Pyrolysis-field ionization mass spectrometry of agricultural soils and humic substances: Effect of cropping systems and influence of the mineral matrix

Whole soil samples, extracted humic substances, the corresponding fulvic (FA) and humic acids (HA) and the extraction residues (humins) from long-term, agricultural test plots were investigated by in-source pyrolysis-field ionization mass spectrometry (Py-FIMS). For the soils distinct differences in the chemical composition of the organic matter in differently managed fields were observed. The FI mass spectra of the extracted humic substances gave complementary chemical information, as they cover a larger mass range compared to the whole soil spectra. The chemical, structural information of the conventional alkaline extraction residues was demonstrated by Py-FIMS spectra to be similar to that of the related soil samples. Influences of mineral matrix to organic matter ratios were studied on mixtures of extracted humic substances with defined mineral components such as quartz, basalt, iron oxide (Fe₂O₃), Ca-montmorillonite, kaolinite and illite. It was shown that in these mixtures the number of mass signals detected and the covered mass range decreased, when organic carbon concentrations (C_org) in this synthetic mineral matrix dropped below 2% (w/w). Limitations in the direct application of Py-FIMS might arise in the case of natural soil samples with C_org concentrations below 0.5% (w/w), high contents of swelling clay minerals and iron oxides. ei]{gnR}{fnMerckx}     

Quantitative analysis of C60 fullerene in blood and tissues by high-performance liquid chromatography with photodiode-array and mass spectrometric detection

A high-performance liquid chromatographic (HPLC) assay method for C₆₀ fullerene, in blood, liver and spleen using photodiode-array detection or mass spectrometric detection (LC–MS) and C₇₀ fullerene, as the internal standard, is described. The recovery from mouse blood and tissues spiked with micronized C₆₀ exceeds 90%. The method is linear from 0.05 to 200 mg of C₆₀ per liter of blood and from 0.05 to 5.00% of C₆₀ per tissue weight. The limit of detection of the method is 0.1 ng of C₆₀ per injection. This method was applied to mouse blood and tissue samples after intraperitoneal administration of a micronized C₆₀ suspension.     

New spectrophotometric procedure for determining cefotaxime based on derivatization with 1,2-naphthoquinone-4-sulphonate into solid-phase extraction cartridges — application to pharmaceutical and urine samples

Cefotaxime was derivatised with 1,2-naphthoquinone-4-sulphonate (NQS), extracted into solid-phase cartridges (C₁₈) and detected using a UV–visible detection system. Optimum conditions for this new procedure were: hydrogencarbonate–carbonate buffer, pH 10.5, 5-min reaction time at 25°C and an NQS concentration of 7.1·10⁻³ mol l⁻¹. The accuracy and the precision of the liquid–solid procedure were tested. The procedure was used to measure cefotaxime in pharmaceutical and urine samples. The results obtained were contrasted with those reported for a HPLC method for urine samples. The generalized H-point standard additions method was used to measure cefotaxime in urine samples.     

Identification and quantification of five macrolide antibiotics in several tissues, eggs and milk by liquid chromatography–electrospray tandem mass spectrometry

We present an electrospray high-performance liquid chromatographic tandem mass spectrometric (HPLC–MS–MS) method capable of determining in several tissues (muscle, kidney, liver), eggs and milk the following five macrolides: tylosin, tilmicosin, spiramycin, josamycin, erythromycin. Roxithromycin was used as an internal standard. The method uses extraction in a Tris buffer at pH 10.5, followed by protein precipitation with sodium tungstate and clean-up on an Oasis solid-phase extraction column. The HPLC separation was performed on a Purospher C₁₈ column (125×3 mm I.D.) protected by a guard column, with a gradient of aqueous 0.1 M ammonium acetate–acetonitrile as the mobile phase at a flow-rate of 0.7 ml min⁻¹. Protonated molecules served as precursor ions for electrospray ionisation in the positive ion mode and four product ions were chosen for each analyte for multiple reaction monitoring (MRM). A validation study was conducted to confirm the five macrolides by MRM HPLC–MS–MS analysis of a negative control and fortified samples. All of the samples analysed were confirmed with four ions. The ion ratio reproducibility limit ranged from 2.4 to 15%. All compounds could be detected and quantified at half-maximum residue limits (MRLs). The method is specific, quantitative and reproducible enough to conform to European Union recommendations within the concentration range 0.5 MRL–2 MRL (accuracy: 80 to 110%, relative standard deviation: 2 to 13%). This whole method allows extraction and analysis of up to 50 samples per day.     

Occurrence of aflatoxin M<Subscript>1</Subscript> in commercial pasteurized milk samples in Sari,Mazandaran province,Iran

The frequency and levels of aflatoxin M₁ (AFM₁) in pasteurized milk samples in Sari, located in Mazandaran province, Iran, were determined by enzyme immunoassay. Seventy-six samples of pasteurized milk from different retail stores were randomly collected over four seasons during the year 2015. AFM₁ contamination was detected in all milk samples. The mean concentration of aflatoxin M₁ was 65.8 ng/l, with a range of 11.7–106.6 ng/l. The highest AFM₁ level was detected in milk samples collected during spring. Forty-six (60.53 %) samples had AFM₁ levels that exceeded the maximum acceptable levels (50 ng/l) recommended by the European Union (EU). Comparison of these results with previously published data for AFM₁ in milk in Iran shows that the percentage of samples exceeding the EU maximum level is consistently high over the years, indicating a general problem related to AFB₁ contamination in dairy feedingstuff.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultraperformance liquid chromatography-tandem mass spectrometry

A simple and sensitive analytical method was developed for the simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Samples were directly purified through HLB cartridge. The organic phase was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by UPLC–MS/MS on an Acquity UPLC^® BEH C₁₈ column with gradient elution. The samples were quantified using clenbuterol-D₉, chloramphenicol-D₅ and diethylstilbestrol-D₈ as internal standards. The proposed method was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk. The total time required for the analysis of one sample was about 50 min.     

Occurrence of aflatoxin M1 in conventional and organic milk offered for sale in Italy

In the present study, 58 samples of milk were analyzed for the presence of aflatoxin M₁ (AFM₁). The samples were purchased during the period April–May 2013 in a random manner from local stores (supermarkets, small retail shops, small groceries, and specialized suppliers) located in the surrounding of Bologna (Italy). The commercial samples of milk were either organic (n = 22) or conventional (n = 36); fresh milk samples and UHT milk samples, whole milk samples, and partially skim milk samples were present in both the two considered categories. For the quantification of AFM₁ in milk, the extraction-purification technique based on the use of immunoaffinity columns was adopted and analyses were performed using HPLC-FD. AFM₁ was detected in 35 samples, 11 from organic production and 24 from conventional production. No statistically (P > 0.05) significant differences were observed in the concentration of AFM₁ in the two categories of product. The levels of contamination found in the positive samples ranged between 0.009 and 0.026 ng mL^?1. No sample exceeded the limit defined at community level for AFM₁ in milk (0.05 μg kg^?1). This demonstrates the effectiveness of the checks before the placing on the market of these food products. Thus, the “aflatoxins” problem that characterized the summer of 2012 does not seem to have had effect on the contamination level of the considered milk samples.     

A rapid and robust liquid chromatography/tandem mass spectrometry method for simultaneous analysis of anti-tuberculosis drugs—Ethambutol and pyrazinamide in human plasma

Ethambutol and pyrazinamide are two first-line anti-tuberculosis drugs. Though they are normally combined for the treatment, their highly different polarity complicates simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of these two drugs in human plasma with decent peak shape and retention. Here we report a rapid and robust LC/MS/MS method for the simultaneous determination of these two drugs in human plasma. Human plasma samples, together with the isotopically labeled internal standards were extracted using protein precipitation, and then separated on a Chromolith SpeedROD RP-18e column and detected with mass spectrometry. The mobile phase is 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in methanol. Addition of trifluoroacetic acid in the mobile phases was found to be able to improve peak shape as well as to increase the retention of ethambutol, thus being able to analyze these two drugs at the same time with both drugs having decent peak shape and enough retention on a C₁₈ column. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The standard curve range was 10.0–5000 ng/mL for ethambutol and 50.0–25,000 ng/mL for pyrazinamide using a plasma sample volume of 50.0 μL. This method has a very short run time of 3.8 min. The method has been fully validated, and <15% relative standard deviation was obtained for both analytes.     

The identification and rapid extraction of hydrocarbons from Nicotiana glauca: A potential advanced renewable biofuel source

Declining fossil fuels reserves, a need for increased energy security and concerns over carbon emissions from fossil fuel use are the global drivers for alternative, renewable, biosources of fuels and chemicals. In the present study the identification of long chain (C₂₉–C₃₃) saturated hydrocarbons from Nicotiana glauca leaves is reported. The occurrence of these hydrocarbons was detected by gas chromatography–mass spectrometry (GC–MS) and identification confirmed by comparison of physico-chemical properties displayed by the authentic standards available. A simple, robust procedure was developed to enable the generation of an extract containing a high percentage of hydrocarbons (6.3% by weight of dried leaf material) higher than previous reports in other higher plant species consequently, it is concluded that N. glauca could be a crop of greater importance than previously recognised for biofuel production. The plant can be grown on marginal lands, negating the need to compete with food crops or farmland, and the hydrocarbon extract can be produced in a non-invasive manner, leaving remaining biomass intact for bioethanol production and the generation of valuable co-products.     

Determination of chlorpromazine in human breast milk and serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of chlorpromazine in serum and human breast milk. Chlorpromazine in serum and human breast milk was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction, and a reversed-phase HPLC separation technique was developed. Chlorpromazine and levomepromazine as the internal standard were detected by ultraviolet absorbance at 254 nm. Determination was possible for chlorpromazine in the concentration range 10–300 ng/ml. The recoveries of chlorpromazine added to serum and human breast milk were 80.1–87.6 and 80.3–84.4%, respectively, with coefficients of variation of less than 10.2 and 7.8%. The method is applicable to drug level monitoring in the serum and human breast milk of patients treated with chlorpromazine.     

Discovery of the oldest known biomarkers provides evidence for phototrophic bacteria in the 1.73 Ga Wollogorang Formation,Australia

The discovery of mid‐Proterozoic (1.8–0.8 billion years ago, Ga) indigenous biomarkers is a challenge, since biologically informative molecules of such antiquity are commonly destroyed by metamorphism or overprinted by drilling fluids and other anthropogenic petroleum products. Previously, the oldest clearly indigenous biomarkers were reported from the 1.64 Ga Barney Creek Formation in the northern Australian McArthur Basin. In this study, we present the discovery of biomarker molecules from carbonaceous shales of the 1.73 Ga Wollogorang Formation in the southern McArthur Basin, extending the biomarker record back in time by ~90 million years. The extracted hydrocarbons illustrate typical mid‐Proterozoic signatures with a large unresolved complex mixture, high methyl alkane/n‐alkane ratios and the absence of eukaryotic steranes. Acyclic isoprenoids, saturated carotenoid derivatives, bacterial hopanes and aromatic hopanoids and steroids also were below detection limits. However, continuous homologous series of low molecular weight C₁₄–C₁₉ 2,3,4‐ and 2,3,6‐trimethyl aryl isoprenoids (AI) were identified, and C₂₀–C₂₂ AI homologues were tentatively identified. Based on elevated abundances relative to abiogenic isomers, we interpret the 2,3,6‐AI isomer series as biogenic molecules and the 2,3,4‐AI series as possibly biogenic. The biological sources for the 2,3,6‐AI series include carotenoids of cyanobacteria and/or green sulphur bacteria (Chlorobiaceae). The lower concentrated 2,3,4‐AI series may be derived from purple sulphur bacteria (Chromatiaceae). These degradation products of carotenoids are the oldest known clearly indigenous molecules of likely biogenic origin.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.     

The bioavailability of colloidal and dissolved organic phosphorus to the alga Pseudokirchneriella subcapitata in relation to analytical phosphorus measurements

The speciation of phosphorus (P) in freshwater affects its bioavailability. Analytical detection methods were compared for different colloidal and dissolved organic forms of phosphorus (P) in relation to the potential P bioavailability to Pseudokirchneriella subcapitata, determined with a 14 day growth response to P. Growth on these P-forms was referenced to supplies of inorganic P (P_i) as operational definition of the relative potential bioavailability. The bioavailability of ten organic P molecules ranged 1–70% of P_i while these forms were generally not detected by colorimetric method (CM, malachite green) or ion chromatography (IC). The bioavailability of P associated with Fe- and Al oxides ranged 55–85% of P_i and these forms were completely detected by CM and partially by IC. The bioavailability of total dissolved P in the environmental samples ranged 7–85% (mean 43%) of P_i. The P detected by IC underestimates bioavailable P while CM and total dissolved P (inductively coupled plasma, ICP) overestimate P bioavailability by, on average 44% (CM) or 57% (ICP) in the environmental samples. We conclude that CM is the best index among the three tested for predicting long-term availability of environmental dissolved P in which colloidal P contributes more importantly than organic P.     

Determination of clofilium, a new antifibrillatory agent, in plasma by gas chromatography—mass spectrometry

A sensitive and selective method for the assay of the new quaternary amine antifibrillatory agent clofilium is described. Plasma samples were extracted with dichloromethane (98.5 ± 0.2% recovery) and analyzed by gas chromatography—mass spectrometry operating in the electron-impact mode. The method involves a Hofmann elimination of an N-alkyl radical from clofilium and the internal standard in the presence of a strong nucleophile in the injector of the gas chromatograph. The resulting tertiary amines are chromatographed and detected by selective ion monitoring. The ratio of the clofilium base peak (m/z 224) to the internal standard peak (m/z 210) was linear relative to the plasma clofilium concentration over the range of 25–1000 ng/ml plasma.     

A re-examination of corn (Zea mays L.) ear volatiles

Helicoverpa zea (Boddie) is a major insect pest of corn and other agricultural crops. An improved understanding of semiochemcials that control adult behavior is needed to develop alternative control measures. In this study, overnight SPME collection of volatiles from corn ears enclosed in Teflon bags in the field at two stages of development were made. C₈–C₁₀ aldehydes, a C₈-alcohol, C₆–C₉ alcohol acetates, and numerous monoterpenes, sesquiterpenes, sequiterpene alcohols, and geosmin were identified after thermal desorption and GC/MS. Structural assignments of the alcohol acetates, Z-3-hexenyl acetate, 2-heptyl acetate, 2-nonyl acetate, and 4-nonyl acetate, the monoterpenes, α- and β-ocimene, and geosmin were made by analysis of standards that were purchased or prepared in the laboratory. All other assignments were based on published Kovat’s retention time indices (KI) and mass spectra. Pair-wise comparison of the relative amounts of each component between two groups of corn ears defined by silk weight did not identify significant differences, thus it is unknown whether or not silk weight impacted volatile emission composition and rate. To our knowledge three compounds detected in SPME collections, 2-heptyl acetate, 2-nonyl acetate, and 4-nonyl acetate have not been previously reported in corn ear or silk volatiles. Their impact on the flight response of gravid earworm females was evaluated in a flight chamber. No significant response to the individual compounds or a blend of all three was observed. Thus, their impact on moth behavior remains uncertain.     

Amphetamine and methamphetamine determination in urine by reversed-phase high-performance liquid chromatography with sodium 1,2-napthoquinone 4-sulfonate as derivatizing agent and solid-phase extraction for sample clean-up

A rapid method is described for the identification and determination of amphetamine and methamphetamine in human urine samples by liquid chromatography with UV-Vis detection. The samples were transferred onto a C₁₈ solid-phase extraction column and chromatographed on a Hypersil ODS RP C₁₈, 5 μm (250 × 4 mm I.D.) with an acetonitrile-water elution gradient containing propylamine. Under these conditions, the amines are eluted with a short retention time. The procedure has been applied to the determination of amphetamine and methamphetamine in the range 0.3–4.0 μg/ml in spiked urine samples. The detection limits at 280 nm were 4 and 2 ng/ml for amphetamine and methamphetamine, respectively. The intra-day and inter-day precision and accuracy of the method were studied.     

Effects of intravenous infusions of acetate, β-hydroxybutyrate, triglyceride and other metabolites on the composition of the milk fat and blood in cows

1. The effects in the cow of intravenous infusions of sodium acetate, butyrate, propionate, β-hydroxybutyrate, malonate, citrate or succinate, of glucose or of an emulsion of cottonseed oil on the secretion of the component fatty acids of milk fat and on the composition of the blood plasma of the jugular vein have been studied. 2. Glucose and cottonseed oil were the only metabolities consistently to affect the yield of milk fat. Glucose decreased the yield of milk fat through a diminished secretion of the C₁₈ fatty acids and in two out of three cows also of the steam-volatile fatty acids (C₄–C₁₀). The cottonseed oil caused an increase in the yield of milk fat through an increased secretion of linoleic acid, the major component acid of the cottonseed oil. In three out of four cows, acetate caused an increase in the yield of milk fat through an increased secretion of mainly palmitic acid. 3. The effects of the infusions on milk-fat secretion are discussed in relation to existing knowledge on the origin of the fatty acids of milk fat.     

Effect in the cow of intraruminal infusions of volatile fatty acids and of lactic acid on the secretion of the component fatty acids of the milk fat and on the composition of blood

1. The effects in the cow of intraruminal infusions of acetic acid, propionic acid or butyric acid on the secretion of the component fatty acids of the milk fat, and of these acids and of lactic acid on the composition of the blood plasma of the jugular vein, have been studied. 2. The infusion of acetic acid or butyric acid increased the yield of the C₄–C₁₆ acids of milk fat but decreased the yield of C₁₈ acids. The infusion of propionic acid decreased the yields of all major component acids except palmitic acid and possibly lauric acid. 3. The changes in the concentrations in blood plasma of glucose and of ketone bodies were consistent with the glucogenic effect of propionic acid and the ketogenic effects of butyric acid and acetic acid. The effects of lactic acid were not consistent from cow to cow. Only with the infusion of acetic acid was a significant increase in the concentration of total volatile fatty acids in blood plasma found. Infusions of butyric acid and of propionic acid tended to depress the concentration of citric acid in the blood plasma and infusion of acetic acid increased it. No consistent effects of the infused acids on the concentration in blood plasma of esterified cholesterol, free cholesterol, triglyceride or phospholipid were observed. 4. The possibility is discussed that the effects of the infused acids on milk-fat secretion are caused through an alteration of the concentrations of precursors of milk fat in mammary arterial blood.     

Determination of gentamicin in bovine milk using liquid chromatography with post-column derivatization and fluorescence detection

A method capable of separating and quantifying the three major and one minor components of gentamicin in milk has been developed. The method is capable of detecting 15 ng/ml gentamicin, based on a total of the four components. Milk samples are centrifuged at 4°C, the fat layer removed, and the samples deproteinated with 30% trichloracetic acid. After a second centrifugation, the supernatant is passed through a C₁₈ solid-phase extraction column. The column is washed with water, water-methanol (50:50) and methanol. Ammonium hydroxide (16%) in methanol is used to elute the gentamicin. The eluent is evaporated to near dryness and taken up with water. An aliquot of the sample is then mixed with an ion-pairing reagent for chromatography. Separation is achieved using pentanesulfonic acid in a water-methanol mobile phase on a C₁₈ reversed-phase column. The o-phthalaldehyde fluorescence derivatives of gentamicin are formed post-column and are detected with excitation at 340 nm and emission at 430 nm. The percent recovery of gentamicin averaged 72, 78 and 88% from milk samples fortified at 15, 30 and 60 ng/ml, respectively.