Analysis of oligonucleotides by hydrophilic interaction liquid chromatography coupled to negative ion electrospray ionization mass spectrometry

Hydrophilic interaction liquid chromatography (HILIC) is here successfully coupled to negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of synthetic and chemically modified oligonucleotides. Separation was performed on a 2.1 mm × 100 mm PEEK ZIC^® HILIC column packed with hydrophilic stationary phase with a permanent zwitterionic functional group and a particle size of 3.5 μm with an average pore diameter of 200 Å. A method was developed to separate homogeneous and heterogeneous oligonucleotides as well as methylated oligonucleotides using a quaternary pumping system containing ammonium acetate and water with an acetonitrile gradient. Analyses of oligonucleotides were performed by LC/MS with a detection limit of 2.5 picomole (20 mer) with signal to noise ratio (S/N) of 4.12. The influence of the eluent composition, type of buffer and its concentration, and organic modifier were also evaluated. The HILIC LC/MS method presented in this paper used common, ‘MS friendly’, mobile phases achieving sensitive and selective oligonucleotide analysis.     

Screening for anabolic steroids and related compounds in illegal cocktails by liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

Findings of illegal hormone preparations such as syringes, bottles, cocktails, and so on, are an important information source for the nature of the current abuse of anabolic steroids and related compounds as growth-promoting agents in cattle. A new screening method for steroids in cocktails is presented based on liquid chromatography (LC) with diode-array UV-absorbance detection and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS). Accurate mass measurements were performed at a mass resolution of 4000 using continuous introduction of a lock mass through a second (electro)sprayer. Similar experiments were carried out using dual-sprayer quadrupole time-of-flight mass spectrometry (ESI-QTOFMS/MS) at a mass resolution of 10 000 with data-dependent MS/MS acquisition; i.e. beyond an intensity threshold for the [M + H](+) ions, MS/MS spectra were automatically acquired at three different collision energies. Elemental compositions were calculated for precursor and product ions and it is shown that the combined information from LC retention behavior, UV spectra, elemental compositions, and accurate mass MS/MS spectra yield a fast impression of the steroids present in the complex mixture. Using a new software tool for structure elucidation of MS/MS spectra, an additional non-steroidal additive was identified as well.     

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

Interferon α‐2b produced by Escherichia coli consists of 165 amino acids and contains two disulphide bonds; its purity was confirmed by LC‐UV (DAD)‐FLD and LC‐MS techniques. A C₄ column was used with UV detection at 214 nm; diode array detector (DAD) spectra were recorded from 200–400 nm and fluorescence detection was performed at specific wavelengths of trypthophan emission and excitation. Peptide mapping was performed with trypsin. Peptides produced by trypsin digestion were analysed by LC‐UV (DAD)‐FLD, LC‐MS, and LC‐MS/MS using a C₁₈ column. Amino acid sequence coverage was about 95%. UV spectra in the range from 200 nm to 400 nm, emission (Em) and excitation (Ex) spectra of each separated peptide were additionally compared with spectra of the same peptide produced by digestion of European Pharmacopaeia interferon α‐2b standard (spectral matching). The chromatogram of any interferon α‐2b (drug substance or certificated standard) sample produced in the same manner with the same amino acid composition should be similar to the chromatogram obtained by the method described in this paper. Molecular masses of peptides were obtained from MS experiments and MS/MS experiments gave additional structural information. The molecular mass of interferon α‐2b was obtained by MALDI‐TOF MS analysis in linear mode, with an accuracy comparable to the theoretical average mass ± 5 atomic mass units. The molecular mass was obtained from the deconvoluted ESI mass spectrum.     

液相色谱-二极管阵列检测器/离子阱质谱法检测水中的5种微囊藻毒素

建立了一种液相色谱-二极管阵列检测器(LC-DAD)/离子阱质谱(IT MS)对水中5种微囊藻毒素(microcystins)的分析方法。水中的微囊藻毒素经固相萃取富集和净化,经LC分离后,采用DAD和IT MS定性分析,DAD定量分析。在优化的条件下,水中5种微囊藻毒素的检出限为0.1 μg/L; 3个质量浓度加标水平(0.2、0.8和5 μg/L)的平均回收率为52.2%～115.2%,相对标准偏差为1.2%～10.0%。该方法从紫外吸收光谱和质谱角度同时进行定性定量分析,可用于地表水和饮用水中多种微囊藻毒素的检测。     

On-Line HPLC-UV-mass spectrometry and tandem mass spectrometry for the rapid delineation and characterization of differences in complex mixtures: a case study using toxic oil variants

An integrated differential approach to the characterization of complex mixtures is presented which includes the targeting of liquid chromatography (LC) peaks for identification using characteristic UV adsorption of the LC peak, subsequent molecular weight and formula determination using accurate mass LC mass spectrometry (MS), and structure characterization using accurate mass LC-tandem mass spectrometry. The use of differential UV adsorption aids in narrowing the scope of the study to only specific peaks of interest. Accurate mass measurement of the molecular ion species provides molecular weight information as well as atomic composition information. The tandem MS (MS/MS) spectra provide fragmentation information which allows for structural characterization of each component. Accurate mass assignment of each of the fragment ions in the MS/MS spectrum provides atomic composition for each of the fragment ions and thus further aids in the structural characterization. These experiments are facilitated through the use of on-line LC-MS and LC-MS/MS with in-line UV detection. A synthetic toxic oil (STO) related to Toxic Oil Syndrome is studied with a focus on possible contaminants resulting from the interaction of aniline, used as a denaturant, with the normal components of the oil. A differential analysis between the STO and a control oil is performed. LC peaks were targeted using UV absorbance to indicate the possible presence of the aniline moiety. Further differential analysis was performed through the determination of the MS signals associated with each component separated on the LC. Finally, the MS/MS data was also used to determine if the fragmentation of the targeted components indicated the presence of aniline. The MS/MS and accurate mass data were used to assign the structures for the targeted components.     

Capillary electrophoresis time-of-flight mass spectrometry of an opium powder

Summary Intensive work has been invested in recent years to evaluate the performance of capillary electrophoresis (CE) in forensic analysis. Tremendous progress has also been achieved in interfacing CE to sensitive and specific detection systems such as the mass spectrometer (MS). We have recently developed an electrospray time-of-flight mass spectrometer (ESI-TOFMS) for use as a detector for fast and efficient liquid phase separations. In the present paper we investigated ESI-TOFMS for the analysis of an opium powder. Both continuous infusion and CE were studied for direct sample introduction into the TOFMS and mixture separation, respectively. CEMS analysis of the opium was performed in a citrate buffer, using aqueous or mixed aqueous/organic eluents. Low fmol detection was achieved.     

Determination of selected non-authorized insecticides in peppers by liquid chromatography time-of-flight mass spectrometry and tandem mass spectrometry

In this work, two analytical methods based on liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOFMS) and tandem mass spectrometry (LC/ESI-MS/MS) are described for the identification, confirmation and quantitation of three insecticides non-authorized in the European Union (nitenpyram, isocarbophos and isofenphos-methyl) but detected in recent monitoring programmes in pepper samples. The proposed methodologies involved a sample extraction procedure using liquid-liquid partition with acetonitrile followed by a cleanup step based on dispersive solid-phase extraction. Recovery studies performed on peppers spiked at different fortification levels (10 and 50 microg kg(-1)) yielded average recoveries in the range 76-100% with relative standard deviation (RSD) (%) values below 10%. Identification, confirmation and quantitation were carried out by LC/TOFMS and LC/MS/MS using a hybrid triple quadrupole linear ion trap (QqLIT) instrument in multiple-reaction monitoring (MRM) mode. The obtained limits of quantitation (LOQs) were in the range 0.1-5 microg kg(-1), depending on each individual technique. Finally, the proposed methods were successfully applied to the analysis of suspected pepper samples.     

Evaluation of matrix solid‐phase dispersion extraction for the determination of polycyclic aromatic hydrocarbons in household dust with the aid of experimental design and response surface methodology

A simple, fast, and inexpensive procedure for sample preparation based on matrix solid‐phase dispersion was developed for the determination of Environmental Protection Agency 16 priority polycyclic aromatic hydrocarbons in indoor dust samples. Parameters that affect the extraction efficiency such as type of dispersant, elution solvent, and solvent volume were evaluated and optimized with the aid of experimental design and response surface methodology. Analysis was performed by HPLC coupled with UV‐Vis diode array detector (UV‐DAD). For verification, a GC coupled with a mass spectrometer in SIM mode was also applied. Recoveries obtained were from 53 to 120% for all target analytes with detection limits ranging from 0.2 to 10 ng/g and 0.2 to 2 ng/g for LC‐UV‐DAD and GC‐MS, respectively. The optimized method was used for the analysis of 11 household dust samples collected from private houses.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Diagnostic protein discovery using liquid chromatography/mass spectrometry for proteolytic peptide targeting

A peptide targeting method has been developed for diagnostic protein discovery, which combines proteolytic digestion of fractionated plasma proteins and liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOFMS) profiling. Proteolysis prior to profiling overcomes molecular weight limitations and compensates for the poor sensitivity of matrix-assisted laser desorption/ionization (MALDI) protein profiling. LC/MS increases the peak capacity compared to crude fractionation techniques or single sample MALDI analysis. Differentially expressed peptides are targeted in the mass chromatograms using bioinformatic techniques and subsequently sequenced with MALDI tandem MS. In a model study comparing pancreatic cancer patients to controls, 74% of the peptide targets were successfully sequenced. This profiling method was superior to previous experiments using single sample MALDI analysis for protein profiling or proteolytic peptide profiling, because more potential protein markers were identified.     

Screening non-colored phenolics in red wines using liquid chromatography/ultraviolet and mass spectrometry/mass spectrometry libraries

Liquid chromatography/ultraviolet (LC/UV) and mass spectrometry/mass spectrometry (MS/MS) libraries containing 39 phenolic compounds were established by coupling a LC and an ion trap MS with an electrospray ionization (ESI) source, operated in negative ion mode. As a result, the deprotonated [M-H]- molecule was observed for all the analyzed compounds. Using MS/MS hydroxybenzoic acid and hydroxycinnamic acids showed a loss of CO2 and production of a [M-H-44]- fragment and as expected, the UV spectra of these two compounds were affected by their chemical structures. For flavonol and flavonol glycosides, the spectra of their glycosides and aglycones produced deprotonated [M-H]- and [A-H]- species, respectively, and their UV spectra each presented two major absorption peaks. The UV spectra and MS/MS data of flavan-3-ols and stilbenes were also investigated. Using the optimized LC/MS/MS analytical conditions, the phenolic extracts from six representative wine samples were analyzed and 31 phenolic compounds were detected, 26 of which were identified by searching the LC/UV and MS/MS libraries. Finally, the presence of phenolic compounds was confirmed in different wine samples using the LC/UV and LC/MS/MS libraries.     

Quantitative determination of coenyzme Q10 by liquid chromatography and liquid chromatography/mass spectrometry in dairy products

The dietary sources of CoQ10 and the evaluation of CoQ10 in dairy products were characterized. For quantitation of CoQ10 in food samples, 2 liquid chromatography (LC) methods with UV and mass spectrometry (MS) detections were developed. LC with UV detection was performed at 25 degrees C on a Hyperclone ODS 5 microm 150 x 4.6 mm column with mobile phase consisting of methanol-ethanol-2-propanol (70 + 15 + 15, v/v/v). Flow rate was 1.0 mL/min. Retention time of CoQ10 was 10.9 +/- 0.1 min. The method was sensitive [limit of detection (LOD) = 0.2 mg/kg], reproducible [relative standard deviation (RSD) = 3:0%), and linear up to 25 mg/kg (R > 0.999). LC/MS analysis was performed on a LUNA C18 3 microm, 150 x 4.6 mm column, using mobile phase consisting of ethanol-dioxane-acetic acid (9 + 1 + 0.01, v/v/v), flow rate was 0.6 mL/min, and the retention time of CoQ10 was 4.1 +/- 0.1 min. Identification and quantitation were performed with a Finnigan-LCQ mass detector in positive atmospheric pressure chemical ionization mode. Mass spectra were obtained in selected-ion monitoring mode; molecular mass (M+H)+ m/z 863.4 +/- 1 was used for quantitative determination. MS detection is more sensitive than UV detection (LOD = 0.1 mg/kg), less reproducible (RSD = 4.0%), and linear in selected range. Analytical recoveries are 75-90% and depend on the ratio between the amount of fat in the matrix and the concentration of CoQ10 in the sample. Some soybean milk products were analyzed together with different cow, goat, and sheep milk products. Concentrations obtained with LC and LC/MS were compared with a few accessible results available from the literature. Concentrations varied from 0 ppm in soybean milk to nearly 2 ppm in fresh milk from local farms.     

Determination and confirmation of the amnesic shellfish poisoning toxin, domoic acid, in shellfish from Scotland by liquid chromatography and mass spectrometry

During 1998 and early 1999, shellfish samples from sites in Scotland were found to contain the amnesic shellfish poisoning toxin, domoic acid (DA). Two different techniques, liquid chromatography (LC) with UV diode-array detection and LC with mass spectrometric (MS) detection, were used to detect and confirm DA in shellfish extracts. The LC/UV method was validated for routine monitoring by recovery experiments on spiked mussel and scallop tissues with a certified mussel tissue used as reference material. Crude extracts of selected samples as well as extracts cleaned with strong anion exchange (SAX) were analyzed by both LC/UV and LC/MS. Good correlation (linear regression r2 = 0.996, slope = 0.93) between the 2 methods was found for cleaned extracts. Analyses of crude extracts by LC/UV produced false-positive results in 2 crab samples, whereas LC/MS analyses gave accurate results. It was concluded that LC/UV is a valid approach for routine monitoring of DA in shellfish when cleanup is performed with a SAX cartridge to prevent false positives. A variety of shellfish species were surveyed for DA content, including Pecten maximus (king scallops), Chlamys opercularis (queen scallop), Mytilus edulis (blue mussels), Cancer pugaris (crab), and Ensis ensis (razor fish). The highest concentration of DA was 105 microg/g in Pecten maximus.     

Comparison of different liquid chromatography methods for the determination of corticosteroids in biological matrices

Various extraction techniques can be combined with column liquid chromatography (LC) and ultraviolet (UV) or mass spectrometric (MS) detection for the determination of synthetic corticosteroids in biological matrices. Target analysis of low concentrations of 25 microg/kg of dexamethasone in feed can be performed by combining immunoaffinity chromatography (IAC) and LC with UV detection. A straightforward multi-analyte procedure is obtained by tandem solid-phase extraction (SPE) and subsequent LC-UV. However, the limits of detection for feed samples are then relatively poor, viz. 100 microg/kg. A multi-analyte method which meets modern demands of about 5 microg/kg detection limit requires one-step SPE combined with LC-MS analysis. As regards urine corticosteroids can be determined down to a level of 0.5 microg/l by either SPE-LC-MS- MS or SPE(IAC)-LC-MS.     

Simultaneous Qualitative Analysis of Coumarins in the Rhizome of Angelica polymorpha Maxim. by LC?CDAD?CESI?CMS

A novel method has been developed for simultaneous qualitative analysis of coumarins in the rhizome of Angelica polymorpha Maxim. with the use of high-performance liquid chromatography (LC)?Cphotodiode array detection (DAD)?Celectrospray ionization (ESI)?Ctandem mass spectrometry (MS). LC analysis was performed on an Agilent Eclipse XDB-C₁₈ analytical column with a gradient solvent system of water and methanol, ultraviolet (UV) absorption detection was at 300?nm and peak UV spectrum and (+) ESI?CMS data were acquired by DAD and a quadrupole mass spectrometer, respectively. Eight peaks were identified as linear furocoumarins including aviprin, isopimpinellin, bergapten, isooxypeucedanin, gosferol, imperatorin, phellopterin and isoimperatorin by comparing their retention time, UV, and MS spectra with that of the authentic standards, and the analysis of UV spectra and ESI?CMS data of the standards gave the characteristics of UV absorption and ESI?CMS fragmentation. On the basis of the above characteristics, the possible structures of four other linear furocoumarins were deduced in LC. The other chemical structures were proposed as three angular furocoumarins, a simple coumarin, two chromones, and a cinnamic acid by comparing their UV and MS data with that of literature reports. Accordingly, the method we established can simply and accurately identify the key chemical constituents in the rhizome of A. polymorpha.     

Analysis of polar hydrophilic aromatic sulfonates in waste water treatment plants by CE/MS and LC/MS

The present work describes the development and optimization of a capillary (zone) electrophoresis/mass spectrometric (CE/MS) analysis method for polar hydrophilic aromatic sulfonates (ASs). The compounds were detected by negative ion electrospray ionization (NIESI) and selected ion monitoring (SIM). In comparison with CE/UV, for CE/MS a lower-concentration volatile ammonium acetate buffer (5 mM) without organic modifier and a higher separation voltage were better suited for separation. Sensitivity of CE/MS was slightly better than for CF/UV, with the limit of detection (LOD) ranging between 0.1 and 0.4 mg l(-1). For verification of the CE/MS results, ASs were also analysed by ion-pair liquid chromatography/diode array UV detection coupled in series with electrospray mass spectrometry (IPC/DAD/ESI-MS). Real water samples of different waste water treatment plants (WWTPs) in Catalonia (NE Spain) were extracted by solid-phase extraction (SPE) with LiChrolut EN and analysed with CE/MS and LC/MS. ASs were found in influent and effluent water samples of the WWTPs in the microg l(-1) concentration range. LC/MS offered a higher separation efficiency and sensitivity than CE/MS. Therefore with LC/MS more compounds could be identified in the WWTPs. The persistency of the ASs was distinct: some compounds were well degraded during the water treatment process, while others were quite persistent.     

Determination of a series of quinolones in pig plasma using solid-phase extraction and liquid chromatography coupled with mass spectrometric detection Application to pharmacokinetic studies

A simple and sensitive liquid chromatography (LC) method was developed for the simultaneous determination of eight quinolones in pig plasma samples. The following two methods of detection were used: ultraviolet (UV) and mass spectrometry with electrospray ionization (ESI/MS). Sample preparation consisted of solid-phase extraction (SPE) on Strata X cartridges prior to the analysis by LC/UV or LC/ESI/MS. The recovery, linearity, limit of detection (LOD) and limit of quantification (LOQ), precision and accuracy of the method were evaluated using spiked pig plasma samples. The suitability of the method for pharmacokinetic studies was evaluated by determining the concentrations of enrofloxacin (ENR) and ciprofloxacin (CIP) also in pig plasma, after administration of 200mg of enrofloxacin per kilogram of fodder during 5 consecutive days.     

Analyses of pesticide residues in fruit-based baby food by liquid chromatography/electrospray ionization time-of-flight mass spectrometry

A liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) method has been developed for the determination of 12 pesticides (namely, carbendazim, thiabendazole, imazalil, tridemorph, triadimefon, bitertanol, prochloraz, flutriafol, myclobutanil, iprodione, diphenylamine and procymidone) in fruit-based baby food (multi-fruit jars and juices intended for infant consumption). The developed method consists of a sample treatment step based on liquid-liquid extraction using acetonitrile, followed by a clean-up step based on dispersive solid-phase extraction (SPE) with a primary-secondary amine (PSA). Multi-fruit and apple juices were processed by a SPE procedure using Oasis HLB cartridges. Subsequent identification and quantitation was accomplished by LC/ESI-TOFMS analysis: the confirmation of the target pesticides was based on accurate mass measurements of selected ions (protonated molecules ([M+H]+) and fragment ions). Confirmation studies were accomplished at low concentration levels (10 microg kg-1) and accuracy errors lower than 2 ppm were obtained in most cases. Baby food extracts spiked at 10 microg kg-1 fortification level yielded average recoveries in the range 78-105% with relative standard deviations less than 10% for most of the analytes. Limits of detection (LODs) were between 0.1 and 4 microg kg-1 depending on the pesticide studied. Finally, the proposed method was applied to a total of 33 baby food samples from Spain and the United Kingdom. Although imazalil, thiabendazole and carbendazim were detected in a high number--over 60%- of baby food samples, none of the samples tested were found to be above the 0.01 mg kg-1 EU standard.     

High-throughput liquid chromatography ultraviolet/mass spectrometric analysis of combinatorial libraries using an eight-channel multiplexed electrospray time-of-flight mass spectrometer

We report a high-throughput liquid chromatography/mass spectrometry (LC/MS) protocol for analyzing large combinatorial libraries using an eight-channel parallel LC/UV/MS (MUX-LCT) system. System configuration, linear response range in UV absorbance, LC column selection, and flow rate were optimized for 24 h/7 day unattended operations. Combinatorial libraries were analyzed on this system at a rate of 3200 compounds per day for a 3.5 min cycle time per injection. This parallel system is compared with a single-channel system in terms of performance and operation.     

Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples

Anabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid-phase microextraction (LPME) sample clean-up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean-up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2-20 ng ml(-1) for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses.