Sequential Elution Research Articles

Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

A novel strategy for sequential analysis of gold nanoparticles and gold ions in water samples by combining magnetic solid phase extraction with inductively coupled plasma mass spectrometry

In this work, Al3+ immobilized Fe3O4@SiO2@iminodiacetic acid (IDA) was prepared and its extraction performance for gold nanoparticles (AuNPs) and gold ions (Au ions) was investigated. It was found that AuNPs and Au ions could be simultaneously retained on this adsorbent and their separation was achieved by sequential elution of Au ions and AuNPs with Na2S2O3 and NH3·H2O, respectively. Based on it, a novel strategy by coupling magnetic solid phase extraction (MSPE) with inductively coupled plasma mass spectrometry (ICP-MS) was developed for the speciation of AuNPs and Au ions in environmental water samples. Various experimental parameters affecting MSPE of AuNPs and Au ions have been investigated carefully. AuNPs in a size range of 14–140 nm and with different coatings, including citrate, 11-mercaptoundecanoic acid (MUA), polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB), could be quantitatively determined by the proposed method. And the size and shape of the AuNPs were found to remain unchanged during the extraction process; thus the elution solutions could be directly introduced into ICP-MS without digestion. The developed MSPE-ICP-MS method has been applied in the analysis of AuNPs in natural water, sewage water and even sea water samples and Au ions in natural water samples with satisfactory results. Compared with the established methods for metal nanoparticle analysis, the developed method is more sensitive, faster, easier-to-operate, requires no digestion, and provides more valuable information for environmental monitoring and the assessment of nanoparticles' toxicity.

Accurate Measurement of Isotope Amount Ratios of Lead in Bronze with Multicollector Inductively Coupled Plasma Mass Spectrometry

Isotope amount ratios of lead in a bronze sample have been successfully determined using multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS). Matrix separation conditions were tested and optimized using ion exchange chromatography with anion-exchange resin, AG1-X8, and sequential elution of the 0.5 M HBr and 7 M HNO3 to separate lead from very high contents of copper and tin in bronze matrix. Mercury was also removed efficiently in the optimized separation condition. The instrumental isotope fractionation of lead in the MC-ICP-MS measurement was corrected by the external stan- dard sample bracketing method using an external standard, NIST SRM 981 lead common isotope ratio standard followed by cor- rection of procedure blank to obtain reliable isotope ratios of lead. The isotope ratios, 206 Pb/ 204 Pb, 207 Pb/ 204 Pb, 208 Pb/ 204 Pb, and 208 Pb/ 206 Pb, of lead were determined as 18.0802 ± 0.0114, 15.5799 ± 0.0099, 38.0853 ± 0.0241, and 2.1065 ± 0.0004, respec- tively, and the determined isotope ratios showed good agreement with the reference values of an international comparison for the same sample within the stated uncertainties

Sequential elution liquid chromatography can significantly increase the probability of a successful separation by simultaneously increasing the peak capacity and reducing the separation disorder

This paper demonstrates that sequential elution liquid chromatography (SE-LC), an approach in which two or more elution modes are employed in series for the separation of two or more groups of compounds, can be used to separate not only weak acids (or weak bases) from neutral compounds, but weak acids and weak bases from neutral compounds (and each other) by the sequential application of either of two types of an extended pH gradient prior to a solvent gradient. It also details a comparison, based on peak capacity and separation disorder, of the probability of success of this approach with the unimodal elution approach taken by conventional column liquid chromatography. For an HPLC peak capacity of 120 and samples of moderate complexity (e.g., 12 components), the probability of success (Rs≥1) increases from 37.9% (HPLC) to 85.8% (SE-LC). Different columns were evaluated for their utility for SE-LC using the following criteria: (1) the prediction of the elution order of the groups based on the degree of ionization of the compounds; and (2) the closeness of the peak shape to the ideal Gaussian distribution. The best columns overall were the Zorbax SB-AQ and Waters XBridge Shield columns, as they provided both between-class and within-class separations of all compounds, as well as the lowest degree of tailing of 4-ethylaniline using the pH 2 to pH 8 gradient.

Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds.

Practical Isolation of Bullatacin from <i>Annona muricata</i> Leaves Extract Using an Open Column Chromatography Technique

Annonaceous acetogenins are bioactive compounds present in the leaves of Annona muricata (soursop) with significant anti-cancer activities. In this study, acetogenin-rich ethanol fraction extracted from soursop leaves was further separated and isolated using a simple open column chromatography technique with silica gel as the stationary phase. The separation procedure consists of three steps, each step employing sequential elution with organic solvents having different polarity as the eluent. Eluted fractions rich in acetogenin compounds were identified using the Kedde reagent which formed a distinctive dark green complex with the unsaturated γ-lactone group present in all of the annonaceous acetogenin compounds. HPLC analysis showed that the initial methanol fraction, as well as isolates from the second and the third isolation steps, contain bullatacin, squamocin, squamostatin-A, and squamostatin-D. Bullatacin produced from this study can be used further as a standard compound for quantitative analysis of other acetogenin compounds contained in soursop leaves extract.

Evaluation on the effect of different in-gel peptide isoelectric focusing parameters in global proteomic profiling

Peptide isoelectric focusing (IEF) is a common technique used in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) proteomic workflow, in which the tryptic peptide is first pre-fractionated based on pI values before being subjected to reverse phase LC–MS analysis. Although this method has been widely used by many research groups, a systemic study on the optimal conditions and fundamental parameters influencing the experimental outcomes has been lacking, including the effect of peptide extraction methods, the extent of pre-fractionation, and the choice of pH range. In this study, we compared the effect of different parameters on the numbers of peptides and proteins identified using two complex mouse proteomes. The results indicated that extraction of peptides from immobilized pH gradient (IPG) strips by sequential elution of increasingly organic solvents provided the highest number of peptide identification. In addition, we showed that approximately 45 more unique proteins were identified for every additional fraction collected during peptide IEF. Although narrow pH ranges provided higher resolution in peptide separation as expected, different pH ranges yielded similar numbers of peptide and protein identification. Overall, we demonstrated that the extraction solvent influenced the numbers of peptide and protein identification and quantitatively demonstrated the advantage of extensive fractionation and the performance of different pH ranges in practice.

18F]Fallypride: Metabolism studies and quantification of the radiotracer and its radiometabolites in plasma using a simple and rapid solid-phase extraction method

[(18)F]Fallypride, a fluorinated and substituted benzamide with high affinity for D2/D3 receptors, is a useful PET radioligand for the study of striatal/extrastriatal areas. Since [(18)F]fallypride is extensively metabolized in vivo and since PET examinations are long lasting in humans, the rapid measurement of the unchanged radiotracer in plasma is essential for the quantification of images. The present study aims: i) to evaluate if the radiometabolites of [(18)F]fallypride cross the blood-brain barrier in rodents, ii) to identify these radiometabolites in baboon plasma and iii) to develop a rapid solid phase extraction method (SPE) suitable for human applications to quantify both [(18)F]fallypride and its radiometabolites in plasma. The metabolites P450-dependant in rat and human liver microsomes were characterized by LC-MS-MS and compared to those detected in vivo. Sequential solvent elution on Oasis®-MCX-SPE cartridges was used to quantify [(18)F]fallypride and its radiometabolites. In rat microsomal incubations, five metabolites generated upon N/O-dealkylation or hydroxylation at the pyrrolidine and/or at the benzamide moiety were identified. No radiometabolite was detected in the rat brain. N-dealkylated and hydroxylated derivatives were detected in human microsomal incubations as well as in baboon plasma. The use of SPE (total recovery 100.2%± 2.8%, extraction yield 95.5%± 0.3%) allowed a complete separation of [(18)F]fallypride from its radiometabolites in plasma and evaluate [(18)F]fallypride at 150 min pi to be 22%± 5% of plasma radioactivity. The major in vivo radiometabolites of [(18)F]fallypride were produced by N-dealkylation and hydroxylation. Allowing the rapid analysis of multiple plasma samples, SPE is a method of choice for the determination of [(18)F]fallypride until late images required for quantitative PET imaging in humans.

Distinguishing metal bioconcentration from particulate matter in moss tissue: Testing methods of removing particles attached to the moss surface

Accurate differentiation of the proportion of bioconcentrated metals (i.e. incorporated into cells) and the proportion that is not bioconcentrated (i.e. adsorbed at the surface) would lead to a better understanding of the uptake processes and would represent an advance in the use of mosses as biomonitors. Traditionally the methods used to remove contaminants that are not bioconcentrated were to wash the plant material with water or to apply the sequential elution technique, but nowadays both options are considered inaccurate for these purposes. The remaining possibilities are to clean the moss samples with a nitrogen jet or by power ultrasound. Samples of terrestrial moss Pseudoscleropodium purum (Hewd.) Fleisch. were collected from five sampling stations. Different nitrogen jet cleaning procedures and ultrasound cleaning procedures were applied to the mosses. To determine whether any of the treatments altered the membrane integrity of the moss samples, the concentrations of K were determined. The shoots were observed under a scanning electron microscope, and the size and number of particles were determined. Nitrogen jet cleaning was determined to be unacceptable because it damaged the phyllids and/or altered the membrane permeability and did not eliminate the particles from the moss surface. Moreover, ultrasound cleaning treatment should also discarded because of the loss of extracellular metals that are transferred to the water in which the moss is cleaned.

Microchip Emitter for Solid-Phase Extraction–Gradient Elution–Mass Spectrometry

A microchip electrospray emitter with a magnetic bead trap has been designed for solid-phase extraction-gradient elution-mass spectrometry (SPE-GEMS). The goal of this method is the detection of analytes at low concentrations and it is here demonstrated using reverse phase coated magnetic beads (Mbs) for the preconcentration and detection of the peptides. The sample is passed through the chip, and the peptides are retained and enriched in the trap. After washing, the peptides are released sequentially by stepwise gradient elution and electrosprayed for mass spectrometry analysis. This approach allows effective sample desalting, enrichment, sequential elution, and MS detection without the introduction of an additional separation step after SPE. Efficient preconcentration of model peptides by SPE and sequential release and analysis of peptides by GEMS were demonstrated for diluted sample solutions within the range of 1 μM to 10 nM. Fortified human blood serum, protein digest and fractions collected after protein digest OFFGEL separation were analyzed by SPE-GEMS allowing the detection of low abundance peptides usually not observed by direct mass spectrometry analysis. A mathematical model for gradient elution is proposed.

Hyphenated Flash Chromatographic Separation and Isolation of Coumarins and Polymethoxyflavones from Byproduct of Citrus Juice Processing Industry

The present study describes an efficient hyphenated flash chromatographic (FC) for the purification of coumarins and polymethoxy flavones (PMF's) from cold pressed grapefruit peel oil (GFO), a byproduct from citrus juice processing plant. GFO was subjected to silica column chromatography using sequential isocratic elution by hexane and chloroform to separate the waxes and phytochemicals enriched fractions, respectively. The chloroform extract was used for hyphenated FC separation using a gradient mobile phase of hexane and acetone. The peak separations were monitored at dual wavelengths of λ210 nm and λ340 nm. The individual fractions were analyzed by HPLC, pooled and concentrated resulting in isolation of two coumarins (auraptene and bergapten) and three PMF's (tangeretin, heptamethoxyflavone, and nobiletin). The purity and identity of the isolated compounds was confirmed by spectral analysis using high performance liquid chromatography, matrix-assisted laser desorption/Ionization-time of flight-mass spectrometry, and nuclear magnetic resonance. The hyphenated FC separation method was further validated by test of repeatability resulting in low RSD for the yield of the phytochemicals. Utilization of the developed separation method for isolation of value added products from grapefruit byproducts could be beneficial to the citrus processing industry. Supplemental materials are available for this article. Go to the publisher's online edition of Separation Science & Technology to view the supplemental file.

High Temperature Simulated Distillation of Bitumen Fractions with Open Tubular Capillary Columns Depleted in Silicone/Siloxane Stationary Phases

High Temperature Simulated Distillation (HTSD) is routinely carried out in petroleum laboratories for characterization of heavy hydrocarbon fractions. However, thermal cracking from HTSD high operating temperatures (up to 430 °C) has been reported to affect analytes during the chromatographic process. This work showed the feasibility of using silicone/siloxane depleted stationary phase wide bore capillary columns for HTSD carried out under lower setup temperatures to avoid cracking. Sequential low temperature elution for analysis followed by high temperature oven cycles for injector cleaning were found mandatory for successful operation. Depleted stationary phase metal capillary columns were determined as a feasible interesting alternative, advantageous from the point of view of column life span and retention times constancy. Present findings suggest that these columns can have useful life in the greater than or equal to a year span. Heavy hydrocarbon upgrading monitoring with standard and depleted stationary phase metal columns was found equivalent, further supporting the feasibility of using the latter for routine applications. X-ray photoelectron spectroscopy (XPS) analysis of the internal wall of metal columns highlighted differences brought by stationary phase depletion, particularly on the silicone/siloxane groups. Finally, preliminary results indicated that one further alternative to avoid “on column” thermal cracking is to elute standard columns to temperature limits <370 °C.

Development of a Single Chain Antibody Using a Phage Display Cloning Method for the Detection of 2,4-Dinitrotoluene

Single-chain variable fragments of antibodies (scFv) specific to 2,4-dinitrotoluene (DNT) were isolated from a phage library displaying synthetic human scFv fragments with 6 diversified complementary determining regions (CDRs). A DNT derivative that contained an extended amine group was synthesized and conjugated to the NHS-group that was linked to magnetic beads. Phages specific to the immobilized DNT derivatives were isolated from the library after 4 rounds of sequential binding and elution processes. The displayed scFv fragments from the isolated phages showed consensus CDR sequences. One DNT-specific scFv was expressed in E. coli and purified using Ni-affinity chromatography. The purified DNT-specific scFv binds specifically to the immobilized DNT-derivative with value of M. The scFv and DNT interaction was not disrupted by the addition of 4-nitrotoluene or benzoic acid. These data demonstrate that the screened scFv from the phage displayed library could be used for selective and sensitive detection of explosives such as TNT.

Multiplexed electrokinetic sample fractionation, preconcentration and elution for proteomics

Both 6 and 8-channel integrated microfluidic sample pretreatment devices capable of performing "in space" sample fractionation, collection, preconcentration and elution of captured analytes via sheath flow assisted electrokinetic pumping are described. Coatings and monolithic polymer beds were developed for the glass devices to provide cationic surface charge and anodal electroosmotic flow for delivery to an electrospray emitter tip. A mixed cationic ([2-(methacryloyloxy)ethyl] trimethylammonium chloride) (META) and hydrophobic butyl methacrylate-based monolithic porous polymer, photopolymerized in the 6- or 8-fractionation channels, was used to capture and preconcentrate samples. A 0.45 wt% META loaded bed generated comparable anodic electroosmotic flow to the cationic polymer PolyE-323 coated channel segments in the device. The balanced electroosmotic flow allowed stable electrokinetic sheath flow to prevent cross contamination of separated protein fractions, while reducing protein/peptide adsorption on the channel walls. Sequential elution of analytes trapped in the SPE beds revealed that the monolithic columns could be efficiently used to provide sheath flow during elution of analytes, as demonstrated for neutral carboxy SNARF (residual signal, 0.08% RSD, n = 40) and charged fluorescein (residual signal, 2.5% n = 40). Elution from monolithic columns showed reproducible performance with peak area reproducibility of ~8% (n = 6 columns) in a single sequential elution and the run-to-run reproducibility was 2.4-6.7% RSD (n = 4) for elution from the same bed. The demonstrated ability of this device design and operation to elute from multiple fractionation beds into a single exit channel for sample analysis by fluorescence or electrospray mass spectrometry is a crucial component of an integrated fractionation and assay system for proteomics.

Separation and Recovery of Cs from High Active Waste Simulant using Resorcinol Formaldehyde Polycondensate Resin: Batch and Column Studies

Resorcinol formaldehyde polycondensate resin has been extensively used for the separation of 137Cs from low and medium active alkaline waste solutions. The present study examines the suitability of the resin for separation of Cs from high active solutions. Batch uptake of Cs by RFPR has been evaluated from test solutions containing varying Na (0.6–3.0 M) and Cs (1.00 × 10−4 to 0.10 M) concentrations. Batch results have been used to establish Cs exchange isotherms of the resin and were found to follow Freundlich as well as Dubinin-Radushkevich (D-R) isotherm equations. The loading behavior of Cs on RFPR column has been evaluated by conducting five column runs using different Cs bearing solutions. Based on these results, it is concluded that the D-R equation can be used to predict saturated loading of Cs in the column. The saturation loading of Cs on 1 L bed was estimated to be as high as 13 g and 34 g from feed solution containing Cs concentration 2.57 × 10−4 and 4.30 × 10−3 mol/L respectively. Sequential elution of Na and Cs resulted in a high Cs bearing concentrate containing very low concentration of sodium. This study is relevant in the preparation of gamma source for radiation technology applications after immobilization of the concentrate in glass.

Assessment of Different Methods To Determine the Total Wax Content of Crude Oils

Wax formation by temperature decrease is a serious issue in the petroleum industry, because it makes the production difficult and can lead to important economic losses. As a result, a large effort has been made to predict the occurrence of this phenomenon as accurately as possible. Thermodynamic models are commonly used to predict the main parameters involved in the wax precipitation process. Models with a wide basis require as most common input information the n-paraffin distribution and the total wax content (C20+ fraction). The aim of this work is to check the capabilities of different experimental techniques to determine the C20+ content of different crude oils. For that purpose, high-temperature gas chromatography (HTGC) and differential scanning calorimetry (DSC) analyses (direct analysis of the raw crude oil in both cases) were used, showing reasonable agreement. Likewise, the wax precipitation of the raw crude oils was performed to obtain the direct C20+ content. Such experiments lead to clearly higher values than those obtained by HTGC and DSC analyses because of the presence of trapped crude oil in the precipitated solid. After that porosity correction carried out by 1H nuclear magnetic resonance (NMR), DSC, and sequential elution chromatography techniques, the obtained results indicate that both 1H NMR and DSC analyses yield more consistent results. An empirical correlation was developed to determine the C20+ content from the American Petroleum Institute (API) gravity and the pour point of the selected crude oils. The correlation was applied to a number of crude oils, obtaining a reasonable agreement between experimental and calculated C20+ values, which shows that the validity of that correlation is wider than previous proposed equations.

Acoustophoretic microfluidic chip for sequential elution of surface bound molecules from beads or cells

An acoustophoresis-based microfluidic flow-chip is presented as a novel platform to facilitate analysis of proteins and peptides loosely bound to the surface of beads or cells. The chip allows for direct removal of the background surrounding the beads or cells, followed by sequential treatment and collection of a sequence of up to five different buffer conditions. During this treatment, the beads/cells are retained in a single flow by acoustic radiation force. Eluted peptides are collected from the outlets and subsequently purified by miniaturized solid-phase extraction and analyzed with matrix assisted laser desorption mass spectrometry. Fundamental parameters such as the system fluidics and dispersion are presented. The device was successfully applied for wash and sequential elution of peptides bound to the surface of microbeads and human spermatozoa, respectively.

TiSH — a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC

Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO(2) pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~6600 unique phosphopeptides from 300 μg of peptides/condition (22 unique phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity.

Nanodiamond‐therapeutic complexes embedded within poly(ethylene glycol) diacrylate hydrogels mediating sequential drug elution

AbstractThe versatility of nanodiamonds (NDs) as a polymer‐embedded therapeutic vehicle was demonstrated by the development of a hybrid material consisting of a poly(ethylene glycol) diacrylate (PEGDA) hydrogel embedded with ND‐drug complexes. A wide range of PEGDA structures, including single and multiple drug reservoirs, exhibited high loading capabilities and stable slow‐release, augmented with the addition of ND conjugates. ND‐PEGDA hydrogels were compared to unmodified, drug‐embedded PEGDA hydrogels to demonstrate their robust drug sequestering capacity in vitro. Remarkably, ND‐embedded hydrogels were capable of mitigating the harmful effects of burst‐release by displaying slow and controlled administration of therapeutic compounds, an improvement over standard diffusion‐based release profiles. The tunable customization of PEGDA hydrogel, both in structure and content, as well as its facile methods of synthesis, make this material an effective multi‐therapeutic complement to the drug delivery abilities of NDs. A hybrid material that utilizes the synergy of PEGDA and ND components has the potential for applications toward a diverse array of biomedical challenges. magnified imageAn artistic rendering of a multi‐layered PEGDA patch. The center of the patch (purple) contains drug‐bound ND particles that are being released (gray truncated octahedra with purple glow), while the outer ring depicts the molecule Temozolomide (green). This image illustrates the ability for tiered patches to simultaneously administer multiple drugs, as well as the drug‐sequestering ability of NDs that mediates sustained delivery and reduced burst release. The nested structure of the two drugs was designed so that the therapeutics could be released sequentially, at different peak‐elution time intervals.

Screening and Selective Quantification of Illicit Drugs in Wastewater by Mixed-Mode Solid-Phase Extraction and Quadrupole-Time-of-Flight Liquid Chromatography–Mass Spectrometry

For the first time, a mixed-mode solid-phase extraction with fractionation of basic analytes from neutral and acidic species during cartridge elution and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) was combined for the quantitative determination of 24 illicit drugs and metabolites in urban sewage samples. The effects of several sample preparation and instrumental parameters in the sensitivity and selectivity of the quantitative method are thoroughly discussed. Under final working conditions, recoveries above 63% and 82% were attained for all species in raw and treated sewage, respectively; whereas, the limits of quantification of the method, defined for a signal-to-noise of 10 (S/N = 10), ranged from 2 to 50 ng L(-1). Sequential elution of mixed-mode cartridges allowed a significant reduction of matrix effects observed during electrospray ionization of basic drugs versus those measured for hydrophilic balance reversed-phase sorbents and the same mixed-mode polymer without fractionated elution. Analysis of raw wastewater samples confirmed the ubiquity of cocaine (COC), benzoylecgonine (BE), and 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in this matrix. The capability of the above methodology to identify new illicit drugs and/or metabolites in sewage samples is also discussed. With this aim, a two step strategy is proposed. First, high-resolution MS chromatograms, acquired throughout each chromatographic run, are automatically searched against an in-house built database, a reduced list of candidate drugs is generated, and the corresponding extracted ion chromatograms are obtained. In a further LC run, the tandem mass spectrometry (MS/MS) spectra of unknown peaks are acquired using different collision energies and compared with those existing in public libraries, or interpreted, to assign the unknown peak to one of the previously selected candidates.