Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

High-temperature solid-phase microextraction procedure for the detection of drugs by gas chromatography–mass spectrometry

High-temperature headspace solid-phase microextraction (SPME) with simultaneous (“in situ”) derivatisation (acetylation or silylation) is a new sample preparation technique for the screening of illicit drugs in urine and for the confirmation analysis in serum by GC–MS. After extraction of urine with a small portion of an organic solvent mixture (e.g., 2 ml of hexane–ethyl acetate) at pH 9, the organic layer is separated and evaporated to dryness in a small headspace vial. A SPME-fiber (e.g., polyacrylate) doped with acetic anhydride–pyridine (for acetylation) is exposed to the vapour phase for 10 min at 200°C in a blockheater. The SPME fiber is then injected into the GC–MS for thermal desorption and analysis. After addition of perchloric acid and extraction with n-hexane to remove lipids, the serum can be analysed after adjusting to pH 9 as described for urine. Very clean extracts are obtained. The various drugs investigated could be detected and identified in urine by the total ion current technique at the following concentrations: amphetamines (200 μg/l), barbiturates (500 μg/l), benzodiazepines (100 μg/l), benzoylecgonine (150 μg/l), methadone (100 μg/l) and opiates (200 μg/l). In serum all drugs could be detected by the selected ion monitoring technique within their therapeutic range. As compared to liquid–liquid extraction only small amounts of organic solvent are needed and larger amounts of the pertinent analytes could be transferred to the GC column. In contrast to solid-phase extraction (SPE), the SPME-fiber is reusable several times (as there is no contamination by endogenous compounds). The method is time-saving and can be mechanised by the use of a dedicated autosampler.     

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on “bryndza”, a traditional Slovak dairy product from sheep milk

“Bryndza” is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in “bryndza” was investigated with the aim to reduce the contaminant agents. “Bryndza” was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, “bryndza” was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 °C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in “bryndza” before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10³ cfu/ml/g, Staphylococcus aureus (10² cfu/ml/g) and enterococci (10⁴ cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10² cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10³ cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in “bryndza”. However, bacteriocin activity was not determined by laboratory analyses.     

Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land

Marine pufferfish (family Tetraodontidae) are believed to accumulate tetrodotoxin (TTX) mainly in liver and ovary through the food chain by ingesting TTX-bearing organisms such as starfish, gastropods, crustacean, flatworms, ribbonworms, etc. Consequently, it is hypothesized that non-toxic pufferfish can be produced if they are cultured with TTX-free diets in netcages at sea or aquaria on land, where the invasion of TTX-bearing organisms is completely shut off. To confirm this hypothesis, more than 5000 specimens of the pufferfish (“torafugu”, Takifugu rubripes) cultured in such manners for 1–3 years were collected from several locations in Japan during 2001–2004, and toxicity of their livers and some other parts was examined according to the Japanese official mouse assay method for TTX. In addition, typical specimens were submitted to LC/MS analysis. The results showed that all the livers and other parts tested were ‘non-toxic’ in both of the mouse assay (less than 2 MU/g) and LC/MS analysis (less than 0.1 MU/g). Thus, it is undoubtedly confirmed that pufferfish are intoxicated through the food chain, and non-toxic pufferfish can be successfully produced by netcage or land culture. The livers from these fish can be used with safety as a Japanese traditional food “fugu-kimo” (puffer liver).     

Valproic acid analysis in saliva and serum using selected ion monitoring (electron ionization) of the tert.-butyldimethylsilyl derivatives

A highly sensitive ion monitoring method for the determination of valproic acid in saliva and in serum has been developed based on the gas chromatographic—mass spectrometric analysis of the tert.-butyldimethylsilyl derivatives. Extraction methods are simple and the techniques for derivatization are rapid and convenient. Selected ion monitoring was carried out using electron ionization conditions and a common ion m/z 201 (M⁺ − 57) present in valproic acid and the internal standard octanoic acid. The lower limit of sensitivity that has acceptable precision for assay purposes is 0.1 mg/l based on a 200-μl sample size. The ion monitoring method (derivatized) was compared to a gas chromatographic method (underivatized) for serum valproate assays and found to be essentially identical.The assay methodology was used in a kinetic study of valproic acid in two normal subjects. Saliva levels of drug were found to give reasonably good correlations with serum total and with serum free concentrations of drug in both individuals.     

Some connections between physiognomic traits and intelligence

A sample of 84 female subjects (students) was taken and split up on the basis of the IST-ratings (“Intelligence Structure Test” by Amthauer) into two extreme groups of “more intelligent” (N = 14) and “less intelligent” (N = 11) subjects with an aim to testing Kozeny's findings. Using his photographic-statistical method, Kozeny had observed that “more intelligent” people have a more open eye, a smaller and straighter mouth, a stronger chin, and—above all in its side dimensions—a better developed forehead. These physiognomic indicators of differences in intelligence were confirmed to a large extent by the measurements taken from the portraits of the subjects.     

Liquid chromatographic–electrospray tandem mass spectrometric method for the simultaneous quantitation of the prodrug fosinopril and the active drug fosinoprilat in human serum

A sensitive, specific, accurate and reproducible LC–MS–MS method was developed and validated for the simultaneous quantitation of the prodrug fosinopril and its active drug fosinoprilat in human serum. The method employed acidification of the serum samples to minimize the hydrolysis of fosinopril to fosinoprilat prior to purification by solid-phase extraction to isolate the two analytes and the two internal standards from human serum. The extracted samples were analyzed by turbo ionspray LC–MS–MS in the positive ion mode. Chromatography was performed on a polymer-based C₁₈ column (Asahipak™ ODP PVA-C₁₈, 2×50 mm) using gradient elution with methanol and 10 mM ammonium acetate, pH 5.5. The calibration curve, 1.17 to 300 ng/ml, was fitted to a weighted (l/x) linear regression model. Serum quality control (QC) samples used to gauge the accuracy and precision of the method were prepared at concentrations of 5.00, 100, 250 and 500 ng/ml of each analyte. The inter-assay accuracies were within 6% (DEV) for both analytes. The intra- and inter-assay precisions were within 7% and 11% (RSD), respectively, for both analytes. The hydrolysis of fosinopril to fosinoprilat during sample processing was ≤6%. This degree of conversion would cause little error in the analysis of post-dose serum samples since such samples are known to contain low levels of the prodrug compared to the drug.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.     

Rapid assay for γ-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the analysis of γ-carboxyglutamic acid (Gla) in urine and bone protein hydrolyzates is described. The method employs precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Gla was quantified by reference to an internal standard (β-carboxyaspartic acid). The “within-run” coefficient of variation of the assay for Gla in urine was between 2.1 and 3.4%, and that for bone protein hydrolyzates was 3.2%. The “between-run” coefficient of variation ranged from 4.1 to 5.5%. There was good agreement between the measurement of urinary Gla by high-performance liquid chromatography and amino acid analyzer. Free Gla could not be detected in serum.     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.     

A note on the effectiveness of behavioural rehabilitation for reducing inter-dog aggression in shelter dogs

The effectiveness of a rehabilitation program for reducing inter-dog aggression was evaluated at the municipal animal shelter. Sixteen dogs (of 60 examined) met the study criteria of medium inter-dog aggression as determined by an inter-dog aggression test. These dogs received a 10-day treatment of daily rehabilitation for 30 min (rehabilitation group, n = 9) or daily release into an outdoor enclosure for 30 min (control group, n = 7). Rehabilitation consisted of desensitising and counter-conditioning dogs to the approach of other “stimulus” dogs. Most dogs in the rehabilitation group showed a decline in aggression scores when re-tested after the last treatment (day 11), and differed significantly from the control dogs which showed either an increase or no change in aggression scores (U = 8.5, P < 0.01). Rehabilitation dogs also showed lower frequencies of aggressive body postures (“facing the stimulus dog”, P < 0.05, and “stiff posture”, P < 0.10) and higher frequencies of less assertive postures (“ears back”, P < 0.05, and “lowered neck”, P < 0.10) on day 11. The differences between groups were no longer significant when a reduced sample of dogs was tested 1 week after rehabilitation ended (day 18). The study shows short-term reduction of inter-dog aggression through rehabilitation, but further work is needed on effective ways of maintaining the behavioural change.     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

Therapeutic drug monitoring of flecainide in serum using high-performance liquid chromatography and electrospray mass spectrometry

High-performance liquid chromatography with electrospray mass spectrometry (LC–MS) was used for analysis of the drug flecainide in serum. The clean-up was performed by solid-phase extraction, and an aromatic ring positional isomer was used as internal standard. Results from method validation on spiked serum samples showed excellent reproducibility; intra- and inter-assay variations (C.V.% and %Bias) were less than 6% within the therapeutic concentration range of the drug (0.2–1.0 μg/ml). Linearity was demonstrated from 0.05 to 2.0 μg/ml. The limit of detection and quantification was 0.025 and 0.05 μg/ml, respectively. Due to the high selectivity of the mass spectrometric detection, no interferences were observed. Results from clinical samples (n=18) from patients in treatment with Tambocor (flecainide acetate) showed excellent correlation with parallel data obtained from a method based on high-performance liquid chromatography (HPLC) with fluorescence detection after liquid/liquid extraction. The chromatographic separation of flecainide and internal standard was improved compared to earlier HPLC methods. The methodology is simple, accurate and requires only 0.25 ml of sample. It is a well suited method for routine therapeutic drug monitoring in a hospital or clinical chemistry laboratory.     

A label-free mass spectrometry method for the quantification of protein isotypes

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIII-tubulin represented 3.2% of the total HeLa β-tubulin.     

Rapid monitoring of cell size, vitality and lipid droplet development in the oleaginous yeast Waltomyces lipofer

The aim of this work was the development of rapid methods suitable for monitoring the growth of the oleaginous yeast Waltomyces lipofer by means of cell size, vitality and the development of internal lipid droplets throughout different growth phases. Oleaginous yeasts are of interest for the industrial production of lipids and therefore precise monitoring of growth characteristics is needed.This paper provides information about both the method development as well as about examples for their use in monitoring applications. Cell size and shape were determined using FPIA (Flow Particle Image Analysis). Vitality and internal lipid droplets were measured using two independent staining methods for Flow Cytometry. Double staining with cFDA & PI was used for the distinction between “vital”, “sublethal” and “dead” subpopulations, whereas Nile Red allowed the monitoring of lipid accumulation. In this approach the method for vitality measurement was optimized focussing on the staining buffer. An addition of 25 mM citric acid and pH 4.8 revealed to be optimal. The cells in the growth experiment showed a constantly high vitality, which was always above 90%, but slowly decreasing over time. In the course of lipid droplet development it could be seen that the cell size and the Nile Red fluorescence intensity increased. It was demonstrated that the tested method combination provides a powerful tool for rapid fermentation monitoring of the oleaginous yeast W. lipofer, which allows gaining information about the desired growth characteristics in less than 45 min. Further applications for the two methods will be discussed in this article.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 μm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5±2.8% with a linear range of 0.1–100 μg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

Correlation between Serum Levels of 3,3?,5?-Triiodothyronine and Thyroid Hormones Measured by Liquid Chromatography-Tandem Mass Spectrometry and Immunoassay

ObjectiveFor measuring serum 3,3′,5′-triiodothyronine (rT3) levels, radioimmunoassay (RIA) has traditionally been used owing to the lack of other reliable methods; however, it has recently become difficult to perform. Meanwhile, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been attracting attention as a novel alternative method in clinical chemistry. To the best of our knowledge, there are no studies to date comparing results of the quantification of human serum rT3 between LC-MS/MS and RIA. We therefore examined the feasibility of LC-MS/MS as a novel alternative method for measuring serum rT3, thyroxine (T4), and 3,5,3′-triiodothyronine (T3) levels.MethodsAssay validation was performed by LC-MS/MS using quality control samples of rT3, T4, and T3 at 4 various concentrations which were prepared from reference compounds. Serum samples of 50 outpatients in our department were quantified both by LC-MS/MS and conventional immunoassay for rT3, T4, and T3. Correlation coefficients between the 2 measurement methods were statistically analyzed respectively.ResultsMatrix effects were not observed with our method. Intra-day and inter-day precisions were less than 10.8% and 9.6% for each analyte at each quality control level, respectively. Intra-day and inter-day accuracies were between 96.2% and 110%, and between 98.3% and 108.6%, respectively. The lower limit of quantification was 0.05 ng/mL. Strong correlations were observed between the 2 measurement methods (correlation coefficient, T4: 0.976, p < 0.001; T3: 0.912, p < 0.001; rT3: 0.928, p < 0.001).ConclusionsOur LC-MS/MS system requires no manual cleanup operation, and the process after application of a sample is fully automated; furthermore, it was found to be highly sensitive, and superior in both precision and accuracy. The correlation between the 2 methods over a wide range of concentrations was strong. LC-MS/MS is therefore expected to become a useful tool for clinical diagnosis and research.     

The Phylogenetics of Mycotoxin and Sclerotium Production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called “S” and “L.” The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as “S” or “L” based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the “S” sclerotium phenotype, whereas group I strains consisted of both “S” and “L” isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

Liquid chromatographic–tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum

An LC–MS–MS method for the analysis of the neuraminidase inhibitor, zanamivir, in human serum is described. Zanamivir was extracted from protein precipitated human serum samples using Isolute SCX solid-phase extraction cartridges and analysed using reversed-phase chromatography with TurboIonSpray atmospheric pressure ionisation followed by mass spectrometric detection. The method uses a stable isotope internal standard, is highly specific and sensitive for a compound of this type and has been used for the analysis of human serum and urine samples from clinical studies. The method was extended to the analysis of serum and plasma samples from pre-clinical studies involving the rat, ferret and cell culture media. The method has been shown to be robust and valid over a concentration range of 10–5000 ng/ml using a 0.2-ml sample volume. The main advantages of this method compared to earlier procedures are primarily specificity, sensitivity, ease of sample preparation, small sample volume and short analysis time (ca. 5 min).     

Endocranial volumes of early African hominids, and the role of the brain in human mosaic evolution

Volumetric data are presented for 16 of the early hominids from both South and East Africa. Although the sample sizes are small, the statistical data support the conclusion that at least three taxa are represented; Australopithecus africanus, A. robustus, and Homo habilis. These data, plus certain morphological attributes, indicate that the brains of early hominids were reorganized to a human pattern, regardless of their small endocranial capacities. Some speculative suggestions are made regarding the possible relationship between brain and body weights, as well as Stephan's (1972) “progression indices”. If the speculations are correct, they provide additional support for the idea that brain reorganization occurred early in human evolution, and that concepts which regard the brain as having a more terminal role in human mosaic evolution are incorrect, as all of the fossil encephalization or “progression indices” are in the range of modern Homo sapiens.