Preparation Of Calibration Curves Research Articles

Fluorescent PCR Based Gene Dose Assessment for Detection of Deletion Mutations in the FIX Gene Among Carriers of Hemophilia B.

Abstract 3486Poster Board III-423Haemophilia B (HB), an X linked inherited disorder is caused by heterogeneous mutations in the F9 gene. Approximately 3% of hemophilia B patients have major deletions in the F9 gene. Gross and small deletions in the F9 gene in HB affected males are easily detected by PCR but detecting the carrier state of females in the family is challenging due to the presence of the normal allele. Different methods like linkage analysis, real time PCR and MLPA have been used to assess the carrier status in this situation. Linkage analysis is limited by the availability of informative markers and adequate number of family members. Real time PCR involves standardisation and preparation of calibration curves for each run. Although MLPA is a better alternative, it can be time consuming and involves multiple steps. We have therefore developed a fluorescent PCR based gene dosage approach which is simple, rapid and cost-effective for determining the carrier status of females in families with deletions in the F9 gene. 200ng of DNA extracted by standard protocols was used for amplification with primers designed to amplify a 160bp product from exon h in the F9 gene. One of the primers was fluorescently labelled. Amplification was carried out using these primers for 20 cycles only and the amplified product was subjected to capillary electrophoresis on an ABI 310 genetic analyser. A 230 bp fragment in the albumin gene was used as the control. Analysis was done using Genescan and Genotyper software. The ratio between the peak heights of the exon h in the F9 and control genes in the patient samples were normalised to a normal control. Five families with deletional HB were analysed (in toto deletion-1; Ex g-h – 1; Ex g-poly A-1; Ex h-poly A-2). The ratios in the probands and the family members are presented in the table. Out of eight females analysed, 6 were carriers and 2 were normal. The mean ratio in the carriers was 0.49±0.08 and 0.75±0.05 in the normal. Deletions are not uncommon in HB and deletions involving the exon g and h constitute a major group. Among 212 families with HB assessed at our centre, we have identified large deletions in 8 families (3.7%). It is interesting to note that all except one of these deletional mutations involved exon h. This method confirmed the presence of these deletions in the males and helped us to identify the carrier status of the females in the family.Identification of carrier status of females with deletions in F9 gene by gene dosageSubject IDPeak height of Exh in F9Peak height of albuminNormalised RatioInterpretationHB52844420.59CarrierHB63054890.57CarrierHB221883720.47CarrierHB63851650.48CarrierHB1292472950.78NormalHB238943260.4CarrierHB2803726790.77NormalHB3842026700.4Carrier [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Air Sampling and Analysis Method for Volatile Organic Compounds (VOCs) Related to Field-Scale Mortality Composting Operations

In biosecure composting, animal mortalities are so completely isolated during the degradation process that visual inspection cannot be used to monitor progress or the process status. One novel approach is to monitor the volatile organic compounds (VOCs) released by decaying mortalities and to use them as biomarkers of the process status. A new method was developed to quantitatively analyze potential biomarkers--dimethyl disulfide, dimethyl trisulfide, pyrimidine, acetic acid, propanoic acid, 3-methylbutanoic acid, pentanoic acid, and hexanoic acid--from field-scale biosecure mortality composting units. This method was based on collection of air samples from the inside of biosecure composting units using portable pumps and solid phase microextraction (SPME). Among four SPME fiber coatings, 85 microm CAR/PDMS was shown to extract the greatest amount of target analytes during a 1 h sampling time. The calibration curves had high correlation coefficients, ranging from 96 to 99%. Differences between the theoretical concentrations and those estimated from the calibration curves ranged from 1.47 to 20.96%. Method detection limits of the biomarkers were between 11 pptv and 572 ppbv. The applicability of the prepared calibration curves was tested for air samples drawn from field-scale swine mortality composting test units. Results show that the prepared calibration curves were applicable to the concentration ranges of potential biomaker compounds in a biosecure animal mortality composting unit.

Hydrogen-Bonding Interactions between Water and the One- and Two-Electron-Reduced Forms of Vitamin K1: Applying Quinone Electrochemistry To Determine the Moisture Content of Non-Aqueous Solvents

Vitamin K(1) (VK(1)) was shown by voltammetry and coulometry to undergo two chemically reversible one-electron reduction processes in acetonitrile (CH(3)CN) containing 0.2 M Bu(4)NPF(6) as the supporting electrolyte. The potential separation between the first and second electron-transfer steps diminished sequentially with the addition of water, so that at a H(2)O concentration of approximately 7 M (approximately 13% v/v) only one process was detected, corresponding to the reversible transfer of two electrons per molecule. The voltammetric behavior was interpreted on the basis of the degree of hydrogen bonding between the reduced forms of VK(1) with water in the solvent. It was found that the potential separation between the first and second processes was especially sensitive to water in the low molar levels (0.001-0.1 M); therefore, by measuring the peak separation as a function of controlled water concentrations (accurately determined by Karl Fischer coulometric titrations) it was possible to prepare calibration curves of peak separation versus water concentration. The calibration procedure is independent of the type of reference electrode and can be used to determine the water content of CH(3)CN between 0.01 and 5 M, by performing a single voltammetric scan in the presence of 1.0 mM VK(1). The voltammetry was also investigated in dichloromethane, dimethylformamide, and dimethyl sulfoxide. The reduction processes were monitored by in situ electrochemical UV-vis spectroscopy in CH(3)CN over a range of water concentrations (0.05-10 M) to spectroscopically identify the hydrogen-bonded species.

Preparation of Calibration Curves for the Direct Determination of Trace Level of Zinc in High Purity Iron and Steel Samples by Graphite Furnace Atomic Absorption Spectrometry Using Solid Sampling Technique

The preparation of calibration curves using an aqueous standard solution was studied for the direct determination of trace levels of zinc in high-purity iron and steels by graphite furnace atomic absorption spectrometry (GF-AAS) using solid sampling technique. Certified reference materials of high-purity iron and steel samples were used to confirm the trueness of the proposed methods. The standard addition method for the solid sampling technique that we have proposed gave accurate analytical results. The calibration curve prepared by the standard addition method can be used as a kind of iron-matrix matching calibration curve, and it provides rapid and accurate determinations of zinc. The detection limit of zinc in iron and steel samples was 0.86 mg kg−1 when 15 mg of one sample was measured.

Detection of Food Additives by Voltammetry at the Liquid−Liquid Interface

Electrochemistry at the liquid-liquid interface enables the detection of nonredoxactive species with electroanalytical techniques. In this work, the electrochemical behavior of two food additives, aspartame and acesulfame K, was investigated. Both ions were found to undergo ion-transfer voltammetry at the liquid-liquid interface. Differential pulse voltammetry was used for the preparation of calibration curves over the concentration range of 30-350 microM with a detection limit of 30 microM. The standard addition method was applied to the determination of their concentrations in food and beverage samples such as sweeteners and sugar-free beverages. Selective electrochemically modulated liquid-liquid extraction of these species in both laboratory solutions and in beverage samples was also demonstrated. These results indicate the suitability of liquid-liquid electrochemistry as an analytical approach in food analysis.

Colloidal-based localized surface plasmon resonance (LSPR) biosensor for the quantitative determination of stanozolol

This work reports the systematic preparation of biosensors through the use of functionalized glass substrates, noble metal gold colloid, and measurement by localized surface plasmon resonance (LSPR). Glass substrate was modified through chemical silanization, and the density of gold colloid was carefully controlled by optimizing the conditions of silanization through the use of mixed silanes and selective mixing procedures. At this point, samples were exposed to bioreagents and changes in the shallow dielectric constant around the particles were observed by dark-field spectroscopy. Biological binding of high affinity systems (biotin/streptavidin and antigen/antibody) was subsequently investigated by optimizing coating layers, receptor concentration profiling, and finally quantitative determination of the analyte of interest, which in this case was a small organic molecule-the widely used, synthetic anabolic steroid called stanozolol. For this system, high specificity was achieved (>97%) through extensive nonspecific binding tests, with a sensitivity measurable to a level below the minimum required performance level (MRPL) as determined by standard chromatographic methods. Analytical best-fit parameters of Hillslope and regression coefficient are also commented on for the final LSPR biosensor. The LSPR biosensor showed good reproducibility (<5% RSD) and allowed for rapid preparation of calibration curves and determination of the analyte (measurement time of each sample ca. 2 min). As an alternative method for quantitative steroidal analysis, this approach significantly simplifies the detection setup while reducing the cost of analysis. In addition the system maintains comparable sensitivity to standard surface plasmon resonance methods and offers great potential for miniaturization and development of multiplexed devices.

In Vitro Synthesis of Curcuminoids by Type III Polyketide Synthase from Oryza sativa

Curcuminoids, major components of the spice turmeric, are used as a traditional Asian medicine and a food additive. Curcumin, a representative curcuminoid, has received a great deal of attention because of its anti-inflammatory, anticarcinogenic, and antitumor activities. Here we report a novel type III polyketide synthase named curcuminoid synthase from Oryza sativa, which synthesizes bisdemethoxycurcumin via a unique mechanism from two 4-coumaroyl-CoAs and one malonyl-CoA. The reaction begins with the thioesterification of the thiol moiety of Cys-174 by a starter molecule, 4-coumaroyl-CoA. Decarboxylative condensation of the first extender substrate, malonyl-CoA, onto the thioester of 4-coumarate results in the formation of a diketide-CoA intermediate. Subsequent hydrolysis of the intermediate yields a beta-keto acid, which in turn acts as the second extender substrate. The beta-keto acid is then joined to the Cys-174-bound 4-coumarate by decarboxylative condensation to form bisdemethoxycurcumin. This reaction violates the traditional head-to-tail model of polyketide assembly; the growing diketide intermediate is hydrolyzed to a beta-keto acid that subsequently serves as the second extender to form curcuminoids. Curcuminoid synthase appears to be capable of the synthesis of not only diarylheptanoids but also gingerol analogues, because it synthesized cinnamoyl(hexanoyl)methane, a putative intermediate of gingerol, from cinnamoyl-CoA and 3-oxo-octanoic acid.

Determination of Water in 1-Ethyl-3-methylimidazolium tetrafluoroborate

Determination of water in 1-ethyl-3-methylimidazolium tetrafluoroborate has been carried out using both electrochemical and infrared spectroscopic methods. At platinum electrodes, voltammetric responses for water oxidation and reduction have been used to prepare calibration curves. It has been found that the water oxidation current at +2.4V vs Ag/AgCl is suitable for water determination to at least 40 mM. Infrared spectroscopy of water in quartz cells in the OH stretching region revealed an IR feature at 3550 cm- 1 for water additions to 20 mM, with another feature at 3650 cm-1 appearing at higher water concentrations. For the low- frequency feature, a calibration curve is linear to 14 mM. Both electrochemical and IR methods provide a lower limit of detection of approximately 1 mM water in this ionic liquid.

Utilization of AC Impedance Measurements for Electrochemical Glucose Sensing Using Glucose Oxidase to Improve Detection Selectivity

Abstract The electrochemical detection of glucose using glucose oxidase (GOx) has been conducted by means of AC impedance measurements. In the absence of glucose, the Nyquist plots were straight line due to a reversible redox reaction involving the electron mediator. On the other hand, the Nyquist plots became a circular arc in the presence of glucose, and its radius decreased with an increase in the glucose concentration. The shape of the Nyquist plots were explained with assistance of numerical calculations. Since the reaction impedance due to direct oxidation of interfering species was significantly higher than that of glucose oxidation catalyzed by glucose oxidase, unfavorable influence from interfering species could be eliminated without needing a complicated sensor device. When ferrocenecarboxylic acid was used as an electron mediator, the calibration curve obtained from the Nyquist plots was not affected by the presence of ascorbic acid. Based on those findings, a sensing system, which has no interference from both ascorbic acid and uric acid, was fabricated by using [Os(bpy)3]Cl2, which has a highly positive redox potential (0.65 V), as an electron mediator. A method to prepare calibration curves from AC impedance measurements with a fixed frequency was established so that this technique can be put to practical use.

Nondestructive and rapid determination of nitrate in soil, dry deposits and aerosol samples using KBr-matrix with diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS)

This paper presents the development of a new, rapid and precise analytical method for submicrogram levels of nitrate (NO 3 −) in environmental samples like soil, dry deposit samples, and coarse and fine aerosol particles. The determination of submicrogram levels of nitrate is based on the selection of a quantitative analytical peak at 1385 cm −1 among the three observed vibrational peaks and preparing calibration curves using different known concentrations of nitrate by diffuse reflectance Fourier transform infra red spectrometric (DRIFTS) technique. Pre-weighed and ground infrared (IR) grade KBr was used as substrate over which remarkably wide range of known concentration of nitrate was sprayed and dried. The dried sample was analyzed by DRIFTS and absorbance was measured. Eight calibration curves for four different concentration ranges of nitrate for absorbance as well as peak area were prepared for samples containing lower and relatively higher values of nitrate. The relative standard deviation ( n = 8) for the nitrate concentration ranges, 0.05–40, 0.05–1.5, 1.5–25, 5–40 μg/0.1 g KBr were in the range 1.6–2.3% for the above calibration curves. The limit of detection (LOD) of the method is 0.07 μg g −1 NO 3 −. The F- and t-tests were performed to check the analytical quality assurance test. The noteworthy feature of the reported method is the noninterference of any of the associated cations. The results were compared with that of ion-chromatographic method with high degree of acceptability. The method can be applied in wide concentration ranges. The method is reagent less, nondestructive, very fast, repeatable, and accurate and has high sample throughput value.

Direct and rapid determination of sulphate in environmental samples with diffuse reflectance Fourier transform infrared spectroscopy using KBr substrate

The feasibility of employing diffuse reflectance Fourier transform infrared spectroscopy (DRS-FTIR) as a sensitive tool in the submicrogram level determination of sulphate (SO 4 2−) was checked in this work. This paper presents the development of a new, rapid and precise analytical method for ppb levels of sulphate (SO 4 2−) in environmental samples like coarse and fine aerosol particles, dry deposits and soil. The determination of submicrogram levels of sulphate is based on the selection of a quantitative analytical peak at 617 cm −1 among the three observed vibrational peaks and preparing calibration curve using different known concentrations of sulphate by diffuse reflectance-Fourier transform infra red spectrometric (DRS-FTIR) technique. Pre-weighed and ground IR grade KBr was used as substrate over which remarkably wide range of known concentration of sulphate was sprayed and dried. The dried sample was analysed by DRS-FTIR. Three calibration curves for three different concentration ranges of sulphate were prepared for samples containing low and relatively higher sulphate contents. The relative standard deviation ( n = 8) for the sulphate concentration ranges, 2.5–35.5, 25.5–165, 55–1000 μg/0.5 g KBr, as used to prepare calibration curves, were 2.4%, 2.1% and 1.5%, respectively. The relative standard deviation for the sulphate concentration in real samples were found to be in the range, 3.11–5.76% ( n = 16), 4.05–7.75% ( n = 16) and 1.48–3.52% ( n = 10) for aerosol, dry deposits and soil, respectively. The LOD of the method is 0.20 μg/g SO 4 2−. The F- and t-tests were performed to check the analytical quality assurance test. The noteworthy feature of the reported method is the non-interference of any of the associated anions and cations. The results were compared with that of ion-chromatographic method with high degree of acceptability. The method can be applied in wide concentration ranges. A method for sulphate determination was introduced that did not require pretreatment of samples. This method employed the direct determination of the sulphate. The method is reagent less, nondestructive, very fast, repeatable, and accurate and has high sample throughput value.

Comment on: Pharmacokinetics of inhaled colistin in patients with cystic fibrosis

Sir, Ratjen et al. recently published an important paper in JAC reporting the pharmacokinetics of colistin after inhalation of colistin methanesulphonate in patients with cystic fibrosis (CF). HPLC was used to measure the concentrations of the formed colistin. The paper raises a number of important issues that deserve comment. It is important not to use the terms colistin and colistin methanesulphonate (sodium salt, or colistimethate-Na in the paper) interchangeably. The chemistry, pharmacokinetics, pharmacodynamics and toxicity of these two entities are substantially different; most importantly, colistin is formed from colistin methanesulphonate both in vitro and in vivo. Even though some manufacturers (such as Grunenthal, Germany) label the content of injection vials of colistin methanesulphonate with ‘158 mg colistin methanesulphonate is equal to 66 mg colistin’, this labelling is misleading because it is based on antibacterial activity measured with microbiological assays and does not mean that there is 66 mg of colistin in each vial. Therefore, we strongly recommend that any pharmacokinetic calculations on colistin or colistin methanesulphonate should not be based on ‘66 mg colistin’. The HPLC assay used by Ratjen et al. was very similar to the one reported previously from another laboratory using derivatization with 9-fluorenylmethyl chloroformate on solid-phase extraction C18 cartridges; in the previously reported HPLC assay, colistin sulphate was employed to prepare calibration curves for measurement of colistin (base) in biological fluids. In the paper of Ratjen et al., it is not clear whether colistin sulphate, colistin A or colistin methanesulphonate (sodium) was used for the calibration curves of the HPLC assay. Ratjen et al. reported the amount (0.18–16.13 mg) of ‘colistin methate’ recovered in urine was equal to a mean 4.3 – 1.3% (range 0.3–24.2%) of the inhaled dose. Unfortunately, it is not clear how the concentrations of ‘colistin methate’ in urine were measured. It appears that the urinary recovery was calculated based on the recovered amount of colistin (base) and the dose of ‘66 mg colistin’. Considering the hydrolysis of colistin methanesulphonate to colistin in vivo and in vitro, such calculation of urinary recovery using the dose of ‘66 mg colistin’ is inappropriate. In addition, it is unclear whether the authors have allowed for the molecular weight difference between colistin methanesulphonate (sodium, average molecular weight 1743), colistin sulphate (average molecular weight 1403, if they used it for calibration curves for colistin) and colistin (average molecular weight 1163) when the concentrations of ‘colistin’ were calculated. Ratjen et al. used the HPLC peak for polymyxin E1 to generate calibration curves for colistin concentrations. Usually, the two major components of colistin, colistin A (polymyxin E1) and colistin B (polymyxin E2), account for more than 85% of colistin, but the ratio of these two components may vary substantially between 3:1 and 1:1. It is not clear how the authors calculated the concentrations of polymyxin E1 in the HPLC assay as the commercially available colistin sulphate is a mixture of many components. In addition, we are not aware of any literature indicating that polymyxin E2 has no antibacterial activity; hence, the claim by Ratjen et al. that ‘colistin is a pro-drug that is converted into its active component polymyxin E1’ is questionable. Indeed, polymyxin E1 is one of the constituents of colistin. It should be noted that the clearance of colistin reported by Ratjen et al. should be regarded as an apparent clearance because the amount of the inhaled dose of colistin methanesulphonate reaching the systemic circulation in the form of colistin is unknown. In summary, Ratjen et al. provided some potentially useful information on the pharmacokinetics of colistin after inhalation of colistin methanesulphonate. However, because of lack of clarity regarding the quantification of colistin in biological fluids and the pharmacokinetic analysis, caution is required when these data are used as reference for inhalation therapy of colistin methanesulphonate in CF patients.

Calorimetric determination of amorphous content in lactose: A note on the preparation of calibration curves

Calorimetric methods (isothermal or solution calorimetry) offer the ability to detect amorphous contents to 0.5% (w/w) or better in processed pharmaceuticals and calorimetric data are becoming more widely accepted in regulatory submissions. However, both methods require the construction of calibration curves, prepared using quantitative physical mixtures of entirely amorphous and entirely crystalline material. If the sample under investigation exists in two or more isomers or polymorphs, and the enthalpy of solution (solution calorimetry) or the enthalpy of crystallisation (isothermal calorimetry) are different for the isomers or polymorphs, then it must be ensured that the batch of material used to prepare the calibration samples has the same isomeric or polymorphic composition as the (processed) material to be tested. Here, we demonstrate the problems that may arise using lactose as a pharmaceutically important model substance. Calibration curves were prepared from solution calorimetry and isothermal gas perfusion calorimetry data using two batches of lactose (one predominantly anhydrous α-lactose and one predominantly β-lactose). The calibration curves are shown to be significantly different for the two batches, and it is shown that quantification of the amorphous content of a processed sample of unknown isomeric composition is impossible, unless the calibration curve is prepared from the same batch of material as the processed sample. In addition, some of the other problems inherent in using isothermal gas perfusion calorimetry for amorphous content determination, such as wetting issues and the preparation of calibration standards that mimic processes samples, are discussed.

In vitro degradation of the antimicrobial human peptide HEM-γ 130–146 in plasma analyzed by a validated quantitative LC–MS/MS procedure

In stability studies during preclinical drug development, the human antimicrobial peptide hHEM-γ 130–146 shows progressive N-terminal degradation in plasma. To determine this effect, we developed and validated a selective and quantitative μHPLC–MS/MS procedure for this compound. Following deproteinization by precipitation, reversed-phase separation is performed with a time-saving two-column design online coupled to an ion trap mass spectrometer for electrospray ionization MS detection. Using a linear calibration curve obtained with synthetic external standards ranging nearly two orders of magnitude, we achieved good precision (repeatability and reproducibility: 5–15%), accuracy (−3 to 15%), and ruggedness with a lower limit of quantification at 0.29 μg/ml plasma (0.15 μM). Because of good linearity ( r 2 > 0.999), the recovery (84 ± 3%) and ion suppression (86 ± 4% remaining intensity) were calculated from specifically prepared calibration curves. The developed procedure was applied to human and animal plasma samples. Incubations in the presence and absence of proteinase inhibitors revealed at least an aminopeptidase M activity for the initial N-terminal truncation of tryptophan (W 130) and a putative glutaminyl-peptide cyclotransferase activity for the resulting intermediate starting with the bared glutamine residue (Q 131). The calculated periods of half-change demonstrated exceeding interspecies variations, whereas the intraspecies variations were only between 20 and 30%. The current procedure is valuable as a generic method for pharmaceutical purposes, and data give important information for further development toward a potential natural drug candidate.

Dichloropentafluoropropanes as solvents for size exclusion chromatography

We have found that HCFC225s (HCFC225ca: 3,3-dichloro-1,1,1,2,2-pentafluoropropane, HCFC225cb: 1,3-dichloro-1,1,2,2,3-pentafluoropropane) are superior mobile phases for size exclusion chromatography (SEC). As alternatives of CFC113, they have been shown to possess a number of excellent properties, such as low flammability, low viscosity, low cost, high purity, and environmental and operational friendliness. In addition, they have distinct advantages for the SEC measurement, because they solubilize some kinds of acrylate such as poly(methyl methacrylate) (PMMA) and commercial monodispersed PMMA can be used to prepare calibration curves necessary for the measurement of equivalent molecular weight of some polymers. Furthermore, we propose an HCFC225/1,1,1,3,3,3-hexafluoroisopropanol mixed solvent for use in the SEC of poly(ethylene terephthalate) (PET) and polyamides. Poly(2-(perfluorooctyl)ethyl acrylate), whose PMMA equivalent weight average molecular weight was 118,100 Da, was evaluated by a multi-angle laser light scattering (MALLS) detector to have an absolute molecular weight of 439,000 Da. The difference can be attributed to the molecular size of the polyfluorinated polymer compared to the non-fluorinated one. The possible application of this novel mobile phase system for molecular size and molecular weight characterization of perfluoropolyethers, PET, nylon 6 and nylon 6,6 are also discussed.

Separation and quantification of chicken and bovine growth plate cartilage matrix vesicle lipids by high-performance liquid chromatography using evaporative light scattering detection

Matrix vesicles (MV) are lipid bilayer-enclosed nanoscale structures that initiate extracellular mineral formation in most vertebrate species. Little attention has been given to differences between species in membrane lipid composition or to how new mineral is formed in MV. To explore more precisely the lipids of MV isolated from avian and bovine species, we developed a new high-performance liquid chromatography (HPLC) method used in combination with evaporative light scattering detection (ELSD) to quantify their lipid composition. HPLC analyses were performed on a Lichrosorb silica column using separate binary gradient elution systems for analyzing polar and nonpolar lipids. Standard mixtures of both phospholipids and nonpolar lipids were used to prepare calibration curves for each lipid, which were analyzed mathematically by polynomial regression for accurate quantitation. This new methodology provides high-resolution separations and quantitation of both the polar and the nonpolar lipids typically present in biological membranes, including lyso- (monoacyl-) phospholipids. We have applied this method to quantitate the phospholipid and nonpolar lipid composition of MV isolated from chicken and bovine growth plate cartilage. We also compared the ability of these MV to induce mineral formation. While the ability to induce mineralization and the lipid composition were generally similar, some significant differences between MV from these two disparate species were seen.

Estimation of the main dill seeds odorant carvone by solid-phase microextraction and gas chromatography.

Solid-phase microextraction (SPME) was examined for its suitability for isolation of volatiles from seeds of dill in comparison with the traditional steam distillation procedure. Two main dill seeds volatiles, carvone and limonene, were taken into consideration. Two Supelco SPME fibers were used for the extraction: polyacrylic (PAc) and polydimethylsiloxane (PDMS). The time required to saturate the fibers was 3 min, while distillation took 3 h. Gas chromatography (GC) separation was reduced to 5 min by use of microcapillary column HP-5 cross-linked 5% Ph Me Siloxane. The standards of limonene and carvone were used to prepare calibration curves. PAc fiber responses were described by quadratic curves while PDMS responded linearly. Six varieties of dill were examined by distillation and SPME with both fibers. The good results were achieved for carvone by SPME-PDMS with significant regression between distillation and SPME. This compound can be measured in dill seeds samples within 10 min. The SPME-PDMS were also tested for its application to chiral resolution of carvone and limonene enantiomers in dill seeds oil. The enantiomeric separation was done with two chiral columns. The enantiomeric ratios measured by SPME were just the same as with distillation.

Determination of 2-Agonists in Bovine Urine: Comparison of Two Extraction/Clean-Up Procedures for High-Resolution Gas Chromatography-Mass Spectrometry Analysis

Two extraction/clean-up analytical procedures were investigated and compared regarding their recovery and matrix-purification efficiency for screening beta2-agonist residues in fortified bovine urine by high-resolution gas chromatography-mass spectrometry (GC-MS). The first procedure, based on an analytical method originally developed for detecting anabolic steroids, consists of the employment of the nonionic resin, Amberlite XAD-2, a styrene-divinylbenzene copolymer for solid-phase extraction (SPE), followed by liquid-liquid extraction with diethyl ether. The second focuses on the use of a mixed SPE cartridge (reversed-phase and ion-exchange sorbent, Bond Elut Certify). In both cases, the trimethylsilylated derivatives were analyzed by GC-MS with an ion-trap detector. Clenbuterol, salbutamol, and terbutaline were used to spike urine samples during the comparison experimental phase. Afterwards, tulobuterol, mabuterol, mapenterol, cimbuterol, and brombuterol were included in the evaluation of the second procedure (the Bond Elut Certify procedure). At this stage, the detection was accomplished by GC-MS (quadrupole mass analyzer) with selective ion monitoring acquisition. The isotopic dilution method with the hexadeuterated analogues of clenbuterol and salbutamol was applied to prepare calibration curves and calculate recovery percentages. With XAD-2 resin, terbutaline and salbutamol (resorcinol and phenol-type beta2-agonists, respectively) could not be detected at 20 ng/mL or at 40 ng/mL. In spite of clenbuterol having been detected at 20 ng/mL, the results obtained were not reproducible. The use of the reversed-phase and ion-exchange sorbent Bond Elut Certify allowed multiresidue detection and showed several advantages for the screening of clenbuterol such as higher recoveries, cleaner final extracts, reduced sample preparation time, less labor intensive, and easier solvent consumption and disposal. Recoveries over 88% (concentrations ranging from 0.5 to 10 ppb) and limits of detection equal to 0.5 ppb were met for all the beta2-agonists studied with the last method.

Quantification of the effect of the hyperparasite Trichoderma harzianum on the saprotrophic growth dynamics of Rhizoctonia solani in compost using a monoclonal antibody-based ELISA

This paper describes the use of a monoclonal antibody-based ELISA for the quantification of the effects of Trichoderma harzianum on the saprotrophic growth dynamics of Rhizoctonia solani in unsterile compost-based microcosms. Quantification of R. solani was based on the immunological detection of a constitutive, extracellular, water soluble glycoprotein antigen (the copper-containing enzyme catechol oxidase) which is secreted by actively growing mycelium of the fungus only. Extracts derived from lyophilized mycelium (FDM) were used as a quantifiable and repeatable source of the antigen in order to prepare calibration curves that accounted for incomplete extraction of the antigen from compost samples. These curves were used to convert the absorbance values of extracts in ELISA to FDM biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of R. solani to be made in the presence and absence of T. harzianum. The high pH (9.6) of the buffer used to extract the antigen from microcosm samples also resulted in the extraction of humic components of the compost which interfered with the sensitivity of the assay. A protocol, based on the determination of optimal ELISA absorbance: extract dilution ratios, was used in order to reduce the interference and improve the sensitivity of the quantitative assay.

Matrix Representation of Solution Mixing by Aliquot Exchange

We present a mathematical description of mixing two solutions by exchanging aliquots back and forth between them. We propose that this method of aliquot exchange can be used to automate calibration curve preparation in a way that produces less solvent waste than conventional serial dilution methods. We also show its use in quickly mixing solutions. The process of aliquot exchange is represented mathematically by a 2 × 2 symmetric matrix, A, that is a function of the volume or percentage of liquid, p, that is exchanged. Each cycle of aliquot exchange is represented by operating the matrix, A, on the previous concentrations. That is, after n mixing cycles, the final concentrations (C(i)(n)()) are given by A(p(n))A(p(n)(-1))...A(p(1)) operating on the initial concentrations (C(i)(0)), or (A(p))(n)()C(i)(0) if the same amount of liquid is exchanged in each step. We observe close agreement between theory and experiment. For solutions that have equal initial volumes, both the matrix A(p) and the product of any number of such matrices that may have the same or different values of p have equal diagonal and equal off-diagonal elements (they are symmetric), the sum of the elements in any row or column sums to unity, and the operation of any of these matrices on a set of concentrations produces two new concentrations that sum to the same value as the sum of the initial concentrations. We follow the mixing process for two solutions of equal volume by plotting the matrix element A(12)(n)() of A(n)(), which approaches 0.5 as n increases. As expected, the larger the exchanged aliquot, the more quickly the solutions mix. By varying the fraction, p, that is exchanged, we show that it should be possible to produce a calibration curve with values that vary in concentration over at least 3 orders of magnitude from just two solutions.