Application of Mixed-Mode Oasis MCX Adsorbent for Chromatographic Separation of Selenomethionine from Antarctic Krill After Enzymatic Digestion

The present communication describes the novel application of mixed-mode adsorbents for bifunctional chromatography. Oasis MCX mixed-mode adsorbent (Waters GmbH, Eschborn, Germany) shows a high binding capacity for Se-Met. The adsorbed selenoamino acid is easily eluted by pH change and addition of organic solvent. Determined adsorption isotherm parameters are suitable for its analytical and preparative chromatographic isolation. A chromatographic column packed with the Oasis MCX adsorbent, designed for a single use solid phase extraction, was successfully applied for bifunctional chromatographic separation of Se-Met from hydrolysed Antarctic krill samples with 85 ± 5% recovery. The MCX-packed column was used for more than 60 runs without any change in its performance.     

Simultaneous determination of 24 or more acidic and alkaline phytohormones in femtomole quantities of plant tissues by high-performance liquid chromatography–electrospray ionization–ion trap mass spectrometry

Phytohormones act at relatively low concentrations as major regulatory factors of plant growth and development, and cross talk of phytohormones is currently of great interest throughout the plant science community. To meet this demand, a method that is capable of simultaneously analyzing diverse plant hormones is essential. This paper introduces a high-performance liquid chromatographic separation technique coupled with sensitive and selective ion trap mass spectrometry to simultaneously determine 24 or more acidic and alkaline phytohormones, including auxin, cis- and trans-abscisic acid, 11 cytokinins, and 10 gibberellins, in a single injection of sample. A binary solid-phase extraction using Oasis MCX cartridges for cations and Oasis MAX cartridges for anions was used to prepurify more than 24 acidic and alkaline phytohormones from a single plant extract. The method showed good linearity for all 24 phytohormones with R ² values ranging from 0.9903 to 0.9997. Limits of detection for most of the phytohormones were in the femtomole range with some extending into the sub-femtomole range. This method was applied to hundreds of plant samples comprising different tissues from various plants, including herbaceous, woody climbing, and woody plants to demonstrate feasibility and to validate the methodology.     

Silver-phase high-performance liquid chromatography–electrospray mass spectrometry of triacylglycerols

Silver-phase chromatography hyphenated with on-line electrospray mass spectrometry can be applied to characterize triacylglycerols present in vegetable oils with respect to their degree of unsaturation, the position of the most unsaturated fatty acid and the carbon number (CN). The CN information obtained with silver-phase HPLC–ESP–mass spectrometry is complementary to the unsaturation information obtained by silver-phase HPLC-flame ionization detection. Both information is essential to monitor or study modified vegetable oils on the presence of non-natural triacylglycerols. The quantitative results obtained with the method are in agreement with the results obtained in the silver-phase HPLC-flame ionization detection and with theoretical values calculated from the fatty acid distribution of the oil. Silver-phase HPLC–ESP-mass spectrometry gives direct information on fatty acid position and triacylglycerol CN, for each of the triacylglycerols in the sample. This in contrast with non-aqueous reversed-phase HPLC hyphenated with on-line atmospheric pressure chemical ionization-mass spectrometry, which requires a more extensive data processing. The results obtained with silver-phase HPLC–ESP-mass spectrometry can be presented in a three-dimensional overview (relative amount, CN, fatty acid position) serving as a fingerprint for the oil.     

Analysis of some cytokinins in coconut (Cocos nucifera L.) water by micellar electrokinetic capillary chromatography after solid-phase extraction

Micellar electrokinetic capillary chromatography (MECC) was developed for the separation of cytokinins including trans-zeatin, trans-zeatin-O-glucoside, dihydrozeatin, dihydrozeatin-O-glucoside, meta-topolin riboside, N6-isopentenyladenine and N6-benzylaminopurine. Under the optimum conditions, i.e. a combination of 10 mM phosphate and 10 mM borate as the running buffer containing 50 mM sodium dodecyl sulphate at pH 10.4, the separation of seven cytokinin standards was accomplished within 11 min. The C18 solid-phase extraction (SPE) method was used to pre-concentrate the putative cytokinins present in the coconut water. Following which, the eluate was further purified using mixed mode Oasis MCX SPE columns and this additional step helps to reduce matrix interference during MECC. After the two solid-phase extraction steps, the optimized MECC method was able to screen for certain cytokinins (zeatin-O-glucoside and dihydrozeatin-O-glucoside) present in coconut water. After this screening, the presence of zeatin-O-glucoside and dihydrozeatin-O-glucoside in coconut water was further confirmed by independent high-performance liquid chromatography and liquid chromatography-mass spectrometry experiments.     

Determination of UV filters in river water samples by in‐line SPE‐CE‐MS

The use of SPE coupled in‐line to CE using electrospray MS detection (in‐line SPE‐CE‐ESI‐MS) was investigated for the preconcentration and separation of four UV filters: benzophenone‐3, 2,2‐dihydroxy‐4‐methoxybenzophenone, 2,4‐dihydroxybenzophenone and 2‐phenylbenzimidazole‐5‐sulphonic acid. First, a CE‐ESI‐MS method was developed and validated using standard samples, obtaining LODs between 0.06 μg/mL and 0.40 μg/mL. For the in‐line SPE‐CE‐ESI‐MS method, three different sorbents were evaluated and compared: Oasis HLB, Oasis MCX, and Oasis MAX. For each sorbent, the main parameters affecting the preconcentration performance, such as sample pH, volume, and composition of the elution plug, and sample injection time were studied. The Oasis MCX sorbent showed the best performance and was used to validate the in‐line SPE‐CE‐ESI‐MS methodology. The LODs reached for standard samples were in the range between 0.01 and 0.05 ng/mL with good reproducibility and the developed strategy provided sensitivity enhancement factors between 3400‐fold and 34 000‐fold. The applicability of the developed methodology was demonstrated by the analysis of UV filters in river water samples.     

Serial mixed-mode cation- and anion-exchange solid-phase extraction for separation of basic,neutral and acidic pharmaceuticals in wastewater and analysis by high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry

A novel solid-phase extraction (SPE) method is presented whereby 15 basic, neutral and acidic pharmaceuticals in wastewater were simultaneously extracted and subsequently separated into different fractions. This was achieved using mixed-mode cation- and anion-exchange SPE (Oasis MCX and MAX) in series. Analysis was performed by high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC/QTOF-MS). A fast separation was achieved, with all compounds eluting within 6 min, narrow chromatographic peaks, with a peak base width of 6 s on average, and a high mass accuracy of quantified wastewater sample ions, with average mass errors in absolute value of 0.7 mDa or 2.7 ppm. The recovery of the SPE method in the analysis of sewage treatment plant (STP) influent and effluent wastewater was on average 80% and the ion suppression 30%. For less demanding samples Oasis MCX used alone may be an alternative method, although for STP influent waters containing high loads of organic compounds the clean-up achieved using only Oasis MCX was insufficient, leading to unreliable quantitation. Furthermore, serial SPE separation according to molecular charge added an additional degree of analyte confirmation. For quantitation, an approach combining external standard calibration curves, isotopically labelled surrogate standards and single-point standard addition was used. The applicability of the method was demonstrated in the analysis of influent and effluent wastewater from an STP, using small sample volumes (25–50 mL). The effluent wastewater had been subjected to three different treatments; activated sludge, activated sludge followed by ozonation, and a membrane bioreactor (MBR). Ozone treatment proved superior in removal of the analysed pharmaceuticals, while the MBR provided higher removal efficiencies than the activated sludge process.     

Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry

In recent plant hormone research, there is an increased demand for a highly sensitive and comprehensive analytical approach to elucidate the hormonal signaling networks, functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive and quantitative analytical method developed with nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring (MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and the detection limits (S/N = 3) of several plant hormones were in the sub-fmol range. The results showed excellent linearity (R² values of 0.9937-1.0000) and reproducibility of elution times (relative standard deviations, RSDs, <1.1%) and peak areas (RSDs, <10.7%) for all target compounds. Further, sample purification using Oasis HLB and Oasis MCX cartridges significantly decreased the ion-suppressing effects of biological matrix as compared to the purification using only Oasis HLB cartridge. The optimized nanoflow-LC-ESI-IT-MS/MS method was successfully used to analyze endogenous plant hormones in Arabidopsis and tobacco samples. The samples used in this analysis were extracted from only 17 tobacco dry seeds (1 mg DW), indicating that the efficiency of analysis of endogenous plant hormones strongly depends on the detection sensitivity of the method. Our analytical approach will be useful for in-depth studies on complex plant hormonal metabolism.     

Analysis of cytokinin nucleotides in coconut (Cocos nucifera L.) water using capillary zone electrophoresis-tandem mass spectrometry after solid-phase extraction

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is described for the separation and determination of six cytokinin nucleotides in coconut water. The best CZE separation for the six cytokinin nucleotide standards was achieved using a 25 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) methanol with an applied gradient separation voltage (25 kV for 32 min, and then a linear gradient to 30 kV in 5 min, finally 30 kV to the end of separation) in less than 60 min. MS/MS with multiple reaction monitoring (MRM) detection was carried out to obtain sufficient selectivity and sensitivity for the cytokinin nucleotides. The combined use of on-line sample stacking and CZE-MS/MS achieved limits of detection (LODs) in the range of 0.06-0.19 microM for the six cytokinin nucleotides at a signal-to-noise ratio of 3. Furthermore, a novel dual-step SPE procedure was developed for the pre-concentration and purification of cytokinin nucleotides using Oasis HLB and Oasis MAX cartridges. The recoveries of the cytokinin nucleotides after the dual-step SPE were in the range of 44-71%. The combination of off-line SPE, on-line sample stacking and CZE-MS/MS approach was successfully applied to screen for endogenous cytokinin nucleotides present in coconut water sample. trans-Zeatin riboside-5'-monophosphate (ZMP) was detected and quantified in coconut water by CZE-MS/MS after SPE and on-line sample stacking.     

Interaction of abscisic-acid-binding cotton (Gossypium hirsutum) protein and phytohormones

The interaction of representatives of three classes of phytohormones, abscisic acid (ABA), the synthetic auxin 1-NAA, and the synthetic cytokinin 6-BAP, with ABA-binding cotton protein was studied. Binding of ABA with its protein was shown to be specific and receptor-like. Competitive protein binding showed that 1-NAA (15±3%), 6-BAP (82±3%), and ABA (95±3%) replace³H-ABA from the protein complex. The possibility of reacting ABA-binding protein with various classes of intracellular phytohormones is discussed.Institute of Genetics and Experimental Biology of Plants, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (99871) 64 22 30. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 252–253, May–June, 2000.     

Simultaneous determination of peimine and peiminine in rat plasma by LC‐ESI‐MS employing solid‐phase extraction

A simple and reliable LC‐ESI‐MS method for the determination of peimine and peiminine in rat plasma was developed for the first time. The method was proven to be specific and sensitive by carrying out validation. The analytes were extracted from rat plasma via solid‐phase extraction on Waters Oasis MCX cartridges. Chromatography separation was achieved on a C₁₈ column using 10 mM ammonium acetate (adjusted to pH 3.0 with glacial acetic acid)–acetonitrile (85:15, v/v) as mobile phase. The linear range was 1–100 ng/mL for peimine and peiminine. Intra‐ and inter‐day precisiond were less than 10%. Accuracies were within 85–115% of their nominal concentrations. The limit of quantification was 1 ng/mL for both analytes. The developed assay was successfully applied to pharmacokinetic study of peimine and peiminine in rats orally administered the alkaloids extracts from Bulbus Fritillariae, demonstrating a possible broader spectrum of applications of this method. Copyright © 2009 John Wiley & Sons, Ltd.     

Assignment of Anomeric Configuration and Identification of Carbohydrate Residues by 13C NMR: Arabino- and Ribopyranosides and Furancsides

Abstract

A ¹³C NMR fingerprint method previously developed for galactosides and glucosides is extended to arabinosides and ribosides. This approach demonstrates the capability of ¹³C NMR to determine ring size and anomeric configuration in four isomeric arabinosides and ribosides.     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Simultaneous separation and identification of limonoids from citrus using liquid chromatography-collision-induced dissociation mass spectra

Limonoids are considered as potential cancer chemopreventive agents and are widely distributed in the Citrus genus as aglycones and glucosides. In the present study, reversed-phase HPLC coupled with CID mass spectra was developed for the simultaneous separation and identification of aglycones and glucosides of limonoids from citrus. Five aglycones such as limonin, deacetyl nomilin, ichangin, isolimonoic acid and nomilin were identified by positive ion CID MS/MS, whereas five glucosides, viz. limonin glucoside, isoobacunoic acid glucoside, obacunone glucoside, deacetyl nomilinic acid glucoside and nomilinic acid glucoside were analyzed by negative ion CID mass spectra. The developed method was successfully applied to complex citrus samples for the separation and identification of aglycones and glucosides. Citrus seeds were extracted with methanol and partially purified and analyzed by LC-CID mass spectra. The separation was achieved by C-18 column; eight limonoids were identified by comparing the retention times and mass spectral fragmentation. To the best of our knowledge, this is the first report on the identification of citrus limonoids using CID technique.     

Purification and determination of plant hormones auxin and abscisic acid using solid phase extraction and two-dimensional high performance liquid chromatography

A method for separation and purification of plant hormones auxin and abscisic acid based on mixed mode reversed-phase anion-exchange solid phase extraction and two-dimensional HPLC was developed. Two-dimensional HPLC in "heart cutting" mode was very efficient in the purification of these two hormones. Its purification power is high enough to allow reliable on-line quantification of both hormones even with non-selective detectors.     

Rapid analysis of three β‐agonist residues in food of animal origin by automated on‐line solid‐phase extraction coupled to liquid chromatography and tandem mass spectrometry

An automated online solid‐phase extraction with liquid chromatography and tandem mass spectrometry method was developed and validated for the detection of clenbuterol, salbutamol, and ractopamine in food of animal origin. The samples from the food matrix were pretreated with an online solid‐phase extraction cartridge by Oasis MCX for <5 min after acid hydrolysis for 30 min. The peak focusing mode was used to elute the target compounds directly onto a C₁₈ column. Chromatographic separation was achieved under gradient conditions using a mobile phase composed of acetonitrile/0.1% formic acid in aqueous solution. Each analyte was detected in two multiple reaction monitoring transitions via an electrospray ionization source in a positive mode. The relative standard deviations ranged from 2.6 to 10.5%, and recovery was between 76.7 and 107.2% at all quality control levels. The limits of quantification of three β‐agonists were in the range of 0.024–0.29 μg/kg in pork, sausage, and milk powder, respectively. This newly developed method offers high sensitivity and minimum sample pretreatment for the high‐throughput analysis of β‐agonist residues.     

离子液体作添加剂对高效液相色谱分离植物激素的影响

探讨了以离子液体作为液相色谱流动相添加剂,对植物激素赤霉素GA3、生长素IAA和脱落酸ABA的分离的影响,以及离子液体的烷烃链长度,阴离子及离子液体的浓度对分离的影响。结果表明:咪唑阳离子和植物激素通过静电作用而保留;植物激素本身的pKa值影响其保留因子,pKa值增大,离子液体浓度对植物激素保留因子影响增大;另外随[BMIM]对应的阴离子电负性的减小,植物激素的保留因子明显地增大;同时植物激素的空间位阻也影响其分离。     

Comparison of different chiral selectors for the enantiomeric determination of amphetamine-type substances in human urine by solid-phase extraction followed by capillary electrophoresis-tandem mass spectrometry

The present study develops a method for the enantioseparation of a group of amphetamines and their metabolites in urine by CE coupled to MS/MS (CE-MS/MS). Amphetamines present a chiral center and thus two enantiomers, which is important from a toxicological point of view because they may have different pharmacokinetic and pharmacological properties. It is therefore essential to find suitable methods to distinguish both enantiomers. Today the use of CE is becoming more important in this field since, with the simple addition of a chiral selector to the background electrolyte, the enantioseparation can easily be achieved. However, when CE is coupled to MS, the use of volatile chiral selectors and compatible background electrolytes or other strategies such as the countercurrent migration approach are required to avoid contamination of the ion source from nonvolatile species. In the present study, we use the latter strategy to evaluate six different chiral selectors using CE-MS/MS. As a sample pre-treatment, two cationic-exchange sorbents—Oasis WCX and Oasis MCX—are compared for the urine pre-treatment. Using this method, it was possible to achieve the complete chiral separation of the amphetamines under study with detection limits ranging between 0.8 and 1.5 ng/mL and method quantification limits between 2.0 and 8.0 ng/mL. Matrix-matched calibration curves up to 150 ng/mL were used to cover the usual concentration ranges at which amphetamines have generally been found in toxicological and forensic analyses.     

High-performance liquid chromatographic stationary phases based on poly(methyloctylsiloxane) immobilized on silica. II. Chromatographic evaluation

An integrated on-line SPE–HPLC–MS/MS system has been developed for the rapid analysis of various trace level priority pesticides in surface and drinking water. Eleven pesticides were included in this study, with various phenylureas, triazines and organophosphorous species among them. Use of turbulent-flow chromatography columns (TFC, 50×1 mm, 30–50 μm particle size) as extraction cartridges enables fast on-line SPE at high sampling flow-rate (5 ml/min). Polymeric and carbon based TFC columns (Oasis HLB, Cyclone, Hypercarb) allow complete extraction with good recoveries from water volumes up to 50 ml. On-line coupling to HPLC is performed with re-mixing of the organic TFC eluate with water in front of the analytical column to ensure efficient band focussing. For fast HPLC analysis, a short monolithic column is applied in combination with highly selective API–MS/MS detection. Matrix effects on the APCI–MS/MS signal were found to be reduced by the system to an acceptable minimum. Limits of detection, determined for 10-ml samples of river water were in the range between 0.4 and 13 ng/l typically, except trifluralin (approximately 280 ng/l), which is less susceptible to ionization under atmospheric pressure conditions. At an enriched water volume of 10 ml, the whole SPE–HPLC–MS/MS procedure requires less than 14 min. The method was successfully applied to the analysis of drinking and surface water samples taken from several sampling sites around the city of Leipzig, Germany. Concentrations measured (maximum: 16 ng/l simazine in river water) were far below the concentration limits scheduled by law.     

Determination of trimethylselenonium iodide, selenomethionine, selenious acid, and selenic acid using high-performance liquid chromatography with on-line detection by inductively coupled plasma mass spectrometry or flame atomic absorption spectrometry

An analytical method has been developed for the determination of selenious acid, selenic acid, trimethylselenonium ion, and selenomethionine. The four selenium compounds were separated by HPLC on a column (25 cm×4 mm I.D.) of the anion-exchanger ESA Anion III with a mobile phase (1.5 ml/min) of 0.0055 M ammonium citrate (pH 5.5). Detection was carried out using an on-line inductively coupled plasma mass spectrometer (ICP-MS) or a flame atomic absorption spectrometer (FAAS) as the selenium-specific detector. The chromatographic parameters and the chemical factors affecting the separation of the selenium species were optimized. The four selenium compounds could be separated within 8 minutes. The detection limits of the coupled HPLC–FAAS system were approximately 1 mg Se/l for each compound (100 μl injection), estimated as three times the base-line noise of the chromatograms. More powerful selenium detection was achieved with an ICP-MS. Selenium was measured at m/z 78. To increase the nebulization efficiency, the Meinhard concentric glass nebulizer was replaced by an ultrasonic nebulizer. The ICP-MS signal intensity was increased with the ultrasonic nebulization by a factor of 7 times for selenious acid and 24 to 31 times for trimethylselenonium ion, selenomethionine, and selenic acid compared to that with the Meinhard nebulization. The detection limits achieved by the HPLC–ICP-MS with the ultrasonic nebulization were 0.08 μg Se/l for trimethylselenonium ion, 0.34 μg Se/l for selenious acid, 0.18 μg Se/l for selenomethionine, and 0.07 μg Se/l for selenic acid, respectively.     

Temperature dependence of Kováts indices in gas chromatography revisited

The performance of Fourier transform infrared spectroscopy (FT-IR) detection coupled to high-performance liquid chromatography for the analysis of C₆₀ and C₇₀ fullerenes was investigated. The isocratic separation method involved an octadecylsilane (ODS) column and an acetonitrile–toluene (1:1) mobile phase. The hyphenated system was designed with a split valve to control eluent volume leading to the FT-IR detector; this allowed for additional coupling of the liquid chromatograph to ultraviolet–visible detection. On-line FT-IR spectra of C₆₀ and C₇₀ were matched with standard off-line FT-IR spectra from the literature. In addition, with band chromatograms individual fullerenes can be identified using FT-IR active modes known specifically for each fullerene. Few changes to a pre-existing HPLC–UV method were necessary for the HPLC–FT-IR method, and there was no need for fraction collection to identify the fullerenes C₆₀ and C₇₀.