Development and validation of dried blood spots technique for quantitative determination of topiramate using liquid chromatography–tandem mass spectrometry

An LC‐MS/MS method for determination of the anti‐epileptic drug topiramate (TPM) in dried blood spot (DBS) samples was developed and validated. DBS samples were prepared by spotting 30 μL of spiked whole blood onto FTA^TM DMPK‐C cards and drying for at least 3 h. Six‐millimetre punched spots were then extracted by using a mixture of methanol and water (90:10, v/v) with deuterated internal standard (topiramate‐d₁₂). The extracted samples were injected into a liquid chromatograph equipped with a tandem mass spectrometric detector. Negative ions were monitored in the selected reaction monitoring mode and transitions m/z 338.2 → 78.1 and m/z 350.3 → 78.1 were used for the quantitative evaluation of TPM and internal standard, respectively. The results obtained from validation were statistically evaluated according to the requirements of the European Medicines Agency and US Food and Drug Administration regulatory guidelines. The linearity of the method was checked within a concentration range from 10 to 2000 ng/mL. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible. Copyright © 2013 John Wiley & Sons, Ltd.     

Energetics of flavone and flavanone

In this work, we have determined the experimental standard (p° = 0.1 MPa) molar enthalpies of formation, in gas phase, of flavone and flavanone.These results were obtained by combining the standard molar enthalpies of formation in the condensed phase with the standard molar enthalpies of sublimation. The former values were derived from combustion experiments in oxygen, at T = 298.15 K, in a static bomb calorimeter. The values of the standard molar enthalpies of sublimation were obtained by Calvet microcalorimetry and corrected to T = 298.15 K.High-level density functional theory calculations using the B3LYP hybrid exchange–correlation energy functional with extended basis sets and more accurate correlated computational techniques of the MCCM/3 suite have been performed for the compounds.The obtained results, experimental and computational, for flavone and flavanone were compared with those obtained for chromone and chromanone, respectively.     

Differential Pulse Polarographic Determination of Nitrate in AUT Solution

A differential pulse polarographic method for the determination of nitrate ion has been developed. With 0.5 M CaCl₂ as supporting electrolyte, NO^?₃ is reduced to give a peak with E_1/2=–1.836 Volt vs. the Ag/AgCl electrode. The differential pulse polarographic peak height is proportional to the nitrate concentration from 20 to 60 ppm. The detection limit for nitrate is 2 ppm in pure aqueous solution. In the determination of 40 ppm nitrate a relative precision (relative standard deviation) of less than 2% was achieved. Nitrite interferes seriously and should be absent if accurate results are required. The method has been applied to the determination of nitrate in Ammonium Uranyl Tricarbonate (AUT) Solution, results obtained by this method are compared to those obtained by ion chromatography. The agreement between the two sets of results suggests that the DPP method can be used with a fair degree of confidence.     

Extraction,derivatization, and determination of metabolome in human macrophages

A GC/TOF‐MS was applied to the determination of metabolites in human macrophages. The extraction conditions and quenching conditions were investigated and optimized. The results indicated that 0.9% w/v sodium chloride at 4°C was the most favorable condition to quench macrophage, 1 mL 50% ACN for 2 min in ice bath was the optimal condition to extract 5 × 10⁶ cells. Two hundred six peaks could be detectable with peak area over 50 using this method. Among these peaks, 45 peaks with the similarity over 700 were identified using standard compounds for endogenous metabolites. Thirty‐seven out of 45 metabolites could be quantified directly by this method. Twenty metabolites were selected randomly, and 15 amino acids were used for method validation. The correlation coefficients (r) ranging from 0.9902 to 0.9977 were obtained for 15 amino acids in the range of 2.35–150.20 μg/mL. The intraday and interday precisions were lower than 19.90% for the randomly selected 20 endogenous metabolites. Using this development method and multivariate statistical technique, several potential biomarkers were found from human macrophages infected by different Mycobacterium tuberculosis (M. tuberculosis) strains. The results suggest that the method could be applied to the investigation of the pathogenicity of tuberculosis.     

The dosage of small volumes for chromatographic quantifications using a drop-on-demand dispenser system

A commercially available piezo-driven drop-on-demand dispenser was tested for its suitability for the preparation of analytical calibration standards and in a standard addition approach prior to quantitative ultra performance liquid chromatography (UPLC) analysis of homoserines. The reproducibility of the drop-on-demand dosing system was tested and the verification of the droplet volume was performed by preparing a series of 1.0 mg/L caffeine standard solutions from a 1,000.0 mg/L stock solution and analysis of the concentrations obtained by UPLC. The reproducibility was better than 1% relative standard deviation from measurement to measurement and the highest was 1.6% from day to day. The results were compared with the conventional way of generating standard solutions (pipetting). A gravimetric method and a photography-based method for the determination of the average single droplet volume were compared and found to be in very good agreement. The system was employed for the quantification of N-decanoyl homoserine by standard addition in bacterial culture supernatants containing this analyte. The agreement with conventional quantification techniques was high. The paper shows the feasibility of the approach with advantages in low sample and solvent volume consumption and very good reproducibility and reliability combined with easy usage. Figure Ejected droplet, 60 μs after application of the pulse     

Evaluation of the rate coefficients and arrhenius parameters of hydrogen atom transfer reactions. II. Application of the method

The bond-strength–bond-length (BSBL) method was used to estimate Arrhenius parameters and rate coefficients for various hydrogen atom transfer reactions of the type A + BC → AB + C. In most cases predicted activation energies agreed within 1.2 kcal/mol with the experimental values (average error = 1.12 and standard deviation = 1.42), and the calculated log A agreed within 0.55 log units with the literature data (average error = 0.47 and standard deviation = 0.57). Predicted activated complex properties were shown to agree very well with potential energy surface properties obtained from ab initio quantum mechanical calculations. Detailed comparison of the BSBL results and the results of bond-energy–bond-order (BEBO)-type calculations are presented.     

Determination of monensins A and B in the fermentation broth ofStreptomyces cinnamonensis by high performance liquid chromatography

Summary High performance liquid chromatography on an octadecyl silica column has been used to determine both the monensin A: monensin B ratio and by the method of standard addition, the concentration of both in the fermentation broth ofStreptomyces cinnamonensis. Refractive index detection was preferred to ultraviolet owing to the presence of UV-absorbing components which could not be completely separated from the substances of interest. A linear relationship was obtained from the calibration data. The coefficients of variation for the estimation both of the ratio and the concentrations of the compounds were better then 5%. The estimated limit of detection for both substances was about 1 g/ml. The results obtained from the determination of the ratios of monensins were compared with those obtained by chemical ionization mass spectrometry. Chromatographic separation of monensins on the silica gel column is also described.     

Ab initio molecular orbital studies for compounds of magnesium

Using a 6-311G** basis set with estimation of correlation energy at the MP2 level, structural and energetic data for 40 molecular species containing magnesium have been calculated. For about half the species studied, further energetic data were obtained using Pople's G2 method. Enthalpy changes at 298.15 K were obtained for isogyric reactions and standard enthalpies of formation were derived from these. Comparison of the standard enthalpies of formation with the sparse literature data suggests the MP2/6-311G** standard enthalpies of formation are accurate to ± 15 kJ mol^?1 and the corresponding G2 enthalpies accurate to ± 10 kJ mol^?1. The calculated ΔH [MgN, g] revealed a gross error in the currently accepted value for this function. It is intended that these results will be used to parameterize the semiempirical molecular orbital package, MOPAC, for the element magnesium. © John Wiley & Sons, Inc.     

Electroanalytical method for determination of the pesticide dichlorvos using gold-disk microelectrodes

This paper reports the use of laboratory-prepared gold microelectrodes and square-wave voltammetry for analytical determination of low concentrations of the pesticide dichlorvos in pure and natural water samples. After optimization of the experimental and voltammetric conditions, the best voltammetric responses—current intensity and voltammetric profile—were obtained in 0.1 mol L^–1 NaClO₄ with f=100 s^–1, a=50 mV, and E_s=2 mV. The observed detection and quantification limits in pure water were 7.8 and 26.0 g L^–1, respectively. The reproducibility and repeatability of the method were also determined; the results were 1.4% (n=5) and 1.2% (n=10), respectively. Possible interfering effects were evaluated in natural water samples collected at different points with different levels of contamination from agricultural, domestic, or industrial waste from an urban stream. Results showed that the detection and quantification limits increased as a function of the quantity of organic matter present in the samples. Nonetheless, the values observed for these method characteristics were below the maximum value allowed by the Brazilian code for organophosphorus pesticides in water samples. Recovery curves constructed using the standard addition method were shown to be satisfactory compared with those obtained from high-performance liquid chromatography, confirming the suitability of the method for analysis of natural water samples. Finally, when the method was used to determine dichlorvos in spiked cows milk samples, satisfactory recovery and relative standard deviations were obtained.     

Determination of Phytate in Urine by High-Performance Liquid Chromatography–Mass Spectrometry

An indirect method is described for determination of phytate in human urine. The method is based on hydrolysis of the phytate and determination of myo-inositol, one of the hydrolysis products. Chromatographic separations were performed on an Aminex HPX-87C column with Milli-Q water as mobile phase; 5 mM ammonium acetate was added post-column. The detector counted positive ions by monitoring m/z=198, which corresponds to the adduct of myo-inositol with the ammonium cation. The relative standard deviations obtained for standards containing 0.5, 1, and 1.5 mg L^–1 phytate were 4.1, 3.0, and 2.7% respectively (n=5). The limit of detection was 60 g L^–1. Different urine samples were analyzed both by this method and by an alternative analytical method based on GC–MS. The results from both methods were comparable.     

A novel headspace solid-phase microextraction method for the exact determination of organochlorine pesticides in environmental soil samples

A novel method of determining organochlorine pesticides (OCPs) is described. It is based on solid-phase microextraction (SPME) and gas chromatography–electron capture detection. During the development of the method, soil samples were prepared, spiked with standard solution, and then aged for some time. Extraction conditions such as the extraction time, the NaCl content, the volume of water, the extraction temperature and the desorption time were investigated and optimized. The limits of detection obtained using the method ranged from 0.10 to 0.51 ng g⁻¹, and relative standard deviations were lower than 10% for most organochlorine pesticides. Real soil samples were successfully analyzed using the proposed method. The results from the method developed here were in good agreement with those obtained using ultrasonic extraction. The result demonstrates that aging soils spiked with standard solution is an important method development step, because the soil samples obtained using this approach are more like real soils than those obtained when aging is not used.      

Simultaneous determination of isomeric substituted anilines by imidization with benzaldehyde and gas chromatography–mass spectrometry

The chromatographic separation of several isomeric anilines is a challenging issue. Herein, a simple method for the simultaneous determination of four groups of isomeric primary aromatic amines, including chloroanilines, methylanilines, methoxylanilines, and dimethylanilines, was presented. In this method, all of the 15 primary aromatic amines were easily transformed into the corresponding imine derivative by treatment with benzaldehyde under mild conditions. The formed isomeric imine derivatives were completely separated on a commercial capillary gas chromatography column. The effects of several derivatization parameters were investigated and optimized. Linearity in the optimized method ranged from 0.050 to 50 μg/mL with the squared correlation coefficients (R²) between 0.9981 and 0.9999. Reasonable reproducibility was obtained, with the intraday relative standard deviation (N = 5) ranging from 0.89 to 4.57% and interday relative standard deviation ranging from 2.26 to 7.69% at the concentration of 5.0 μg/mL. The developed method has been successfully applied to determine these isomeric aromatic amines in real samples.     

An evaluation of solid-phase microextraction for analysis of volatile organic compounds in drinking water

Solid-phase microextraction (SPME) has been applied to the quantitative analysis of 60 volatile organic compounds (VOCs) in drinking water. Equilibration curves for the partitioning of the VOCs between the fiber coating and fortified water obtained at 20, 50, and 80 °C are found between the theoretical curves for completely agitated and non-agitated samples. Two important factors for the amount adsorbed by the SPME fiber coating are the extraction time and the fiber coating/water distribution coefficient, K_FW . Both depend on the sample temperature, but in a counteracting manner: Increasing the temperature shortened the equilibration times, especially for the heavier VOCs, but also lead to lower K_FW values, and consequently a lower sensitivity of the method. K_FW values are determined for 33 of the VOCs at 40, 60, and 80°C and the heats of adsorption,–ΔH, are calculated. The nature of the adsorption is found to be exothermic which explains the decreasing sensitivity of the method with increasing temperature. Detection limits were typically from 20 ng/l to 200 ng/l, except for the very light VOCs with which detection difficulties were encountered. For all of the VOCs the linear range extended from the lowest concentration at which they were actually detected to at least 5 mg/l. The precision, 3% average standard deviation when an internal standard was used, was satisfactory for most quantitative routine analysis. SPME was also applied to head-space (HS) analysis of drinking water through the coupled equilibrium between water/head-space/fiber coating. HS-SPME is demonstrated to have shorter equilibration times than SPME directly from the water and equal sensitivities, except for the very light VOCs. Water samples from a drinking water plant contaminated in the low μg/l range with 1,1,1-trichloroethane, trichloroethene and tetrachloroethene were analyzed. There seems to be a reasonable agreement between results obtained by SPME and purge & trap. It is concluded that SPME has a great potential for drinking water analysis.     

Determination of tetrakis(hydroxymethyl)phosphonium sulfate in commercial formulations and cooling water by capillary electrophoresis with contactless conductivity detection

A novel capillary electrophoresis method using capacitively coupled contactless conductivity detection is proposed for the determination of the biocide tetrakis(hydroxymethyl)phosphonium sulfate. The feasibility of the electrophoretic separation of this biocide was attributed to the formation of an anionic complex between the biocide and borate ions in the background electrolyte. Evidence of this complex formation was provided by ¹¹B NMR spectroscopy. A linear relationship (R² = 0.9990) between the peak area of the complex and the biocide concentration (50–900 μmol/L) was found. The limit of detection and limit of quantification were 15.0 and 50.1 μmol/L, respectively. The proposed method was applied to the determination of tetrakis(hydroxymethyl)phosphonium sulfate in commercial formulations, and the results were in good agreement with those obtained by the standard iodometric titration method. The method was also evaluated for the analysis of tap water and cooling water samples treated with the biocide. The results of the recovery tests at three concentration levels (300, 400, and 600 μmol/L) varied from 75 to 99%, with a relative standard deviation no higher than 9%.     

Gas chromatographic technique for determination of Irgafos 168 in polyolefin samples after conversion to the related phenolic compound by saponification

A gas chromatographic technique is reported for the determination of a secondary antioxidant, Irgafos 168, in polymeric samples. Irgafos 168 [tris(2,4-di-tert-butyl phenyl)phosphite] is extracted by dissolution/precipitation, saponified to 2,4-di-tert-butyl phenol by refluxing in the presence of methanolic potassium hydroxide, and determined by gas chromatography-flame ionization detection. The method’s repeatability is good, and the relative standard deviation is 7.5% (between runs) and 15.5% (between days). This method was applied to the determination of Irgafos 168 in commercial polymers, and the obtained results were in relatively good agreement with those obtained by the previously reported spectrophotometric method. Correspondence: Mir Ali Farajzadeh, Department of Chemistry, Faculty of Science, Urmia University, Urmia, Iran     

Quantitative Analysis of Clove Oil by NMR Spectrometry

A procedure utilizing NMR spectrometry in the quantitative analysis of clove oil is described. The ¹H-NMR spectrum is determined with acetophenone as internal standard, the ¹³C-NMR spectrum is determined with chloroform as internal standard and 0.1M Cr(acac)₃ as relaxation reagent. The contents of eugenol are compared to the results obtained from G.C. (90.8%, s.d. 0.7%) and from chemical method (89.6% s.d. 1.2%). The result obtained from ¹H-NMR method is 90.4% (s.d. 0.3%) and from ¹³C-NMR method is 90.5% (s.d. 0.7%). The time required for the ¹H-NMR method is about 20mins and for the ¹³C-NMR method is about 2-3hours. Apparently the ¹H-NMR method proves to be more simple, rapid and accurate.     

Polyvinylimidazole/sol–gel composite as a novel solid‐phase microextraction coating for the determination of halogenated benzenes from aqueous solutions

A polyvinylimidazole/sol–gel composite is proposed as a novel solid‐phase microextraction fiber to extract five halobenzenes from the headspace of aqueous solutions in combination with gas chromatography with mass spectrometry. The prepared fiber was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. The obtained results showed that porous polyvinylimidazole/sol–gel composite was chemically deposited on fused silica fiber. The effect of important extraction parameters including extraction temperature, extraction time, and salt content were investigated. The optimum conditions were as follows: extraction temperature 25°C, extraction time 20 min, and salt concentration 30 w/v%. Detection limits and relative standard deviations of the developed method for halogenated benzenes were below 0.1 pg/mL and 15%, respectively. Repeatability of the proposed method, explained by relative standard deviation, varied between 5.48 and 9.15% (n = 5). The limits of detection (S/N = 3) ranged between 0.01 and 0.10 ng/L using gas chromatography with mass spectrometry with selected ion monitoring mode. For real sample analysis, three types of water samples with different matrices (ground, surface, and tap water) were studied. The optimized procedure was applied to extraction and method validation of halogenated benzenes in spiked water samples.     

Quantification of serum apolipoprotein B by infrared spectroscopy

While the conventional approach to assessing both the risk of coronary artery disease and the adequacy of therapy is LDL cholesterol testing, there is compelling evidence to suggest that apolipoprotein B (apoB) is superior to LDL cholesterol for both of these purposes. However, the measurement of apoB requires techniques that can be expensive and difficult to standardize. The aim of this study was, therefore, to develop a new method, based on infrared (IR) spectroscopy, for the routine quantification of apoB in human serum. A total of 366 serum samples were obtained from patients with various disorders. Small volumes (2 μl) of serum specimens were dried to films, and duplicate IR absorption spectra measured. The reference apoB concentrations were determined separately using a standard method, and the proposed IR method was then calibrated using partial least squares (PLS) regression analysis to quantitatively correlate the IR spectra with the reference results. The apoB concentrations predicted from the IR spectra of serum were highly correlated and in excellent agreement with those determined by the reference method. The correlation coefficient (r) for apoB was 0.94, with the standard error between IR-predicted and reference values was 0.10 g/L. In combination with earlier work demonstrating the accurate determination of LDL-C, HDL-C, total cholesterol, and triglycerides from a single infrared spectroscopic measurement, the addition of accurate apoB determination from the same spectrum makes the method very attractive for laboratory use in the routine evaluation of coronary artery disease risk.      

Rapid Determination of Gallamine Triethiodide (Flaxedil®) and Pancuronium Bromide (Pavulon®) in Pharmaceutical and Urine Matrices by Means of Modified‐Carbon‐Paste Ion‐Selective Electrodes

A new analytical method for the determination of gallamine triethiodide (Flaxedil^®; 1 ) and pancuronium bromide (Pavulon^®; 2 ), two muscle relaxants used in surgical operations and in pain relief, has been developed. Our approach relies on rapid, precise, and sensitive potentiometric sensors based on modified‐carbon‐paste ion‐selective electrodes (CP‐ISEs). Linear calibration graphs in the working ranges of ca. 4.5–892 and 7.3–733 μg/ml (in H₂O, pH 7.0, T=25°) were established for 1 and 2 , respectively; and Nernst slopes corresponding to three‐ or two‐electrons transfers, respectively, were obtained. The method works best in a pH range of 7–9. Average relative errors of 2.12 and 2.14%, with average standard deviations of 1.98–2.47 and 2.64–3.45, respectively, were obtained for urine samples of 1 and 2 . The corresponding relative errors for the pharmaceutical samples were 1.59 and 1.64%, with standard deviations of 0.54–1.34 and 0.52–1.67, respectively. Statistical Student and F tests were applied to the data, and satisfactory results were obtained.