Determination of hexabromocyclododecane diastereoisomers in air and soil by liquid chromatography-electrospray tandem mass spectrometry

Hexabromocyclododecanes (HBCDs), used as additive brominated flame retardants, are of high concern due to their widespread use and increasing levels in various environmental systems. High-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed for the determination of HBCD diastereoisomers. A detailed study was carried out to optimize the composition of the mobile phase involving methanol/acetonitrile/water, and the values of MS/MS parameters. It was found that the mobile phase could simultaneously affect the chromatographic separation and sensitivity. The instrumental limits of detection (LODs) on column in this study were 0.5, 0.3 and 0.3 pg for alpha-HBCD, beta-HBCD and gamma-HBCD, respectively. The effects of extracted matrix components on HBCD determination were investigated by spiking air and soil sample extracts with three 13C-labelled individual stereoisomers. The results indicated that the responses of the HBCD analysis in air and soils were not significantly affected by matrix effects. The method reported here was further applied to the air and soil samples. Three HBCD diastereoisomers were detected in all the air and soil samples, with levels ranging from 1.2 to 1.8 pg/m3 and 1.7 to 5.6 ng/g dry weight, respectively.     

Factors influencing enantiomeric fractions of hexabromocyclododecane measured using liquid chromatography/tandem mass spectrometry

Hexabromocyclododecane (HBCD), the most heavily produced of the cycloaliphatic brominated flame retardants (BFRs), is a mixture of three predominant diastereomers (alpha-, beta-, and gamma-HBCD), each with a corresponding pair of enantiomers. We have investigated the liquid chromatography/mass spectrometry (LC/MS) behaviour of the HBCD enantiomers, and demonstrated that enantiomeric fractions (EFs) calculated using data uncorrected for instrument and/or matrix effects can result in potentially inaccurate EF values. However, use of labelled surrogates effectively corrects for these effects. Experiments with racemic HBCD standards indicate that chromatographic factors, including mobile phase composition and column bleed from chiral stationary phases, may be contributors to variations in the mass spectrometric response of the HBCD enantiomers.     

A liquid chromatography-tandem mass spectrometry method for the quantitative determination of diastereomers of a phosphoramidate nucleotide prodrug (PSI-7851) in human plasma

A rapid and stereospecific method using high performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) for the separation and determination of PSI‐7851 diastereomers in human K₂EDTA plasma has been developed. The analytical method involves direct protein precipitation with acetonitrile, followed by separation of the diastereomers on a Luna C₁₈ column, positive mode electrospray ionization and selected reaction monitoring mode mass spectrometry detection. The mobile phase composition and pH were investigated for the resolution of the two diastereomers of PSI‐7851. The optimized method showed good resolution (R_s = 4.8) within short analysis time (approximately 8 min). The assay range was 5–2500 ng/mL for both diastereomers using a 1/x² weighted linear regression analysis for standard curve fitting. Replicate sample analysis indicated that intra‐ and inter‐day accuracy and precision were within ±15.0%. The recovery of diastereomers from human plasma was greater than 85% and no significant matrix effect was observed. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Cyanobiphenyl-Mesogened Liquid Crystalline Polymer Bonded on Silica as the Stationary Phase with Shape and Polarity Recognition for LC

Cyanobiphenyl-mesogened liquid crystalline polymer is bonded on silica by surface-initiated atom transfer radical polymerization and is used as the stationary phase for liquid chromatography. Various instrumental analyses such as elemental analysis, X-ray photoelectron spectroscopy and differential scanning calorimetry were used for its characterization. The stationary phase exhibits multiple characteristics of low hydrophobicity, low hydrophobic selectivity, polarity recognition and shape selectivity in the separation of polyaromatic hydrocarbons and polar neural aromatic compounds. Temperature and mobile phase composition were confirmed to have effects on the chromatographic behavior. Isomers of polyaromatic hydrocarbons and carotenes are well separated on the stationary phase.     

Comparison of electrospray ionization, atmospheric pressure photoionization, and anion attachment atmospheric pressure photoionization for the analysis of hexabromocyclododecane enantiomers in environmental samples

Anion attachment atmospheric pressure photoionization (AA-APPI) has been suggested as a means of expanding the range of compounds that may be analyzed by LC-MS, and has been found to enhance the ionization of some macromolecules (e.g., peptides, polymers) that were unable to be ionized by other techniques. In this study, AA-APPI was compared to APPI, using hexabromocyclododecane (HBCD) enantiomers as a model compound, to provide proof of principle of the use of AA-APPI for small molecule analysis. The use of AA-APPI, with 1,4-dibromobutane in toluene as a bromide source, offered increased sensitivity and lower limits of detection than APPI. Minimal matrix effects were found with AA-APPI in sediment extracts spiked with HBCD post-extraction, with less than a 6% enhancement in the ion signal. Furthermore, enantiomer fractions of HBCD enantiomers were racemic in spiked sediment extracts, in contrast to the more commonly used technique of electrospray ionization, for which matrix effects caused ion signal modification to cause non-racemic measurement artifacts. The use of AA-APPI offers a simple means of further extending the range of compounds ionizable by AA-APPI while maintaining minimal matrix effects.     

Porous graphitic carbon chromatography/tandem mass spectrometric determination of cytarabine in mouse plasma

A high-performance liquid chromatography (HPLC) system using a porous graphitic carbon (PGC) stationary phase interfaced with an electrospray ionization (ESI) source and a tandem mass spectrometer (MS/MS) for the analysis of cytarabine (ara-C) in mouse plasma samples has been developed in support of a pharmacodynamic study. The graphitized carbon column was adopted for the separation of ara-C and endogenous peaks from mouse plasma samples under the reversed-phase phase mode in liquid chromatography. The retention characteristics of the PGC column and the ionization efficiencies of all analytes based on the experimental factors such as the composition of mobile phases were investigated. The potential of ionization suppression resulting from the endogenous biological matrices on the PGC column during HPLC/ESI-MS/MS was investigated using post-column infusion. The concentrations of ara-C in mouse plasma obtained by using PGC-HPLC/MS/MS and ion-pairing HPLC/MS/MS were found to be in good agreement in terms of analytical accuracy.     

超高效液相色谱-串联四极杆质谱法检测小型家电外壳中的六溴环十二烷

建立了加热回流萃取-超高效液相色谱-串联四极杆质谱检测小型家用电器中六溴环十二烷( Hexabromocyclododecane,HBCD)的方法.实验优化了电子电器类产品的前处理方法,以甲苯-甲醇(10∶1,V/V)为萃取剂,加热回流4h.萃取出的溶液经N2吹干,初始流动相复溶,涡旋、离心、过膜,经ACQUITYTMUPLC BEH C18色谱柱分离;以甲醇-乙腈(4∶1,V/V)-10 mmol/L醋酸铵为流动相,质谱的多反应监测模式(MRM)进行检测,HBCD的3种同分异构体在3 min内完全分开.该物质的检山限为0.014 mg/L:定量限为0.068 mg/L;标准曲线的线性范围为1.6～ 32.4 mg/L,线性相关系数大于0.996,萃取回收率为68.0％～75.2％.通过外标法定量,并将本方法应用于实际样品(电视机外壳、电子相框、电磁炉外壳等)的检测.     

Molecularly imprinted monolithic stationary phases for liquid chromatographic separation of enantiomers and diastereomers

The method for preparation of molecularly imprinted monolithic stationary phase has been improved to achieve liquid chromatographic separation of enantiomers and diastereomers. By adopting low polar porogenic solvents of toluene and dodecanol and optimal polymerization conditions, the molecularly imprinted monolithic stationary phases with good flow-through properties and high resolution were prepared. Enantiomers of amino acid derivatives and diastereomers of cinchona alkaloids were completely resolved using the monolithic stationary phases. The influence of porogenic composition, monomer-template ratio and polymerization conditions on the chromatographic performance was investigated. Some chromatographic conditions such as the composition of the mobile phase and the temperature were characterized. Scanning electron microscopy showed that the molecularly imprinted monolithic stationary phase has a large through-pore structure to allow the mobile phase to flow through the column at very low backpressure. Accelerated separations of enantiomers and diastereomers were therefore achieved at elevated flow rates. Finally, the chiral recognition performance of the prepared stationary phase in aqueous media was investigated. Hydrophobic interaction, and ionic and/or hydrogen bonding interactions were proposed to be responsible for the recognition mechanism.     

Selectivity enhancement for trans-2-(2,3-anthracenedicarboximido)-cyclohexane-derived diastereomers in HPLC by using an ordered organic stationary phase.

2-(2,3-anthracenedicarboximido)cyclohexane derivatives (AC) have been known as the evolutionary diastereomerizing reagents for enantiomer discrimination in HPLC with ODS. However, a substantial separation of diastereomers can be observed only at lower temperatures, such as -40 degrees C. Therefore, in this work, poly(octadecyl acrylate)-grafted silica, ODAn was applied as an alternative stationary phase to ODS for the separation of AC-derived diastereomers. As a result, complete separation was achieved even under the conventional condition: for example, methanol as the mobile phase and 0 degrees C as the column temperature. An investigation on the temperature dependency of the selectivity demonstrated that ODAn shows a remarkable increase in selectivity at temperatures below 30 degrees C, which almost agreed with the peak-top temperature of the endothermic peak in a DSC thermogram for ODA35 immersed in a mobile phase. The better separation would be derived from a highly ordered structure of ODAn and a carbonyl-pi interaction with AC-derived diastereomers.     

Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry

The chromatographic behaviour of 44 polar compounds (23 beta-adrenergic agents, 11 stimulants, 4 narcotics and 6 phenolalkylamines) included in the list of prohibited substances and methods of the World Anti-Doping Agency, has been investigated under hydrophilic interaction liquid chromatography conditions by application of different mobile phase compositions (percentage of the organic solvent, type and amount of mobile phase additive and ionic strength) and column temperatures. Detection of analytes was performed by a triple quadrupole mass spectrometer in positive ionization mode and selected reaction monitoring acquisition mode after liquid/liquid extraction. Data collected using as stationary phase type-B silica materials from different producers, showed that the best chromatographic conditions in terms of peak shape, selectivity and chromatographic retention were obtained using an initial percentage of acetonitrile of 90%, a column temperature of 35 °C, a mobile phase pH of 4.5 and ammonium acetate (5 mM) and acetic acid (0.1%) as mobile phase additives. The selected chromatographic conditions were used to develop screening and confirmation analytical procedures to detect polar compounds in human urine for antidoping purpose. The developed methods were validated in terms of specificity, matrix effect, linearity, precision, accuracy, sensitivity, robustness and repeatability of retention times and relative ion abundances. Such methods offer attractive alternatives and considerable advantages over traditional approaches especially for the analysis of the phenolalkylamines.     

Preconcentration and sensitive determination of hexabromocyclododecane diastereomers in environmental water samples using solid phase extraction with bamboo charcoal cartridge prior to rapid resolution liquid chromatography-electrospray tandem mass spectrometry

In this paper, a simple and cheap method for the simultaneous preconcentration and sensitive determination of three hexabromocyclododecane (HBCD) diastereomers (α-, β-, and γ-HBCD) in environmental water samples has been developed. It was based on solid phase extraction (SPE) and rapid resolution liquid chromatography-electrospray tandem mass spectrometry. Bamboo charcoal, one kind of cheap material, was investigated and used as SPE adsorbent for the enrichment and determination of HBCD diastereomers. Related important parameters affecting extraction efficiencies, including type and volume of eluant, amount of sorbent, sample pH, flow rate, and sample volume, were investigated and optimized in detail. Under the optimum conditions, experimental data exhibited excellent linear relationships between peak area and concentrations over the range 0.1-10?μg?L(-1). The limits of detection and precision were in the range of 0.005-0.015?μg?L(-1) and 4.59-7.47%, respectively. The proposed method has been successfully applied for the trace analysis of HBCD diastereomers in real-world environmental water samples.     

Sheath liquid interface for the coupling of normal-phase liquid chromatography with electrospray mass spectrometry and its application to the analysis of neoflavonoids

A novel interface that allows normal-phase liquid chromatography to be coupled with electrospray ionization (ESI) is reported. A make-up solution of 60 mM ammonium acetate in methanol, infused at a 5 microl min(-1) flow-rate at the tip of the electrospray probe, provides a sheath liquid which is poorly miscible with the chromatographic effluent, but promotes efficient ionization of the targeted analytes. Protonated molecules generated in the ESI source were subjected to tandem mass spectrometric experiments in a triple-quadrupole mass spectrometer. The main fragmentation reactions were characterized for each analyte and specific mass spectral transitions were used to acquire chromatographic data in the multiple reaction monitoring detection mode. Results obtained during optimization of the sheath liquid composition and flow-rate suggest that the electrospray process was mainly under the control of the make-up solution, and that it forms an external charged layer around a neutral chromatographic mobile phase core. This sheath liquid interface was implemented for the analysis of some neoflavonoid compounds and its performance was evaluated. Limits of detection were established for calophillolide, inophyllum B, inophyllum P and inophyllum C at 100, 25, 15 and 100 ng ml(-1), respectively.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

Chromatographic shape selectivity with carbon dioxide-acetonitrile mobile phases. Effect of mobile phase composition and density

Trends in chromatographic shape selectivity with mobile phases consisting of mixtures of carbon dioxide and acetonitrile are investigated. Selectivity is evaluated as a function of mobile phase composition, temperature, and column bonding chemistry. SRM (standard reference material) 869a is used as a probe of shape selectivity, while the selectivity between triphenylene and o-terphenyl is used to investigate planarity selectivity. Four molecular mass 228 polyaromatic hydrocarbon isomers are used to investigate shape selectivity based on differences in length-to-breadth ratio. Shape selectivity trends as a function of temperature and column type are found to be similar to what is seen in reversed-phase liquid chromatography, while the trend seen as the amount of acetonitrile in the mobile phase increases is found to be different than in reversed-phase liquid chromatography. In addition, the effect of mobile phase density, i.e., solvent strength, on shape selectivity is investigated by examining shape selectivity as a function of density with neat carbon dioxide as the mobile phase.     

Facile separation of enantiomers,geometrical isomers,and routine compounds on stable cyclodextrin LC bonded phases

The effectiveness of employing stationary phases composed of chemically bonded cyclodextrin molecules in the high performance liquid chromatographic separation of a variety of different types of compounds is summarized. Over one hundred compounds, including optical, geometrical, and structural isomers, diastereomers, and epimers were successfully separated from each other via use of beta- or gamma-cyclodextrin bonded phases and aqueous methanolic mobile phases. The mechanism of separation is based upon inclusion complex formation between the compounds being separated and the cyclodextrin molecules bonded to the stationary phase. The effects of temperature, mobile phase composition and flow rate upon the chromatographic selectivity and resolution are described. The results indicate that the cyclodextrin columns may be more versatile, flexible, and effective compared to the conventional normal or reversed phase columns.     

Optimization of micro‐HPLC peak focusing for the detection and quantification of low hepcidin concentrations

Micro‐high‐performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 μL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile‐phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.     

Open-tubular capillary electrochromatography-mass spectrometry with sheathless nanoflow electrospray ionization for analysis of amino acids and peptides

A novel, rugged sheathless capillary electrochromatography-electrospray ionization (CEC-ESI) device, in which an open-tubular separation capillary and an electrospray tip are integrated with a Nafion tubing junction, is coupled to mass spectrometry (MS) for the analysis of amino acids and peptides. A stable electrospray was generated at nanoflow rates by applying a positive electrical potential at the Nafion membrane junction. To sustain the stable spray, an electroosmotic flow (EOF) to the spray was supported by coating the fused silica capillary with Lupamin, a high-molecular-weight linear positively charged polyvinylamine (PVAm) polymer, which also minimizes analyte adsorption. Electrochromatographic separation of amino acids and peptides was further enhanced by the chromatographic selectivity of Lupamin stationary phase for these molecules. The device was very reliable and reproducible for CEC-ESI-MS analyses of amino acids and peptides for over a hundred injections. The separation and detection behaviors of amino acids and peptides under different conditions including pH, concentration, and composition of mobile phases on Lupamin-coated and uncoated capillaries have been investigated. The relationship between nano electrospray stability and EOF is discussed.     

Analysis of derivatized and underivatized theanine enantiomers by high-performance liquid chromatography/atmospheric pressure ionization-mass spectrometry

Theanine, a naturally occurring non-proteinic amino acid found in tea leaves, has demonstrated wide-ranging physiological activity, from lowering blood pressure to enhancing the anti-tumor activity of chemotherapeutic drugs. The chiral nature of theanine suggests that enantiospecificity plays a significant role in its various pharmacological functions. Using the Chirobiotic T (teicoplanin) chiral stationary phase, native and derivatized theanine enantiomers were separated and detected via high-performance liquid chromatography (HPLC) coupled to atmospheric pressure ionization mass spectrometry (API-MS). With the use of flow rates compatible with each ionization source, native theanine standards achieved excellent sensitivity and detection limits (10 ng/mL) for both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Optimum sensitivity and detection limits for derivatized theanine standards were achieved using ESI-MS. The enantiomeric composition of six commercially available L-theanine samples was evaluated using the high-flow APCI-MS method and confirmed with photodiode array detection. Five of the six products contained significant amounts of D-theanine. Only one product, SunTheanine, appeared to contain only the L-theanine enantiomer.     

Simultaneous Quantification of 3′- and 6′-Sialyllactose in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Study

Sialyllactose (SL), an acidic oligosaccharide, has immune-protective effects against pathogens and helps with the development of the immune system and intestinal microorganisms. To elucidate the pharmacokinetic characterization after oral administration to rats, the simultaneous quantification method for 3′-SL and 6′-SL in rat plasma was validated, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an electrospray ionization (ESI) mode. Several types of columns [C18, amide, and hydrophilic interaction liquid chromatography (HILIC) phase] were used to separate the peaks of 3′-SL and 6′-SL, which improved chromatographic selectivity. Ultimately, the HILIC phase column had a good peak shape and quick resolution, with a mobile phase comprising ammonium acetate buffer and acetonitrile obtained by gradient elution. In addition, the simultaneous quantification of 3′-SL and 6′-SL in rat plasma samples were adequately applied to pharmacokinetic study.     

Optimization of electrospray interface and quadrupole ion trap mass spectrometer parameters in pesticide liquid chromatography/electrospray ionization mass spectrometry analysis

Optimization of both the ionization process and ion transportation in the mass spectrometer is of crucial importance in order to achieve high sensitivity and low detection limits and acceptable accuracy in liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) analysis. In this paper four optimization procedures of electrospray interface and quadrupole ion‐trap mass spectrometer parameters (ESI‐MS) (nebulizer gas and drying gas flow rate, end plate voltage, capillary voltage, skimmer voltage, octopoles direct current and radio frequency, trap drive and lens voltages) were studied on three pesticides – thiabendazole, aldicarb and imazalil. The results demonstrate that the methodology of optimization strongly influences the effectiveness of finding true optima of the operating parameters. Both eluent flow rate and composition during optimization have to mimic the situation during real analysis as closely as possible in order to achieve parameters giving the highest sensitivity. Therefore, post‐column addition of analyte to the mobile phase identical in composition to the one in which analyte elutes during real analysis combined with software‐based optimization was found to be the most effective and fastest method for achieving intensity maxima. The parameters most strongly affecting ion formation and transportation, hence sensitivity, were capillary voltage, direct current of the first octopole, trap drive and the second lens for all pesticides under study. In addition to sensitivity and detection limit matrix effect was considered in the optimization process. It was found that the matrix effect can be reduced but not eliminated by adjusting the ESI and MS parameters. The optimal parameters from the point of view of the matrix effect can only be found with factorial design. Parameters giving higher sensitivity tended to be more affected by matrix effect causing higher ionization suppression by co‐eluting compounds. Copyright © 2010 John Wiley & Sons, Ltd.