Multiantigen print immunoassay (MAPIA) for the diagnosis of neurocysticercosis: a single-center diagnostic optimization and accuracy study in Lima, Peru.

Neurocysticercosis (NCC) is the most common helminthic infection of the human central nervous system. The antibody detection assay of choice is the enzyme-linked immunoelectrotransfer blot assay using lentil-lectin purified parasite antigens (LLGP-EITB, Western blot), an immunoassay with exceptional performance in clinical samples. However, its use is mainly restricted to a few research laboratories because the assay is labor-intensive and requires sophisticated equipment, expertise, and large amounts of parasite material for preparation of reagents. We report a new immunoprint assay (MAPIA) that overcomes most of these barriers. We initially compared the performance of five different antigen combinations in a subset of defined samples in the MAPIA format. After selecting the best-performing assay format (a combination of rGP50 + rT24H + sTs14 antigens), 148 archived serum samples were tested, including 40 from individuals with parenchymal NCC, 40 with subarachnoid NCC, and 68 healthy controls with no evidence of neurologic disease. MAPIA using three antigens (rGP50 + rT24H + sTs14) was highly sensitive and specific for detecting antibodies in NCC. It detected 39 out of 40 (97.5%) parenchymal NCC cases and 40/40 (100%) subarachnoid cases and was negative in 67 out of 68 (98.53%) negative samples. MAPIA using three recombinant and synthetic antigens is a simple and economical tool with a performance equivalent to the LLGP-EITB assay for the detection of specific antibodies to NCC. The MAPIA overcomes existing barriers to adoption of the EITG LLGP and is a candidate for worldwide use.

Effects of organic modifier and temperature on the enantiomeric separation of several azole drugs using supercritical fluid chromatography and the Chiralpak AD column.

The effects of organic modifier and temperature on the enantioseparation of 10 triazoles and eight imidazoles, using supercritical fluid chromatography with the Chiralpak AD column, have been investigated in this work. For this purpose four different organic modifiers (methanol, ethanol, 2-propanol and acetonitrile) were evaluated. Only in the case of two compounds could the enantiomeric separation not be achieved with any of the modifiers tested; the rest of compounds were baseline or partially resolved with at least one of the modifiers. The alcohol-type modifiers provided the best results in terms of retention time and resolution. In general, retention increased in the order methanol < ethanol < 2-propanol; moreover it was possible to establish a relationship between the retention and the number of aromatic rings and dioxolane groups in the molecule, that is, the higher the number is, the higher the retention time. From the study of the temperature effect, the enthalpy-entropy compensation was demonstrated for all the compounds, except for bifonazole using methanol and miconazole using acetonitrile. This suggested that both analytes are enantiomerically resolved through different mechanisms.

Supercritical fluid extraction of free amino acids from broccoli leaves.

The extraction of free amino acids (AAs) from broccoli leaves using supercritical fluid extraction (SFE) with CO(2) modified with methanol, is presented in this work. The effect of the different variables was studied, showing the percentage of methanol a strong influence on the extraction. The best results in terms of extraction yield were obtained at 250 bar, 70°C, 35% methanol as organic modifier, a flow rate of 2 mL/min, and 5 min and 30 min as static and dynamic extraction times, respectively. The extraction yield obtained with the SFE method was comparable to that obtained employing conventional solvent extraction with methanol-water (70:30) and minor than using water, but the relative proportion of the AAs in the extracts was very different. For example, the use of SFE allowed the enrichment in proline and glutamine of the extracts. The selected conditions were applied to obtain SFE extracts of broccoli leaves from different varieties (Naxos, Nubia, Marathon, Parthenon and Viola). The highest levels of AAs were found in the SFE extracts from the Nubia variety.

Determination of tylosins A, B, C and D in bee larvae by liquid chromatography coupled to ion trap-tandem mass spectrometry.

A LC-MS/MS method has been developed to simultaneously quantify tylosins A, B, C and D in bee larvae, compounds currently used to treat one of the most lethal diseases affecting honey bees around the world, American Foulbrood (AFB). The influence of different aqueous media, temperature and light exposure on the stability of these four compounds was studied. The analytes were extracted from bee larvae with methanol and chromatographic separation was achieved on a Luna C(18) (150 × 4.6 mm i.d.) using a ternary gradient composed of a diluted formic acid, methanol and acetonitrile mobile phase. To facilitate sampling, bee larvae were initially dried at 60°C for 4h and afterwards, they were diluted to avoid problems of pressure. MSD-Ion Trap detection was employed with electrospray ionization (ESI). The calibration curves were linear over a wide range of concentrations and the method was validated as sensitive, precise and accurate within the limits of quantification (LOQ, 1.4-4.0 ng/g). The validated method was successfully employed to study bee larvae in field tests of bee hives treated with two formulations containing tylosin. In both cases it was evident that the minimal inhibitory concentration (MIC) had been reached.

Study of the enantiomeric separation of an acetamide intermediate by using supercritical fluid chromatography and several polysaccharide based chiral stationary phases.

Four chiral stationary phases, based on the phenylcarbamate derivatives of amylose or cellulose: Chiralcel OD-H, Chiralpak AD, Lux Cellulose-2 and Lux Amylose-2, were evaluated for the enantiomeric separation of an acetamide chiral intermediate, the (4S-trans)-4-(ethylamino)-4-(N-acetamide)-5,6-dihydro-(6S)-methyl-4H-thieno-[2,3-b]thiopyran-7,7-dioxide, using SFC. The effect of the different modifiers and temperatures, on the separation, was also studied. The chiral separation could not be achieved using the Chiralpak AD column, nevertheless the other columns provided excellent results with analysis times close to 6 min and resolutions higher than 2. The highest enantioresolutions and retentions were obtained with the Lux Cellulose-2 column and 2-propanol as organic modifier. The isoelution temperatures were estimated from the van't Hoff plots, and in all the cases they were above the temperature range studied which means that the enantiomeric separation was enthalpy driven.

Profile and relative concentrations of fatty acids in corn and soybean seeds from transgenic and isogenic crops.

In this work 44 fatty acids, which were analyzed as methyl esters by GC/MS in scan mode, have been determined in genetically modified corn and soybean seeds. Their relative concentrations have been compared with those of isogenic lines grown in the same conditions. Studied compounds comprised saturated and unsaturated fatty acids, including cis/trans isomers and minor fatty acids. A classical soxhlet extraction and an accelerated solvent extraction have been assayed to extract the fatty compounds from seeds and the GC separation has been carried out on a biscyanopropylpolysiloxane chromatographic column. Soxhlet extraction was selected as the most convenient and applied to compare the samples. Specific compounds, which could denote the origin of the crop have not been observed, but for some sample pairs, significant differences have been found in relation to the percentage of certain acids; the highest differences for major acids were 4.1% in corn and 4.8% in soybean. The concentrations of long chain acids such as 24:0, 26:0 and 28:0 were higher in some isogenic lines whereas the concentrations of short chain acids such as 6:0, 8:0, 9:0, 10:0 and 12:0 were higher in their transgenic counterparts.

Use of supercritical fluid extraction and gas chromatography-mass spectrometry to obtain amino acid profiles from several genetically modified varieties of maize and soybean.

A method using supercritical fluid extraction (SFE) and gas chromatography-mass spectrometry (GC/MS) for obtaining the amino acid profiles of genetically modified maize and soybean is proposed. SFE is carried out in a homemade modular system where amino acids are extracted with carbon dioxide modified with 35% of methanol, in conditions optimized by a central composite design. Once extracted, the amino acids are determined by GC/MS. The method has been applied to three samples of maize derived from MON810, other from NK 603 and a Roundup ready soybean sample. The profiles are compared with those obtained from their corresponding isogenic non-transgenic varieties. Although differences are directly observed in some cases by visual comparison of the chromatograms, the application of ANOVA shows more significant differences. In general terms, isogenic varieties seem to have higher content of several amino acids.

Semipreparative enantiomeric separation of omeprazole by supercritical fluid chromatography.

Omeprazole, a widely used antiulcer drug, has been enantiomerically separated at semipreparative scale on a polysaccharide based chiral stationary phase by supercritical fluid chromatography (SFC). For this work, a modular supercritical fluid chromatograph was adapted to operate at semipreparative scale and a Chiralpak AD (250 mm x 10 mm) column was used. The effect of two organic modifiers (ethanol and 2-propanol) was studied, and different injection volumes and concentrations of the omeprazole racemic mixture were evaluated in order to obtain high enantiomeric purities and production rates. Better results were achieved using concentration overloading instead of volume overloading. The recoveries decreased when the requirements of enantiomeric purity or the load increased, but it was possible to recover 100% of both enantiomers at an enantiomeric purity higher than 99.9% under some loading conditions, like injecting 1 and 2 ml of a solution of 3g/l. As far as production rates are concerned, the best result for S-(-)-omeprazole at that purity (27.2mg/h) was achieved with sample concentrations of 10 g/l and the injection of 2 ml, while a volume of 4 ml was better in the case of R-(+)-omeprazole (20.5mg/h).

Study of the enantiomeric separation of oxfendazole and cetirizine using subcritical fluid chromatography on an amylose-based column.

The enantiomeric separation of cetirizine and oxfendazole on a Chiralpak AD column using subcritical fluid chromatography has been studied in this work. The enantioseparation of cetirizine was only possible when 2-propanol was used as a modifier, obtaining better results in presence of the additives triethylamine (TEA) and trifluoroacetic acid (TFAA). On the contrary, 2-propanol provided the lowest enantioresolutions for oxfendazole, in this case the best results in terms of high resolution and short analysis time were obtained with ethanol. The study of the temperature effect revealed that in the case of cetirizine using 2-propanol, and oxfendazole using methanol, the separation was enthalpy-driven and the isoelution temperature was above the working range. Using ethanol or 2-propanol, the results showed that the oxfendazole enantioseparation was entropically driven and the isoelution temperatures were below the range studied.

Comparative study of the enantioselective separation of several antiulcer drugs by high-performance liquid chromatography and supercritical fluid chromatography.

A comparative study of the enantiomeric separation of several antiulcer drugs such as omeprazole, lansoprazole, rabeprazole and pantoprazole using HPLC and supercritical fluid chromatography (SFC) on the Chrialpak AD column is presented in this work. The results show that employing the above mentioned column only two compounds (omeprazole and pantoprazole) could be enantiomerically resolved using HPLC, on the contrary SFC allowed the enantiomeric separation of all the compounds studied with higher resolutions and lower analysis times.

Chiral separation of some triazole pesticides by supercritical fluid chromatography.

The enantiomeric separation of six triazole pesticides: cyproconazole, propiconazole, diniconazole, hexaconazole, tebuconazole and tetraconazole, using supercritical fluid chromatography and the Chiralpak AD column, is presented in this work. The effect of different organic modifiers such as methanol, ethanol and 2-propanol on the retention and resolution was studied as well as the presence of additives in the mobile phase. The results obtained were highly successful, all the compounds were enantiomerically separated and in most of the cases the analysis time was close to 10 min. The type of organic modifier that provided the best results depended on the compound.

Use of semipreparative supercritical fluid chromatography to obtain small quantities of the albendazole sulfoxide enantiomers.

The semipreparative separation of the albendazole sulfoxide enantiomers using chiral supercritical fluid chromatography is presented in this work. For this purpose, a modular SFC chromatograph was adapted to work at semipreparative scale and a Chiralpak AD (250 x 10 mm) column was used. Different injection volumes were evaluated in order to obtain high purities and throughputs. Using the maximum load, it was possible to obtain 37 mg/h of the first eluted enantiomer with a purity of 99.9%, and 36.5 mg/h of the second eluted enantiomer with a purity of 95%.

Quality assurance of commercial beeswax II. Gas chromatography-electron impact ionization mass spectrometry of alcohols and acids.

Gas chromatography with mass spectrometric detection was used to find the fraction of alcohols and acids present in pure beeswax from Apis mellifera. Some new compounds not described till now were found, such as a family of unsaturated linear fatty acids, several hydroxyacids and 1,2,3-propanetriol monoesters. The chromatographic profiles obtained from pure beeswax and bee-rejected foundation beeswax can be used to discriminate them; they mainly differ in the amount of some acids and alcohols.

Albendazole sulphoxide enantiomers in pregnant rats' embryo concentrations and developmental toxicity.

Three single oral doses (8.5, 10, and 14 mg/kg) of a racemic formulation of albendazole sulphoxide (ABZSO) were administered to pregnant rats on day 10 of gestation. Mother plasma and embryo concentrations of ABZSO enantiomers and albendazole sulphone (ABZSO(2)) were determined 9 h after administration. The (-)-ABZSO enantiomer showed higher peak concentrations in both maternal plasma and embryo than the (+) enantiomer. An increase in embryo concentrations of ABZSO enantiomers and ABZSO(2) was only observed when dose rose to 14 mg/kg. There was an increase in resorption when the dose increased, but significant differences were only found in the higher dose group when compared with the other groups. The incidence of external and skeletal malformations (mostly of the tail, vertebrae and ribs) rose significantly in the 10 mg/kg group, producing almost 20% and 90% of malformed fetuses, respectively, and gross external and skeletal abnormalities in the thoracic region and limbs were also found.

Separation of triadimefon and triadimenol enantiomers and diastereoisomers by supercritical fluid chromatography.

The enantiomeric separation of triadimenol and triadimefon on a Chiralpak AD column using supercritical fluid chromatography, was studied in this work. The effect of different modifiers (methanol, ethanol and 2-propanol) was tested, with methanol and ethanol providing the best results for the enantiomeric separation of the two compounds. The enantioseparation of a mixture of triadimenol and triadimefon (six stereoisomers) was achieved in only 15 min using a gradient of ethanol, 200 bar, 35 degrees C and a flow-rate of 2 ml/min. The separation of triadimenol diastereoisomers on different achiral columns (diol, silica and ODS) was also investigated. In this case, the type of organic modifier to be used depended on the stationary phase, the Spherex Diol being the column that gave the best separation. Using this column, resolutions higher than 3 were obtained in analysis times of 5 min with any of the modifiers checked.

Development and validation of an LC assay for sumatriptan succinate residues on surfaces in the manufacture of pharmaceuticals.

A high performance liquid chromatographic (HPLC) method for the assay of sumatriptan succinate residues in swabs collected from manufacturing equipment surfaces was developed and validated in order to control a cleaning procedure. The swabbing procedure using two cotton swabs moistened with water was validated applying a wipe-test and a HPLC method developed to determine low quantities of the drug. The HPLC method involves a C18 column at 25 degrees C, a mixture of ammonium phosphate monobasic (0.05 M)-acetonitrile (84:16, v/v) as a mobile phase and UV detection at 228 nm. Using the proposed method, the average recoveries obtained are of 88.5% for vinyl, 94.2% for glass and 95.2% for stainless steel plates with RSD of 5.5 (n=36), 2.3 (n=36), 2.2% (n=36), respectively. The method was successfully applied to the assay of real swab samples collected from the equipment surfaces.

Capillary gas chromatography with mass spectrometric and atomic emission detection for characterization and monitoring chlordimeform degradation in honey.

Capillary gas chromatography-mass spectrometry and capillary gas chromatography-atomic emission detection have been successfully used to identify and monitor the main degradation products of chlordimeform when this compound is initially present in honey. The analysis of laboratory-spiked honey samples over 28 weeks revealed the occurrence of two degradation products: 4-chloro-o-toluidine (I) and N-formyl-4-chloro-o-toluidine (III). During this period the concentration of chlordimeform decreased to 7.5% of its initial value; the concentration of compound I increased gradually whereas compound III was present in a larger proportion and reached a maximum around the 14th week.

Separation of antifungal chiral drugs by SFC and HPLC: a comparative study.

The enantiomeric separation of several compounds, including an antifungal drug and several of its precursors, using HPLC and SFC is described in this work. The columns employed were based on polysaccharide derivatives and the results show that most of the separations obtained by SFC are better, in terms of high resolution and short analysis time, than those obtained by HPLC. Only one compound could not be resolved using SFC but, in this case, HPLC provided baseline resolution.

Separation of albendazole sulfoxide enantiomers by chiral supercritical-fluid chromatography.

The enantioseparation of albendazole sulfoxide (ABZSO) by chiral supercritical-fluid chromatography (SFC) on two columns, based on the polysaccharide derivatives Chiralpak AD and Chiralcel OD, was studied. The effect of different modifiers, methanol, ethanol, 2-propanol, and acetonitrile, was examined. The results showed that ABZSO can be separated on both columns, using an alcohol-type modifier. Using the Chiralpak AD column, the best results were obtained with 2-propanol and, in the case of the Chiralcel OD, with methanol.

Use of deactivated fused-silica capillary precolumns in pesticide analysis by gas chromatography with electron-capture detection.

The advantages and disadvantages of coupling a retention gap of fused-silica between the injection port and the chromatographic column are discussed. The influence on the peak width and height of several factors such as the solvent (n-hexane, acetone, ethyl acetate and methanol), the gap (length, inner diameter, deactivation mode), the injection volume and the pesticide concentration has been examined. Those factors have very different incidences so, it is not possible to extract a general recommendation about the use of gaps. For this reason, checking its viability in each particular case is more advisable.