoxicillin, and six related penicillins (ampicillin, cloxacillin, dicloxacillin, penicillin G, penicillin V and oxacillin), have been carefully addressed, including sample treatment, sample stability, chromatographic analysis and mass spectrometric detection by triple quadrupole. Given the low recoveries obtained using different solid-phase extraction cartridges, we applied the direct injection of water samples using a reversed-phase chromatographic column that allowed working with 100% aqueous mobile phase. Matrix effects were evaluated and corrected using the isotopically labelled internal standard or correction factors based on signal suppression observed in the analysis of spiked samples.The methodology developed was satisfactorily validated at 50 and 500 ng L − 1 for the seven penicillins studied, and it was applied to different types of water matrices, revealing the presence of ampicillin in one surface water sample and cloxacillin in three effluent wastewater samples.

Comprehensive two-dimensional temperature-responsive × reversed phase liquid chromatography for the analysis of wine phenolics

Phenolic compounds are an interesting class of natural products because of their proposed contribution to health benefits of foods and beverages and as a bio-source of organic (aromatic) building blocks. Phenolic extracts from natural products are often highly complex and contain compounds covering a broad range in molecular properties. While many 1D-LC and mass spectrometric approaches have been proposed for the analysis of phenolics, this complexity inevitably leads to challenging identification and purification. New insights into the composition of phenolic extracts can be obtained through online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to photodiode array and mass spectrometric detection. However, several practical hurdles must be overcome to achieve high peak capacities and to obtain robust methods with this technique. In many LC × LC configurations, refocusing of analytes at the head of the 2D column is hindered by the high eluotropic strength of the solvent transferred from the 1D to the 2D, leading to peak breakthrough or broadening. LC × LC combinations whereby a purely aqueous mobile phase is used in the 1D and RPLC is used in the 2D are unaffected by these phenomena, leading to more robust methods. In this contribution, the combination of temperature-responsive liquid chromatography (TRLC) with RPLC is used for the first time for the analysis of phenolic extracts of natural origin to illustrate the potential of this alternative combination for natural product analyses. The possibilities of the combination are investigated through analysis of wine extracts by TRLC × RPLC-DAD and TRLC × RPLC-ESI-MS.

Assessment of the greenness of micellar HPLC method for rapid separation and simultaneous estimation of chlorpheniramine maleate in presence of some co-administered drugs in three pharmaceutical dosage forms using single run

Abstract Sustainable chemistry established one of kind standards to maintain protection of environment through using safer mobile phase composition and/or lower solvent consumption. A fast green micellar HPLC method was developed and applied for the first time aiming at simultaneous determination of chlorpheniramine maleate, one of the most widely used antihistamine in combination with levochlopersatine fenodizoate or dextromethorphan hydrobromide or dexamethasone, in their pure forms, laboratory prepared mixtures and pharmaceutical dosage forms used in alleviating the symptoms of cough resulting from common colds and allergy. The separation was achieved on Kinetex C18 column (100 mm × 4.6 mm i.d., 2.6-μm particle size) using micellar aqueous mobile phase consisting of (30 mM sodium dodecyl sulfate and 50 mM sodium dihydrogen phosphate, pH 5) and ethanol (85:15) with UV detection at 230 nm. The four drugs were successfully separated using isocratic elution in a single run not exceeding 7 min. According to ICH guidelines, the method was confirmed to be linear, accurate and precise over the concentration ranges of 5–60 μg mL−1 for chlorpheniramine maleate, 10–100 μg mL−1 for levocloperastine fenodizoate and dextromethorphan hydrobromide and 5–30 μg mL−1 for dexamethasone. In addition, the greenness of the developed method was assessed using two different tools indicating their least hazardous effect on the environment.

Robust optimization of gradient RP HPLC method for simultaneous determination of ivabradine and its eleven related substances by AQbD approach

Abstract This paper is aimed at developing a gradient elution reversed-phase high-performance liquid chromatography (RP-HPLC) method for the separation of a complex mixture composed of ivabradine and its eleven impurities, in a reasonable timeframe. In order to obtain a robust and reliable HPLC method for separation of this mixture, Analytical Quality by Design (AQbD) was applied. This approach demonstrated to be useful in development of a long lasting life cycle methods. Four chromatographic variables were defined as key method parameters (KMPs) and optimized towards the analytical target profile (ATP). Designated KMPs were initial and final amount of acetonitrile in the mobile phase, pH value of the aqueous phase and gradient time, while resolutions of critical peak pairs were denoted as critical method attributes (CMAs). Relationships between KMPs and CMAs were obtained with the aid of Design of Experiments (DoEs) methodology among which Box-Behnken design (BBD) was employed to gain valid mathematical models. Obtained mathematical equations were used to construct the Design Space (DS) and select reliable optimal separation conditions. They included 11% (v/v) and 34% (v/v) of initial and final amount of acetonitrile, respectively, as well as 45 min of gradient elution time and 20 mM ammonium acetate as aqueous mobile phase with pH set to 7.35. The possibility to separate the diastereoisomers of impurity X was also evaluated. It was demonstrated that this separation could not be achieved in gradient elution mode within the defined variable domains and in a reasonable time span. The developed method was validated according to ICH Q2 (R1) guideline and met all the required criteria.

Method development and validation of sildenafil, N-desmethylsildenafil and N1,N4-desmethylsildenafil by LC-MS/MS and its application

This study aimed to develop an LC-MS/MS method for determining sildenafil and its metabolites N-desmethylsildenafil and N1,N4-desmethylsildenafil in human plasma and applying it to a pharmacokinetic study of sildenafil in healthy volunteers. Sildenafil-d8 was used as the internal standard. Plasma samples were pretreated via protein precipitation with acetonitrile. The extractives were then separated on an ACQUITY UPLC BEH C18 (50-mm × 2.1-mm, 1.7-μm) column using gradient elution. The aqueous and organic mobile phases were ammonium formate 2 mM supplemented with 0.1% formic acid in water and acetonitrile, respectively, and the flow rate was 0.3 mL/min. An electrospray ionization source was applied, and multiple reaction monitoring was operated in the positive mode with selective channels at m/z 475.30 → 100.10, 461.20 → 283.30, 483.30 → 108.10, and 449.00 → 283.00 for sildenafil, sildenafil-d8, N-desmethylsildenafil, and N1,N4-desmethylsildenafil, respectively. The linear calibration curves of sildenafil and its metabolites spanned 1.0–1000 ng/mL. The lower limit of quantification was 1.0 ng/mL. The extractive recovery of analytes from the biological matrix was more than 90% and the matrix effect complied with relevant provisions. The intra- and inter-day precisions of sildenafil and its metabolite were <10%. The intra- and inter-day accuracy of sildenafil, N-desmethylsildenafil, and N1,N4-desmethylsildenafil was more than 99%. The method is highly sensitive and selective, and it was successfully applied to the bioequivalence studies of 100-mg sildenafil citrate tablets in 40 healthy Chinese volunteers.

LC-ESI-tandem MS and in silico ADMET analysis of polyphenols from Rhus coriaria L. and Micromeria fruticosa (L.) Druce ssp. brachycalyx P. H. Davis

BackgroundMicromeria fruticosa (L.) Druce ssp. brachycalyx P. H. Davis and Rhus coriaria L., which are Lamiaceae species, are used both as spices in food and medicinally. Lamiaceae species are known to contain high amounts of polyphenols. In this study, liquid chromatography–quadrupole time-of-flight–tandem mass spectrometry (LC-QTOF-MS/MS) was used for analysis of polyphenols in the plants. Under gradient elution with using 0.1% aqueous acetic acid solution and acetonitrile mobile phases, an Agilent Poroshell C18 reversed phase column was used for the simultaneous determination of 18 polyphenols, and separation was performed in 30 min. Pharmacokinetic properties of these polyphenols such as drug-like and toxicity were estimated using open-source software, pkCSM and SwissADME.ResultsThese compounds were determined to represent different classes of polyphenols, including phenolic acids, flavonoids, coumarin and tannins. ADMET predictions of polyphenols indicated that these compounds are easily absorbed and do not have toxic effects.ConclusionWhile the Rhus coriaria L. includes anthocyanidins, tannins, phenolic acid and flavonoids, the Micromeria fruticosa (L.) Druce ssp. brachycalyx P. H. Davis has phenolic acid, coumarin and flavonoids, according to these results. In silico ADME/Tox predictions revealed that these bioactive components are to be drug-like and non-mutagenic. These data are supportive for future analysis that can lead to their therapeutic use of the plants, suggesting that this species may be used as a natural medicinal source in the future after detailed analysis tests.Graphical abstract

Solid-phase extraction of alkylphosphonic and O-alkyl alkylphosphonic acids followed by HPLC separation using porous graphitic carbon sorbent

An HPLC separation of alkyl phosphonic acids on porous graphitic carbon adsorbent Hypercarb, based upon a step gradient of formic acid concentration in an aqueous mobile phase, was evaluated in this study. Analytes were detected by single quadrupole and triple quadrupole MS. Good separation was achieved for methyl phosphonic acid, ethylphosphonic acid, n-propylphosphonic acid, isopropylphosphonic acid, ethyl methylphosphonic acid, isopropyl methylphosphonic acid, isobutyl methylphosphonic acid, pinacolyl methylphosphonic acid. Solid-phase extraction of these analytes on Hypercarb was also proposed, desorption was carried out with aqueous ammonium formate and water-methanol mixtures. LODs were 0.1–0.2 ng mL−1 for these analytes without SPE; solid-phase extraction resulted in the decrease of LODs at least 100-fold. Accuracy was proven by analyses of spiked samples of river water and snow meltwater.

Single run analysis of glutathione and its disulfide in food samples by liquid chromatography coupled to on-line post-column derivatization

Glutathione and its disulfide were determined in a single run using liquid chromatography with on-line post-column derivatization and fluorimetric detection (340 nm/425 nm). The analytes were separated using a reversed-phase column capable of operating at 100% aqueous mobile phase and detected following direct on-line reaction with o-phthalaldehyde (7.5 mmol L−1) in highly basic medium (0.37 mol L−1 NaOH). The instrumental and chemical variables were carefully investigated towards high sensitivity and throughput, while special attention was paid to validating potential matrix effects. Glutathione and its disulfide could be selectively determined with respective LODs of 0.10 and 0.30 μmol L−1 in the absence of matrix effect (<6%). The endogenous content of the analytes was accurately determined in various food samples with recoveries ranging between 80 and 120% in all cases. The proposed method is reliable and promising as a generic analytical tool for the convenient estimation of the redox status of glutathione in various food matrices.

Rapid, sensitive, and reliable quantitation of nicotine and its main metabolites cotinine and trans-3′-hydroxycotinine by LC-MS/MS: Method development and validation for human plasma

New nicotine delivery products are gaining market share. For evaluation of their characteristics, toxicokinetic investigations are in current research focus. For reliable determination of blood plasma levels of nicotine and its main metabolites cotinine and trans-3′-hydroxycotinine, a quantitation method based on LC-ESI-MS/MS was developed and validated. Addition of isotope labeled internal standards prior to rapid sample preparation using protein precipitation with methanol was chosen for sample preparation. Different stationary phases were tested and phenyl-hexyl separation was found to be superior to HILIC, C18, and C8 stationary phases. Ion suppression effects caused by hydrophilic early eluting matrix were eliminated by the adjustment of an adequate retention utilizing a phenyl-hexyl separation stationary phase. Exchange of acetonitrile as organic mobile phase by methanol and elevation of pH value of aqueous mobile phase containing 5 mM NH4Ac to 4.50 improved the chromatographic resolution. The limits of quantitation for nicotine, cotinine, and hydroxycotinine were 0.15, 0.30, and 0.40 ng/mL, respectively. Linearity was proven by matrix matched calibration for the whole working range from 0.50 ng/mL to 35.0 ng/mL for nicotine and from 6.00 to 420 ng/mL for cotinine and hydroxycotinine (Mandel’s fitting test with R2 > 0.995). Quality control samples at four different levels (0.50, 1.50, 17.5, 28.0 ng/mL for nicotine and 6.00, 18.0, 210, 336 ng/mL for cotinine and hydroxycotinine) in plasma were analyzed six times on three days. Mean accuracies ranged from 87.7% to 105.8% for nicotine, from 90.3% to 102.9% for cotinine, and from 99.9% to 109.9% for hydroxycotinine. Intra- and inter-day precisions (RSD %) were below 15% for all analytes (<20% for LLOQ). As proof of concept, the method was successfully applied to a real plasma sample from a cigarette smoking volunteer.