Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Whole Proteome Analysis of the Protozoan Parasite Trypanosoma brucei using Stable Isotope Labeling by Amino Acids in Cell Culture and Mass Spectrometry

The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time allowed for the characterization of the proteome from several different life stages of the parasite (1-3). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC; (4)) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knock-out approaches.

Combined Secondary Ion Mass Spectrometry Depth Profiling and Focused Ion Beam Analysis of Cu Films Electrodeposited under Oscillatory Conditions

The recrystallization behavior of Cu films electrodeposited under oscillatory conditions in the presence of plating additives was studied by means of secondary ion mass spectrometry (SIMS) and focused ion beam analysis. When combined with bis-(sodium-sulfopropyl)-disulfide (SPS), Imep levelers (polymerizates of imidazole and epichlorohydrin) show characteristic oscillations in the galvanostatic potential/time transient measurements. These are related to the periodic degradation and restoration of the active leveler ensemble at the interface. The leveler action relies on adduct formation between the Imep and MPS (mercaptopropane sulfonic acid)-stabilized CuI complexes that appear as intermediates of the copper deposition when SPS is present in the electrolyte. SIMS depth profiling proves that additives are incorporated into the growing film preferentially under transient conditions during the structural breakdown of the leveler ensemble and its subsequent restoration. In contrast, Cu films electrodeposited in the presence of a structurally intact Imep–CuI–MPS ensemble remain largely contamination free.

Characterization and source apportionment of organic aerosol using offline aerosol mass spectrometry

Field deployments of the Aerodyne Aerosol Mass Spectrometer (AMS) have significantly advanced real-time measurements and source apportionment of non-refractory particulate matter. However, the cost and complex maintenance requirements of the AMS make its deployment at sufficient sites to determine regional characteristics impractical. Furthermore, the negligible transmission efficiency of the AMS inlet for supermicron particles significantly limits the characterization of their chemical nature and contributing sources. In this study, we utilize the AMS to characterize the water-soluble organic fingerprint of ambient particles collected onto conventional quartz filters, which are routinely sampled at many air quality sites. The method was applied to 256 particulate matter (PM) filter samples (PM1, PM2:5, and PM10, i.e., PM with aerodynamic diameters smaller than 1, 2.5, and 10 μm, respectively), collected at 16 urban and rural sites during summer and winter. We show that the results obtained by the present technique compare well with those from co-located online measurements, e.g., AMS or Aerosol Chemical Speciation Monitor (ACSM). The bulk recoveries of organic aerosol (60–91 %) achieved using this technique, together with low detection limits (0.8 μg of organic aerosol on the analyzed filter fraction) allow its application to environmental samples. We will discuss the recovery variability of individual hydrocarbon ions, ions containing oxygen, and other ions. The performance of such data in source apportionment is assessed in comparison to ACSM data. Recoveries of organic components related to different sources as traffic, wood burning, and secondary organic aerosol are presented. This technique, while subjected to the limitations inherent to filter-based measurements (e.g., filter artifacts and limited time resolution) may be used to enhance the AMS capabilities in measuring size-fractionated, spatially resolved longterm data sets.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Determination of cyclic and linear siloxanes in wastewater samples by ultrasound-assisted dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry

A fast, simple and environmentally friendly ultrasound-assisted dispersive liquid-liquid microextraction (USA-DLLME) procedure has been developed to preconcentrate eight cyclic and linear siloxanes from wastewater samples prior to quantification by gas chromatography-mass spectrometry (GC-MS). A two-stage multivariate optimization approach has been developed employing a Plackett-Burman design for screening and selecting the significant factors involved in the USA-DLLME procedure, which was later optimized by means of a circumscribed central composite design. The optimum conditions were: extractant solvent volume, 13 µL; solvent type, chlorobenzene; sample volume, 13 mL; centrifugation speed, 2300 rpm; centrifugation time, 5 min; and sonication time, 2 min. Under the optimized experimental conditions the method gave levels of repeatability with coefficients of variation between 10 and 24% (n=7). Limits of detection were between 0.002 and 1.4 µg L−1. Calculated calibration curves gave high levels of linearity with correlation coefficient values between 0.991 and 0.9997. Finally, the proposed method was applied for the analysis of wastewater samples. Relative recovery values ranged between 71–116% showing that the matrix had a negligible effect upon extraction. To our knowledge, this is the first time that combines LLME and GC-MS for the analysis of methylsiloxanes in wastewater samples.

Development of a novel pyrolysis-gas chromatography/mass spectrometry method for the analysis of poly(lactic acid) thermal degradation products

Thermal degradation of PLA is a complex process since it comprises many simultaneous reactions. The use of analytical techniques, such as differential scanning calorimetry (DSC) and thermogravimetry (TGA), yields useful information but a more sensitive analytical technique would be necessary to identify and quantify the PLA degradation products. In this work the thermal degradation of PLA at high temperatures was studied by using a pyrolyzer coupled to a gas chromatograph with mass spectrometry detection (Py-GC/MS). Pyrolysis conditions (temperature and time) were optimized in order to obtain an adequate chromatographic separation of the compounds formed during heating. The best resolution of chromatographic peaks was obtained by pyrolyzing the material from room temperature to 600 °C during 0.5 s. These conditions allowed identifying and quantifying the major compounds produced during the PLA thermal degradation in inert atmosphere. The strategy followed to select these operation parameters was by using sequential pyrolysis based on the adaptation of mathematical models. By application of this strategy it was demonstrated that PLA is degraded at high temperatures by following a non-linear behaviour. The application of logistic and Boltzmann models leads to good fittings to the experimental results, despite the Boltzmann model provided the best approach to calculate the time at which 50% of PLA was degraded. In conclusion, the Boltzmann method can be applied as a tool for simulating the PLA thermal degradation.

The measurement of nicotine in human plasma by high-performance liquid chromatography-electrospray-tandem mass spectrometry

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 muL) with added deuterium-labeled d(3)-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 muL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium fortriate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2 --> 130.2; IS m/z 166.2 --> 87.2). The assay was linear from 0.5 to 100 mug/L (r > 0.993, n = 9). The accuracy and imprecision of the method for quality control sampleswere 87.5% to 113% and < 10.2%, respectively. Interday accuracy and imprecision at the limit of quantification (0.5 mug/L) was 113% and 7.2% (n = 4). The process efficiency for nicotine in plasma was > 75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.

Assessment of the occurrence and potential risks of antibiotics and their metabolites in South Florida waters using liquid chromatography tandem mass spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. ^ The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can't be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications.^ Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. ^ Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.^

Online solid phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of sucralose in reclaimed and drinking waters and its photo degradation in natural waters from South Florida

Background Sucralose has gained popularity as a low calorie artificial sweetener worldwide. Due to its high stability and persistence, sucralose has shown widespread occurrence in environmental waters, at concentrations that could reach up to several μg/L. Previous studies have used time consuming sample preparation methods (offline solid phase extraction/derivatization) or methods with rather high detection limits (direct injection) for sucralose analysis. This study described a faster and sensitive analytical method for the determination of sucralose in environmental samples. Results An online SPE-LC–MS/MS method was developed, being capable to quantify sucralose in 12 minutes using only 10 mL of sample, with method detection limits (MDLs) of 4.5 ng/L, 8.5 ng/L and 45 ng/L for deionized water, drinking and reclaimed waters (1:10 diluted with deionized water), respectively. Sucralose was detected in 82% of the reclaimed water samples at concentrations reaching up to 18 μg/L. The monthly average for a period of one year was 9.1 ± 2.9 μg/L. The calculated mass loads per capita of sucralose discharged through WWTP effluents based on the concentrations detected in wastewaters in the U. S. is 5.0 mg/day/person. As expected, the concentrations observed in drinking water were much lower but still relevant reaching as high as 465 ng/L. In order to evaluate the stability of sucralose, photodegradation experiments were performed in natural waters. Significant photodegradation of sucralose was observed only in freshwater at 254 nm. Minimal degradation (<20%) was observed for all matrices under more natural conditions (350 nm or solar simulator). The only photolysis product of sucralose identified by high resolution mass spectrometry was a de-chlorinated molecule at m/z 362.0535, with molecular formula C12H20Cl2O8. Conclusions Online SPE LC-APCI/MS/MS developed in the study was applied to more than 100 environmental samples. Sucralose was frequently detected (>80%) indicating that the conventional treatment process employed in the sewage treatment plants is not efficient for its removal. Detection of sucralose in drinking waters suggests potential contamination of surface and ground waters sources with anthropogenic wastewater streams. Its high resistance to photodegradation, minimal sorption and high solubility indicate that sucralose could be a good tracer of anthropogenic wastewater intrusion into the environment.

Improved dynamic headspace sampling and detection using capillary microextraction of volatiles coupled to gas chromatography mass spectrometry

Sampling and preconcentration techniques play a critical role in headspace analysis in analytical chemistry. My dissertation presents a novel sampling design, capillary microextraction of volatiles (CMV), that improves the preconcentration of volatiles and semivolatiles in a headspace with high throughput, near quantitative analysis, high recovery and unambiguous identification of compounds when coupled to mass spectrometry. The CMV devices use sol-gel polydimethylsiloxane (PDMS) coated microglass fibers as the sampling/preconcentration sorbent when these fibers are stacked into open-ended capillary tubes. The design allows for dynamic headspace sampling by connecting the device to a hand-held vacuum pump. The inexpensive device can be fitted into a thermal desorption probe for thermal desorption of the extracted volatile compounds into a gas chromatography-mass spectrometer (GC-MS). The performance of the CMV devices was compared with two other existing preconcentration techniques, solid phase microextraction (SPME) and planar solid phase microextraction (PSPME). Compared to SPME fibers, the CMV devices have an improved surface area and phase volume of 5000 times and 80 times, respectively. One (1) minute dynamic CMV air sampling resulted in similar performance as a 30 min static extraction using a SPME fiber. The PSPME devices have been fashioned to easily interface with ion mobility spectrometers (IMS) for explosives or drugs detection. The CMV devices are shown to offer dynamic sampling and can now be coupled to COTS GC-MS instruments. Several compound classes representing explosives have been analyzed with minimum breakthrough even after a 60 min. sampling time. The extracted volatile compounds were retained in the CMV devices when preserved in aluminum foils after sampling. Finally, the CMV sampling device were used for several different headspace profiling applications which involved sampling a shipping facility, six illicit drugs, seven military explosives and eighteen different bacteria strains. Successful detection of the target analytes at ng levels of the target signature volatile compounds in these applications suggests that the CMV devices can provide high throughput qualitative and quantitative analysis with high recovery and unambiguous identification of analytes.