Mass spectroscopy with two different ionization techniques, electrospray ionization and atmospher... more Mass spectroscopy with two different ionization techniques, electrospray ionization and atmospheric pressure chemical ionization, optimized for cocaine, was applied to study 170 molecules. For each of the molecules, 429 molecular descriptors were calculated. The aim of this work is, on one hand, to determine whether it is possible to identify which is the better ionization technique, electrospray ionization or atmospheric pressure chemical ionization, to analyse a given molecule, based on the calculated descriptors, and on the other hand to correlate the response of each of the techniques individually with these descriptors. For each molecule, the ratio between the corrected responses of electrospray ionization and atmospheric pressure chemical ionization was used as criterion to decide which ionization technique was the better for the analysis. The chemometrical methods used to explore and model the data were Principal Component Analysis, Projection Pursuit, Classification and Regression Trees, Partial Least Squares and Stepwise Multiple Linear Regression.
Methods in molecular biology (Clifton, N.J.), 2008
This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-r... more This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry for the single-step desalting, and separation, as well as characterization of oligonucleotides in the framework of quality control after synthesis. Separation is performed using a 25 mM ammonium carbonate buffer supplemented with 0.2 mM trans-1,2-diaminocyclohexane-N, N, N', N' id (CDTA) (pH 9.7). During the electrophoretic process, sodium and potassium ions are removed from the polyanionic backbone of the oligonucleotides by exchange of these ions with ammonium ions or by chelation on CDTA, thus eliminating a sample preparation step. A sample stacking procedure used to concentrate the samples on the CZE capillary is described. After analysis, the obtained spectrum is deconvoluted to the zero charge spectrum to yield the molecular mass of the oligonucleotide. A misincorporation of one nucleotide can be ...
Journal of Chromatography B: Biomedical Sciences and Applications, 1997
The potentials and limitations of high-performance liquid chromatography-photodiode array detecti... more The potentials and limitations of high-performance liquid chromatography-photodiode array detection are highlighted in respect to its use in the analysis of different biological matrices followed by the identification of unknowns. The logical analytical approach used in clinical and forensic toxicology, vital for the identification of one or more toxic substances as a cause of intoxication, is largely based on both simple and fast "general unknown screening" methods which cover most relevant drugs and potentially hazardous chemicals. In this field of systematic toxicological analysis, a literature overview shows that HPLC can play a substantial role. Both column packing material and eluent composition have their impact on intra- and interlaboratory reproducibility. In view of the sometimes different retention characteristics of various HPLC columns, several possibilities are addressed to enhance the discriminating power of primary retention parameters. The advantages of photodiode array detection as compared to UV detection have been of paramount importance to the success of HPLC in toxicological analysis. Dedicated libraries with spectral information and searching software are powerful tools in the process of identification of an unknown substance. In the present paper, these aspects are also verified in a number of real cases, i.e., trazodone and dothiepin, azide, chloroquine and cocaine, in which we illustrate from our own experience the potentials of HPLC-photodiode array detection in systematic toxicological analysis.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2006
An exploratory analysis was performed in order to evaluate the feasibility of building of neural ... more An exploratory analysis was performed in order to evaluate the feasibility of building of neural network (NN) systems automating the identification of amphetamines necessary in the investigation of drugs of abuse for epidemiological, clinical and forensic purposes. A first neural network system was built to distinguish between amphetamines and nonamphetamines. A second, more refined system, aimed to the recognition of amphetamines according to their toxicological activity (stimulant amphetamines, hallucinogenic amphetamines, nonamphetamines). Both systems proved that discrimination between amphetamines and nonamphetamines, as well as between stimulants, hallucinogens and nonamphetamines is possible (83.44% and 85.71% correct classification rate, respectively). The spectroscopic interpretation of the 40 most important input variables (GC-FTIR absorption intensities) shows that the modeling power of an input variable seems to be correlated with the stability and not with the intensity of the spectral interaction. Thus, discarding variables only because they correspond to spectral windows with weak absorptions does not seem be not advisable.
The work presented here deals with the development of a quantitative tool for the simultaneous de... more The work presented here deals with the development of a quantitative tool for the simultaneous determination of sulfamethoxypyrazine (sulfalene)/pyrimethamine in plasma. The chromatography used only takes 12.5 min, allowing a fast sample turnover time. Relative standard deviation of retention times was never above 3.48% (n = 66). Adequate sample clean-up was achieved by a simple and relatively fast liquid/liquid extraction. In this way, ionisation suppression effects, typical for more simple sample clean-up procedures, could be avoided resulting in absolute plasma effects of maximum -17.1% for sulfalene, -16.1 for the internal standard (IS), and 12% for pyrimethamine. For both pyrimethamine and sulfalene, quadratic calibration curves from 0.00101 to 0.807 microg/mL for pyrimethamine and from 0.271 to 216 microg/mL for sulfalene gave the best fit. Mean coefficients of determination (R2) were 0.9951 (n = 6, CV% 0.39) for pyrimethamine and 0.9942 (n = 6, CV% 0.13) for sulfalene. Precision was below 9.35% for pyrimethamine and 13.9% for sulfalene. Inaccuracy remained below 15% at all cases. The optimised method was used for a time-course study of the sulfalene/pyrimethamine combination concentration in plasma of patients treated with Co-Arinate, a new curative antimalaria-medicine.
... Mortier, KA, Clauwaert, KM, Lambert, WE, Van Bocxlaer, JF, Van den Eeckhout, EG, Van Peteghem... more ... Mortier, KA, Clauwaert, KM, Lambert, WE, Van Bocxlaer, JF, Van den Eeckhout, EG, Van Peteghem, CH and De Leenheer, AP (2001), Pitfalls associated with liquid chromatography/ electrospray tandem mass spectrometry in quantitative bioanalysis of drugs of abuse in saliva. ...
A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) ... more A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.
This paper describes the investigation of the potential of a quadrupole orthogonal acceleration t... more This paper describes the investigation of the potential of a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF) equipped with an atmospheric pressure ionisation interface for quantitative measurements of small molecules separated by reversed phase liquid ...
This article describes a simple method to perform lock mass corrected accurate mass measurements ... more This article describes a simple method to perform lock mass corrected accurate mass measurements in tandem mass spectrometry (MS/MS) with a quadrupole time-of-flight (Q-TOF) mass spectrometer. The experimental approach consists of using the protonated molecule of a known compound, which is measured in a MS/MS function using low collision energy (no fragmentation), as mass calibrator. The unknown compound is acquired in MS/MS mode albeit using high collision energy. After the acquisition, the two MS/MS spectra of unknown and mass calibrator are combined, and the fragments of the unknown are lock mass corrected by using the protonated molecule of the mass calibrator. To prove this concept, 10 compounds were analyzed using this approach, the fragments interpreted and, where possible, related to structural data available in the literature. All the unequivocally assigned fragments were accurately mass measured with mass errors within appropriate limits, i.e. for m/z values <200 with a mass tolerance of 3 mDa while for m/z > 200 the mass tolerance is expressed as 10 ppm.
Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light... more Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light irradiation in the presence of flavins. The sulfur moiety seems pivotal for interaction, as was determined from kinetic analyses using laser flash photolysis spectroscopy. After photooxidation, the resulting radical intermediates were characterized by addition to a spin trap, followed by electron paramagnetic resonance spectroscopy and evaluation of the coupling constants. The presence of the proposed radical intermediates was strongly supported by the identification of the reaction products using mass spectrometry. Accordingly, feasible degradation pathways for various sulfur-containing amino acids and derivatives were proposed. It was finally proven that flavin-induced photoproduction of sulfhydryl radicals and recombination with a 3-methylbut-2-enyl radical, derived from the photodegradation of hop-derived isohumulones, are decisive in the formation of beer lightstruck flavor.
Isohumulones, the main bittering agents in beer, are decomposed by light-induced reactions, there... more Isohumulones, the main bittering agents in beer, are decomposed by light-induced reactions, thereby leading to radical precursors on the pathway to lightstruck flavour formation. Excited flavins, formed on visible-light irradiation, readily interact with isohumulones, as well as with reduced and oxidized derivatives thereof. From identification of both volatile and non-volatile reaction products thus formed, feasible degradation mechanisms are proposed.
Opioid drugs, including the newly developed peptides, should penetrate the blood-brain barrier (B... more Opioid drugs, including the newly developed peptides, should penetrate the blood-brain barrier (BBB) for pain management activity. Although BBB transport is fragmentarily described for some mu-opioid peptides, a complete and comparative overview is currently lacking. In this study, the BBB transport of eight opioid peptides (EM-1, EM-2, CTAP, CTOP, DAMGO, dermorphin, TAPP and TAPS) is described and compared. In addition, the metabolic stability in plasma and brain was evaluated. The highest influx rate was obtained for dermorphin (K(in)=2.18 microl/(g x min)), followed by smaller rates for EM-1, EM-2 and TAPP (K(in)=1.06-1.14 microl/(g x min)). Negligible influx was observed for DAMGO, CTOP and TAPS (K(in)=0.18-0.40 microl/(g x min)) and no influx for CTAP. Capillary depletion revealed that all peptides reached brain parenchyma for over 75%. Efflux was shown for TAPP (t(1/2)=2.82 min) and to a lesser extent for EM-1, EM-2 and DAMGO (t(1/2)=10.66-21.98 min), while no significant efflux was observed for the other peptides. All peptides were stable in mouse plasma and brain, with generally higher stability in brain, except for EM-1 and EM-2 which showed plasma half-life stabilities of a few minutes only.
Liquid chromatography-mass spectrometry has evolved from a topic of mainly research interest into... more Liquid chromatography-mass spectrometry has evolved from a topic of mainly research interest into a routinely usable tool in various application fields. With the advent of new ionization approaches, especially atmospheric pressure, the technique has established itself firmly in many areas of research. Although many applications prove that LC-MS is a valuable complementary analytical tool to GC-MS and has the potential to largely extend the application field of mass spectrometry to hitherto "MS-phobic" molecules, we must recognize that the use of LC-MS in forensic toxicology remains relatively rare. This rarity is all the more surprising because forensic toxicologists find themselves often confronted with the daunting task of actually searching for evidence materials on a scientific basis without any indication of the direction in which to search. Through the years, mass spectrometry, mainly in the GC-MS form, has gained a leading role in the way such quandaries are tackled. The advent of robust, bioanalytically compatible combinations of liquid chromatographic separation with mass spectrometric detection really opens new perspectives in terms of mass spectrometric identification of difficult molecules (e.g., polar metabolites) or biopolymers with toxicological relevance, high throughput, and versatility. Of course, analytical toxicologists are generally mass spectrometry users rather than mass spectrometrists, and this difference certainly explains the slow start of LC-MS in this field. Nevertheless, some valuable applications have been published, and it seems that the introduction of the more universal atmospheric pressure ionization interfaces really has boosted interests. This review presents an overview of what has been realized in forensic toxicological LC-MS. After a short introduction into LC-MS interfacing operational characteristics (or limitations), it covers applications that range from illicit drugs to often abused prescription medicines and some natural poisons. As such, we hope it can act as an appetizer to those involved in forensic toxicology but still hesitating to invest in LC-MS.
This paper describes the surplus value of a quadrupole-orthogonal acceleration TOF mass spectrome... more This paper describes the surplus value of a quadrupole-orthogonal acceleration TOF mass spectrometer, coupled to a liquid chromatographic separation system, for the unequivocal identification and structural elucidation of an unknown compound in the field of designer drugs. In a patient sample set (blood, tissues, vitreous humor, etc.), analyzed with a dedicated liquid chromatographic-fluorescence detection method for the determination of methylenedioxy amphetamine, methylenedioxy methamphetamine, and methylenedioxy ethylamphetamine (MDEA), a "strange" inexplicable peak appeared at a retention time not corresponding to any of our reference materials. Based on the identical excitation and emission wavelengths in detection, and a retention behavior comparable to MDEA, it was assumed that this unknown compound was an isomer of the recreational drug MDEA. With a simple and straightforward methodological crossover between LC fluorescence detection and LC-MS/MS, additional information for structural elucidation was easily obtained. Chromatographic separation was achieved on a Hypersil BDS C18 column (fluorescence detection part) and on a Hypersil BDS phenyl column (mass spectrometric detection part). MS showed that the unknown compound's molecular mass was identical to that of MDEA, and, in addition, its fragmentation pattern too proved quite similar to that of MDEA. A thorough literature overview and study of the fragmentation pattern by means of the MS/MS spectrum led to an evidence-based hypothesis of 3,4-methylenedioxy N,N-dimethylamphetamine (MDDM) being the unknown compound. To confirm this hypothesis, MDDM was synthesized and its presence in our biological sample was finally demonstrated by co-injection with alternatively synthesized MDDM and MDEA. This application shows the synergism between LC and MS in the elucidation of unknown compounds, nevertheless emphasizing the essence of chromatographic separation when dealing with isomers.
Fentanyl, norfentanyl, alfentanil, sufentanil, remifentanil and 3-methylfentanyl are potent, shor... more Fentanyl, norfentanyl, alfentanil, sufentanil, remifentanil and 3-methylfentanyl are potent, short-acting, synthetic narcotic analgesics that are not revealed in standard opiate immunoassays. In this article, a fully validated analytical method for the determination of these fentanyl-type compounds in plasma and urine is presented, consisting of a liquidliquid extraction followed by a LC-MS/MS analysis using electrospray ionisation in the positive ionisation mode. Fentanyl-d 5 and norfentanyl-d 5 were used as internal standards. The lower LOQ in plasma and urine was 0.1 ng/mL for fentanyl, norfentanyl, alfentanil, remifentanil and 3-methylfentanyl, and 0.2 ng/mL for sufentanil. The method proved linear over a concentration range of 0.2-50 ng/mL for sufentanil and 0.1-50 ng/mL for all other analytes, with correlation coefficients of 0.998 or better. The analytical procedure showed excellent selectivity and precision (all CVs below 15%) for all analytes. Accuracy was good, except for sufentanil, where deviations of more than 15% from nominal concentrations were observed. No matrix effects were observed, and stability of stock and internal standard solutions was within acceptability limits.
A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cereb... more A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cerebrospinal fluid (CSF). On column focusing on a C18 trapping column, in-line with the analytical column, was used for preconcentration. Quantification was performed with a triple quadrupole instrument in the multiple reaction monitoring mode. Weighted linear regression analysis proves to be a good linearity in a dynamic range of two orders of magnitude. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9914. Assay precision and accuracy were evaluated by direct injection of enkephalin fortified artificial CSF (aCSF) samples at three concentration levels. Mean accuracy of analysed concentrations was between 97.63 and 107.6%. LOD and LOQ were assessed at, respectively, 0.5 and 1 pmol/mL. Validation results show that it is feasible, with a capillary LC-MS/MS system, to quantify neuropeptides in the low femtomole range in aCSF. The obtained coefficients of variation, however, indicate that the use of appropriate isotopically labelled internal standards in neuropeptide quantification using narrow bore LC, combined with ESI-MS, may be highly beneficial.
A hydrophilic interaction LC method with MS/MS was developed and validated for the simultaneous q... more A hydrophilic interaction LC method with MS/MS was developed and validated for the simultaneous quantification of omeprazole and lansoprazole in human plasma. Chromatographic separation was achieved on a Betasil silica column using a high organic mobile phase (eluent A: ACN/formic acid 997.5:2.5 v/v; eluent B: water/formic acid 997.5:2.5 v/v) and gradient elution. The mass spectrometer was operated in the Multiple Reaction Monitoring mode. Prior to chromatography, liquid-liquid extraction with ethyl acetate was used and the organic layer was diluted with ACN, allowing direct injection on column. The method showed acceptable linearity, high precision (RSD%<10.5%), accuracy (88.9-109.3%) and selectivity in the two concentration ranges studied: 1.5-100 and 5-2000 ng/mL. The LOQ was established at 1.5 and 5 ng/mL for the two concentration ranges. Lack of variability in matrix effects was demonstrated and mean extraction recovery for omeprazole and lansoprazole was determined in the low (56.3-67.7%) and high (45.3-44.3%) concentration range, respectively. Additionally, plasma samples were found to be stable after three freeze-thaw cycles and for at least 15 h after extraction. This assay was successfully applied to a pharmacokinetic omeprazole study in children with cerebral palsy and mental retardation.
In metabolomics, major efforts are invested in the development of suitable analytical approaches.... more In metabolomics, major efforts are invested in the development of suitable analytical approaches. A tendency towards the use of LC-MS is nowadays very obvious. A great majority of the metabolites of interest are polar to highly polar in nature. We focus on the so-called 'extended polarity' reversed LC phases, developed specifically to allow better retention characteristics for polar compounds. Several of these phases (Atlantis dC18, Inertsil ODS-3, Zorbax XDB, Alltima HP C18) are tested for different column dimension variations (0.5, 1.0, 2.1 mm id) in a specific LC-MS metabolomics setting. Important chromatographic and mass spectrometric quality parameters such as capacity factor, separation efficiency, peak symmetry, sensitivity, and mass accuracy are taken into account. All phases show adequate retention of polar compounds and also perform well with highly aqueous mobile phase compositions. On comparing 1.0 and 2.1 mm id columns, it is clear that the potential gain in sensitivity is not achieved. Using a Lockspray device, accurate mass measurement with a Q-TOF micro is feasible within a mass range of 12 ppm if signal intensities of compound and lockmass are equated. Finally, the extended polarity RP approach in metabolomics experiments is endorsed using real plant extracts.
The use of marker-peptides, measured by LC-MS/MS, is investigated for the quantitative analysis o... more The use of marker-peptides, measured by LC-MS/MS, is investigated for the quantitative analysis of proteins. To that end, cystatin C is chosen as a model protein. It not only functions as a proof of concept protein but the growing interest in cystatin C as a new marker of kidney failure provides a practical application at the same time. The use of trypsin-based proteolysis, to obtain so-called marker-peptides, simplifies the quantification of a protein to the quantification of a single or a number of peptides. Reproducibility of the trypsin proteolysis procedure is vital and has been optimised. A number of the marker-peptides obtained are selected for LC-MS(/MS) analysis. They are completely separated by high-pressure LC allowing maximum selectivity and mass spectrometric multiple reaction monitoring sensitivity. By doing so, linear calibration curves can be obtained for cystatin C over two orders of magnitude. Experiments have been performed on a triple quadrupole mass spectrometer by single ion monitoring (maximum sensitivity) as well as by multiple reaction monitoring (maximum specificity).
Mass spectroscopy with two different ionization techniques, electrospray ionization and atmospher... more Mass spectroscopy with two different ionization techniques, electrospray ionization and atmospheric pressure chemical ionization, optimized for cocaine, was applied to study 170 molecules. For each of the molecules, 429 molecular descriptors were calculated. The aim of this work is, on one hand, to determine whether it is possible to identify which is the better ionization technique, electrospray ionization or atmospheric pressure chemical ionization, to analyse a given molecule, based on the calculated descriptors, and on the other hand to correlate the response of each of the techniques individually with these descriptors. For each molecule, the ratio between the corrected responses of electrospray ionization and atmospheric pressure chemical ionization was used as criterion to decide which ionization technique was the better for the analysis. The chemometrical methods used to explore and model the data were Principal Component Analysis, Projection Pursuit, Classification and Regression Trees, Partial Least Squares and Stepwise Multiple Linear Regression.
Methods in molecular biology (Clifton, N.J.), 2008
This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-r... more This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry for the single-step desalting, and separation, as well as characterization of oligonucleotides in the framework of quality control after synthesis. Separation is performed using a 25 mM ammonium carbonate buffer supplemented with 0.2 mM trans-1,2-diaminocyclohexane-N, N, N', N' id (CDTA) (pH 9.7). During the electrophoretic process, sodium and potassium ions are removed from the polyanionic backbone of the oligonucleotides by exchange of these ions with ammonium ions or by chelation on CDTA, thus eliminating a sample preparation step. A sample stacking procedure used to concentrate the samples on the CZE capillary is described. After analysis, the obtained spectrum is deconvoluted to the zero charge spectrum to yield the molecular mass of the oligonucleotide. A misincorporation of one nucleotide can be ...
Journal of Chromatography B: Biomedical Sciences and Applications, 1997
The potentials and limitations of high-performance liquid chromatography-photodiode array detecti... more The potentials and limitations of high-performance liquid chromatography-photodiode array detection are highlighted in respect to its use in the analysis of different biological matrices followed by the identification of unknowns. The logical analytical approach used in clinical and forensic toxicology, vital for the identification of one or more toxic substances as a cause of intoxication, is largely based on both simple and fast "general unknown screening" methods which cover most relevant drugs and potentially hazardous chemicals. In this field of systematic toxicological analysis, a literature overview shows that HPLC can play a substantial role. Both column packing material and eluent composition have their impact on intra- and interlaboratory reproducibility. In view of the sometimes different retention characteristics of various HPLC columns, several possibilities are addressed to enhance the discriminating power of primary retention parameters. The advantages of photodiode array detection as compared to UV detection have been of paramount importance to the success of HPLC in toxicological analysis. Dedicated libraries with spectral information and searching software are powerful tools in the process of identification of an unknown substance. In the present paper, these aspects are also verified in a number of real cases, i.e., trazodone and dothiepin, azide, chloroquine and cocaine, in which we illustrate from our own experience the potentials of HPLC-photodiode array detection in systematic toxicological analysis.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2006
An exploratory analysis was performed in order to evaluate the feasibility of building of neural ... more An exploratory analysis was performed in order to evaluate the feasibility of building of neural network (NN) systems automating the identification of amphetamines necessary in the investigation of drugs of abuse for epidemiological, clinical and forensic purposes. A first neural network system was built to distinguish between amphetamines and nonamphetamines. A second, more refined system, aimed to the recognition of amphetamines according to their toxicological activity (stimulant amphetamines, hallucinogenic amphetamines, nonamphetamines). Both systems proved that discrimination between amphetamines and nonamphetamines, as well as between stimulants, hallucinogens and nonamphetamines is possible (83.44% and 85.71% correct classification rate, respectively). The spectroscopic interpretation of the 40 most important input variables (GC-FTIR absorption intensities) shows that the modeling power of an input variable seems to be correlated with the stability and not with the intensity of the spectral interaction. Thus, discarding variables only because they correspond to spectral windows with weak absorptions does not seem be not advisable.
The work presented here deals with the development of a quantitative tool for the simultaneous de... more The work presented here deals with the development of a quantitative tool for the simultaneous determination of sulfamethoxypyrazine (sulfalene)/pyrimethamine in plasma. The chromatography used only takes 12.5 min, allowing a fast sample turnover time. Relative standard deviation of retention times was never above 3.48% (n = 66). Adequate sample clean-up was achieved by a simple and relatively fast liquid/liquid extraction. In this way, ionisation suppression effects, typical for more simple sample clean-up procedures, could be avoided resulting in absolute plasma effects of maximum -17.1% for sulfalene, -16.1 for the internal standard (IS), and 12% for pyrimethamine. For both pyrimethamine and sulfalene, quadratic calibration curves from 0.00101 to 0.807 microg/mL for pyrimethamine and from 0.271 to 216 microg/mL for sulfalene gave the best fit. Mean coefficients of determination (R2) were 0.9951 (n = 6, CV% 0.39) for pyrimethamine and 0.9942 (n = 6, CV% 0.13) for sulfalene. Precision was below 9.35% for pyrimethamine and 13.9% for sulfalene. Inaccuracy remained below 15% at all cases. The optimised method was used for a time-course study of the sulfalene/pyrimethamine combination concentration in plasma of patients treated with Co-Arinate, a new curative antimalaria-medicine.
... Mortier, KA, Clauwaert, KM, Lambert, WE, Van Bocxlaer, JF, Van den Eeckhout, EG, Van Peteghem... more ... Mortier, KA, Clauwaert, KM, Lambert, WE, Van Bocxlaer, JF, Van den Eeckhout, EG, Van Peteghem, CH and De Leenheer, AP (2001), Pitfalls associated with liquid chromatography/ electrospray tandem mass spectrometry in quantitative bioanalysis of drugs of abuse in saliva. ...
A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) ... more A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.
This paper describes the investigation of the potential of a quadrupole orthogonal acceleration t... more This paper describes the investigation of the potential of a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF) equipped with an atmospheric pressure ionisation interface for quantitative measurements of small molecules separated by reversed phase liquid ...
This article describes a simple method to perform lock mass corrected accurate mass measurements ... more This article describes a simple method to perform lock mass corrected accurate mass measurements in tandem mass spectrometry (MS/MS) with a quadrupole time-of-flight (Q-TOF) mass spectrometer. The experimental approach consists of using the protonated molecule of a known compound, which is measured in a MS/MS function using low collision energy (no fragmentation), as mass calibrator. The unknown compound is acquired in MS/MS mode albeit using high collision energy. After the acquisition, the two MS/MS spectra of unknown and mass calibrator are combined, and the fragments of the unknown are lock mass corrected by using the protonated molecule of the mass calibrator. To prove this concept, 10 compounds were analyzed using this approach, the fragments interpreted and, where possible, related to structural data available in the literature. All the unequivocally assigned fragments were accurately mass measured with mass errors within appropriate limits, i.e. for m/z values <200 with a mass tolerance of 3 mDa while for m/z > 200 the mass tolerance is expressed as 10 ppm.
Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light... more Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light irradiation in the presence of flavins. The sulfur moiety seems pivotal for interaction, as was determined from kinetic analyses using laser flash photolysis spectroscopy. After photooxidation, the resulting radical intermediates were characterized by addition to a spin trap, followed by electron paramagnetic resonance spectroscopy and evaluation of the coupling constants. The presence of the proposed radical intermediates was strongly supported by the identification of the reaction products using mass spectrometry. Accordingly, feasible degradation pathways for various sulfur-containing amino acids and derivatives were proposed. It was finally proven that flavin-induced photoproduction of sulfhydryl radicals and recombination with a 3-methylbut-2-enyl radical, derived from the photodegradation of hop-derived isohumulones, are decisive in the formation of beer lightstruck flavor.
Isohumulones, the main bittering agents in beer, are decomposed by light-induced reactions, there... more Isohumulones, the main bittering agents in beer, are decomposed by light-induced reactions, thereby leading to radical precursors on the pathway to lightstruck flavour formation. Excited flavins, formed on visible-light irradiation, readily interact with isohumulones, as well as with reduced and oxidized derivatives thereof. From identification of both volatile and non-volatile reaction products thus formed, feasible degradation mechanisms are proposed.
Opioid drugs, including the newly developed peptides, should penetrate the blood-brain barrier (B... more Opioid drugs, including the newly developed peptides, should penetrate the blood-brain barrier (BBB) for pain management activity. Although BBB transport is fragmentarily described for some mu-opioid peptides, a complete and comparative overview is currently lacking. In this study, the BBB transport of eight opioid peptides (EM-1, EM-2, CTAP, CTOP, DAMGO, dermorphin, TAPP and TAPS) is described and compared. In addition, the metabolic stability in plasma and brain was evaluated. The highest influx rate was obtained for dermorphin (K(in)=2.18 microl/(g x min)), followed by smaller rates for EM-1, EM-2 and TAPP (K(in)=1.06-1.14 microl/(g x min)). Negligible influx was observed for DAMGO, CTOP and TAPS (K(in)=0.18-0.40 microl/(g x min)) and no influx for CTAP. Capillary depletion revealed that all peptides reached brain parenchyma for over 75%. Efflux was shown for TAPP (t(1/2)=2.82 min) and to a lesser extent for EM-1, EM-2 and DAMGO (t(1/2)=10.66-21.98 min), while no significant efflux was observed for the other peptides. All peptides were stable in mouse plasma and brain, with generally higher stability in brain, except for EM-1 and EM-2 which showed plasma half-life stabilities of a few minutes only.
Liquid chromatography-mass spectrometry has evolved from a topic of mainly research interest into... more Liquid chromatography-mass spectrometry has evolved from a topic of mainly research interest into a routinely usable tool in various application fields. With the advent of new ionization approaches, especially atmospheric pressure, the technique has established itself firmly in many areas of research. Although many applications prove that LC-MS is a valuable complementary analytical tool to GC-MS and has the potential to largely extend the application field of mass spectrometry to hitherto "MS-phobic" molecules, we must recognize that the use of LC-MS in forensic toxicology remains relatively rare. This rarity is all the more surprising because forensic toxicologists find themselves often confronted with the daunting task of actually searching for evidence materials on a scientific basis without any indication of the direction in which to search. Through the years, mass spectrometry, mainly in the GC-MS form, has gained a leading role in the way such quandaries are tackled. The advent of robust, bioanalytically compatible combinations of liquid chromatographic separation with mass spectrometric detection really opens new perspectives in terms of mass spectrometric identification of difficult molecules (e.g., polar metabolites) or biopolymers with toxicological relevance, high throughput, and versatility. Of course, analytical toxicologists are generally mass spectrometry users rather than mass spectrometrists, and this difference certainly explains the slow start of LC-MS in this field. Nevertheless, some valuable applications have been published, and it seems that the introduction of the more universal atmospheric pressure ionization interfaces really has boosted interests. This review presents an overview of what has been realized in forensic toxicological LC-MS. After a short introduction into LC-MS interfacing operational characteristics (or limitations), it covers applications that range from illicit drugs to often abused prescription medicines and some natural poisons. As such, we hope it can act as an appetizer to those involved in forensic toxicology but still hesitating to invest in LC-MS.
This paper describes the surplus value of a quadrupole-orthogonal acceleration TOF mass spectrome... more This paper describes the surplus value of a quadrupole-orthogonal acceleration TOF mass spectrometer, coupled to a liquid chromatographic separation system, for the unequivocal identification and structural elucidation of an unknown compound in the field of designer drugs. In a patient sample set (blood, tissues, vitreous humor, etc.), analyzed with a dedicated liquid chromatographic-fluorescence detection method for the determination of methylenedioxy amphetamine, methylenedioxy methamphetamine, and methylenedioxy ethylamphetamine (MDEA), a "strange" inexplicable peak appeared at a retention time not corresponding to any of our reference materials. Based on the identical excitation and emission wavelengths in detection, and a retention behavior comparable to MDEA, it was assumed that this unknown compound was an isomer of the recreational drug MDEA. With a simple and straightforward methodological crossover between LC fluorescence detection and LC-MS/MS, additional information for structural elucidation was easily obtained. Chromatographic separation was achieved on a Hypersil BDS C18 column (fluorescence detection part) and on a Hypersil BDS phenyl column (mass spectrometric detection part). MS showed that the unknown compound's molecular mass was identical to that of MDEA, and, in addition, its fragmentation pattern too proved quite similar to that of MDEA. A thorough literature overview and study of the fragmentation pattern by means of the MS/MS spectrum led to an evidence-based hypothesis of 3,4-methylenedioxy N,N-dimethylamphetamine (MDDM) being the unknown compound. To confirm this hypothesis, MDDM was synthesized and its presence in our biological sample was finally demonstrated by co-injection with alternatively synthesized MDDM and MDEA. This application shows the synergism between LC and MS in the elucidation of unknown compounds, nevertheless emphasizing the essence of chromatographic separation when dealing with isomers.
Fentanyl, norfentanyl, alfentanil, sufentanil, remifentanil and 3-methylfentanyl are potent, shor... more Fentanyl, norfentanyl, alfentanil, sufentanil, remifentanil and 3-methylfentanyl are potent, short-acting, synthetic narcotic analgesics that are not revealed in standard opiate immunoassays. In this article, a fully validated analytical method for the determination of these fentanyl-type compounds in plasma and urine is presented, consisting of a liquidliquid extraction followed by a LC-MS/MS analysis using electrospray ionisation in the positive ionisation mode. Fentanyl-d 5 and norfentanyl-d 5 were used as internal standards. The lower LOQ in plasma and urine was 0.1 ng/mL for fentanyl, norfentanyl, alfentanil, remifentanil and 3-methylfentanyl, and 0.2 ng/mL for sufentanil. The method proved linear over a concentration range of 0.2-50 ng/mL for sufentanil and 0.1-50 ng/mL for all other analytes, with correlation coefficients of 0.998 or better. The analytical procedure showed excellent selectivity and precision (all CVs below 15%) for all analytes. Accuracy was good, except for sufentanil, where deviations of more than 15% from nominal concentrations were observed. No matrix effects were observed, and stability of stock and internal standard solutions was within acceptability limits.
A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cereb... more A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cerebrospinal fluid (CSF). On column focusing on a C18 trapping column, in-line with the analytical column, was used for preconcentration. Quantification was performed with a triple quadrupole instrument in the multiple reaction monitoring mode. Weighted linear regression analysis proves to be a good linearity in a dynamic range of two orders of magnitude. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9914. Assay precision and accuracy were evaluated by direct injection of enkephalin fortified artificial CSF (aCSF) samples at three concentration levels. Mean accuracy of analysed concentrations was between 97.63 and 107.6%. LOD and LOQ were assessed at, respectively, 0.5 and 1 pmol/mL. Validation results show that it is feasible, with a capillary LC-MS/MS system, to quantify neuropeptides in the low femtomole range in aCSF. The obtained coefficients of variation, however, indicate that the use of appropriate isotopically labelled internal standards in neuropeptide quantification using narrow bore LC, combined with ESI-MS, may be highly beneficial.
A hydrophilic interaction LC method with MS/MS was developed and validated for the simultaneous q... more A hydrophilic interaction LC method with MS/MS was developed and validated for the simultaneous quantification of omeprazole and lansoprazole in human plasma. Chromatographic separation was achieved on a Betasil silica column using a high organic mobile phase (eluent A: ACN/formic acid 997.5:2.5 v/v; eluent B: water/formic acid 997.5:2.5 v/v) and gradient elution. The mass spectrometer was operated in the Multiple Reaction Monitoring mode. Prior to chromatography, liquid-liquid extraction with ethyl acetate was used and the organic layer was diluted with ACN, allowing direct injection on column. The method showed acceptable linearity, high precision (RSD%<10.5%), accuracy (88.9-109.3%) and selectivity in the two concentration ranges studied: 1.5-100 and 5-2000 ng/mL. The LOQ was established at 1.5 and 5 ng/mL for the two concentration ranges. Lack of variability in matrix effects was demonstrated and mean extraction recovery for omeprazole and lansoprazole was determined in the low (56.3-67.7%) and high (45.3-44.3%) concentration range, respectively. Additionally, plasma samples were found to be stable after three freeze-thaw cycles and for at least 15 h after extraction. This assay was successfully applied to a pharmacokinetic omeprazole study in children with cerebral palsy and mental retardation.
In metabolomics, major efforts are invested in the development of suitable analytical approaches.... more In metabolomics, major efforts are invested in the development of suitable analytical approaches. A tendency towards the use of LC-MS is nowadays very obvious. A great majority of the metabolites of interest are polar to highly polar in nature. We focus on the so-called 'extended polarity' reversed LC phases, developed specifically to allow better retention characteristics for polar compounds. Several of these phases (Atlantis dC18, Inertsil ODS-3, Zorbax XDB, Alltima HP C18) are tested for different column dimension variations (0.5, 1.0, 2.1 mm id) in a specific LC-MS metabolomics setting. Important chromatographic and mass spectrometric quality parameters such as capacity factor, separation efficiency, peak symmetry, sensitivity, and mass accuracy are taken into account. All phases show adequate retention of polar compounds and also perform well with highly aqueous mobile phase compositions. On comparing 1.0 and 2.1 mm id columns, it is clear that the potential gain in sensitivity is not achieved. Using a Lockspray device, accurate mass measurement with a Q-TOF micro is feasible within a mass range of 12 ppm if signal intensities of compound and lockmass are equated. Finally, the extended polarity RP approach in metabolomics experiments is endorsed using real plant extracts.
The use of marker-peptides, measured by LC-MS/MS, is investigated for the quantitative analysis o... more The use of marker-peptides, measured by LC-MS/MS, is investigated for the quantitative analysis of proteins. To that end, cystatin C is chosen as a model protein. It not only functions as a proof of concept protein but the growing interest in cystatin C as a new marker of kidney failure provides a practical application at the same time. The use of trypsin-based proteolysis, to obtain so-called marker-peptides, simplifies the quantification of a protein to the quantification of a single or a number of peptides. Reproducibility of the trypsin proteolysis procedure is vital and has been optimised. A number of the marker-peptides obtained are selected for LC-MS(/MS) analysis. They are completely separated by high-pressure LC allowing maximum selectivity and mass spectrometric multiple reaction monitoring sensitivity. By doing so, linear calibration curves can be obtained for cystatin C over two orders of magnitude. Experiments have been performed on a triple quadrupole mass spectrometer by single ion monitoring (maximum sensitivity) as well as by multiple reaction monitoring (maximum specificity).
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Papers by Jan Bocxlaer