Measuring insulin in human vitreous humour using LC-MS/MS

Besides its particular importance as a widely used therapeutic agent, insulin (and its synthetic derivatives) has been suspected, purported, and proven to be a lethal weapon in numerous cases of attempted or successful homicide and suicide. In addition to blood and urine as common matrices for clinical diagnosis and post-mortem analysis, vitreous humour has gained considerable attention in autopsy and follow-up investigations due to its ability to provide valuable information on cause and time of death. However, post-mortem insulin analyses using such specimens have been rare due to the limited penetration of peptide hormones into the vitreous body, and immunoassays were exclusively employed in those studies. In the present communication, the determination of insulin(s) from vitreous humour by means of immunopurification combined with ultrahigh performance liquid chromatography--high resolution/high accuracy (tandem) mass spectrometry is reported. Exploiting the constantly increasing sensitivity and robustness of modern mass-spectrometry-based instruments, the option to identify insulin in post-mortem vitreous samples is demonstrated with a specimen collected from a non-diabetic victim who died from an insulin overdose. This communication represents the first successful mass-spectrometry-based analysis of post-mortem material related to an insulin poisoning case.     

Detection and quantification of endogenous cyclic DNA adducts derived from trans-4-hydroxy-2-nonenal in human brain tissue by isotope dilution capillary liquid chromatography nanoelectrospray tandem mass spectrometry

A sensitive and selective capillary liquid chromatography nanoelectrospray isotope dilution mass spectrometric method was developed to identify and quantify the endogenous cyclic DNA adducts derived from trans-4-hydroxy-2-nonenal with 2'-deoxyguanosine (HNE-dG) in human brain tissues. Authentic and 13C and 15N stable isotope-labeled HNE-dG were synthesized to serve as standards. The in vitro HNE-modified calf-thymus DNA as well as the DNA samples isolated from human brain tissues of normal and Alzheimer's disease subjects were enzymatically digested to nucleosides in vitro with the presence of internal standard (HNE-dG-13C10, 15N5). The enzymatic digests were cleaned up by solid phase extraction. Only 1-2 microg of DNA digests was loaded on a laboratory-constructed reversed phase capillary chromatography column, and the HNE-dG adducts were separated from intact nucleosides and quantified by a high capacity ion trap mass spectrometer in the MS/MS mode. This method was able to quantify an adduct level of approximately 40 lesions/10(9) normal DNA nucleosides. The detected level of HNE-dG adducts in hippocampus/parahippocampal gyrus and inferior parietal regions of postmortem brains from AD subjects were 556 +/- 379 and 238 +/- 72 adducts per 10(9) normal nucleosides, respectively. These results were consistent with the 32P postlabeling results, which detected 400-600 adducts per 10(9) normal nucleotides in the hippocampus.     

The absolute quantification of endogenous levels of brain neuropeptides in vivo using LC-MS/MS

Neuropeptides seem to play an important role when the CNS is challenged. In order to obtain better insights into the central peptidergic effects, it is essential to monitor their concentration in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to glass and polymeric materials. The quantification of neuropeptides in dialysates therefore necessitates the use of very sensitive nano-LC-MS/MS methods. A number of LC-MS/MS and microdialysis parameters need to be optimized to achieve maximal sensitivity. The optimized and validated methods can be used to investigate the in vivo neuropeptide release during pathological conditions, in this way initiating new and immense challenges for the development of new drugs.     

Quantitative determination of underivatized polyamines by using isotope dilution RP-LC-ESI-MS/MS

A rapid, sensitive and selective method using LC-MS/MS was developed and validated for the simultaneous quantitative determination of five polyamines N(1),N(12)-diethylspermine (DESpm), N-ethylspermine (EtSpm), N(1)-ethylspermidine (EtSpd), spermidine (Spd) and N(1)-ethyl-1,3-diaminopropane (EtDAP) without any derivatization steps. The LC-MS/MS system was operated using the positive electrospray ionization (ESI) mode. The chromatographic separation only took 10min and was performed on a reversed phase C18 column with 0.1% heptafluorobutyric acid as the ion-pairing agent and acetonitrile gradient. Stable, deuterium labelled internal reference compounds of the five analytes were included in the quantification. The lower limit of quantification for all of the five analytes was 0.03muM and the method was linear for DESpm, EtSpd, Spd and EtDAP over the range of 0.03-60muM and for EtSpm over the range of 0.03-30muM. Correlation coefficients (R(2)) were always >0.995 for all the analytes. The precision of the overall method ranged from 0.2 to 9.7% as intra-day variability and from 0.9 to 6.8% as inter-day variability. The intra-day and inter-day accuracy of the assay ranged between 87.6-109.8% and 89.6-106.6%, respectively. The method has been applied successfully to quantify metabolites of DESpm as a substrate for recombinant human polyamine oxidase.     

Carbon isotope ratio of endogenous urinary steroids of the Cuban population of athletes studied for doping purposes

The aim of this work was to validate the gas chromatography/combustion/isotope ratio mass spectrometry method in Havana Antidoping Laboratory and verify its implementation with a study of the Cuban population. The method was precise and accurate inside the linear working range; the limit of quantification and the uncertainty were compliant with TD2019IRMS. The study of the Cuban population showed no differences in δ¹³C values between females and males. Only three values of Δδ¹³C showed significant differences between sexes (PD‐T, OHA‐T, and 11‐keto‐Et‐T). The values of δ¹³C between ?17.8‰ and ?21.2‰ (upper and lower limits based on normal distribution) were consistent with other populations where C4 plant derivatives prevail in the diet.     

Simultaneous detection and quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography tandem mass spectrometry

Nitration and bromination of proteins, giving rise to the respective 3-nitrotyrosine (3NT) and 3-bromotyrosine (3BT), are implicated in asthma, allergic inflammatory disorders, and cancer. We have developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits (S/N = 3) were 10 pg (44 fmol) for 3NT and 5.0 pg (19 fmol) for 3BT injected on-column. The average levels of protein-bound 3NT and 3BT in 23 healthy individuals were 9.7 ± 11.0 (mean ± S.D.) in 10⁵ tyrosine and 4.4 ± 3.9 (mean ± S.D.) in 10³ tyrosine, respectively, using this highly sensitive LC/MS/MS under the selective reaction monitoring mode. Furthermore, the levels of urinary 3NT and 3BT show a statistically significant correlation (R² = 0.55, p = 0.0065, n = 23). The high specificity and accuracy of this LC/MS/MS method render it a valuable tool in measurement of 3NT and 3BrT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.     

Atriopeptin, sodium azide and cyclic GMP reduce secretion of aqueous humour and inhibit intracellular calcium release in bovine cultured ciliary epithelium.

This study examined the involvement of cyclic GMP, protein kinase G and intracellular Ca2+ movements in the modulation of aqueous humour formation. Using the bovine arterially-perfused eye preparation, drug effects on intraocular pressure and aqueous humour formation rate were measured by manometry and fluorescein dilution, respectively. Drug effects on intracellular [Ca2+] were determined by fura-2 fluorescence ratio technique in nontransformed, cultured ciliary epithelium. Intra-arterial injection of atriopeptin (50 pmol) or sodium azide (10 nmol) produced significant reduction in aqueous humour formation (>38%). This was blocked by selective inhibition (KT-5823) of protein kinase G, but not by selective inhibition (KT-5720) of protein kinase A. Reductions of intraocular pressure produced by atriopeptin or azide were almost completely blocked by KT-5823. ATP (100 microM) caused rapid, transient increase in intracellular Ca2+ followed by a slow decline and prolonged plateau. This response showed concentration-dependent inhibition by atriopeptin, azide or 8-bromo cyclic GMP, and this inhibition of the rapid (peak) Ca2+ increase was enhanced by zaprinast (100 microM; phosphodiesterase inhibitor). KT-5823 blocked the suppression of the peak Ca2+ response but not suppression of the plateau. Arterial perfusion of ATP (0.1-100 microM) produced a concentration-dependent decrease in aqueous humour formation. Aqueous humour formation in the bovine eye can be manipulated through cyclic GMP, operating via protein kinase G. Close parallels appear when Ca2+ movements are modified by similar manipulations of cyclic GMP, suggesting that Ca2+ transients may play an important role in aqueous humour formation and that interplay occurs between cyclic GMP and Ca2+.     

Detection and quantification of 5-chlorocytosine in DNA by stable isotope dilution and gas chromatography/negative ion chemical ionization/mass spectrometry

Hypochlorous acid (HOCl) is generated from activated phagocytes during infections and inflammation. One of the major products of HOCl reaction with DNA was 5-chlorocytosine (5Cl-Cyt). In this report, a gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay with stable isotope dilution was developed for detection and quantification of 5Cl-Cyt in DNA. During hydrolysis of DNA, 5Cl-Cyt undergoes spontaneous deamination quantitatively forming 5-chlorouracil (5Cl-Ura). The stable isotope of 5Cl-Ura with six mass units higher than the normal 5Cl-Ura was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selected ion monitoring at [M - 181](-) fragments of bispentafluorobenzylated 5Cl-Ura and its isotope analogue. The limit of detection was 20 amol (S/N = 8) of bispentafluorobenzylated 5Cl-Ura injected on column with selective ion monitoring mode and the limit of quantification for the entire assay was 14 fmol of 5Cl-Cyt. Analysis of hypochlorous acid-treated calf thymus DNA by both GC/NICI/MS and HPLC/UV detection provided similar adduct levels and thus verified this new GC/NICI/MS assay. Using this highly specific and ultrasensitive GC/NICI/MS method, the levels of 5Cl-Cyt in untreated calf thymus DNA and human placental DNA were determined as 0.6 and 6.6 adducts per 10(7) normal cytosine, respectively. Peroxynitrite also contributed to 5Cl-Cyt formation in DNA. Level of 5Cl-Cyt in DNA treated with peroxynitrite in the presence of chloride was higher than that without addition of chloride. Thus, quantification of 5Cl-Cyt in DNA by this isotope dilution GC/NICI/MS assay may facilitate research on the role of DNA chlorination in carcinogenesis and in cancer development.     

Vancomycin levels in human aqueous humour after intravenous and subconjunctival administration.

The purpose of the present study was to evaluate the level of vancomycin in human aqueous humour after intravenous (i.v.) and subconjunctival administration. One hundred patients scheduled to undergo cataract extraction participated in the study. Fifty-three of them received 20 mg vancomycin subconjunctivally and 47 received two doses of vancomycin i.v. (1 g b.i.d.). Specimens of aqueous humour from the first group were collected 1, 2, 2.5, 3, 3.5, 5, 6, 7, and 8 h after the subconjunctival injection. In the second group, specimens of blood and aqueous humour were collected 1, 2, 4, 6, 8, 10, and 12 h after the end of infusion of the second dose of the antibiotic. The levels of vancomycin were determined by fluorescent polarization immunoassay. In the first group peak levels of 24.82+/-3.55 microg/ml were achieved in aqueous humour at 5 h, whereas in the second group peak levels of 1.42+/-0.47 microg/ml were detected at 6 h. The latter levels, although higher than the MICs of most of the Gram-positive pathogens causing eye infections, are inadequate for the treatment of infections in vivo. These results support the need for subconjunctival instead of i.v. administration of vancomycin in order to achieve therapeutic levels of the drug in human aqueous humour or for prophylactic use whenever indicated.     

Quantitation of organophosphorus nerve agent metabolites in human urine using isotope dilution gas chromatography-tandem mass spectrometry

An isotope dilution gas chromatography-tandem mass spectrometric (GC-MS-MS) method was developed for quantitating the urinary metabolites of the organophosphorus nerve agents sarin, soman, tabun (GA), VX, and GF. Urine samples were concentrated by codistillation with acetonitrile, derivatized by methylation with diazomethane, and analyzed by GC-MS-MS. The limits of detection were less than 4 microg/L for all the analytes except for the GA metabolite, which had a limit of detection of less than 20 microg/L.     

The action of sympathomimetic amines on the outflow of aqueous humour from the eye

The actions of adrenaline, noradrenaline and isoprenaline on the resistance to outflow of aqueous humour from the rabbit eye are described. In some experiments, correlations have been made with pupil diameter. Intravitreous injections of adrenaline, noradrenaline and large doses of isoprenaline decreased the resistance to outflow of fluid from the eye and dilated the pupil. Intravitreous injection of phentolamine was without effect on the intraocular pressure and resistance to outflow of aqueous humour, although such injections resulted in miosis. The actions of adrenaline, noradrenaline and large doses of isoprenaline on the outflow resistance were antagonized by phentolamine. Postganglionic sympathetic denervation did not affect the aqueous humour dynamics when the eyes where examined 2 weeks later, but all the denervated eyes exhibited miosis at this time. Much smaller doses of noradrenaline were required to lower the intraocular pressure and decrease the resistance to outflow of aqueous humour in the denervated eyes; in addition, the dose/response curve for the effect of noradrenaline on the outflow resistance was shifted to the left in these experiments. These results support the view that adrenaline α-receptors are associated with the resistance to outflow of aqueous humour from the rabbit eye.     

Determination of digoxin in human serum using stable isotope dilution liquid chromatography/electrospray ionization-tandem mass spectrometry

A method for the determination of digoxin in human serum using a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) technique is reported. Digoxin and the internal standard, [21,21,22-(2)H(3)]digoxin, were extracted from 250 mul of human serum using a solid phase extraction cartridge (Oasis HLB) and analyzed by LC/ESI-MS/MS in the selected reaction monitoring mode. The intra- and inter-assay reproducibility and accuracy were satisfactory within the quantification range of 0.20-3.20 ng/ml. The concentrations of digoxin in the serum samples obtained from digitalized patients (n=19) were in the range of 0.25-2.84 ng/ml, which were compared to those obtained by radioimmunoassay.     

A reverse isotope dilution method for determining benzene and metabolites in tissues

A method utilizing reverse isotope dilution for the analysis of benzene and its organic soluble metabolites in tissues of rats and mice is presented. Tissues from rats and mice that had been exposed to radiolabeled benzene were extracted with ethyl acetate containing known, excess quantities of unlabeled benzene and metabolites. Butylated hydroxytoluene was added as an antioxidant. The ethyl acetate extracts were analyzed with semipreparative reversed-phase HPLC. Isolated peaks were collected and analyzed for radioactivity (by liquid scintillation spectrometry) and for mass (by UV absorption). The total amount of each compound present was calculated from the mass dilution of the radiolabeled isotope. This method has the advantages of high sensitivity, because of the high specific activity of benzene, and relative stability of the analyses, because of the addition of large amounts of unlabeled carrier analogue.     

Actions of synthetic and endogenous steroids on the GABAA receptor

Traditionally steroid hormones have been thought to exert their effects on the mammalian CNS via an interaction with intracellular receptors, producing gene-regulated changes in protein synthesis. Whilst this is undoubtedly the mechanism of action for many steroids, some endogenous and synthetic steroids induce hypnosis/anaesthesia rapidly, suggesting an alternative mechanism of action. Jeremy Lambert, John Peters and Glen Cottrell review recent evidence which clearly demonstrates that some steroids are potent stereoselective potentiators of the actions of GABA at GABA_A receptors in vitro. Furthermore, at higher concentrations these steroids can directly activate the GABA_A receptor. As a number of the active steroids occur endogenously, the fascinating possibility exists that they may influence GABA_A receptors under physiological and pathophysiological conditions and so modulate the activity of the CNS.     

Direct quantification in bioanalytical LC-MS/MS using internal calibration via analyte/stable isotope ratio

The possibility to rationalize and simplify bioanalysis, without compromising the analytical quality, by omitting the calibration curves was studied. Using mass spectrometry (MS) and a stable isotope labeled internal standard it was possible to get equally good results by calculating the results directly from the analyte/internal standard area ratio and a predetermined response factor as by the traditional way, using a calibration curve run at the same occasion. To be able to use this simplified quantification method, that we call internal calibration, in its most simple form there are some prerequisites that must be considered: (1) The relative response should not be concentration dependent. (2) The relative response should be constant between batches/days. (3) The level of analyte in the internal standard should not be detectable. (4) There should be no influence from naturally occurring isotopes of the analyte on the internal standard peak area. A bioanalytical LC-MS/MS method for a research compound was validated both with and without calibration curves and no significant differences were found regarding precision and accuracy. It was shown that all four prerequisites above were fulfilled. Validation data were very good for the whole concentration range, 0.010-30 micromol/L. Long-term data for QC samples showed excellent precision and accuracy.     

Development and validation of an ESI‐LC‐MS/MS method for simultaneous identification and quantification of 24 analytes of forensic relevance in vitreous humour,whole blood and plasma

Detection and quantification of drugs from various biological matrices are of immense importance in forensic toxicological analysis. Despite the various reported methods, development of a new method for the detection and quantification of drugs is still an active area of research. However, every method and biological matrix has its own limitation, which further encourage forensic toxicologists to develop new methods and to explore new matrices for the analysis of drugs. In this study, an electrospray ionization‐liquid chromatograph‐tandem mass spectrometry (ESI‐LC‐MS/MS) method is developed and validated for simultaneous identification and quantification of 24 drugs of forensic relevance in various body fluids, namely, whole blood, plasma and vitreous humour. The newly developed method has been validated for intra‐day and inter‐day accuracy, precision, selectivity and sensitivity. Absolute recovery shows a mean of 84.5, 86.2, and 103% in the vitreous humour, whole blood and plasma respectively, which is suitable for the screening procedure. Further, the absolute matrix effect (AME) shows a mean of 105, 96.5, and 109% in the vitreous humour, whole blood and plasma, respectively. In addition, to examine the practical utility of this method, it has been applied for screening of drugs in post‐mortem samples of the vitreous humour, whole blood and plasma collected at autopsy from ten cadavers. Experimental results show that the newly developed method is well applicable for screening of analytes in all the three matrices. Copyright © 2015 John Wiley & Sons, Ltd.