Pharmacokinetics of pioglitazone after multiple oral dose administration in horses.

Pioglitazone is a thiazolidinedione class of antidiabetic agent with proven efficacy in increasing insulin sensitivity in humans with noninsulin-dependent diabetes mellitus, a syndrome of insulin resistance sharing similarities with equine metabolic syndrome. The purpose of this study was to determine the pharmacokinetics of pioglitazone in adult horses following multiple oral dose administration. Pioglitazone hydrochloride (1 mg/kg) was administered orally for 11 doses at 24-h intervals, and plasma samples were collected. Initially, a pilot study was performed using one horse; and thereafter the drug was administered to six horses. Samples were analyzed by liquid chromatography with tandem mass spectrometry, and pharmacokinetic parameters were calculated using noncompartmental modeling. The maximum plasma concentration was 509.1 ± 413.5 ng/mL achieved at 1.88 ± 1.39 h following oral administration of the first dose, and 448.1 ± 303.5 ng/mL achieved at 2.83 ± 1.81 h (mean ± SD) following the eleventh dose. Apparent elimination half-life was 9.94 ± 4.57 and 9.63 ± 5.33 h after the first and eleventh dose, respectively. This study showed that in healthy horses, pioglitazone administered at a daily oral dose of 1 mg/kg results in plasma concentrations and total drug exposure approximating, but slightly below, those considered therapeutic in humans.

Isolation and Purification of Pyrazines Produced by Reaction of Cellulosic-Derived Sugars with NH4OH and Selected Amino Acids.

Various pyrazines have been synthesized via reaction of selected cellulosic-derived sugars, ammonium hydroxide and amino acids at 110°C for 2 hours. Different methods of sample cleanup such as liquid-liquid extraction (LLE), liquid-solid extraction, column chromatography and distillation were employed to isolate pyrazines from the reaction mixture. Effective LLE of pyrazines from aqueous solution using either hexane, methyl-t-butyl ether (MTBE) or ethyl acetate required multiple extraction steps with fresh solvent each time. When hexane was used as the extraction solvent, no imidazole derivatives were extracted with the pyrazines. However, when MTBE or ethyl acetate was employed, 4-methyl imidazole was co-extracted and further cleanup was required. Passing the organic solvent extracts through a column of silica revealed that the silica retained the undesirable imidazoles, such as 4-methyl imidazole. A mixture of 90/10 hexane/ethyl acetate as eluting solvent provided the desirable pyrazines, but it also provided a desirable separation of pyrazines as a function of total alkyl substituent content. Distillation of the aqueous reaction mixture was also used to isolate the pyrazines, leaving the undesirable imidazoles in the undistilled portion of the reaction. Additional chromatographic methods were used to isolate pyrazines from the aqueous distillate including a column packed with C18-bonded silica.

Isolation of tetra-acyl sucrose esters from turkish tobacco using supercritical fluid CO2 and comparison with conventional solvent extraction.

Pure supercritical CO2 at various pressures and temperatures was used to effect the fractionation of tetra-acyl sucrose esters (SE) from dried, ground Turkish tobacco without any further pretreatment of the matrix. It was determined that SE cannot be extracted using low density CO2 (150 atm, 60 degrees C, and 0.62 gm/mL or 200 atm, 100 degrees C, and 0.49 gm/mL), whereas other analytes, which strongly interfere with the conventional solvent extraction of SE, can be easily removed under the same conditions. At the higher temperature (100 degrees C), these same analytes that interfere with the conventional solvent extraction of SE are even more readily removed, while the very poor extractability of SE is not affected. It was demonstrated, however, that SE can be removed from the pre-extracted tobacco with supercritical CO2 if the density is greater than (or equal to) 0.73 gm/mL. The supercritical fluid extraction method has been compared with other previous extraction methods that employ conventional solvents. This study provides one of the clearest examples of how the variable density property of a supercritical fluid can be utilized to effect the fractionation of a complex mixture.

Feasibility of correlating separation of ternary mixtures of neutral analytes via thin layer chromatography with supercritical fluid chromatography in support of green flash separations.

Method development for normal phase flash liquid chromatography traditionally employs preliminary screening using thin layer chromatography (TLC) with conventional solvents on bare silica. Extension to green flash chromatography via correlation of TLC migration results, with conventional polar/nonpolar liquid mixtures, and packed column supercritical fluid chromatography (SFC) retention times, via gradient elution on bare silica with a suite of carbon dioxide mobile phase modifiers, is reported. Feasibility of TLC/SFC correlation is individually described for eight ternary mixtures for a total of 24 neutral analytes. The experimental criteria for TLC/SFC correlation was assumed to be as follows: SFC/UV/MS retention (tR) increases among each of the three resolved mixture components; while, TLC migration (Rf) decreases among the same resolved mixture components. Successful correlation of TLC to SFC was observed for most of the polar organic solvents tested, with the best results observed via SFC on bare silica with methanol as the CO2 modifier and TLC on bare silica with a methanol/dichloromethane mixture.

Study of UltraHigh Performance Supercritical Fluid Chromatography to measure free fatty acids with out fatty acid ester preparation.

Most lipids are best characterized by their fatty acids which may differ in (a) chain length, (b) degree of unsaturation, (c) configuration and position of the double bonds, and (d) the presence of other functionalities. Thus, a fast, simple, and quantitative analytical technique to determine naturally occurring free fatty acids (FFA) in different samples is very important. Just as for saponified acylglycerols, the determination of FFA's has generally been carried out by high resolution gas chromatography (HRGC). The use of an open tubular capillary column coupled with a flame ionization or mass spectrometric detector provides for both high resolution and quantification of FFA's but only after conversion of all free fatty acids to fatty acid methyl esters (FAME) or pentafluorobenzyl esters. Unfortunately, volatilization of labile ester derivatives of mono- and poly-unsaturated FFA's can cause both thermal degradation and isomerization of the fatty acid during HRGC. The employment of a second generation instrument (here referred to as UltraHigh Performance Supercritical Fluid Chromatograph, UHPSFC) with high precision for modified flow and repeated back pressure adjustment in conjunction with sub-2µm various bonded silica particles (coupled with evaporative light scattering, ELSD, and mass spectrometric, MS, detection) for separation and detection of the following mixtures is described: (a) 31 free fatty acids, (b) isomeric FFA's, and (c) lipophilic materials in two real world fish oil samples. Limits of detection for FFA's via UHPSFC/MS and UHPSFC/ELSD versus detection of FAME's via HRGC/MS are quantitatively compared.

Ultrahigh performance supercritical fluid chromatography of lipophilic compounds with application to synthetic and commercial biodiesel.

Ultrahigh performance supercritical fluid chromatography (UHPSFC) in combination with sub-2µm particles and either diode array ultraviolet (UV), evaporative light scattering, (ELSD), or mass spectrometric (MS) detection has been shown to be a valuable technique for the determination of acylglycerols in soybean, corn, sesame, and tobacco seed oils. Excellent resolution on an un-endcapped single C18 column (3.0mm×150mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10min served greatly as an improvement on first generation packed column SFC instrumentation. Unlike high resolution gas chromatography and high performance liquid chromatography with mass spectrometric detection, UHPSFC/MS was determined to be a superior analytical tool for both separation and detection of mono-, di-, and tri-acylglycerols as well as free glycerol itself in biodiesel without derivatization. Baseline separation of residual tri-, di-, and mono-acylglycerols alongside glycerol at 0.05% (w/w) was easily obtained employing packed column SFC. The new analytical methodology was applied to both commercial B100 biodiesel (i.e. fatty acid methyl esters) derived from vegetable oil and to an "in-house" synthetic biodiesel (i.e. fatty acid ethyl esters) derived from tobacco seed oil and ethanol both before and after purification via column chromatography on bare silica.

Feasibility of supercritical fluid extraction with on-line coupling of reversed-phase liquid chromatography for quantitative analysis of polymer additives.

An on-line analytical method for determining polymer additives which incorporates (a) sample preparation and concentration, (b) chromatographic separation, and (c) UV detection is presented. The on-line system which combines supercritical fluid extraction (SFE) and high-performance liquid chromatography (LC) allows analytes to first be extracted and transferred to the SFE sorbent trap, then desorbed from the trap and presented to the LC system. No UV detector interference from residual dissolved CO2 in the mobile phase was observed since CO2 is eliminated by a pre-wash of the sorbent trap with a small amount of water. Reversed-phase LC with a mobile phase gradient of acetonitrile-water was employed. The described method allows a complete analysis to be processed in less than 30 min.

Comparison of methods for quantitative analysis of additives in low-density polyethylene using supercritical fluid and enhanced solvent extraction.

On-line supercritical fluid extraction-supercritical fluid chromatography (SFE-SFC) with cryogenic trapping was used to extract and separate five additives from a low-density polyethylene (LDPE) sample. A glass tube filled with glass wool afforded excellent collection efficiency for the extracted analytes. Additive spiked sand was employed to optimize the various parameters of the on-line SFE-SFC system. Calibration curves from the spiked sand studies for on-line SFE-SFC were obtained with good linearities for quantitation. Results obtained on additives in LDPE from on-line SFE-SFC were comparable to those from off-line SFE-HPLC and off-line enhanced solvent extraction (ESE)-HPLC for all additives except Irganox 1076. However, the precision obtained with on-line SFE-SFC was lower than that from off-line SFE-HPLC and off-line ESE-HPLC due to the small sample size employed in the on-line system. Considerable clean-up of the ESE extract was required prior to chromatographic analysis. On-line SFE-SFC minimized the sample handling and eliminated the use of organic solvent. Despite the lower than expected precision, the on-line SFE-SFC method for quantitation of polymer additives appears to be reliable and robust for application in routine quality control analysis.

Analysis of the sulfomycin component of alexomycin in animal feed by enhanced solvent extraction and supercritical fluid chromatography.

Enhanced solvent extraction (ESE) was used to isolate components of alexomycin from different types of animal feed samples. It was demonstrated that ESE was more economical as regards money and, for this particular matrix, twice as fast as supercritical fluid extraction (SFE) even though it was demonstrated that alexomycin can be extracted from feed quantitatively using both ESE and SFE. Supercritical fluid chromatography was used to separate alexomycin from other co-extractives of feed since liquid chromatography was unable to isolate the alexomycin for quantification purposes. Our results also showed that the concentration of alexomycin in an extruded sample was approximately half of the concentration which was originally added to the feed.

Comparison of gravimetry and hydrolysis/derivatization/gas chromatography-mass spectrometry for quantitative analysis of fat from standard reference infant formula powder using supercritical fluid extraction.

This paper describes a comparative study of the gravimetric versus hydrolysis/derivatization/gas chromatography-mass spectrometry determination of fat in infant formula. Fat was extracted using supercritical carbon dioxide modified with a small amount of ethanol, the extract was weighed, and the total fat was determined gravimetrically. Subsequently, another sample of the supercritical fluid fat extract was hydrolyzed to yield free fatty acids, which were converted to their methyl ester derivatives (FAMEs). Quantification was performed by GC-MS. NIST Standard Reference Material (SRM-1846) was used to validate both fat determination methods. Results showed that the gravimetric average percent fat was 26.86%, whereas the GC-MS method yielded 24.64%. Some peaks were detected in the ion chromatogram from the GC-MS that were identified as nonfatty acids such as aldehydes, which may account for the higher percentage fat measured as weight of extract rather than measured as FAMEs expressed as triglycerides.

Solubility of metal chelates and their extraction from an aqueous environment via supercritical CO(2).

Solubility of nickel(II), copper(II), and chromium(III) hexafluoroacetylacetone and chromium(III) acetylacetone chelates was measured in supercritical CO(2) at two different pressures (200 and 400 atm) and 60 degrees C. Solubility of fluorinated acetylacetone chelates was at least an order of magnitude higher than the non-fluorinated complexes. These pre-formed metal chelates as well metal diethyldithiocarbamate (DDC) and metal bis(trifluoroethyl)dithiocarbamate (FDDC) have also been extracted from aqueous environment using pure supercritical CO(2). It was demonstrated that metal HFA chelates while exhibiting higher solubility in supercritical CO(2) compared with metal FDDC chelates, exhibited lower extraction efficiency using the same extraction conditions. This behavior of metal HFA chelates is related to their stability in an aqueous environment. Direct extraction of Ni(+2) and Cu(+2) from an aqueous matrix was also achieved via in-situ chelation using diethyldithiocarbamate and bis(trifluoroethyl)dithiocarbamate as the ligands. Bis(trifluoroethyl)dithiocarbamate proved to be a more effective ligand for direct extraction of metal ions from aqueous environment using supercritical CO(2).

HPLC/atmospheric pressure chemical ionization-mass spectroscopy of eight regulated sulfonamides.

Reversed phase high performance liquid chromatography coupled with on-line atmospheric pressure chemical ionization mass spectrometry, HPLC,APCI-MS, has been applied to a mixture of eight sulfonamides. In full scan mode, extracted ion chromatograms produced minimum detectable quantities (MDQ) of 0.8 ng on column, for six of the eight regulated sulfonamides investigated. Selected ion monitoring yielded a 50 pg MDQ for sulfamerazine, sulfadiazine and sulfamethazine, while, the other compounds presented higher values. Analysis of supercritical fluid extracts of chicken liver containing sulfadimethoxine were found to be easily detected by HPLC/APCI-MS. In extracts of chicken liver spiked with 25 microg/kg(-1) (25 ppb) of sulfadimethoxine this compound could be detected in selected ion mode, while 100 pg/microl(-1) was detectable in either full scan or single ion modes. The analysis method for extracted sulfadimethoxine also demonstrated good linearity and reproducibility in both single ion and scan mode.

Demonstrative validation study employing a packed column pressurized fluid chromatography method that provides assay, achiral impurities, chiral impurity, and IR identity testing for a drug substance.

Assay development, assay validation, and documentation are reported here for a single packed column pressurized fluid chromatographic/ultraviolet (UV) method that provides: (1) simultaneous detection and quantification for the chiral drug, the chiral impurity and seven achiral impurities; and (2) a Fourier transform infrared (FT-IR) spectrometric identification test result for the Searle drug substance sample, xemilofiban. The separation is achieved in less than 30 min with three columns in tandem and a gradient of CO2-CH3OH. The post-column flow is split between UV (assay) and FT-IR (identification). Precision and accuracy are consistent within figures of merit obtained by liquid chromatographic-ultraviolet assays on analogous drug substances. The reported procedure combines three typical drug substance tests into one test (e.g. chiral impurities, achiral impurities, and infrared identification).

Comparison of supercritical CHF3 and CO2 and methanol-modified CHF3 and CO2 for extraction of sulfonamides from chicken liver.

Supercritical CHF3 and methanol-modified CHF3 were compared with supercritical CO2 and methanol-modified CO2 for extraction of sulfonamides from fortified chicken liver admixed with Hydromatrix. Results showed that solvating power and selectivity were higher for supercritical and methanol-modified CHF3 than for supercritical and methanol-modified CO2. Visual observation showed that chicken liver extract obtained with methanol-modified CHF3 was cleaner than that obtained with methanol-modified CO2. Fat precipitated in the solvent trap when CO2 was used as the extraction medium. Also, simple off-line collection of fortified chicken liver extract obtained with CO2 in a solid-phase extraction cartridge (packed with either C18 or alumina) followed by phosphate buffer-methanol (50 + 50) rinse yielded an extract that required no further cleanup for analysis.

Feasibility of on-line supercritical fluid extraction of steroids from aqueous-based matrices with analysis via gas chromatography-mass spectrometry.

The solubility of testosterone, boldenone, androstenone, etiocholanolone, and epitestosterone are measured in pure supercritical CO2. Testosterone exhibited the highest solubility in supercritical CO2. The solubility of all steroids except epitestosterone increased by one order of magnitude with increasing pressure from 100 to 400 atm. Epitestosterone had the lowest solubility in supercritical CO2 and its solubility was not affected by pressure. The extraction efficiency of steroids from an aqueous saline environment exceeded 95%. Because of the partial solubility of water in supercritical CO2, the addition of a moisture trap after the aqueous vessel is necessary to prevent the plugging and deterioration of the gas chromatographic (GC) column. It is demonstrated that on-line supercritical fluid extraction-GC-mass spectrometry is feasible for the quantitative extraction and analysis of steroids from both saline and urine solutions. However, it is determined that the adsorbent vessel filled with Hydromatrix is not sufficient to trap all the moisture, and after 3 to 4 extractions, the GC column efficiency lowered.

High-performance liquid chromatography coupled with chemiluminescence nitrogen detection for the study of ethoxyquin antioxidant and related organic bases.

The Chemiluminescent Nitrogen Detector (CLND) for use with high-performance liquid chromatography (HPLC) allows for the low-level detection of nitrogen-containing compounds with simple quantitation. The nitrogen selective detector's equimolar response (i.e., equal response for nitrogen independent of its chemical environment) allows for any nitrogen-containing compound to be quantitated as long as the number of nitrogens are known. The HPLC-CLND provides a new detection method for analytes that are not available in large quantities or have unknown chemical or physical characteristics such as oxidation products, metabolites, or impurities. Ethoxyquin is a primary antioxidant that is used to preserve many food products and animal feeds. HPLC-CLND is used in the study of the oxidation products of ethoxyquin because limited quantities of these compounds are available and subsequent calibration curves are difficult to maintain. HPLC-CLND as a new method of detection has been evaluated for its equimolarity of response, linear range, limit of detection, and limit of quantitation.

Gradient separation of PTH-amino acids employing supercritical CO2 and modifiers.

Twenty-four PTH-amino acids are rapidly and efficiently separated on a packed cyanopropyl Zorbax column by gradient elution of supercritical CO2 and tetramethylammonium hydroxide-modified methanol. Complete or partial resolution of 22 derivatives is observed with only valine coeluting with norleucine and lysine coeluting with asparagine. A wide variety of stationary phases and modifiers are investigated with supercritical CO2 in attempting to achieve the separation in less than 15 min. Critical to achieving a rapid and efficient separation is the control of modifier flow into the CO2. No modifier is required for elution of neutral PTH-amino acids. The addition of base plays a major role in the elution of acidic and basic PTH-amino acids. Peak tailing is minimized and the elution order of several peaks is altered upon incorporation of reagent into the mobile phase.

Evolution of strategies to achieve baseline separation of ten anionic, water-soluble sulfated estrogens via achiral packed column supercritical fluid chromatography.

Near baseline separation of ten sulfated sodium salts of various structurally related estrogens employing a variety of bonded stationary phase packed columns was obtained using a conventional supercritical fluid chromatograph coupled with UV detection. Critical pairs 2/3 (8,9-dehydroestrone/17ß-dihydroequilin) and 6/7 (17α-estradiol or 17α-dihydroequilin/estrone), however, failed to baseline separate. In all preliminary separations, 10mM ammonium acetate and variable percentages of H2O were initially used as co-additives in conjunction with methanol as a modifier. Different modifier programs and temperatures were employed to optimize the separation in a timely manner. A 2-ethylpyridine column provided the best separation compared to bare silica, diol, and cyano-based bonded phase columns. The employment of both salt and water as additives to the methanol-modified CO2 mobile phase suggested a mixed mode separation mechanism involving both ion pairing of each anionic sulfated estrogen with ammonium ion and hydrophilic interaction facilitated by partitioning of analyte between the aqueous solvated stationary phase and the aqueous component of the mobile phase. Upon more extensive study with either iso-propylamine or formic acid-ammonium formate buffer, the critical anionic pairs were 95% baseline resolved.

Screening strategies for achiral supercritical fluid chromatography employing hydrophilic interaction liquid chromatography-like parameters.

A thorough evaluation of 5 µm bare silica from two major vendors for achiral supercritical fluid chromatography of polar analytes has been carried out. Columns were the same dimension, and a virgin column was reserved for each modifier-mixture combination. Three mixtures were prepared and chromatographically separated via a gradient of methanol-modified CO(2) that incorporated 5% (w/w) water as a neutral additive. Mixture (A) invoked both trifluoroacetic acid and water as additives. Mixture (B) utilized isopropyl amine and water; while mixture (C) employed either ammonium acetate and water as additives or only water. Regardless of the mixture components and mobile phase composition, duplicate separations with superior selectivity and excellent peak resolution were observed on five analysis days over a 15-day period. Subsequent removal of water (i.e. primary additive) from each of the mobile phases led to lower selectivity for early eluting components but excellent peak resolution prevailed for later eluting peaks during a later 5-day testing period with only the secondary additive. The re-introduction of 5% water into the mobile phase (after allowing the bare silica columns that were used with no water to sit for 30 days) slowly yielded the original separation after approximately five injections. A hydrophilic interaction liquid chromatography (HILIC)-like mechanism for SFC whereby analyte partitions between water absorbed on the silica and water in the mobile phase is proposed. The general utility of this experimental approach with bare silica was subsequently demonstrated by single injection of ten drug-like compounds with each of the four mobile phases that previously were utilized with the three model compound mixtures. In each case, sharp peaks were observed for each drug-like compound regardless of the additive although retention times varied with the additive employed.

Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation.

Both analytical scale and preparative scale packed column supercritical fluid chromatography (SFC) have found widespread applicability for chiral separations of multiple polar pharmaceutical candidates. However, SFC is rapidly becoming an achiral technique. More specifically, ion pair SFC is finding greater utility for separation of ionic analytes such as amine salts and organic sulfonates. The key to this success is, in part, the incorporation of additives such as trifluoroacetic acid and ammonium acetate into the mobile phase in association with a wide variety of both bonded silica stationary phases and high purity bare silica. Ion pairing SFC coupled with evaporative light scattering detection and mass spectrometric detection is presented here for the separation of water soluble, uncapped, isomeric peptide pairs that differ in amino acid arrangement. The separation is best achieved on either diol-bonded silica or bare silica with 1-5% (w/w) water as a significant ingredient in the mobile phase. Nitrogenous stationary phases such as 2-ethylpyridine, which had been very successful for the separation of capped peptides failed to yield the desired separation regardless of the mobile phase composition. A HILIC type retention mechanism is postulated for the separation of both isomeric uncapped peptide pairs.