The composition of chlorinated or oxidized phosphatidylcholine products changes with hypochlorite concentration: Application to abscess lipid analysis

Hypochlorous acid, produced by myeloperoxidase upon neutrophil activation, can oxidize various compounds and exert antimicrobial activity in vivo. To elucidate the mechanisms underlying the reactions of the unsaturated phosphatidylcholines, which abound in cell membranes, with hypochlorous acid, we identified and examined phosphatidylcholine chlorination and oxidation products formed under various reaction conditions. We first investigated the products of unsaturated phosphatidylcholine and hypochlorous acid reaction with respect to hypochlorite concentration and reaction time. Next, we examined the lipids extracted postmortem from human abscesses. For all the analyses, we used liquid chromatography–quadrupole time-of-flight mass spectrometry. Various compounds, including phosphatidylcholine chlorohydrin and phosphatidylcholine hydroxide/epoxide, were detected. Oxidized phosphatidylcholines were mainly detectable upon reaction with low concentrations of sodium hypochlorite, whereas chlorinated phosphatidylcholines formed in the presence of higher concentrations. In human abscesses, oxidized phosphatidylcholines were detected in the cases with high procalcitonin concentration, whereas chlorinated phosphatidylcholines were undetected. The detections of oxidized phosphatidylcholines in human tissues might indicate previous exposure to hypochlorous acid in septic cases. Our results provide insight into the mechanisms underlying pathogen survival following inflammation associated with neutrophil activation and topical myeloperoxidase release and show postmortem biomarkers candidates for sepsis.     

Thermal degradation of a new synthetic cannabinoid QUPIC during analysis by gas chromatography–mass spectrometry

Quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate (QUPIC) is a newly introduced synthetic cannabinoid in the drug market. This drug was found to undergo thermal decomposition during gas chromatography–mass spectrometry (GC–MS), probably because of the presence of an ester bond in its structure. Most notably, when QUPIC dissolved in methanol or ethanol was analyzed by GC–MS, most of the QUPIC decomposed to give thermal degradation products. We identified the products as methyl 1-pentyl-(1H-indole)-3-carboxylate, ethyl 1-pentyl-(1H-indole)-3-carboxylate, and methyl indole-3-carboxylate by comparison of their mass spectra with those of reference standards synthesized in our laboratory. Nonalcoholic solvents such as acetone and chloroform gave a major peak and a minor peak for unchanged QUPIC and the degradation product 8-quinolinol, respectively. Furthermore, we studied the effects of various parameters, such as injection methods (splitless or split, and split ratio), injector temperatures, and injector liners on the thermal degradation of QUPIC. Split injection was effective in avoiding degradation. When performing splitless injection, an injector temperature of 250 °C and a surface deactivated injector liner without glass wool minimized the degradation and enhanced the sensitivity. These results indicate that special attention is required for GC–MS analysis of QUPIC, and the information presented in this study will be very useful for forensic toxicologists using GC–MS.     

Radiation-induced fragmentation of cardiolipin in a model membrane

PURPOSE: To obtain evidence for the possibility of free-radical fragmentation of cardiolipin under the action of ionizing radiation as measured by its aqueous dispersion from liposomes. MATERIALS AND METHODS: Liposomes of tetramyristoylcardiolipin (TMCL) were exposed to gamma-rays from 60Co or 137Cs sources at doses between 1 and 24kGy. Fragmentation products were identified using thin-layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Using MALDI-TOF MS and thin-layer chromatography, it was shown that gamma-irradiation of liposomes consisting of TMCL was accompanied by free-radical fragmentation of the lipid to form dimiristoylphosphatidic acid and dimiristoylphosphatidyl hydroxyacetone. The yields of dimiristoylphosphatidic acid were greater than those of dimiristoylphosphatidyl hydroxyacetone, and formation of the named compounds was inhibited by dissolved oxygen. CONCLUSION: It is shown for the first time that on gamma-irradiation, cardiolipin can undergo free-radical fragmentation in its polar component.     

Analysis of fentanyl and sufentanil in hair by GC/MS/MS

Fentanyl and sufentanil are potent narcotic analgesics used only in hospitals as anaesthetic agents. The dependence potential of fentanyl is known. As they are given in doses at the microgram level and their elimination half-life is in the order of a few hours, detection in body fluids is possible only for a short time after administration. Radioimmunological methods are the only ones capable of detecting fentanyl in hair, as normal GC/MS methods for hair analysis are not sensitive enough to detect the drugs after doses in the order of micrograms. We therefore chose GC/MS/MS to analyse fentanyl and sufentanil in two cases where the drugs were given under controlled conditions over several days. The concentration was in the order of less than 100 pg/mg hair.     

Decomposition of aqueous solutions of phenol using high energy electron beam irradiation—A large scale study

High-energy electron-beam irradiation was used to remove phenol from aqueous solution. The variables that affected phenol decomposition were solute concentration, absorbed dose and total alkalinity. Experiments were conducted at large scale (480 L min⁻¹), at solute concentrations of 10.6, 106 and 531 μmol L⁻¹ (1, 10 and 50 mg L⁻¹) over the pH range 5–9, and in the presence and absence of solids (3% w/w kaolin clay). Absorbed doses ranged from 0–7 kGy (0–700 krad). At low absorbed doses, catechol, hydroquinone and resorcinol were identified as the major reaction byproducts. These compounds are consistent with hydroxyl radical (OH·) addition to phenol. Subsequent ring cleavage of hydroxylated phenolic radicals and continued oxidative processes resulted in the formation of formaldehyde, acetaldehyde, glyoxal and formic acid. At high doses only trace amounts of the carbonyl derivatives were observed. Two recirculation experiments were conducted at higher phenol concentrations (≈950 μmol L⁻¹) and it was shown that phenol was removed while the total organic carbon of the solution decreased only slightly. These results suggest that phenol was not mineralized but, rather, that irradiation resulted in the possible formation of higher molecular weight polymers.     

Fading characteristics of an alanine-polystyrene dosimeter

Alanine dosimetry is useful for transfer dosimetry by long distance mailing, because of its stability. It has the advantage that the measurement of electron spin resonance (ESR) spectral signal is non-destructive to the dosimeter, with the promise that the method may supply archival dosimetry data, depending on the degree of post-irradiation stability of the signal. The effects of temperature during irradiation and storage on fading of the ESR signal were studied using an alanine dosimeter molded with polystyrene (alanine-PS dosimeter). This investigation covered a long range of storage time (up to 160 days) after irradiation to absorbed doses in the range 1 to 100 kGy, for application to transfer dosimetry between Japan and neighboring Asian countries.Dose response of an alanine-PS dosimeter depends on the temperature during irradiation. The same temperature coefficient of +0.24%/°C was measured at different dose levels of 1, 10 and 100 kGy administered at a constant dose rate of 7 kGy/h. Fading of the dose response was measured under storage at various temperatures (5–40°C). The fading curve generally has two phases with fast and slow fading rates. The response of an alanine dosimeter is relatively stable for doses of 1.4 and 14 kGy, when stored at temperatures below 25°C. However, the degree of fading was roughly 3 and 5% under a storage temperature of 40°C for 5 and 100 days, respectively, after irradiation to 14 kGy. The fading percentages at 100 kGy were 2 and 4% (after 5 days) and 6 and 15% (after 100 days) under the storage temperature of 25 and 40°C, respectively. The fading rates have a relatively small dependence on irradiation temperature. This is observed even when irradiation are made at high temperatures (60°C) and for the doses 100 kGy and above. The mechanism of decay of radicals is discussed to explain the fading characteristics of the two phases of fading. The alanine-PS dosimeter is useful for transfer standard dosimetry up to a dose level of 10 kGy when stored after irradiation at temperature below 40°C. However, consideration of temperature effects during and after irradiation is vital for accurate transfer dosimetry of high doses, especially in the southern Asian countries.     

Zum Suchtmittelnachweis in Haaren. VII. Untersuchungen bei Methadonpatienten unter konstanter Wirkstoffeinnahme

Hair samples and perspiration collected from long term drug addicts (n = 18) under constant methadone treatment were investigated. Hair segments representing the individual hair growth of 4 weeks were analyzed for methadone, opiates and cocaine by GC/MS. The results obtained from the transdermal collection devices confirmed the presence of drug substances in variable amounts on the skin surface, methadone being the main analyte. The perspiration test period lasted for 4 days. For six persons illicit drugs similar to those in the corresponding hair samples were found. Overall, a poor relationship (r = 0,602) was found between the methadone dose under steady state conditions and the methadone level in hair. The results are discussed in the light of pharmacokinetic aspects, drug uptake by perspiration and forensic interpretation.     

Radiation-induced decomposition of aqueous trichloroethylene solutions

In air-saturated reagent grade water, 10 ppm trichloroethylene are decomposed by γ radiation in a roughly first-order reaction; the initial G-value being 5.4 molecules/100 eV. At sub-ppm concentrations the kinetics remain roughly first-order; the initial G-values decrease with decreasing concentration. The main decomposition products are Cl⁻, CO₂ and HCOOH. A tentative reaction scheme in accordance with these results is presented.     

Testing of skeletonized human remains using GC/MS – development of a personal environmental profile

ABSTRACT

Gas chromatography/mass spectrometry (GC/MS) is well used in forensic science for the identification of materials present in toxicology, pharmacology and arson investigation. However, GC/MS has been underused in human identification. In this study, highly commingled skeletonized remains from a mass fatality incident involving over 400 individuals were examined. Environmental compounds and those present in the remains themselves were extracted from osseous materials removed from the surface of the skeletal elements using two different solvents, dichloromethane and acetonitrile. Solvent extracts were concentrated and resuspended in methanol prior to injection on the GC/MS instrument. Compounds present from the environment surrounding the individual post-mortem, as well as biological materials present in the individual ante-mortem, are detectable. Traces generated from the GC/MS instrument provide distinctive images, and analysis of those materials shows patterns that are specific to the individual and may be used for individuation. Results indicate that GC/MS analysis of skeletonized remains may be a new tool for human identification.     

Radiation‐induced fragmentation of cardiolipin in a model membrane

Purpose: To obtain evidence for the possibility of free‐radical fragmentation of cardiolipin under the action of ionizing radiation as measured by its aqueous dispersion from liposomes.

Materials and methods: Liposomes of tetramyristoylcardiolipin (TMCL) were exposed to γ‐rays from ⁶⁰Co or ¹³⁷Cs sources at doses between 1 and 24?kGy. Fragmentation products were identified using thin‐layer chromatography and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS).

Results: Using MALDI‐TOF MS and thin‐layer chromatography, it was shown that γ‐irradiation of liposomes consisting of TMCL was accompanied by free‐radical fragmentation of the lipid to form dimiristoylphosphatidic acid and dimiristoylphosphatidyl hydroxyacetone. The yields of dimiristoylphosphatidic acid were greater than those of dimiristoylphosphatidyl hydroxyacetone, and formation of the named compounds was inhibited by dissolved oxygen.

Conclusion: It is shown for the first time that on γ‐irradiation, cardiolipin can undergo free‐radical fragmentation in its polar component.     

Development of a preparation method to produce a single sample that can be applied to both LC–MS/MS and GC–MS for the screening of postmortem specimens

Simple and efficient extraction methods have been developed for the screening of a wide array of drugs in postmortem autopsy specimens. Acidic and basic compounds were targeted with two extraction methods that can be applied to both GC–MS and LC–MS/MS instrumentation. The extractions were achieved by utilizing lipid-removal and solid-phase extraction cartridges while carefully monitoring the pH of the samples to ensure the adequate removal of interfering substances like lipids and amino acid derivatives. These methods were applied to actual autopsy cases, with 94 and 124 compounds detected by GC–MS and LC–MS/MS, respectively. The developed methods could easily be incorporated into a forensic laboratory’s daily routine for screening many different compounds from postmortem samples.     

Supramolecular solvent (SUPRASs) extraction method for detecting benzodiazepines and zolpidem in human urine and blood using gas chromatography tandem mass spectrometry

ObjectiveA high-throughput and sensitive method using supramolecular solvent (SUPRASs) for detecting 9 benzodiazepines and zolpidem in human urine and blood by gas chromatography-tandem mass spectrometry (GC–MS/MS) was newly established and applied to authentic human urine and blood samples in this study.MethodsUrine and blood samples were subjected to liquid–liquid extractions with supramolecular solvent mixture which consists of tetrahydrofuran and 1-hexanol. The solvent layer was evaporated to dryness by stream of nitrogen. The residue was reconstituted with methanol, and subjected to analysis by GC–MS/MS in multiple reaction monitoring (MRM) mode; internal standard method was employed for quantifying of each targeted compound.ResultsThe regression equation has a good linear relationship with correlation coefficients for all tested compounds were not lower than 0.9991. The lower limits of the quantification ranged from 0.20 to 5 ng/mL for tested compounds in urine; Meanwhile, the lower limits of the quantification in this method ranged from 1 to 50 ng/mL for tested compounds in blood. These results showed that excellent reproducibility and satisfactory extraction recovery rates could be obtained for the established analytical method for 10 drugs in both blood and urine samples.ConclusionThe established method in this study was high-throughput, simple and sufficiently sensitive for determining of benzodiazepinesand zolpidem in human urine and blood. Therefore, this newly established method could be of use for qualitative and quantitative determination of such drugs in urine and blood samples either for clinical poisoning monitoring or for forensic identification.     

Changes in digestible energy values of some agricultural residues treated with gamma irradiation

The effects of different doses of gamma irradiation (0, 5, 20, 50, 100 and 150 kGy) on gross energy (GE), in vitro apparent organic matter digestibility (IVOMD) and digestible energy (IVDE), have been evaluated in barley straw, sorghum straw, wheat chaffs and maize cobs. The results indicate that, there were significant (P<0.05) increases in IVOMD and IVDE values, especially, at the dose of 150 kGy. The increases in IVOMD were 22, 21 and 23% for barley straw, sorghum straw and wheat chaffs, respectively; whereas, such an increase was 12% for maize cobs. Digestible energy values increased over the control by 1165, 1621, 1540 and 1130 kJ/kg dry matter for barley straw, sorghum straw, wheat chaffs and maize cobs, respectively. There was no significant effect of gamma irradiation on GE values for the studied agricultural residues.     

Validating a gas chromatography-mass spectrometric method and sample classification procedure for cannabis profiling using cannabinoids from case samples

A gas chromatography–mass spectrometric (GC–MS) method was evaluated for cannabis profiling. Method validation was carried out using four cannabinoids extracted from cannabis case samples. In the presence of the sample matrix, the instrument was able to achieve repeatability and reproducibility with relative standard deviation (RSD) values ≤ 10.99% and ≤ 9.09%, respectively. The MS detector also produced a linear response for each of the target cannabinoids with R² values ranging from 0.976 to 1.000. The method was found to be accurate as it displayed satisfactory recoveries (assessed based on the percentage difference between the measured amount and the expected or calculated amount of the analyte) between –11.35% and 0.96% for the difference. Subsequently, five different batches/groups of cannabis plants that were assumed to have come from different sources were tentatively used to assess the sample classification outcome. These samples were correctly clustered on a dendrogram with 12% errors. Through this preliminary study, it is deduced that cannabis samples could be profiled to estimate their source level relationships by using the proposed GC–MS method.     

Mass spectrometric differentiation of the isomers of mono-methoxyethylamphetamines and mono-methoxydimethylamphetamines by GC–EI–MS–MS

Mass spectrometric differentiation of the six isomers of mono-methoxyethylamphetamines (MeO-EAs) and mono-methoxydimethylamphetamines (MeO-DMAs) by gas chromatography–electron ionization–tandem mass spectrometry (GC–EI–MS–MS) was investigated. Based on their EI-mass spectra, the fragment ions at m/z 121 and 72 were selected as precursor ions for their regioisomeric and structurally isomeric differentiation, respectively. Collision-induced dissociation provides intensity differences in product ions among the isomers, enabling mass spectrometric differentiation of the isomers. Furthermore, high reproducibility of the product ion spectra at the optimized collision energy was confirmed, demonstrating the reliability of the method. To our knowledge, this is the first report on mass spectrometric differentiation of the six isomers of MeO-EAs and MeO-DMAs by GC–EI–MS–MS. Isomeric differentiation by GC–EI–MS–MS has a high potential to discriminate isomers of newly encountered designer drugs, making GC–MS–MS a powerful tool in the forensic toxicology field.     

Screening for hydroxyethyl starch (HES) doping in sport

The artificial colloid hydroxyethyl starch (HES) is among the most frequently used plasma volume expanders in the medical field. However, in 1998, its misuse by the athletic community was officially reported and since 2000, HES is prohibited by the International Olympic Committee (IOC). Therefore, several methods enabling the detection of HES in urine were developed, most based on gas chromatography–mass spectrometry (GC–MS). In the present work, a simple and low-cost screening method, intended to reduce the number of samples to be analysed by GC–MS, was developed. The method is based on the acid hydrolysis of HES and detection of the resulting glucose and hydroxyethyl glucose derivatives by Benedict's reaction (reduction of copper sulfate to brick-red cuprous oxide by glucose and/or derivatives). Samples considered suspect were submitted to GC–MS analysis for identification of HES. The method was successfully applied for screening of HES in 2627 urine samples from 1346 Brazilian soccer players and 1281 athletes from the Pan-American Games (Rio de Janeiro, 2007); 71 (2.7%) samples, considered suspect, were submitted to GC–MS, but no positive results were found. Moreover, a thin layer chromatography (TLC) method was adapted for visualisation of the characteristic band pattern of HES hydrolysis products. The results indicate that the methods are efficient and useful for the screening of HES in urine.     

Identification of a new synthetic cannabinoid in a herbal mixture: 1-butyl-3-(2-methoxybenzoyl)indole

Two unknown cannabimimetic compounds were detected in a seized herbal mixture after gas chromatography–mass spectrometry (GC–MS) screening. To elucidate the chemical structures, 0.3 g of the dried plant material was extracted with methanol and concentrated under reduced pressure. The extract was purified by silica gel column chromatography with methylene chloride and methanol. Pure compounds were isolated by preparative high-performance liquid chromatography (HPLC) and then analyzed by electrospray ionization (ESI) mass spectrometry (MS) with direct flow injection, high-resolution ESI-time-of-flight (TOF)–MS and one-dimensional and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. GC–MS spectra showed that the base ion at m/z 321 for compound 1 was the same as that of 1-pentyl-3-(4-methoxybenzoyl)indole (RCS-4), and the fragment ions were almost the same as those of RCS-4. The GC–MS spectrum of compound 2 was very similar to that of compound 1 except that the mass numbers of the fragment ions at m/z 290, 200, 186, and 173 of compound 2 were equally smaller than those of compound 1 by 14 amu. From these GC–MS results, compound 1 was assumed to be the 2- or 3-methoxy isomer of RCS-4, and compound 2 was assumed to be a 1-butylindole homologue of compound 1. The ESI mass spectra showed a single peak at m/z 322.33 for compound 1 and a single peak at m/z 308.25 for compound 2, which showed the masses of the protonated ions. High-resolution TOF–MS spectra showed the accurate mass numbers of protonated molecular ions at m/z 322.180512 for compound 1 and at m/z 308.164895 for compound 2, suggesting the molecular formulas of C₂₁H₂₃NO₂ and C₂₀H₂₁NO₂, respectively. The ¹H NMR spectra showed signals that suggested 23 and 21 protons for compounds 1 and 2, respectively, while the respective ¹³C NMR spectra showed 21 and 20 carbon signals. All protons and carbons were assigned by their couplings and correlations observed in ¹H–¹H correlation spectroscopy (COSY), ¹H–¹³C heteronuclear multiple bond correlation (HMBC), and ¹H–¹³C heteronuclear single quantum coherence (HSQC) spectra. On the basis of the spectral data, compound 1 was identified as the 2-methoxy isomer of RCS-4; compound 2 was identified for the first time as 1-butyl-3-(2-methoxybenzoyl)indole. Phenazepam and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) were also identified as coexisting drugs in the herbal mixture. The contents of compounds 1 and 2 in the mixture were calculated to be 22.4 and 3.45 mg/g, respectively.     

Simultaneous determination of tryptamine analogues in designer drugs using gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

In recent years, a large number of tryptamine-based designer drugs have been encountered in forensic samples. We have developed simultaneous analytical methods for 14 tryptamine analogues using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). Trimethylsilyl (TMS) derivatives of the analytes were separated on a DB-1ms column within 15 min. The structural isomers could be differentiated by electron ionization GC–MS. LC–MS–MS with a C₁₈ column could separate structural isomers of tryptamines except for a combination of 5-methoxy-N,N-diethyltryptamine and 5-methoxy-N-methyl-N-isopropyltryptamine. Higher collision energy gave different product ion spectra between the structural isomers. The results indicate that GC–MS is the first choice for identification of tryptamines, preferably after TMS derivatization, and LC–MS–MS can be used as a complementary approach for the unequivocal differentiation of tryptamine isomers.     

Phosphorus-specific determination of glyphosate,glufosinate, and their hydrolysis products in biological samples by liquid chromatography–inductively coupled plasma–mass spectrometry

Anion exchange liquid chromatography (LC) with inductively coupled plasma–mass spectrometry (ICP–MS) was used for simultaneous determination of glyphosate and glufosinate (phosphonic and amino acid group-containing herbicides; PAAHs) and their hydrolysis products, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicoacetic acid (MPPA), in biological samples. The target compounds were separated using an anion exchange resin column and gradient elution with sodium carbonate and sodium hydroxide system. The chromatographic eluates were passed through a membrane suppressor system and monitored by phosphorus-specific detection at m/z 31. PAAHs and their hydrolysis products were baseline separated from each other within 40 min of elution time, and phosphoric acid did not interfere with detection. The detection limits in serum samples were 0.1–0.7 µg/ml; those in urine samples, 0.2–1.6 µg/ml for the four compounds. The spiked recoveries for the four compounds were over 91 % in serum and urine samples. The detection of the compounds was not subject to interference from sample matrix components. The present method would be useful for toxicological analysis of PAAHs and their products in actual forensic practice. To our knowledge, this is the first trial to establish an LC–ICP–MS method for analysis of PAAHs and their products in biological samples .