Development and validation of method for analysis of some ototoxic solvents in saliva matrix by headspace gas chromatography/mass spectrometry

The aim of this study was to develop an analytical method to monitor the saliva matrix for ototoxic solvents absorption: the method is based on headspace gas chromatography/mass spectrometry and represents an alternative biological monitoring for investigating low exposure to hazardous ototoxic solvents. Simultaneous determination of toluene, ethylbenzene, xylenes and styrene has been carried out and the method has been optimized for both instrumental parameters and samples treatment. Chromatographic conditions have been set in order to obtain a good separation of xylene isomers due to the interest in p-xylene as ototoxic one. Method validation has been performed on standards spiked in blank saliva by using two internal standards (2-fluorotoluene and deuterated styrene-d₈). This method showed the possibility to detect the target compounds with a linear dynamic range of at least a 2 orders of magnitude characterized by a linear determination coefficient (r²) greater than 0.999. The limit of detection (LOD) ranged between 0.19 ng/mL (styrene) and 0.54 ng/mL (m-xylene) and the lower limit of quantification (LLOQ) ranged between 0.64 ng/mL (styrene) and 1.8 ng/mL (m-xylene). The method achieved good accuracy (from 99 to 105%) and precision for both intra- and inter-assay (relative standard deviation ranging from 1.7 to 13.8%) for all six compounds concerned. The repeatability was improved by adding sodium sulphate to the matrix. Saliva samples resulted stable for at least 7 days after collection, if stored in headspace vials, at the temperature of 4 °C. An evaluation of the main sources of uncertainty of the method is also included: expanded uncertainties ranges between 10 and 16% for all of the target compounds. In summary, the headspace gas chromatography/mass spectrometry method is a highly sensitive, versatile and flexible technique for the biological monitoring of exposure to ototoxic solvents by saliva analysis.     

Quantitation of tetramethylene disulfotetramine in human urine using isotope dilution gas chromatography mass spectrometry (GC/MS and GC/MS/MS)

Tetramethylene disulfotetramine (tetramine) is a rodenticide associated with numerous poisonings was extracted and quantified in human urine using both gas chromatography/mass spectrometry (GC/MS) and GC/tandem mass spectrometry (MS/MS). 1200 μL samples were prepared using a ¹³C₄-labeled internal standard, a 96-well format, and a polydivinyl-benzene solid phase extraction sorbent bed. Relative extraction recovery was greater than 80% at 100 ng/mL. Following extraction, samples were preconcentrated by evaporation at 60 °C, and reconstituted in 50 μL acetonitrile. One-microliter was injected in a splitless mode on both instruments similarly equipped with 30 m × 0.25 mm × 25 μm, 5% phenyl-methylpolysiloxane gas chromatography columns. A quantification ion and a confirmation ion (GC/MS) or analogous selected reaction monitoring transitions (GC/MS/MS) were integrated for all reported results. The method was characterized for precision (5.92–13.4%) and accuracy (96.4–111%) using tetramine-enriched human urine pools between 5 and 250 ng/mL. The method limit of detection was calculated to be 2.34 and 3.87 ng/mL for GC/MS and GC/MS/MS, respectively. A reference range of 100 unexposed human urine samples was analyzed for potential endogenous interferences on both instruments—none were detected. Based on previous literature values for tetramine poisonings, this urinary method should be suitable for measuring low, moderate, and severe tetramine exposures.     

Development of high performance liquid chromatography/electrospray ionization mass spectrometry for assay of ginkgolic acid (15:1) in rat plasma and its application to pharmacokinetics study

A highly sensitive HPLC–ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50 μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C₁₈ column (2.1 × 100 mm, 3 μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8–1000 ng/mL with the square regression coefficient (r²) of 0.996. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n = 6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10 mg/kg. GA (15:1) pharmacokinetic parameters C_max, T_max, t_1/2, AUC_0–12h are 1552.9 ± 241.0 ng/mL, 0.9 ± 0.7 h, 5.5 ± 2.6 h, 3356.0 ± 795.3 ng h/mL, respectively.     

Confirmatory analysis of buprenorphine,norbuprenorphine, and glucuronide metabolites in plasma by LCMSMS. Application to umbilical cord plasma from buprenorphine-maintained pregnant women

An LCMSMS method was developed and fully validated for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine-glucuronide (BUP-Gluc), and norbuprenorphine-glucuronide (NBUP-Gluc) in 0.5 mL plasma, fulfilling confirmation criteria with two transitions for each compound with acceptable relative ion intensities. Transitions monitored were 468.3 > 396.2 and 468.3 > 414.3 for BUP, 414.3 > 340.1 and 414.3 > 326.0 for NBUP, 644.3 > 468.1 and 644.3 > 396.3 for BUP-Gluc, and 590.3 > 414.3 and 590.3 > 396.2 for NBUP-Gluc. Linearity was 0.1–50 ng/mL for BUP and BUP-Gluc, and 0.5–50 ng/mL for NBUP and NBUP-Gluc. Intra-day, inter-day, and total assay imprecision (%RSD) were <16.8%, and analytical recoveries were 88.6–108.7%. Extraction efficiencies ranged from 71.1 to 87.1%, and process efficiencies 48.7 to 127.7%. All compounds showed ion enhancement, except BUP-Gluc that demonstrated ion suppression: variation between 10 different blank plasma specimens was <9.1%. In six umbilical cord plasma specimens from opioid-dependent pregnant women receiving 14–24 mg/day BUP, NBUP-Gluc was the predominant metabolite (29.8 ± 7.6 ng/mL), with BUP-Gluc (4.6 ± 4.8 ng/mL), NBUP (1.5 ± 0.8 ng/mL) and BUP (0.4 ± 0.2 ng/mL). Although BUP biomarkers can be quantified in umbilical cord plasma in low ng/mL concentrations, the significance of these data as predictors of neonatal outcomes is currently unknown.     

Quantitation of sorafenib and its active metabolite sorafenib N-oxide in human plasma by liquid chromatography–tandem mass spectrometry

A simple and rapid method with high performance liquid chromatography/tandem mass spectrometry is described for the quantitation of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma. A protein precipitation extraction procedure was applied to 50 μL of plasma. Chromatographic separation of the two analytes, and the internal standard [²H₃¹³C]-sorafenib, was achieved on a C₁₈ analytical column and isocratic flow at 0.3 mL/min for 4 min. Mean within-run and between-run precision for all analytes were <6.9% and accuracy was <5.3%. Calibration curves were linear over the concentration range of 50–10,000 ng/mL for sorafenib and 10–2500 ng/mL for sorafenib N-oxide. This method allows a specific, sensitive, and reliable determination of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma in a single analytical run.     

Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid–liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C₁₈ column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three “in house” QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.     

Effects of dietary vitamin supplementation and semen collection frequency on hormonal profile during ejaculation in the boar

To evaluate the effect of dietary and management factors on boar hormonal status during ejaculation, 39 boars were canulated to determine the profiles of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17β-estradiol (E₂), and testosterone (T) in blood plasma and seminal fluid. Prior to canulation, 18 boars were fed a basal diet (control), whereas the remainder (n = 21) were fed a basal diet supplemented with extra vitamins (supplemented). Within each dietary treatment, two regimens of semen collection were used over the 3 mo preceding the hormonal evaluation: three times per 2 wk (3/2) or three times per wk (3/1). Plasma E₂ was lower (P < 0.01) before ejaculation (232.5 ± 22.6 pg/mL) than at the onset of ejaculation (255.2 ± 27.1 ng/mL). Plasma T increased from 5.14 ± 0.72, before ejaculation to 5.87 ± 0.86 ng/mL at the onset of ejaculation in supplemented boars, whereas it decreased from 5.15 ± 0.65 to 4.87 ± 0.70 ng/mL in controls (diet by time, P < 0.05). At the onset of ejaculation, plasma FSH was higher in 3/2 boars (0.436 ± 0.06 ng/mL) than in 3/1 boars (0.266 ± 0.04 ng/mL; P < 0.05). During ejaculation, plasma LH increased linearly (P < 0.01) from 0.59 ± 0.07 to 0.97 ± 0.10 ng/mL, and plasma E₂ and T concentrations were correlated (r = 0.62, P < 0.01). Plasma FSH before and during ejaculation was negatively correlated with sperm production (r = −0.60, P < 0.01) and testicular weight (r = −0.50, P < 0.01). In conclusion, dietary and management factors had few impacts on hormonal profiles during ejaculation, but homeostasis of some hormones was related to some criteria of reproductive performance in boars.     

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam,and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D₅) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.     

Determination of TJ0711 hydrochloride in rat plasma by high performance liquid chromatography with fluorescence detection and its application to pharmacokinetics

In the present study, a simple and sensitive high performance liquid chromatography with fluorescence detection (HPLC-FD) method was developed to determine TJ0711 hydrochloride, a novel α- and β-receptor blocker. TJ0711 hydrochloride and verapamil hydrochloride (the internal standard) were separated on Knauer Eurospher C₁₈ (250 mm × 4.0 mm i.d., 5 μm) column at 50 °C. The mobile phase was methanol:perchloric acid (12 nM, aq) (56:44, v:v), with a flow rate of 1.0 mL/min. The wavelengths of FD were set at 246 nm for excitation and 300 nm for emission. For plasma samples of rats, the analytes were extracted with acetic ether from alkalinized plasma, and then back-extracted into 10 mM dilute sulfuric acid. The linearity was over a concentration range of 20–10,000 ng/mL. The intra- and inter-day precisions referred by relative standard deviation were less than 2.0% and 4.3%, respectively. The mean analytical recoveries of TJ0711 hydrochloride at different concentrations (50, 1000 and 8000 ng/mL) ranged from 88.3% to 92.9%. The lower limit of quantification (LLOQ) was 20 ng/mL. Finally, this method was successfully applied to the estimation of pharmacokinetic parameters of TJ0711 hydrochloride after intravenous doses of 4, 8 and 16 mg/kg in rats.     

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC–MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm × 50 mm, 3 μm) using a mobile phase of ACN/H₂O (80/20, v/v) containing 10 mM NH₄Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408 → 235 for sitagliptin and m/z 412 → 239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 μL of plasma is processed. The linear calibration range is 1–1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n = 6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

A fast and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of cudratricusxanthone B in rats

A method for the quantitative analysis of cudratricusxanthone B (CXB) in rat plasma by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) has been developed and validated. The method involved liquid–liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C₁₈ column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the internal standard (I.S.) cudraxanthone H. The standard curves were linear over the concentration range of 1–500 ng/mL for CXB in rat plasma. The intra- and inter-batch accuracy for CXB at four concentrations was 89.4–99.5% and 89.4–100.8%, respectively. The RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/mL using 100 μL of plasma. The average extraction recoveries of CXB ranged from 80.1 to 95.4% at the concentrations of 2, 50 and 500 ng/mL, respectively. This method was successfully applied to the pharmacokinetic study after an intravenous administration of CXB in male Sprague–Dawley (SD) rats.     

HPLC method with fluorescence detection for the quantitative determination of flaxseed lignans

We report a rapid and simple HPLC method with fluorescence detection for the quantification of the major flaxseed lignan, secoisolarisiresinol diglucoside (SDG) and its major metabolites. The method is specific for SDG, secoisolarisiresinol (SECO), enterodiol (ED) and entrolactone (EL) in rat serum. The assay procedure involves chromatographic separation using a Waters Symmetry C₁₈ reversed-phase column (4.6 mm × 150 mm, 5 μm) and mobile phase gradient conditions consisting of acetonitrile (0.1% formic acid) and water (0.1% formic acid). SDG extraction from serum requires the use of Centrifuge filters while SECO, ED and EL are extracted with diethyl ether. The organic layer is evaporated and reconstituted in 100 μL of mobile phase and 50 μL of reconstituted sample or filtrate is injected onto the column. Total run time is 25 min. Calibration curves are linear (r² ≥ 0.997) from 0.05 to 10 μg/mL for SDG and EL and 0.01–10 μg/mL for SECO and ED. Precision and accuracy are within USFDA specified limits. The stability of all lignans is established in auto-injector, bench-top, freeze–thaw and long-term stability at −80 °C for 30 days. The method's reasonable sensitivity and reliance on more widely available HPLC technology should allow for its straightforward application to pharmacokinetic evaluations of lignans in animal model systems such as the rat.     

Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC₅₀ = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC₅₀ = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.     

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied ‘food grade’ sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC₅₀ value of 5.521 mg/mL is high compared with 0.2811 μg/mL (182.5 ρM or 1.825 × 10⁻¹⁰ M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E^® is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%, 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease.     

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C₁₈ column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.     

Isolation and characterization of a novel protein toxin from fire coral

Fire corals (Millepora spp.) cause severe pain and inflammatory effects in humans upon contact, and the organs responsible for these effects are called nematocysts. Here, we isolated an active cytotoxin of ca. 18 kDa (MCTx-1) from nematocysts of Millepora dichotoma var. tenera. MCTx-1 was potently cytotoxic (EC₅₀ value 79 ng/mL) towards L1210 mouse leukemia cells, hemagglutinated a 0.8% suspension of sheep erythrocytes (0.2 μg protein/mL) and was lethal in crayfish (LD₅₀, 106 μg/kg). We deduced the primary structure of MCTx-1 from the corresponding cDNA sequence and found that MCTx-1 is a novel dermatopontin that is an extracellular matrix protein in mammals. This is the first characterization of a proteinaceous toxin from fire coral.     

Effect of feed to inoculum ratios on biogas yields of food and green wastes

Biogas and methane yields of food and green wastes and their mixture were determined using batch anaerobic digesters at mesophilic (35 ± 2 °C) and thermophilic (50 ± 2 °C) temperatures. The mixture was composed of 50% food waste and 50% green waste, based on the volatile solids (VS) initially added to the reactors. The thermophilic digestion tests were performed with four different feed to inoculum (F/I) ratios (i.e., 1.6, 3.1, 4.0 and 5.0) and the mesophilic digestion was conducted at one F/I (3.1). The results showed that the F/I significantly affected the biogas production rate. At four F/Is tested, after 25 days of thermophilic digestion, the biogas yield was determined to be 778, 742, 784 and 396 mL/g VS for food waste, respectively; 631, 529, 524 and 407 mL/g VS for green waste, respectively; and 716, 613, 671 and 555 mL/g VS for the mixture, respectively. About 80% of the biogas production was obtained during the first 10 days of digestion. At the F/I of 3.1, the biogas and methane yields from mesophilic digestion of food waste, green waste and their mixture were lower than the yields obtained at thermophilic temperature. The biogas yields were 430, 372 and 358 mL/g VS, respectively, and the methane yields were 245, 206, and 185 mL/g VS, respectively.     

Simple,sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma

A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C₈ column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]⁺ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.     

Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the RIA-706 and RIA-srPool methods, respectively. From Day 24 to Day 50, using both RIA methods, PAGs could be detected in all pregnant ewes. On Day 24, the best threshold for pregnancy diagnosis was obtained by use of RIA-srPool, maximal concentration in nonpregnant ewes being 0.3 ng/mL and minimal concentration in pregnant ewes being 4.8 ng/mL. In general, progesterone and PAG concentrations were higher in multiple pregnancies than in single pregnancies. However, because of large individual variations, single pregnancies could not be differentiated from multiple pregnancies.