Direct high-performance liquid chromatographic enantioseparation of free α-, β- and γ-aminophosphonic acids employing cinchona-based chiral zwitterionic ion exchangers

We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid chromatography on CHIRALPAK ZWIX(+) and ZWIX(-) (cinchona-based chiral zwitterionic ion exchangers) under polar organic chromatographic elution conditions. Mobile phase characteristics such as acid-to-base ratio, type of counterion, and solvent composition were systematically varied in order to investigate their effect on the separation performance and to achieve optimal separation conditions for the set of analytes. Under the optimized conditions, 32 of 37 racemic aminophosphonic acids studied reached baseline separation when we employed a single generic mass-spectrometry-compatible mobile phase, with reversal of the elution order when we used (+) and (-) versions of the chiral stationary phase.

Effect of mobile phase on resolution of the isomers and homologues of tocopherols on a triacontyl stationary phase

Reversed-phase liquid chromatographic (RPLC) separation of isomers and homologues of similar polarity is challenging. Tocopherol isomers and homologues are one such example. α, β, γ, and δ-tocopherols have been successfully separated by RPLC on triacontyl (C₃₀) stationary phase. System suitability was tested by using four mobile phases, and observed chromatographic separations of β and γ-tocopherols were compared. Comparison indicated that methanol–tert-butyl methyl ether (TBME) 95:5 (v/v) at a flow rate of 0.75 mL min^?1 was the best mobile phase. Detection systems were also evaluated on the basis of limit of quantification; it was concluded that fluorescence detection was best. The method was validated by analysis of two homologues and two isomers of tocopherol in sesame, maize, and soybean samples. MS coupled with an ESI interface in negative-ion mode [M ? H]^? was used for identification of individual components. It was concluded that addition of TBME to methanol was required to enhance the separation of β and γ-tocopherols, although methanol alone provided similar results. The applicability of the method to cereal, pulse, and oilseed samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the range 1.7–3.9 %. Recovery of tocopherols added to sesame samples ranged from 91 to 99 %.

Competitive growth of α- and β-crystals in isotactic polypropylene with versatile nucleating agents under shear flow

Sorbitol derivatives, the conventional α-nucleating agents of isotactic polypropylene (iPP), are discovered to induce β-phase iPP under normal crystalline conditions. Combined effects of shear flow and sorbitol derivatives on the crystallization of iPP were investigated by using differential scanning calorimetry, wide-angle X-ray diffraction, and small-angle X-ray scattering. In the nucleation stage, sorbitol derivatives induce both α- and β-nuclei, while shear flow and the interactions between shear and sorbitol derivatives enhance the amount of α-nuclei. In the growth stage, the epitaxial growth of β-crystals on shear-induced α-row nuclei occurs. As the shear rate increases, more epitaxial β-crystals form due to the increase of α-row nuclei, further increasing the content of β-crystals. Under high shear rate, the presence of sorbitol derivatives and shear flow exhibit a synergistic interaction on increasing the content of β-crystals. Moreover, α-nuclei, which arise from the interaction between shear and sorbitol derivatives, emerge earlier than shear-induced α-row nuclei.

Enantioseparation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate tagged amino acids and other zwitterionic compounds on cinchona-based chiral stationary phases

The fluorescent tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ Fluor reagent kit from Waters) is a commercial N-terminal label for proteinogenic amino acids (AAs), designed for reversed-phase separation and quantification of the AA racemates. The applicability of AQC-tagged AAs and AA-type zwitterionic compounds was tested for enantiomer separation on the tert-butyl carbamate modified quinine and quinidine based chiral stationary phases, QN-AX and QD-AX employing polar-organic elution conditions. The investigated test analytes included the enantiomers of the positional isomers of isoleucine (Ile), threonine, homoserine, and 4-hydroxyproline. Furthermore, β-AAs, cyclic, and heterocyclic AAs including trans-2-amino-cyclohexane carboxylic acid and trans-2-aminocyclohexyl sulfonic acid, phenylalanine derivatives substituted with halides with increasing electronegativity and 3,4-dihydroxyphenylalanine, cysteine-related derivatives including homocysteic acid, methionine sulfone, cysteine-S-acetic acid, and cysteine-S-acetamide as well as a small range of aminophosphonic acids were enantioseparated. A mechanistic interaction study of AQC-AAs in comparison with fluoresceine isothiocyanate-labeled AAs was performed. The chiral and chemoselective recognition processes involved in enantiomer separation and retention was systematically discussed. Special emphasis was set on the influential factors exhibited by the chemistry, branching position, and spatial properties of the investigated zwitterionic analytes. The general interest to separate and distinguish between different types of branched-chained AAs and metabolic side products thereof lies in the toxicity of some of these compounds, which makes for instance allo–Ile an attractive candidate in disease-related biomarker research.

Advantages of Monodisperse and Chemically Robust “SpheriCal” Polyester Dendrimers as a “Universal” MS Calibrant

The utilization of dendrimer calibrants as an alternative to peptides and proteins for high mass calibration is explored. These synthetic macromolecules exhibited a number of attractive advantages, including exceptional shelf-lives, broad compatibility with a wide range of matrices and solvents, and evenly spaced calibration masses across the mass range examined, 700–30,000 u. The exceptional purity of these dendrimers and the technical simplicity of this calibration platform validate their broad relevance for high molecular weight mass spectrometry.

Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein–ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric “oxidized” peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Direct lactonization of α-amino γ,δ-unsaturated carboxylic acid esters via olefin activation: stereo- and regioselective production of homoserine lactone scaffolds having contiguous stereocenters

Triflic acid-mediated stereoselective direct lactonization of a variety of α-amino γ,δ-unsaturated carboxylic acid esters and the construction of new γ-butyrolactone structural motifs are reported. Several α-amino γ,δ-unsaturated carboxylic acid esters underwent stereo- and regioselective 1,5-cyclization and afforded a variety of highly substituted homoserine lactone scaffolds having contiguous stereocenters. The direct lactonization of the chiral α-amino γ,δ-unsaturated carboxylic acid esters with triflic acid led to the enantioselective synthesis of the novel homoserine lactones. A plausible mechanism for the direct lactonization of α-amino γ,δ-unsaturated carboxylic acid esters is presented. The stereochemistry of major isomers 3f, 7a, 7b, and 7d was unambiguously established from the X-ray structure analysis.     

Spectral Reproducibility and Quantification of Peptides in MALDI of Samples Prepared by Micro-Spotting

Previously, we reported that MALDI spectra of peptides became reproducible when temperature was kept constant. Linear calibration curves derived from such spectral data could be used for quantification. Homogeneity of samples was one of the requirements. Among the three popular matrices used in peptide MALDI [i.e., α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapinic acid (SA)], homogeneous samples could be prepared by conventional means only for CHCA. In this work, we showed that sample preparation by micro-spotting improved the homogeneity for all three cases.

Copolymer-grafted silica phase from a cation–anion monomer pair for enhanced separation in reversed-phase liquid chromatography

This work reports a new imidazolium and l-alanine derived copolymer-grafted silica stationary phase for ready separation of complex isomers using high-performance liquid chromatography (HPLC). For this purpose, 1-allyl-3-octadecylimidazolium bromide ([AyImC₁₈]Br) and N-acryloyl-l-alanine sodium salt ([AAL]Na) ionic liquids (IL) monomers were synthesized. Subsequently, the bromide counteranion was exchanged with the 2-(acrylamido)propanoate organic counteranion by reacting the [AyImC₁₈]Br with excess [AAL]Na in water. The obtained IL cation–anion monomer pair was then copolymerized on mercaptopropyl-modified silica (Sil-MPS) via a surface-initiated radical chain-transfer reaction. The selective retention behaviors of polycyclic aromatic hydrocarbons (PAHs), including some positional isomers, steroids, and nucleobases were investigated using the newly obtained Sil-poly(ImC₁₈-AAL), and octadecyl silylated silica (ODS) was used as the reference column. Interesting results were obtained for the separation of PAHs, steroids, and nucleobases with the new organic phase. The results showed that the Sil-poly(ImC₁₈-AAL) presented multiple noncovalent interactions, including hydrophobic, π–π, carbonyl–π, and ion–dipole interactions for the separation of PAHs and dipolar compounds. Only pure water was sufficient as the mobile phase for the separation of the nucleobases. Ten nucleosides and bases were separated, using only water as the mobile phase, within a very short time using the Sil-poly(ImC₁₈-AAL), which is otherwise difficult to achieve using conventional hydrophobic columns such as ODS. The combination of electrostatic and hydrophobic interactions are important for the effective separation of such basic compounds without the use of any organic additive as the eluent on the Sil-poly(ImC₁₈-AAL) column.

Separation and quantification of four isomers of indole-3-acetyl-myo-inositol in plant tissues using high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

Indole-3-acetyl-myo-inositol (IAInos) is one of the most important auxin conjugates for storage and transportation of auxin. The information of its composition, distribution, and metabolism is particularly desired for elucidating the related signal transduction pathways of the plant hormones. However, separation and quantification of the four individual IAInos isomers in plant tissues have not been reported so far. In this work, we first synthesized and isolated four IAInos isomers using semi-preparative high-performance liquid chromatography (HPLC). The IAInos isomer structures were characterized using liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC-QTOF/MS) and nuclear magnetic resonance spectroscopy (NMR). Using these pure compounds as internal or external standards, an efficient LC-MS method was developed for simultaneous detection of indole-3-acetic acid, methyl indole-3-acetic acid ester, and the four IAInos isomers in plant tissue samples. The linear working range and lower limit of detection for the four IAInos isomers are 10–2,000 ng mL^?1 and 5.0 ng mL^?1, respectively. The stabilities and interconversion pathways of IAInos isomers were studied using our synthetic isomers. It was found that two IAInos isomers existed in Zea mays kernels, while all of the four IAInos isomers were present in the roots of Arabidopsis thaliana. The content of IAInos in A. thaliana roots was much lower than in the Z. mays kernels. The methodology in this article provides useful techniques and methods for systematic study on the phytophysiology and phytochemistry of IAA conjugates and other related plant hormones.

Development and validation of a microfluidic chip-based nano-liquid chromatography–triple quadrupole tandem mass spectrometry method for a sensitive and reliable quantification of 7-ethyl-10-hydroxycamptothecin (SN38) in mouse plasma

There is an increasing need for more sensitive analytical methods in pharmacokinetic studies, for example, for phase 0 clinical trials. A novel HPLC Chip–triple quadrupole mass spectrometer method (HPLC Chip-MS/MS method) for the quantification of 7-ethyl-10-hydroxycamptothecin (SN38) was developed, validated, and employed to the pharmacokinetic analysis of SN38 in ICR mice. Protein precipitation with a ratio of plasma/acetonitrile of 1:10 was chosen as the sample processing method. The nano-electrospray inserted in the microfluidic chip operated in positive mode, and selected reaction monitoring was used for quantification. Our bioanalytical method met all essential validation parameters—selectivity, accuracy, precision, dilution integrity, calibration curve, matrix effect, recovery, and different stability tests (benchtop, freeze–thaw, autosampler stability). The calibration curves (weight 1/x ²) were linear for the range 50–10,000 pg/mL. Clogging was not observed until the end of the lifetime of the microfluidic chip (350–400 injections), and carryover was practically eliminated through the introduction of a step gradient elution program. After intraperitoneal injection of 0.1 mg/kg irinotecan, SN38 concentration could be measured up to 6 h with accuracy and precision. Thus, we developed a new, very sensitive HPLC Chip-MS/MS method for the determination of plasma SN38 that has been validated in compliance with guidelines from different regulation authorities.

Application of normal-phase high-performance liquid chromatography followed by gas chromatography for analytics of diesel fuel additives

The paper presents the results of investigations on new procedures of determination of selected cleaning additives in diesel fuel. Two procedures: one-step analysis using gas chromatography with flame ionization detection (GC-FID) or mass spectrometry (GC-MS) and a two-step procedure in which normal-phase high-performance liquid chromatography (NP-HPLC) was used for preliminary separation of the additives, were compared. The additive fraction was collected using either simple elution or eluent backflush. Final determinations were performed by GC-FID and GC-MS. The studies revealed that it was impossible to determine the investigated analytes by one-step procedures, i.e. by using solely HPLC or GC. On the other hand, the use of a two-step procedure ensures reproducible results of determinations, and the limits of quantitation are, depending on the method of fraction collection by HPLC, from 1.4–2.2 ppm (GC-MS in SIM mode) to 9.6–24.0 ppm (GC-FID). Precision and accuracy of the developed procedures are compared, and possible determination errors and shortcomings discussed.

Electrochemical sensor based on a carbon nanotube-modified imprinted sol–gel for selective and sensitive determination of ß2-agonists

We describe a molecularly imprinted electrochemical sensor for selective and sensitive determination of β₂-agonists. It is making use of a combination of single-wall carbon nanotubes (SWNTs) with a molecularly imprinted sol–gel. The SWNTs were introduced in order to enhance electron transport and sensitivity. The imprinted sol–gel film with its specific binding sites acts as a selective recognition element and as a preconcentrator for β₂-agonists. The morphology of the imprinted film was characterized by scanning electron microscopy. The optimized sensor displays high sensitivity and excellent selectivity for the β₂-agonists as shown for their determination in human serum samples.

In-Depth Comparative Characterization of Hemoglobin Glycation in Normal and Diabetic Bloods by LC-MSMS

The glycation level at β-Val-1 of the hemoglobin β chain in human blood (HbA1c%) is used to diagnose diabetes and other diseases. However, hemoglobin glycation occurs on multiple sites on different isoforms with different kinetics, but its differential profile has not been clearly demonstrated. In this study, hemoglobin was extracted from the blood of normal and diabetic individuals by protein precipitation. Triplicate solutions prepared from each sample were directly analyzed or digested with multiple enzymes and then analyzed by nano-LC/MS via bottom-up approach for side-by-side characterization. Intact hemoglobin analysis indicated a single glucose-dominant glycation, which showed good correlation with the HbA1c% values. Moreover, full sequence (100 %) of α/β globin was mapped and seven glycation sites were unambiguously assigned. In addition to β-Val-1, two other major sites at α-Lys-61 and β-Lys-66, which contain the common sequence HGKK, and four minor sites (<1 %) on α-Val-1, β-Lys-132, α-Lys-127, and α-Lys-40 were identified. All sites were shown to exhibit similar patterns of site distribution despite different glucose levels. Both the intact mass measurement and bottom-up data consistently indicated that the total glycation percentage of the β-globin was twice higher than the α-globin. Using molecular modeling, the 3D structure of the consensus sequence (HGKK) was shown to contain a phosphate triangle cavity, which helps to catalyze the glycation reaction. For the first time, hemoglobin glycation in normal and diabetic bloods was comparatively characterized in-depth with 100 % sequence coverage. The results provide insight about the HbA1c parameter and help define the new and old markers.

Simultaneous Determination of Ten Sugars in Tinospora cordifolia by Ultrasonic Assisted Extraction and LC-ELSD

Sugars present in medicinal plants are known for protecting and stimulating the immune system against various biological disorders. Tinospora cordifolia is a reputed Indian herb used for immunity enhancing which is mainly attributed to saccharides. In the present study, a simple, sensitive, and reliable liquid chromatography method based on ultrasonic assisted extraction and evaporative light scattering detection was developed for simultaneous determination of ten sugars comprising of monosaccharides (l-(+)-rhamnose, d-(+)-xylose, d-(?)-arabinose, β-d-(+)-glucose), disaccharides (sucrose, d-(+)-cellobiose, α-lactose), alditols (xylitol, d-(+)-mannitol) and a polyalcohol (myo-inositol) in T. cordifolia. The separation was achieved on Zorbax-NH₂ column (250 mm × 4.6 mm, 5 µm) in gradient elution of acetonitrile: water as mobile phase with flow rate of 0.5 mL min^?1. The drift tube temperature and nitrogen flow-rate were optimized at 70 °C and 2.0 standard litres per minute, respectively. The method was validated for linearity, accuracy, precision, limits of detection and quantification. The calibration equation revealed a good linear relationship (r ²  = 0.959–0.999). The sufficient recovery was observed in the range of 94.1–99.9%. The method showed good reproducibility with intra- and inter-day precision of <0.99 and 0.97% (RSD), respectively. The detection and quantification limits for the compounds were in the range of 8.32–44.29 and 25.23–134.20 μg mL^?1, respectively.     

Enantioselective separation and simultaneous determination of fenarimol and nuarimol in fruits, vegetables, and soil by liquid chromatography–tandem mass spectrometry

A method for simultaneous enantioselective determination of fenarimol and nuarimol in apple, grape, cucumber, tomato, and soil was developed using liquid chromatography–tandem mass spectrometry. The enantioseparation results of the two fungicides through three different cellulose-based chiral columns are discussed. The influence of column temperature on the resolution of the enantiomers of the two fungicides was examined. Complete enantioseparation of the two fungicides’ enantiomers was obtained on a cellulose tris(4-methylbenzoate) column (Lux Cellulose-3) at 25?°C using methanol and 0.1?% formic acid solution (80:20, v/v) as mobile phase. The linearity, matrix effect, recovery, and precision were evaluated. Good linearity was obtained over the concentration range of 1–500?μg?L^?1 for each enantiomer in the standard solution and sample matrix calibration solution. There was no significant matrix effect in apple, grape, cucumber, or tomato samples, but signal suppression was typically observed with the soil extracts. The mean recoveries, repeatability, and reproducibility were 76.5–103?%, 2.1–9.0?%, and 4.2–11.8?%, respectively. The limit of quantification for enantiomers of the two fungicides in fruits, vegetables and soil was 5?μg?kg^?1. Moreover, the absolute configuration of the enantiomers of fenarimol and nuarimol was determined from a combination of experimentally determined and predicted electronic circular dichroism spectra.

A multiresidual method based on ion-exchange chromatography with conductivity detection for the determination of biogenic amines in food and beverages

In the present work a sensitive and accurate method by ion chromatography and conductimetric detection has been developed for the determination of biogenic amines in food samples at microgram per kilogram levels. The optimized extraction procedure of trimethylamine, triethylamine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine from real samples, as well as the separation conditions based on a multilinear gradient elution with methanesulfonic acid and the use of a weak ionic exchange column, have provided excellent results in terms of resolution and separation efficiency. Extended calibration curves (up to 200 mg/kg, r?>?0.9995) were obtained for all the analyzed compounds. The method gave detection limits in the range 23–65 μg/kg and quantification limits in spiked blank real samples in the range 65–198 μg/kg. Recovery values ranged from 82 to 103 %, and for all amines, a good repeatability was obtained with precision levels in the range 0.03–0.32 % (n?=?4). The feasibility and potential of the method were tested by the analysis of real samples, such as tinned tuna fish, anchovies, cheese, wine, olives, and salami.

New validated HPLC methodology for the determination of (−)-trans-paroxetine and its enantiomer in pharmaceutical formulations with use of ovomucoid chiral stationary phase

A new chromatographic method for the enantioseparation and the determination of (?)-trans-paroxetine and (+)-trans-paroxetine has been developed with the aid of amylose ovomucoid-based chiral stationary phase. The method is faster and five times more sensitive than procedures recommended previously: limit of detection and limit of quantification are 5 and 16 ng/mL, respectively [modified (Ferretti et al. in J Chromatogr B 710:157–164, 1998): 20 and 60 ng/mL]. It was carefully validated and applied for the determination of (?)-trans-paroxetine and (+)-trans-paroxetine in Parogen (Mc Dermott Laboratories Ltd.) and Xetanor (Actavis) coated tablets.

Method development for simultaneous analysis of HBCD,TBBPA, and dimethyl-TBBPA in marine biota from Greenland and the Faroe Islands

The brominated flame retardants hexabromocyclododecane (HBCD) and tetrabromobisphenol A (TBBPA) are high-production-volume chemicals. In recent years, their presence has been reported in sediment and biota from the marine environment. In this study, an analytical method was developed for the simultaneous determination of HBCD, TBBPA, and the possible metabolite dimethyl-TBBPA. The method was applied in a preliminary screening of egg, liver, and adipose tissue of marine biota from Greenland and the Faroe Islands. α-HBCD was detected in 35 of 36 analysed samples from the Arctic, indicating a ubiquitous presence of α-HBCD in the environment. β- and γ-HBCD were found in 10 and 14 samples, respectively. TBBPA and dimethyl-TBBPA were not detected in any of the samples indicating limited or no transport of these compounds to remote areas.     

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.