Development and validation of a liquid chromatography-electrosprayionization mass spectrometric method for the determination of dexamethasone in sheep plasma

A quantitative liquid chromatographic-electrospray ionization mass spectrometric method for the determination of dexamethasone in sheep plasma has been developed and validated. The samples were extracted using solid-phase extraction cartridges with mixed reversed-phase materials (oasis-HLB). The chromatographic separation was performed on a reversed-phase XTerrra MS C₁₈ column ( mm; 5 μm) using a mobile phase consisting of 65% methanol in water containing 0.1% (v/v) formic acid, pumped at a flow rate of 0.30 ml min⁻¹. The analyte was detected after positive electrospray ionization using selected ion monitoring (SIM) mode. The probe heater temperature was set at 260 °C, the capillary voltage was set at 3.5 kV and the source block voltage (AQA_max) was set at 30 V. The method was fully validated. Calibration graphs were linear (r better than 0.998, n=11), in concentration ranges 6-1000 ng ml⁻¹ for dexamethasone. The intra- and inter-day RSD values were less than 24.1% (n=6). The limits of detection and quantitation for dexamethasone were found to be 1 and 6 ng ml⁻¹, respectively. The efficiency of the solid phase extraction procedure was found to be 92.4% for dexamethasone. The method was further applied to a pilot kinetic study in order to assess the main pharmacokinetic parameters of dexamethasone in sheep.     

Enantioselective analysis of mirtazapine and its two major metabolites in human plasma by liquid chromatography-mass spectrometry after three-phase liquid-phase microextraction

A three-phase liquid-phase microextraction (LPME) method using porous polypropylene hollow fibre membrane with a sealed end was developed for the extraction of mirtazapine (MRT) and its two major metabolites, 8-hydroxymirtazapine (8-OHM) and demethylmirtazapine (DMR), from human plasma. The analytes were extracted from 1.0 mL of plasma, previously diluted and alkalinized with 3.0 mL 0.5 mol L⁻¹ pH 8 phosphate buffer solution and supplemented with 15% sodium chloride (NaCl), using n-hexyl ether as organic solvent and 0.01 moL L⁻¹ acetic acid solution as the acceptor phase. Haloperidol was used as internal standard. The chromatographic analyses were carried out on a chiral column, using acetonitrile-methanol-ethanol (98:1:1, v/v/v) plus 0.2% diethylamine as mobile phase, at a flow rate of 1.0 mL min⁻¹. Multi-reaction monitoring (MRM) detection was performed by mass spectrometry (MS-MS) using a triple-stage quadrupole and electrospray ionization interface operating in the positive ion mode. The mean recoveries were in 18.3-45.5% range with linear responses over the 1.25-125 ng mL⁻¹ concentration range for all enantiomers evaluated. The quantification limit (LOQ) was 1.25 ng mL⁻¹. Within-day and between-day assay precision and accuracy (2.5, 50 and 100 ng mL⁻¹) showed relative standard deviation and the relative error lower than 11.9% for all enantiomers evaluated. Finally, the method was successfully used for the determination of mirtazapine and its metabolite enantiomers in plasma samples obtained after single drug administration of mirtazapine to a healthy volunteer.     

A rapid determination of propiverine and its N-oxide metabolite in human plasma by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for determination of propiverine and propiverine N-oxide metabolite in human plasma using oxybutynin as internal standard. Instead of extracting propiverine from plasma using organic solvents, which should be separated from the aqueous phase and evaporated before injecting the sample into the chromatograph, plasma sample containing propiverine and N-oxide was directly injected after precipitating proteins with acetonitrile. Numerous compounds in the plasma did not interfere with the highly specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted propiverine, N-oxide and oxybutynin within 2 min (0.1% formic acid in water/acetonitrile, 25:75, v/v). The LC-MS/MS method and an alternative LC-MS method, using methyl-t-butyl ether extraction and selected ion monitoring, were validated over 1-250 ng ml⁻¹ of propiverine and 2 to 500 ng ml⁻¹ of N-oxide, and successfully applied in a pharmacokinetic study. The lower limit of quantitation was 1 ng ml⁻¹ for propiverine and 2 ng ml⁻¹ for N-oxide in both methods.     

Determination of arsenic, iron and selenium in moss samples using hexapole collision cell, inductively coupled plasma-mass spectrometry

Different collision gases (H₂, He and premixed 7% H₂ in He) used in the hexapole collision cell of an inductively coupled plasma-mass spectrometer (ICP-MS) were compared, and the gas-flow rates were optimized for the determination of arsenic (), iron () and selenium (). The study showed that the argon-based interferences at mass-to-charge ratios (m/z) of 56, 75 and 80 can be overcome by the optimized gas flows (7.5 ml min⁻¹ premixed 7% H₂ in He and 2 ml min⁻¹ H₂) in the hexapole collision cell. Detection limits of 15.5 ng l⁻¹ for iron () and 29 ng l⁻¹ for selenium () in 2% (v/v) HNO₃ were obtained under optimized collision cell conditions. The detection limit for arsenic () obtained in difficult hydrochloride acid matrix (5% HCl (v/v)) was 153 ng l⁻¹. The accuracy of the optimized method was confirmed by analyzing two moss reference materials. The results obtained by ICP-MS for arsenic, selenium and iron from both moss reference samples were, in most cases, in good agreement with the certified values.     

A liquid chromatography/positive ion tandem mass spectrometry method for the determination of cilazapril and cilazaprilat in human plasma

A simple, rapid, and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for simultaneous determination of cilazapril levels and its active metabolite, cilazaprilat, in human plasma using enalapril as internal standard. The acquisition was performed in the multiple reaction monitoring mode; monitoring the transitions: m/z 418.4 > 211.1 for cilazapril and m/z 390.3 > 211.1 for cilazaprilat. The method involves a simple single-step liquid-liquid extraction with ethyl acetate. The analyte was chromatographed on an YMC C₈ reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (10:90, v/v; pH 3.2 with formic acid). Numerous compounds did not interfere with specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted cilazapril, cilazaprilat, and enalapril within 2 min. This method was validated over 0.1-500 ng ml⁻¹ of cilazapril and 0.5-50 ng ml⁻¹ of cilazaprilat. Cilazapril and cilazaprilat were stable in standard solution and in plasma samples under typical storage and processing conditions. The assay was successfully applied to a pharmacokinetic study of cilazapril given as a single oral dose (5 mg) to healthy volunteers.     

Determination of chromium, cadmium and lead in food-packaging materials by axial inductively coupled plasma time-of-flight mass spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS) with a time-of-flight (TOF) analyser was used for the determination of chromium, cadmium and lead in six food-packaging materials (paper and paper board). The samples (0.20-0.25 g) were digested in concentrated nitric acid in a high pressure microwave oven at 180 °C within 15 min. Two different plasma conditions were applied: cool plasma conditions (0.76 kW; 0.85, 0.89 and 15.5 l min⁻¹ nebuliser, auxiliary and plasma gas flow rate, respectively) for the determination of chromium and normal plasma conditions (1.21 kW; 0.66, 0.68 and 13.6 l min⁻¹ nebuliser, auxiliary and plasma gas flow rate, respectively) for the determination of cadmium and lead. External calibration was used in combination with rhodium (40 ng g⁻¹) as an internal standard. The detection limits (DL = 3S.D./sensitivity) under the conditions used corresponded to 0.01 ng g⁻¹ (), 0.06 ng g⁻¹ (), 0.07 ng g⁻¹ (), 0.03 ng g⁻¹ () and 0.02 ng g⁻¹ ( and ). The precision (R.S.D.) for six replicate determinations (10 s integration time) of 1 and 10 ng g⁻¹ of each analyte varied from 0.72% () to 4.43% (). The contents of chromium, cadmium and lead in the examined materials were evaluated using the signals of , and . They were in the range: 0.25-0.50 μg g⁻¹ for Cr, not detected (nd) to 0.12 μg g⁻¹ for Cd and 0.28-0.35 μg g⁻¹ for Pb in paper and 0.50-0.64 μg g⁻¹ for Cr, nd to 0.09 μg g⁻¹ for Cd and 0.67-0.99 μg g⁻¹ for Pb in paper board.     

Simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their hydroxy/N(4)-acetyl metabolites with gradient liquid chromatography in chicken plasma, tissues, and eggs

A simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their hydroxy/N⁴-acetyl metabolites in chicken plasma, muscle, liver, and eggs using gradient high-performance liquid chromatography (HPLC) with a photo-diode array detector is developed. All the compounds are extracted by a handheld ultrasonic homogenizer with ethanol followed by centrifugation. The separation is performed by a reversed-phase C4 column with a gradient elution (ethanol:1% (v/v) acetic acid, v/v; 10:90 → 20:80). Average recoveries from samples spiked at 0.1-1.0 μg g⁻¹ or μg ml⁻¹ for each drug were >90% with relative standard deviations within 4%. The limits of quantitation were <30 ng g⁻¹ or ng ml⁻¹.     

Determination of total bilirubin in human serum by chemiluminescence from the reaction of bilirubin and peroxynitrite

Peroxynitrous acid (ONOOH) was produced by the on-line reaction of acidified hydrogen peroxide with nitrite in a flow system, and peroxynitrite (ONOO⁻) was generated from ONOOH in NaOH solution. A weak chemiluminescent (CL) emission was observed due to the production of singlet oxygen ( ) during the decomposition of ONOO⁻. Bilirubin and its conjugate were found to enhance the CL emission of in a suitable micellar medium. For the first time, the feasibility of employing the present CL system for the sensitive and selective determination of total bilirubin contents in human serum was demonstrated and the results were compared with certified values. The present method showed a great improvement on overcoming bis(2,4,6-trichlorophenyl)oxalate CL highly insolubility in aqueous solution and exhibiting higher tolerance towards interferences than redox reaction of bilirubin with various oxidizing agents such as sodium hypochlorite and iodine. The recoveries of bilirubin were found to fall in the range between 95 and 108%. The detection limits (S/N=3) for bilirubin and its conjugate were determined to be 10 and 8 ng ml⁻¹, respectively. The relative standard deviations (R.S.D.) for the consecutive CL detection of a series of bilirubin (30 μg l⁻¹) and bilirubin ditaurite (25 μg l⁻¹) were 3.2 and 2.9% (n=11), respectively.     

Room-temperature luminescence optosensings based on immobilized active principles actives: Application to nafronyl and naproxen determination in pharmaceutical preparations and biological fluids

This paper presents two easy and selective methods for determining the active principles nafronyl (NFL) and naproxen (NAP), using a flow-through fluorescence optosensor based on the on-line immobilization on a nonionic-exchanger (Silica Gel, Davisil™ and Amberlite XAD 7, respectively) solid support. The determination was performed in 5×10⁻³ M HAc/NaAc buffer solution at pH 5 for NFL and 15×10⁻³ M glycine/HCl buffer solution at pH 2.5 for NAP at a working temperature of 20 °C. The fluorescence intensities were measured at λ_ex/em=294/336 nm and λ_ex/em=332/354 nm for NFL and NAP, respectively. The response time for these optosensors were practically instant, obtaining a linear concentration range between 0 and 700.0 ng ml⁻¹ with a detection limit of 20.8 ng ml⁻¹, an analytical sensitivity of 10.1 ng ml⁻¹ and a standard deviation of 1.27% at a 500 ng ml⁻¹ concentration level for NFL and a linear concentration range between 0 and 200.0 ng ml⁻¹ with the detection limit of 13.3 ng ml⁻¹, an analytical sensitivity of 6.0 ng ml⁻¹ and a standard deviation of 3.52% at a 100 ng ml⁻¹ concentration level for NAP. The proposed methods were satisfactorily applied to real samples (three commercial formulations and urine samples). The effects of the possible interferences were evaluated in all cases.     

On-line preconcentration of palladium on alumina microcolumns and determination in urban waters by inductively coupled plasma mass spectrometry

A method for on-line preconcentration of palladium at ultra-trace level on alumina microcolumns and determination by inductively coupled plasma mass spectrometry (ICP-MS) has been developed. A sampling time of 10 min (30 ml) and an eluent (KCN) volume of 300 μl provides a limit of detection of 1 ng l⁻¹. The precision was 4% at the 25 ng l⁻¹ level. The proposed system allows the on-line removal of the primary interferents (Cu and Y) providing the credible accuracy of the results. The proposed method is suitable for palladium determination in urban water samples. Platinum concentration in the analysed urban water samples was also determined.     

Determination of Imidacloprid and metabolites by liquid chromatography with an electrochemical detector and post column photochemical reactor

A procedure for the determination of Imidacloprid and its main metabolites was set up by means of liquid chromatography with an electrochemical detector and post-column photochemical reactor (LC-hν-ED). Sample clean-up was developed for bees, filter paper and maize leaves. Chromatographic conditions were based on a reversed-phase C-18 column operated by phosphate buffer 50 mM/CH₃CN (80/20, v/v) at pH 2.9. Detection of Imidacloprid and its metabolites was performed at a potential of 800 mV after photoactivation at 254 nm. Compared to conventional techniques such as gas chromatography/mass spectrometry (GC/MS) or LC coupled to other detectors, the present method allows simultaneous trace-level determination of both Imidacloprid (0.6 ng ml⁻¹) and its main metabolites (2.4 ng ml⁻¹).     

Determination of sulfur-containing amino acids by capillary electrophoresis dynamic reaction cell inductively coupled plasma mass spectrometry

Capillary electrophoresis dynamic reaction cell™ inductively coupled plasma mass spectrometry (CE-DRC-ICP-MS) for the determination of sulfur-containing amino acids is described. The sulfur-containing amino acids studied include l-cysteine, l-cystine, dl-homocystine and l-methionine. The species studied were well separated using a 70 cm length×75 μm i.d. fused silica capillary while the applied voltage was set at +22 kV and a 10 mmol l⁻¹ disodium tetraborate buffer (pH 9.8) containing 0.1 mmol l⁻¹ EDTA and 0.5 mmol l⁻¹ Triton X-100 was used as the electrophoretic buffer. The sulfur-selective electropherogram was determined at m/z 48 as by using its reaction with O₂ in the reaction cell. The method avoided the effect of polyatomic isobaric interferences at m/z 32 caused by and on by detecting as the oxide ion at m/z 48, which is less interfered. The detection limit of various species studied was in the range of 0.047-0.058 μg S ml⁻¹, which corresponded to the absolute detection limit of 1.3-1.6 pg S based on the injection volume of 27 nl. We determined the concentrations of selected sulfur-containing amino acids in urine and nutritive complement samples. The recovery was in the range of 92-128% for various species.     

High-performance liquid chromatographic method for the simultaneous determination of cyclosporine A and its four major metabolites in whole blood

This study aimed to develop a simple and efficient optimized high-performance liquid chromatograph (HPLC) method for simultaneous determination of cyclosporine A (CyA) and its major, partly active metabolites AM1, AM9, AM4N, and AM19 in whole blood from transplant patients using cyclosporine D (CyD) as internal standard. The method used a CN analytical column maintained at 60 °C with hexan-isopropanol (93:7, v/v) as mobile phase; detection was at 212 nm. Linearity for all five compounds was tested in the range of 31-1500 ng ml⁻¹ for CyA and of 31-1000 ng ml⁻¹ for metabolites. The limit of detection was found to be 15 ng ml⁻¹ for all compounds.This modified, inexpensive method is also suitable for measuring cyclosporine A and metabolite concentrations in routine monitoring of patients undergoing treatment with CyA.     

Quantification of sex pheromone from Anocentor nitens by gas chromatography-mass spectrometry-selected ion monitoring

Two highly sensitive and selective methods based on gas chromatography coupled to mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode have been developed for the quantification of 2,6-dichlorophenol (2,6-DCP), a sex pheromone of the tick females of Anocentor nitens. Standard addition method and calibration curve techniques using 5-bromine-4-hydroxy-3-methoxybenzaldehyde (5-BrV) as internal standard (IS) afforded detection limit of 0.1 ng ml⁻¹. The calibration curve was linear over the concentration range from 0.5 to 500 ng ml⁻¹ for 2,6-DCP. Results show that the concentration range of sex pheromone in the extracts samples was 1.08-10.35 ng ml⁻¹. The methods developed provided reliable procedures to determine amounts of 2,6-DCP present in ticks.     

Ferric nanoparticle-based resonance light scattering determination of DNA at nanogram levels

A novel assay of DNA has been proposed by using ferric nanoparticles as probes coupled with resonance light scattering (RLS) detection. At pH 7.40, the RLS intensity of ferric nanoparticles can be greatly enhanced by the aggregation of positively charged ferric nanoparticles through electrostatic interaction with negatively charged DNA. The enhanced intensity of RLS at 452 nm is proportional to the concentration of DNA in the range of 0.01-0.8 μg ml⁻¹ for calf thymus and salmon sperm DNA and in the range of 0.005-0.3 μg ml⁻¹ for E. coli K12 genomic DNA. Detection limits are 3.6 ng ml⁻¹ for calf thymus DNA, 4.4 ng ml⁻¹ for salmon sperm DNA, and 1.9 ng ml⁻¹ for E. coli K12 genomic DNA, respectively. Compared with the chromophores previously used in RLS assay, the ferric nanoparticles have offered several advantages in easy preparation, good photostability and high sensitivity without being modified or functionalized.     

Group-specific enzyme-linked immunosorbent assay for fenitrooxon and 3-methyl-4-nitrophenol in water samples based on a polyclonal antibody

Fenitrooxon [O,O-dimethyl-O-(4-nitro-m-tolyl)phosphate] is the major metabolite of the organophosphorus insecticide fenitrothion, and 3-methyl-4-nitrophenol is its major degradation product. In the present study, we describe the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of these compounds in water samples based on a group-specific polyclonal antiserum generated with a “bifunctional hapten”, which has two functions: the conventional function of producing an antibody against an antigen and a unique function of promoting the production of the antibodies in rabbit. For application to water samples, the influence of several factors such as organic solvent, pH, and detergent was studied. Under optimized conditions, the quantitative working range of the fenitrooxon ELISA was 0.71-27 ng ml⁻¹ with a limit of detection (LOD) of 0.32 ng ml⁻¹, and the fenitrooxon concentration giving 50% reduction of the maximum signal (IC₅₀) was 4.2 ng ml⁻¹. The quantitative working range of the 3-methyl-4-nitrophenol ELISA was 0.67-27 ng ml⁻¹ with a LOD of 0.38 ng ml⁻¹ and an IC₅₀ of 3.7 ng ml⁻¹. No significant matrix effect originating from the water sample (river water, tap water, purified water, and bottled water) was shown by addition of Tween 20 to the assay buffer. Water samples spiked with each of these compounds at 1, 5, 10, and 20 ng ml⁻¹ were directly analyzed without extraction and clean-up by the proposed ELISA. The mean recovery was 100.9%, and the mean coefficient of variation (CV) was 7.7% for the fenitrooxon ELISA and for the 3-methyl-4-nitrophenol ELISA, the mean recovery was 97.6%, and the mean CV was 7.2%. The proposed ELISA allows precise and accurate determination of these compounds in water at such low levels.     

Determination of clarithromycin in rat plasma by HPLC-UV method with pre-column derivatization

A novel high-performance liquid chromatographic (HPLC) method using pre-column derivatization and UV detection at 275 nm for the determination of clarithromycin in rat plasma has been validated. Clarithromycin was extracted from plasma sample spiked with internal standard (erythromycin) under alkaline condition with ethyl ether and derivatizated with trimethylbromosilane. The analyses were run on a C₁₈ column, maintained at 40 °C during elution, using a mobile phase comprised of potassium dihydrogen phosphate (50 mM, pH 6.8, contained 0.7% triethylamine), acetonitrile, and methanol (30:45:25, v/v/v). The standard calibration curve for clarithromycin was linear (r² = 0.9998) over the concentration range of 0.1-10 μg ml⁻¹ in rat plasma. The limit of detection (LOD) and limit of quantitation (LOQ) was 30 ng ml⁻¹ and 0.1 μg ml⁻¹ respectively. The intra- and inter-day assay variability range was 2.6-7.4% and 3.3-8.5%, respectively. This method has been successfully applied to a pharmacokinetic study of clarithromycin in rats.     

Hetero dimer from tetrakisammonium cavitand and tetratopic crown ether cavitand

Hetero dimer between tetrakis(m-ammonium)cavitand and tetrakis(dibenzo-25-crown-8)cavitand 5 was formed in CDCl₃ at room temperature. The effects of solvent polarity and temperature on the stability of were studied and the thermodynamic parameters for the formation of are , ΔH⁰ = −67.4 kcal mol⁻¹ and ΔS⁰ = −201.6 cal mol⁻¹ K⁻¹.     

Determination of the antidepressant mirtazapine and its two main metabolites in human plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination in human plasma of the recent noradrenergic and specific serotonergic antidepressant (NaSSA) mirtazapine and its two main metabolites, N-desmethylmirtazapine and 8-hydroxymirtazapine, has been developed. Fluorescence detection was used, exciting at λ = 290 nm and monitoring emission at λ = 370 nm. Separation was obtained by using a reversed-phase column (C8, 250 mm × 4.6 mm I.D., 5 μm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 3.0 and 25% acetonitrile. Melatonin was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with phenyl cartridges (100 mg, 1 mL). The calibration curves were linear over a working range of 5-150 ng mL⁻¹ for mirtazapine and of 2.5-75.0 ng mL⁻¹ for N-desmethylmirtazapine and 8-hydroxymirtazapine. The limit of quantitation (LOQ) was 2.5 ng mL⁻¹ and the limit of detection (LOD) was 1.25 ng mL⁻¹ for all analytes. The method was applied with success to plasma samples from depressed patients undergoing treatment with mirtazapine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence the method is suitable for therapeutic drug monitoring of mirtazapine and its metabolites in depressed patients’ plasma.     

Tetramidocavitand: strong anion receptor by well-organized four -(CO)N-H?X interactions

Resorcin[4]arene-based tetramidocavitands containing four secondary amide groups on their upper rim showed strong (R = methyl or ethyl) binding properties. The caviplex formation through hydrogen bonds of -(CO)N-H?X⁻ was supported by ¹H NMR and crystal structure analyses. In a mixture of C₂D₂Cl₄/DMSO/D₂O = 5:15:2 at 25 °C, the thermodynamic parameters for caviplex @1, ΔG (kcal mol⁻¹), ΔH (kcal mol⁻¹), and ΔS (cal K⁻¹ mol⁻¹), are −3.7, −8.6, and −16.7, respectively.