Coupling of Ultrafast LC with Mass Spectrometry by DESI

Recently we reported a desorption electrospray ionization (DESI) interface to combine liquid chromatography (LC) with mass spectrometry (MS) using a new LC eluent splitting strategy through a tiny orifice on LC capillary tube [J. Am. Soc. Mass Spectrom. 25, 286 (2014)]. The interface introduces negligible dead volume and back pressure, thereby allowing “near real-time” MS detection, fast LC elution, and online MS-directed purification. This study further evaluates the LC/DESI-MS performance with focus of using ultra-fast LC. Using a monolithic C₁₈ column, metabolites in urine can be separated within 1.6 min and can be online collected for subsequent structure elucidation (e.g., by NMR, UV, IR) in a recovery yield up to 99%. Using a spray solvent with alkaline pH, negative ions could be directly generated for acidic analytes (e.g., ibuprofen) in acidic LC eluent by DESI, offering a novel protocol to realize “wrong-way around” ionization for LC/MS analysis. In addition, DESI-MS is found to be compatible with ultra-performance liquid chromatography (UPLC) for the first time.      

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.

A micro-discharge photoionization detector for micro-gas chromatography

We report on a small (20?×?10 mm) micromachined device for the detection of gases in micro-gas chromatography (GC). It incorporates a micro-discharge across a 20-μm gap, and a remote electrode in the micro cavity that generates an electrical signal corresponding to the photo-ionization of gaseous analytes in a stream of carrier gas. Multi-component mixtures were detected and the results compared to those obtained with a flame ionization detector. The minimum detectable limit is 350 pg.μL^?1 of n-octane in air when applying a 1.4 mW discharge. The combination of wet etching of glass (as used for microfluidic channels) with a lift-off process for detector electrodes by a robust batch process results in a universal, non-destructive, and sensitive microdetector for micro-GC.

Evaluation of the Potential of GC-APCI-MS for the Analysis of Pesticide Residues in Fatty Matrices

A method based on gas chromatography-atmospheric pressure chemical ionization-mass spectrometry (GC-APCI-MS) has been developed for the analysis of pesticides in meat by using quadrupole-time of flight mass spectrometry (QTOF-MS). Ionization and MS conditions were studied for 71 compounds, although only 51 showed acceptable performance. The protonated form of the analytes was mainly found ([M?+?H]⁺), although some compounds generated the molecular ion (M^+?). A fast and generic extraction procedure was applied in sample pretreatment. The analytical method was suitable for qualitative analysis, and it was also evaluated for quantitative analysis, obtaining adequate recovery and precision values for most of the studied analytes at two concentration levels (50 and 150 μg/kg). Several operational drawbacks were found with this instrument, such as slow stabilization and moderate sensitivity, although the fast switching between LC and GC allows the increase of its applicability.

Development of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry for analysis of halogenated flame retardants in wastewater

Until recently, atmospheric pressure photoionization (APPI) has typically been used for the determination of non-polar halogenated flame retardants (HFRs) by liquid chromatography (LC) tandem mass spectrometry. In this study, we demonstrated the feasibility of utilizing liquid chromatography atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-APCI-MS/MS) for analysis of 38 HFRs. This developed method offered three advantages: simplicity, rapidity, and high sensitivity. Compared with APPI, APCI does not require a UV lamp and a dopant reagent to assist atmospheric pressure ionization. All the isomers and the isobaric compounds were well resolved within 14-min LC separation time. Excellent instrument detection limits (6.1 pg on average with 2.0 μL injection) were observed. The APCI mechanism was also investigated. The method developed has been applied to the screening of wastewater samples for screening purpose, with concentrations determined by LC-APCI-MS/MS agreeing with data obtained via gas chromatography high resolution mass spectrometry.

False Results Caused by Solvent Impurity in Tetrahydrofuran for MALDI TOF MS Analysis of Amines

Tetrahydrofuran (THF) is one of the most frequently used solvents in the MALDI TOF MS analysis of synthetic compounds. However, it should be used with caution because a trace amount of 4-hydroxybutanal (HBA) might be generated and accumulated in THF during storage. Since only a tiny amount of analytes is required in MALDI MS measurements, a trace amount of HBA might have a significant effect on the MS results. It was found that HBA will quickly react with primary and secondary amino compounds, leading to false results about the sample composition with an extra series of ions with additional mass of 70 Da in between. The formation of HBA can be inhibited by butylated hydroxytoluene (BHT) antioxidant. Therefore, when THF is required as the solvent for sample preparation, it is strongly recommended to use a BHT-stabilized one, at least for the analysis of compounds with amino groups.

Simultaneous determination of NNK and its metabolites in mouse tissue for evaluating the effects of chronic alcohol consumption on the metabolism of NNK in mouse liver and lung

A hydrophilic-interaction liquid chromatography–tandem mass spectrometry (HILIC–MS–MS) method was developed for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites in mouse liver and lung. The limits of detection of all analytes were in the range 0.017–0.057 ng mL^?1, and recovery ranged from 88.4–119.8 % with intra and inter-day precision in the range 0.89–6.03 % and 1.01–6.97 %, respectively. This simple and accurate method was used to evaluate the effect of chronic alcohol consumption on NNK bioactivation in mouse tissue. Time-course curves for NNK and its metabolites were generated, and the areas under the curves (AUCs) were compared. It was found that target tissues of NNK carcinogenesis in C57BL/6 mice contained high levels of α-hydroxylation metabolites of NNK and its carbonyl reduction metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The most pronounced effect of alcohol was to enhance α-hydroxylation of NNK in mouse lung and liver, which suggests that chronic alcohol consumption may increase the risk of carcinogenicity associated with NNK in mice.

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.

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

Ambient DESI and LESA-MS Analysis of Proteins Adsorbed to a Biomaterial Surface Using In-Situ Surface Tryptic Digestion

The detection and identification of proteins adsorbed onto biomaterial surfaces under ambient conditions has significant experimental advantages but has proven to be difficult to achieve with conventional measuring technologies. In this study, we present an adaptation of desorption electrospray ionization (DESI) and liquid extraction surface analysis (LESA) mass spectrometry (MS) coupled with in-situ surface tryptic digestion to identify protein species from a biomaterial surface. Cytochrome c, myoglobin, and BSA in a combination of single and mixture spots were printed in an array format onto Permanox slides, followed by in-situ surface digestion and detection via MS. Automated tandem MS performed on surface peptides was able to identify the proteins via MASCOT. Limits of detection were determined for DESI-MS and a comparison of DESI and LESA-MS peptide spectra characteristics and sensitivity was made. DESI-MS images of the arrays were produced and analyzed with imaging multivariate analysis to automatically separate peptide peaks for each of the proteins within a mixture into distinct components. This is the first time that DESI and LESA-MS have been used for the in-situ detection of surface digested proteins on biomaterial surfaces and presents a promising proof of concept for the use of ambient MS in the rapid and automated analysis of surface proteins.

On-line solid-phase microextraction of triclosan, bisphenol A, chlorophenols, and selected pharmaceuticals in environmental water samples by high-performance liquid chromatography–ultraviolet detection

A method using on-line solid-phase microextraction (SPME) on a carbowax-templated fiber followed by liquid chromatography (LC) with ultraviolet (UV) detection was developed for the determination of triclosan in environmental water samples. Along with triclosan, other selected phenolic compounds, bisphenol A, and acidic pharmaceuticals were studied. Previous SPME/LC or stir-bar sorptive extraction/LC-UV for polar analytes showed lack of sensitivity. In this study, the calculated octanol–water distribution coefficient (log D) values of the target analytes at different pH values were used to estimate polarity of the analytes. The lack of sensitivity observed in earlier studies is identified as a lack of desorption by strong polar–polar interactions between analyte and solid-phase. Calculated log D values were useful to understand or predict the interaction between analyte and solid phase. Under the optimized conditions, the method detection limit of selected analytes by using on-line SPME-LC-UV method ranged from 5 to 33 ng?L^?1, except for very polar 3-chlorophenol and 2,4-dichlorophenol which was obscured in wastewater samples by an interfering substance. This level of detection represented a remarkable improvement over the conventional existing methods. The on-line SPME-LC-UV method, which did not require derivatization of analytes, was applied to the determination of TCS including phenolic compounds and acidic pharmaceuticals in tap water and river water and municipal wastewater samples.

Separation of dansylated 17β-estradiol, 17α-estradiol, and estrone on a single HPLC column for simultaneous quantitation by LC–MS/MS

We show here that baseline separation of dansylated estrone, 17β-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs ¹³C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®.

Picoelectrospray Ionization Mass Spectrometry Using Narrow-Bore Chemically Etched Emitters

Electrospray ionization mass spectrometry (ESI-MS) at flow rates below ~10 nL/min has been only sporadically explored because of difficulty in reproducibly fabricating emitters that can operate at lower flow rates. Here we demonstrate narrow orifice chemically etched emitters for stable electrospray at flow rates as low as 400 pL/min. Depending on the analyte concentration, we observe two types of MS signal response as a function of flow rate. At low concentrations, an optimum flow rate is observed slightly above 1 nL/min, whereas the signal decreases monotonically with decreasing flow rates at higher concentrations. For example, consumption of 500 zmol of sample yielded signal-to-noise ratios ~10 for some peptides. In spite of lower MS signal, the ion utilization efficiency increases exponentially with decreasing flow rate in all cases. Significant variations in ionization efficiency were observed within this flow rate range for an equimolar mixture of peptide, indicating that ionization efficiency is an analyte-dependent characteristic for the present experimental conditions. Mass-limited samples benefit strongly from the use of low flow rates and avoiding unnecessary sample dilution. These findings have important implications for the analysis of trace biological samples.

A highly sensitive caffeine immunoassay based on a monoclonal antibody

A new immunoassay has been developed based on a commercially available anti-caffeine monoclonal antibody and a de novo synthesized tracer, using horseradish peroxidase and UV–visible detection. Caffeine, which is frequently found in surface waters, can be quantified with a relative error lower than 20% for concentrations above 0.025 μg L^?1 (limit of quantitation, direct analysis). The limit of detection is 0.001 μg L^?1 and can be reduced by solid-phase extraction (SPE). Moreover, with minor adaptations, the assay can be used to quantify caffeine in several beverages, shampoo, and caffeine tablets. The results obtained by ELISA correlate well with those from liquid chromatography–tandem mass spectrometry (LC–MS–MS) for the tested matrices. Several surface waters from Berlin were analysed and all tested positive for caffeine, with concentrations higher than 0.030 μg L^?1. In one run 66 samples can be analysed within 2 h.

Micro-scale quantitation of ten phthalate esters in water samples and cosmetics using capillary liquid chromatography coupled to UV detection: effective strategies to reduce the production of organic waste

We have extracted ten phthalate esters (C1 to C8) using six different micro-scale methods for extraction, and then separated them by capillary liquid chromatography coupled to UV detection. The methods included liquid-liquid extraction, ultrasonic-assisted extraction, microwave-assisted extraction, dispersive liquid-liquidmicroextraction, dispersive liquid-liquid microextraction solidification of floating organic droplets, and cloud point extraction. The linear range of the analytes is from 0.5 to 50 μg mL^?1, and the detection limits range from 0.02 to ~0.17 μg mL^?1. The precision and accuracy of all intra- and inter-day analyses are <5.5%. We find that dispersive liquid-liquid microextraction solidification of floating organic droplet (DLLME-SFO) is the best method for quantification of most phthalate esters in water samples and cosmetics because of its low limit of detection and high extraction efficiencies.

Detection, quantification, and identification of dermorphin in equine plasma and urine by LC–MS/MS for doping control

Dermorphin is a unique opioid peptide that is 30–40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC–MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20–10,000 pg/mL in plasma and 50–20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

Ultra performance liquid chromatography tandem mass spectrometry performance evaluation for analysis of antibiotics in natural waters

An ultra performance liquid chromatography electrospray tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the determination of 17 antibiotics in natural waters in one single extraction and chromatographic procedure. Gradient separation conditions were optimised for 17 compounds belonging to five different antibiotic groups: quinolones (oxolinic acid, nalidixic acid, pipemidic acid, flumequine), fluoroquinolones (enoxacin, ciprofloxacin, norfloxacin, ofloxacin, enrofloxacin, sarafloxacin, danofloxacin, difloxacin, lomefloxacin), sulphonamides (sulphamethoxazole, sulphamethazine), nitro-imidazole (ornidazole) and diaminopyrimidine (trimethoprim). The separation of all compounds, obtained using a 1.7 μm particle size column (100 mm?×?2.1 mm), was achieved within 10 min time. Water samples were adjusted to pH 7 and extracted using Oasis hydrophilic–lipophilic balance (HLB) solid phase extraction cartridges. After elution with methanol and concentration, extracts were injected in a C18 column (Acquity UPLC BEH C18) and detected by tandem mass spectrometry. Average recovery from 100 ng L^?1 fortified samples was higher than 70% for most of the compounds, with relative standard deviations below 20%. Performances of the method (recoveries, detection limit, quantification limit and relative standard deviation) and matrix effects were studied, and results obtained showed that method was suitable for routine analysis of antibiotics in surface water. Samples analysis from Seine River (France) confirmed the interest of antibiotic contamination evaluation in that area.

Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography–tandem mass spectrometry

Since sarcosine and d,l-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine–alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R ²?=?0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of d,l-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb–1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from d,l-alanine leading to underivatized quantification of sarcosine.

Solid phase ligand-less extraction of cadmium(II) using a silica gel modified with an amino-functionalized ionic liquid

A method was developed for the determination of cadmium(II) by ligand-less solid phase extraction that is based on the direct retention of Cd(II) in a mini-column filled with a silica gel modified with an amino-functionalized ionic liquid. The effects of pH, sample volume and its flow rate, eluent concentration and its volume, the flow rate of eluent, and of potential interferences on extraction and desorption were optimized. Following its determination by electrothermal atomic absorption spectrometry, the detection limit for Cd(II) is 8.9 ng L^?1, and the relative standard deviation is 2.3 % (at 1.0 ng mL^?1; for n?=?5). The method was applied to the analysis of Cd(II) in a certified reference material (laver; GBW10023), and the recoveries ranged from 97.0 to104.0 %