Use of a guard column coupled to mass spectrometry as a fast semi-quantitative methodology for the determination of plasticizer metabolites in urine.

For the first time, a very simple and fast method combining the use of a guard column coupled to tandem mass spectrometry (guard column-MS/MS) has been proposed for the determination of plasticizer metabolites in urine. Briefly, samples (1.0 mL) were submitted to enzymatic hydrolysis for 10 min, filtered, diluted 1/10 v/v with ultrapure water and directly injected into the system. A fast run of only 2 min (3 min including the injection cycle) allowed the determination of 19 analytes. Enzymatic hydrolysis, filtering material, and guard column-MS/MS conditions were optimized. Intra-day precision at the low-level concentration (expressed as relative standard deviation, %RSD) obtained from the analysis of synthetic urine samples varied between 11 and 20%. Limits of quantification ranged from 2.8 to 60 ng/mL. Trueness values, calculated as apparent recoveries, ranged from 70 to 135%. To correct for matrix effects, analyte concentrations in real urine were quantified by the standard addition method. To confirm the results obtained by guard column-MS/MS, an ultra(high)-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was also applied (total chromatographic run time 17 min, including column re-equilibration). Concentrations measured with both methods were in good agreement. Hence, we propose the use of guard column-MS/MS to analyse a large number samples in a very short time (semi-quantification), and apply the chromatographic analysis only to those samples with levels close to/higher than the concentrations equivalent to the safe maximum daily intakes of the parent compounds (confirmation). This double strategy (semi-quantification by guard column-MS/MS and confirmation-when needed-by UHPLC-MS/MS) implies important savings in time and money.

Determination of Hydroxy Polycyclic Aromatic Hydrocarbons in Human Urine Using Automated Microextraction by Packed Sorbent and Gas Chromatography-Mass Spectrometry.

A fast methodology for the determination of monohydroxy polycyclic aromatic hydrocarbons in human urine using a fully automated microextraction by packed sorbent coupled to a gas chromatograph-mass spectrometer is reported. Sample preparation requires simple hydrolysis, centrifugation, filtration, and dilution. The method does not require a derivatization step prior to analysis with gas chromatography and allows the measurement of up to three samples per hour after hydrolysis. Quantitation is carried out by a one-point standard addition allowing the determination of 6 analytes with good limits of detection (10.1-39.6 ng L-1 in water and 0.5-19.4 µg L-1 in urine), accuracy (88-110%) and precision (2.1-23.4% in water and 5.1-19.0% in urine) values. This method has been successfully applied to the analysis of six urine samples (three from smoker and three from non-smoker subjects), finding significant differences between both types of samples. Results were similar to those found in the literature for similar samples, which proves the applicability of the methodology.

Determination of leucine and isoleucine/allo-isoleucine by electrospray ionization-tandem mass spectrometry and partial least square regression: Application to saliva samples.

Herein we propose, for the first time, a rapid method based on flow injection analysis, electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) and multivariate calibration for the determination of l-leucine, l-isoleucine and L-allo-isoleucine in saliva. As far as we know, multivariate calibration has never been applied to the data from this non-separative approach. The possibilities of its use were explored and the results obtained were compared with the corresponding ones when using univariate calibration. Partial least square regression (PLS1) multivariate calibration models were built for each analyte by analyzing different saliva samples, and were subsequently applied to the analysis of another set of samples which had not been used in any calibration step. For Leu, the model worked satisfactorily with root mean square errors in the prediction step of 17%. This error can be considered acceptable and is common in methodologies that do not include a separation step. Results were compared with those obtained when univariate calibration was used, using the m/z transition 132.1 â†’ 43.0 as the quantitation variable. In this case, the obtained results were not acceptable, with RMSEP of 236%, due to the fact that saliva samples contained another compound, different to the target analytes, which also shared the same transition. Ile and aIle have the same fragmentation patterns, so quantification of the sum of both compounds was performed, with RMSEP of 14% using a PLS1 model. Similar results were obtained when a univariate calibration model using the m/z transition 132.1 â†’ 69.0 was employed. However, the use of this transition should be carefully examined when other compounds present in the matrix contribute to the analytical signal. The method increases sample throughput more than one order of magnitude compared to the corresponding LC-ESI-MS/MS method and is especially suitable as screening. When abnormally high or low concentrations of the analytes studied are obtained, the use of the method that includes separation is recommended to confirm the results.

Deregulation of the Purine Pathway in Pre-Transplant Liver Biopsies Is Associated with Graft Function and Survival after Transplantation.

The current shortage of livers for transplantation has increased the use of marginal organs sourced from donation after circulatory death (DCD). However, these organs have a higher incidence of graft failure, and pre-transplant biomarkers which predict graft function and survival remain limited. Here, we aimed to find biomarkers of liver function before transplantation to allow better clinical evaluation. Matched pre- and post-transplant liver biopsies from DCD (n = 24) and donation after brain death (DBD, n = 70) were collected. Liver biopsies were analysed using mass spectroscopy molecular phenotyping. Discrimination analysis was used to parse metabolites differentiated between the two groups. Five metabolites in the purine pathway were investigated. Of these, the ratios of the levels of four metabolites to those of urate differed between DBD and DCD biopsies at the pre-transplantation stage (q < 0.05). The ratios of Adenosine monophosphate (AMP) and adenine levels to those of urate also differed in biopsies from recipients experiencing early graft function (EGF) (q < 0.05) compared to those of recipients experiencing early allograft dysfunction (EAD). Using random forest, a panel consisting of alanine aminotransferase (ALT) and the ratios of AMP, adenine, and hypoxanthine levels to urate levels predicted EGF with area under the curve (AUC) of 0.84 (95% CI (0.71, 0.97)). Survival analysis revealed that the metabolite classifier could stratify six-year survival outcomes (p = 0.0073). At the pre-transplantation stage, a panel composed of purine metabolites and ALT could improve the prediction of EGF and survival.

Determination of polyamines and related compounds in saliva via in situ derivatization and microextraction by packed sorbents coupled to GC-MS.

Here we show the determination of different polyamines (putrescine, cadaverine, spermidine) and related compounds (gamma-aminobutyric acid and l-ornithine) in saliva samples. These compounds are known to be biomarkers for several diseases. We have optimised an in situ derivatization process using ethyl chloroformate, an automated microextraction by packed sorbent and the determination of the corresponding products using a programmed temperature vaporizer coupled to a gas chromatograph - mass spectrometer. After finding that saliva matrix has an effect on the analysis, quantitation was performed using the one-point standard additions method and normalization to IS. This allows the detection of the analytes in the range of µg/L within a matrix obtained by a non-invasive procedure. The method has been successfully validated and it has been used in the determination of these compounds in six saliva samples finding that putrescine and cadaverine present the highest concentrations in the subject diagnosed with rheumatoid arthritis. For ornithine and spermidine, the highest concentrations were found for male subjects, especially heavy smokers. All concentrations found for the compounds were in good agreement with data found in bibliography.

Development of a screening and confirmatory method for the analysis of polar endogenous compounds in saliva based on a liquid chromatographic-tandem mass spectrometric system.

In this paper, a high-throughput approach is proposed for the sensitive screening and the confirmatory analysis of polar compounds in saliva using a two-step approach based on a liquid chromatographic system coupled to a triple quadrupole mass spectrometer. A reversed-phase chromatographic column was used in both steps and changes in the composition of the mobile phase allowed the screening and the confirmatory analyses to be performed with the same instrumental configuration. The proposed strategy has been tested for the determination of a multiclass group of polar endogenous compounds (creatinine, polyamines and amino acids) in saliva samples. The validation of the entire procedure showed consistent results for all the compounds in both steps. Repeatability and reproducibility were evaluated for both procedures, with values below 8% in the case of repeatability and 17% in the case of reproducibility. The instrumental limits of detection were found to be between 1.22 × 10-3 and 46.1 × 10-3 mg/L for creatinine and lysine, respectively, and accuracy of the method was evaluated in terms of apparent recoveries and values were found to be between 80 and 127%. Matrix effects were evaluated and it was found that the analytical outcome was influenced by the matrix of the sample. Thus, a one-point standard addition method was used for quantification. The optimized two-step procedure was applied to saliva samples from apparent healthy volunteers. Overall, satisfactory results were obtained in both steps, demonstrating its applicability for quantitative analysis of polar endogenous compounds in this kind of matrices.

Non-separative mass spectrometry methods for non-invasive medical diagnostics based on volatile organic compounds: A review.

In this review, an assessment of non-separative methods based on mass spectrometry used to analyse volatile organic compounds in the field of bioanalysis is performed. The use of non-separative methods based on mass spectrometry has been established as an attractive option for analysing compounds. These instrumental configurations are suitable for biomedical applications because of their versatility, rapid output of results, and the wide range of volatile organic compounds that can be determined. Here, techniques such as headspace sampling coupled to mass spectrometry, membrane introduction mass spectrometry, selected ion flow tube mass spectrometry, proton transfer reaction mass spectrometry, secondary electrospray ionization mass spectrometry and ion mobility mass spectrometry, are evaluated. Samples involving non-invasive methods of collection, such as urine, saliva, breath and sweat, are mainly considered. To the best of our knowledge, a comprehensive review of all the non-separative instrumental configurations applied to the analysis of gaseous samples from all matrices non-invasively collected has not yet been carried out. The assessment of non-separative techniques for the analysis of these type of samples can be considered a key issue for future clinical applications, as they allow real-time sample analysis, without patient suffering. Any contribution to the early diagnosis of disease can be considered a priority for the scientific community. Therefore, the identification and determination of volatile organic compounds related to particular diseases has become an important field or research.

Use of microextraction by packed sorbent directly coupled to an electron ionization single quadrupole mass spectrometer as an alternative for non-separative determinations.

Sometimes it is not necessary to separate the individual compounds of a sample to resolve an analytical problem, it is enough to obtain a signal profile of the sample formed by all the components integrating it. Within this strategy, electronic noses based on the direct coupling of a headspace sampler with a mass spectrometer (HS-MS) have been proposed. Nevertheless, this coupling is not suitable for the analysis of non-volatile compounds. In order to propose an alternative to HS-MS determinations for non-volatile compounds, here we present the first 'proof of concept' use of the direct coupling of microextraction by packed sorbents (MEPS) to a mass spectrometer device using an electron ionization (EI) and a single quadrupole as ionization source and analyzer, respectively. As target compounds, a set of analytes with different physic-chemical properties were evaluated (2-ethyl-1-hexanol, styrene, 2-heptanone, among others). The use of MEPS extraction present many advantages, such as it is fast, simple, easy to automate and requires small volumes of sample and organic solvents. Moreover, MEPS cartridges are re-usable as samples can be extracted more than 100 times using the same syringe. In order to introduce into the system all the elution volume from the MEPS extraction, a programmable temperature vaporizer (PTV) is proposed as the injector device. Results obtained with the proposed methodology (MEPS-PTV/MS) were compared with the ones obtained based on the separative scheme, i.e. using gas chromatography separation (MEPS-PTV-GC/MS), and both methods provided similar results. Limits of detection were found to be between 3.26 and 146.6µgL-1 in the non-separative scheme and between 0.02 and 1.72µgL-1 when the separative methodology was used. Repeatability and reproducibility were evaluated with values below 17% in all cases.

Use of microextraction by packed sorbents and gas chromatography-mass spectrometry for the determination of polyamines and related compounds in urine.

A novel methodology for the determination of ornithine, putrescine, cadaverine, spermidine and gamma-amino butyric acid in urine samples has been developed. The method uses in situ aqueous derivatization followed by automated microextraction by packed sorbent coupled to a gas chromatography-mass spectrometry system equipped with a programmed temperature vaporizer. This instrumental configuration minimizes sample manipulation due to from the mixing of the reagents, the process is completely automated. The analytes were derivatized using ethyl chloroformate as derivatization reagent. The reaction occurred in aqueous medium and was carried out in 1min in the vial of an autosampler used to perform microextraction by packed sorbent. The parameters affecting derivatization, extraction and separation were optimized in order to obtain maximum sensitivity. Calibration curves were obtained for five calibration levels in three different matrices. All the calibration models displayed good linearity, with R(2) values higher than 0.95. The validity of the models was checked using ANOVA, and it was observed that they did not exhibit any lack of fit. Repeatability and reproducibility was evaluated, with values below 15% in both cases. LOD and LOQ values were found to be in the low µg/L level. Influence of the matrix was confirmed, thus quantification was performed using the standard additions method and normalization to IS. The method developed was applied to the analysis of these compounds in urine samples from healthy individuals and cancer diagnosed patients (Internal Medicine Unit of the Virgen de la Vega Hospital, Salamanca, Spain). Significant differences (Mann-Whitney U test) were observed for putrescine and ornithine concentrations.

The Impact of Ischemia/Reperfusion Injury on Liver Allografts from Deceased after Cardiac Death versus Deceased after Brain Death Donors.

BACKGROUND AND AIMS: The shortage of organs for transplantation has led to increased use of organs procured from donors after cardiac death (DCD). The effects of cardiac death on the liver remain poorly understood, however. Using livers obtained from DCD versus donors after brain death (DBD), we aimed to understand how ischemia/reperfusion (I/R) injury alters expression of pro-inflammatory markers ceramides and influences graft leukocyte infiltration. METHODS: Hepatocyte inflammation, as assessed by ceramide expression, was evaluated in DCD (n = 13) and DBD (n = 10) livers. Allograft expression of inflammatory and cell death markers, and allograft leukocyte infiltration were evaluated from a contemporaneous independent cohort of DCD (n = 22) and DBD (n = 13) livers. RESULTS: When examining the differences between transplant stages in each group, C18, C20, C24 ceramides showed significant difference in DBD (p<0.05) and C22 ceramide (p<0.05) were more pronounced for DCD. C18 ceramide is correlated to bilirubin, INR, and creatinine after transplant in DCD. Prior to transplantation, DCD livers have reduced leukocyte infiltration compared to DBD allografts. Following reperfusion, the neutrophil infiltration and platelet deposition was less prevalent in DCD grafts while cell death and recipients levels of serum aspartate aminotransferase (AST) of DCD allografts had significantly increased. CONCLUSION: These data suggest that I/R injury generate necrosis in the absence of a strong inflammatory response in DCD livers with an appreciable effect on early graft function. The long-term consequences of increased inflammation in DBD and increased cell death in DCD allografts are unknown and warrant further investigation.

Adenosine monophosphate is elevated in the bronchoalveolar lavage fluid of mice with acute respiratory toxicity induced by nanoparticles with high surface hydrophobicity.

Inhaled nanomaterials present a challenge to traditional methods and understanding of respiratory toxicology. In this study, a non-targeted metabolomics approach was used to investigate relationships between nanoparticle hydrophobicity, inflammatory outcomes and the metabolic fingerprint in bronchoalveolar fluid. Measures of acute lung toxicity were assessed following single-dose intratracheal administration of nanoparticles with varying surface hydrophobicity (i.e. pegylated lipid nanocapsules, polyvinyl acetate nanoparticles and polystyrene beads; listed in order of increasing hydrophobicity). Broncho-alveolar lavage (BAL) fluid was collected from mice exposed to nanoparticles at a surface area dose of 220 cm(2) and metabolite fingerprints were acquired via ultra pressure liquid chromatography-mass spectrometry-based metabolomics. Particles with high surface hydrophobicity were pro-inflammatory. Multivariate analysis of the resultant small molecule fingerprints revealed clear discrimination between the vehicle control and polystyrene beads (p < 0.05), as well as between nanoparticles of different surface hydrophobicity (p < 0.0001). Further investigation of the metabolic fingerprints revealed that adenosine monophosphate (AMP) concentration in BAL correlated with neutrophilia (p < 0.01), CXCL1 levels (p < 0.05) and nanoparticle surface hydrophobicity (p < 0.001). Our results suggest that extracellular AMP is an intermediary metabolite involved in adenine nucleotide-regulated neutrophilic inflammation as well as tissue damage, and could potentially be used to monitor nanoparticle-induced responses in the lung following pulmonary administration.

In situ derivatization coupled to microextraction by packed sorbentand gas chromatography for the automated determination ofhaloacetic acids in chlorinated water.

A novel analytical method is reported for the determination of monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, monobromoacetic acid, and dibromoacetic acid. These are the five haloacetic acids (HAAs) for which the U.S. Environmental Protection Agency (US EPA) has regulated a maximum contamination level (MCL) of 0.060 mg L−1for the sum of their concentrations in drinking waters. Themethod uses in situ aqueous derivatization, followed by microextraction by packed sorbent (MEPS) priorto gas chromatography­mass spectrometry (GC­MS). The parameters affecting derivatization and extraction were optimized with a view to obtaining maximum sensitivity. The HAAs were derivatized with 2,2,2-trifluroethylamine (TFEA), using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) as a condensation agent. The reaction occurred in aqueous medium and was carried out in 10 min in the vial of anautosampler used to perform microextraction by packed sorbent. The whole process, from the mixing of the reagents for the derivatization, was automated. Precision values varied from 4.2 to 9.8% (as intra-day relative standard deviation, RSD) and 9.4 to 14% (as inter-day RSD). The recoveries from spiked concentrations ranged from 83 to 117%, revealing the accuracy of the method. The detection limits ranged from 0.36 to 1.2 g L−1, such that it is possible to measure the US EPA MCL in drinking waters. The method developed was applied to the analysis of HAAs in drinking and swimming pool water from Salamanca(North West of Spain).

GC-MS determination of parabens, triclosan and methyl triclosan in water by in situ derivatisation and stir-bar sorptive extraction.

Stir-bar sorptive extraction in combination with an in situ derivatisation reaction and thermal desorption-gas chromatography-mass spectrometry was successfully applied to determine parabens (methylparaben, isopropylparaben, n-propylparaben, butylparaben and benzylparaben), triclosan and methyltriclosan in water samples. This approach improves both the extraction efficiency and the sensitivity in the GC in a simple way since the derivatisation reaction occurs at the same time as the extraction procedure. The in situ derivatisation reaction was carried out with acetic anhydride under alkaline conditions. Thermal desorption parameters (cryofocusing temperature, desorption flow, desorption time, desorption temperature) were optimised using a Box-Behnken experimental design. All the analytes gave recoveries higher than 79%, except methylparaben (22%). The method afforded detection limits between 0.64 and 4.12 ng/L, with good reproducibility and accuracy values. The feasibility of the method for the determination of analytes in water samples was checked in tap water and untreated and treated wastewater.

In situ derivatization reaction and determination of ibuprofen in water samples using headspace generation-programmed temperature vaporization-gas chromatography-mass spectrometry.

The aim of the present work is to propose a method for the determination of ibuprofen, as a typical representative of pharmaceutical compounds, in aqueous samples. To do so, an in situ derivatization reaction in aqueous medium was employed in the vial of a headspace sampler (HS), after which instrumental measurements were made with gas chromatography-mass spectrometry (GC-MS). As the injection system we propose a programmed temperature vaporizer (PTV) where, in solvent vent mode, better results can be obtained than with the conventional split and splitless injection modes. Since the derivatization reaction takes place in the HS vial, after the mixing of reagents and the sealing of the vial, the whole process takes place on-line, with no need for intermediate steps. The simplicity and speed of the method--analysis throughput: 10.5 min--together with the limit of detection obtained (0.23 microg/L), bearing in mind that no preconcentration step or later clean-up step are required, make this a good method for the analysis of ibuprofen in aqueous samples of urban waste water.

Use of a programmed temperature vaporizer and an in situ derivatization reaction to improve sensitivity in headspace-gas chromatography. Application to the analysis of chlorophenols in water.

In the present work we propose the combined use of a derivatization reaction within the vial of a headspace sampler with a programmed temperature vaporizer (PTV) inlet in the solvent vent mode as a new methodology for obtaining an increase in sensitivity in headspace-gas chromatography (HS-GC) for the analysis of sparingly volatile compounds. As test analytes the following chlorophenols were used: 2-chlorophenol (2CP), 2,4-dichlorophenol (24DCP), 4-chloro-3-methylphenol (4C3MP) and 2,4,6-trichlorophenol (246TCP). The derivatization reaction was carried out with acetic anhydride because it can be carried out in situ in aqueous medium. In the programmed temperature vaporizer inlet, three different liners, one of them empty and the others with materials of different trapping strengths (glass wool and Tenax-TA), were compared. The best results were obtained when an empty liner was used, with better repeatability and S/N ratios. In the case of the liner filled with Tenax-TA, a considerable lack of repeatability was observed, this being attributed to interactions between the derivatized compounds and the adsorbent. The proposed methodology affords very low limits of detection, in the range of a few ng/L for all the compounds, with good precision and accuracy values.