Wide-bore Column Research Articles

GC determination of long chain fatty acids that compose D003 in 5-mg film-coated tablets

D003 is a new product which consists of a mixture of aliphatic fatty acids (C 24:0–C 36:0), that shows antiplatelet, antithrombotic and cholesterol-lowering effects in experimental models. A gas chromatographic (GC) method using a DB-5 wide-bore column and 1-nonadecanoic acid as internal standard was developed and validated in order to determine D003 in 5 mg film-coated tablets. The acids were analyzed as methyl esters derivatives, prepared using 5% aqueous HCl–methanol. Developed method was specific for the active principle, even when samples were subjected to stress conditions. Good linearity (correlation coefficient >0.99) and accuracy (total average recovery=99.60%) were proven over a range 38–150% of the nominal concentration. Within-day and intermediate precisions at the nominal dose (100%) were <1.5%. The method was suitable for quality control and stability studies of these tablets.

Design considerations for rapid‐heating columns applied in fast capillary gas chromatography

AbstractNumerical calculation procedures are developed to predict the optimum experimental conditions for rapid‐programming fast capillary gas chromatography (GC). Retention times and peak widths are calculated from experimentally determined thermodynamic compound properties. Errors in the predicted values are below 2 and 15% for the retention times and peak widths, respectively. It is shown that rapid temperature programming is a powerful tool for speeding up the separation of samples containing homologous series of compounds such as, e.g., triglycerides. The optimal programming rate and the minimum required column length to obtain the shortest possible analysis time with sufficient resolution (Rs=1.5) are predicted. Using a wide‐bore column with a length of 10 m, a programming rate of 4°C/s and a carrier gas velocity of approximately 250 cm/s, the shortest possible analysis time for a triglycerides sample can be obtained while still maintaining sufficient resolution. With these instrumental settings the analysis time of a triglycerides separation can be reduced from 15 to approximately 5 minutes. © 2001 John Wiley &amp; Sons, Inc. J Micro Sep 13: 337–345, 2001

Fast gas chromatography using vacuum outlet conditions

In this study the possibility of operating a wide-bore column with an inner diameter of 530 μm at vacuum conditions was investigated as a route towards fast gas chromatography. High analysis speeds can be obtained as a result of the sub-ambient pressure conditions present in the column, and the consequently high diffusion coefficient of the solute in the mobile phase. The advantage of using a wide-bore column is the high sample capacity. To enable vacuum operation through coupling a wide-bore column to a mass spectrometer, three types of restricting the column flow were investigated. The first method uses a narrow bore pre-column (60 cm length×100 μm i.d.), the second method relies on the use of a tapered on column supercritical fluid chromatography restrictor (SFC-restriction), whereas the third method makes use of a micro-injection valve. It was concluded that the best performance was obtained using the narrow-bore column restriction and the SFC-type restriction. Analysis examples in the seconds- and minutes-range are shown, with peak-widths ranging from 0.1 to 2 seconds at half height. It was concluded that with a film-thickness above 1.5 μm, the advantage of the increase in analysis-speed due to low pressures is nullified due to retention time increase on a thick film column. © 2001 John Wiley & Sons, Inc. J Micro Sep 12: 613–622, 2000

Standard on-column injection: A fast and simple method for large volume injection in medium-resolution gas chromatography

In gas chromatography (GC) large volume injection of samples containing very high molecular weight analytes presents unique problems. Standard programmed-temperature vaporizing or on-column large volume injection systems cannot be used, mainly due to instrumental difficulties. A new, simple solution to this problem is presented. If short, wide-bore columns are used at carrier gas velocities above optimum, the cloud of vapor formed upon evaporation of the solvent can be rapidly discharged from the system by the column flow alone. Hence, in medium-resolution GC, large sample volumes (e.g., 50 μL) can be injected using a standard on-column injector. Neither a retention gap nor a solvent vapor exit is needed. The absence of column connectors makes this setup extremely rugged. The optimization of the direct on-column large volume injection is described. The applicability of the new technique for the injection of large volumes of triacylglycerides and heavy mineral oil fractions is shown. It is demonstrated that the new technique is relatively insensitive toward solvent composition. This makes the new method eminently suited for combination with gradient liquid chromatography preseparations. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 523–529, 2000

Determination of N-nitrosodiethanolamine in urine by gas chromatography thermal energy analysis: application in workers exposed to aqueous metalworking fluids.

The aim of this study was to describe a detailed and validated methodology designed for the analysis of carcinogenic N-nitrosodiethanolamine (NDELA) down to sub-microgram/l levels in urine and its application to a number of workers exposed to NDELA-contaminated aqueous metalworking fluids (MWF). Following a work-up procedure based on solid-phase extraction of NDELA, the urinary extracts were analysed without derivatization by gas chromatography on a polar wide-bore column with chemiluminescent detection using a thermal energy analyser (TEA). N-Nitroso-(2-hydroxypropyl)amine was used as an internal standard. The method was applied to 12 workers using "nitrite-free" or "nitrite-formulated" MWF and to 15 unexposed subjects. The NDELA content of the MWF was also determined using a similar, but simpler method able to easily quantify NDELA down to at least 0.1 mg/l. Contamination by NDELA traces of some chemicals used for the sample preparation, particularly ethyl formate, must be carefully checked since it can give rise to false-positive results of up to 1 or 2 micrograms/l. The response was linear in the range of 0-500 micrograms/l. Between 0.5 and 10 micrograms/l, the recovery rate was close to 95%, while repeatability ranged from 12.5 to 6.4% (n = 5). The detection limit was 0.3 microgram/l (Signal/noise = 3). No detectable NDELA could be observed in the control workers. There was no significant increase in NDELA levels at the end of shift spot samples from an exposed worker over 1 week. Higher NDELA concentrations were found in two workers (4.3 and 10.7 micrograms/l) exposed to "nitrite-formulated" fluids (contaminated with 65 and 18 mg NDELA per 1, respectively) than in nine workers (range, 0.4-1.3 micrograms/l exposed to "nitrite-free" fluids with lower levels of NDELA (range, 0.5-6.6 mg/l). The detailed methodology described in this work and applied to a limited industrial situation was found to be suitable for monitoring NDELA in the urine of workers exposed to aqueous MWF. A much larger screening has been undertaken with the aim of obtaining better information on the real exposure of workers sometimes exposed to "nitrite-formulated" fluids that are still used.

High temperature and temperature programming in high-performance liquid chromatography: Instrumental considerations

The successful application of high temperature and temperature programming in high-performance liquid chromatography (HPLC) requires the appropriate configuration of a chromatographic system and selection of proper column dimensions. Knowing the exact column temperature is important, especially if a developed method is to be transferred to another laboratory or is going to be performed on a chromatographic instrument with a different heating system. We have examined the suitability of several columns of different bore diameters in isothermal and temperature programming modes. The effect of different mobile phase flow rates on the overall system performance was also studied. Low volumetric flow rates, which generate high linear velocity in capillary and narrow-bore columns, allow fast heat equilibration between column wall and bulk liquid. The uniformity of the column heating, observed in the narrow-bore columns, is another characteristic that favors small diameter columns over the wide-bore columns. Mobile phase preheating significantly improves system performance, minimizing the effect of axial and radial temperature gradients on separation quality. The use of temperature programming in HPLC can be improved through the appropriate selection of the heating systems and by optimization of operating parameters, such as column dimensions and mobile phase flow rate. ©1999 John Wiley & Sons, Inc. J Micro Sep 11: 403–413, 1999

Dissipation of the Fungicide Tetraconazole from Field-Sprayed Sugar Beets

The reduction of tetraconazole residue levels in field sugar beets (roots and foliage) was investigated, and the effectiveness of this new fungicide against the leaf spot disease of sugar beet was evaluated. The fungicide was used according to the recommended application procedures. Sugar beets received five applications of tetraconazole at rates of 0.05 and 0.10 kg of active ingredient/ha. A multiresidue method was adapted, and tetraconazole residues were determined with gas chromatography using a wide-bore column and an electron capture detector. Recovery of tetraconazole from sugar beet roots and leaves was found in the range of 86−111 and 78−103%, and the detection limits were estimated as 0.001 and 0.002 mg/kg, respectively. A relatively rapid dissipation rate of tetraconazole residues was found with a half-life of 5 days for either sugar beet roots or foliage. Keywords: Dissipation; tetraconazole; residues; analysis; sugar beet

Cyclodextrin Derivatives in GC Separation of Racemic Mixtures of Volatiles. Part XIV: Some Applications of Thick-Film Wide-Bore Columns to Enantiomer GC Micropreparation

Representative examples of preparative GC isolation of pure enantiomers in amounts ranging from 0.5 to 2 mg, with thick-film wide-bore open tubular columns coated with cyclodextrin (CD) diluted in polysiloxanes, are described. Two 25 m×0.53 mm i.d. columns, coated with 3 μm of 30% 2,6-di-O-methyl-3-O-pentyl-β-CD/OV-1701 and 2 μm of 30% 2,3-di-O-acetyl-6-O-tert- butyldimethylsilyl-β-CD/PS-086 as stationary phases, were used. Methyl 3-hydroxyhexanoate, methyl 2-methylbutanoate, δ-hexalactone, δ-octalactone, and γ-decalactone (the latter from a nature-identical mango aroma), were submitted to automated micropreparative GC. Average yields of about 30% were achieved when amounts above 20 μg/μl were injected, and an enantiomeric purity above 90% was obtained.

23] Measurement of pantothenic acid and hopantenic acid by gas chromatography-mass spectroscopy

This chapter investigates the properties of hopantenic acid (HOPA)—a natural homolog of pantothenic acid (PaA) containing γ -aminobutyric acid (GABA) in place of β -alanine. Its calcium salt and pharmacologic properties have been used to improve blood circulation and metabolism in the brain. Because hopantenic acid is used pharmacologically it is important to be able to determine HOPA and PaA separately for pharmacokinetic studies. Gas chromatography–mass fragmentography (GC–MF) has been used to assay HOPA after administration of calcium hopantenate. Other methods for the assay of HOPA have also been developed, such as the GC–MF determination of pantoyllactone, which is a product of HOPA hydrolysis, the colorimetric determination of GABA with sodium 1,2-naphthoquinone 4-sulfate, and a high-performance liquid chromatographic (HPLC) method with 9-anthryldiazomethane as a fluorescent derivative. A bioassay method using Lactobacillus arabinosus for the determination of trimethylsilylated pantoyllactone by GC–MF and for the assay of PaA is developed. The simultaneous rapid microanalysis for PaA and HOPA in biological samples and natural products by GC–MF with a wide-bore column and multiple ion detection (MID) is described in the chapter. 5-[(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)amino]pentanoic acid calcium salt is used as an internal standard (IS) to measure PaA and HOPA simultaneously in plasma samples from humans, monkeys, dogs, pigs, rabbits, mice, rats, chickens, and soft-shelled turtles; in brain samples of chicken and soft-shelled turtle; and also in natural products.

Quantification of toxic hydrocarbon target compounds in engine exhaust and air by aluminum oxide porous-layer open-tubular capillary gas chromatography-mass spectrometry using isotopically labeled internal standards

Abstract A method for the determination of 1,3-butadiene, benzene and toluene in ambient air and engine exhaust is described. Air was sampled with a canister and then analysed by Al 2 O 3 -coated porous-layer open-tubular (PLOT) capillary gas chromatography-mass spectrometry. The PLOT column provides better resolution for light hydrocarbons and also alleviates the need of using a sub-ambient oven temperature. While the retention times might change owing to the adsorption of moisture, the use of mass spectrometry for detection and isotopically labeled analytes as the internal standards greatly improved the confidence of identification. The heating of solid deuterated butadiene sulfone provides a convenient method for the preparation of deuterated butadiene standard gas. To couple a wide-bore PLOT column with a bench-top mass spectrometer, a pressure gradient and open split interface were used in order to maintain the chromatographic resolution and limit the running time within a reasonable range.

Modification of AOAC Multiresidue Method for Determination of Synthetic Pyrethroid Residues in Fruits, Vegetables, and Grains. Part I: Acetonitrile Extraction System and Optimization of Florisil Cleanup and Gas Chromatography

We present a multiresidue method for determination of synthetic pyrethroids in fruits, vegetables, and grains. The method is a modification of AOAC Method 970.52. Residues are extracted with acetonitrile (for fruits and vegetables) or acetonitrile-water (2 + 1) (for grains) and then transferred to hexane. Coextractives are removed by acetonitrile partitioning and open-column chromatography with deactivated Florisil. The final extract is analyzed by gas chromatography with electron capture detection (GC-ECD). An HP-17 wide-bore column is used to determine the individual isomeric contents of each insecticide. The method was used to recover 8 pyrethroids (biphenthrin, fenpropathrin, cyhalothrin, permethrin, cypermethrin, fluvalinate, fenvalerate, and deltamethrin) spiked at 0.01-4.0 mg/kg in 20 crops (apple, pear, peach, banana, grape, strawberry, potato, tomato, cucumber, pepper, cabbage, carrot, celery, polished rice, wheat, green gram, buckwheat, sorghum, maize, and barley). Recoveries of the 8 pyrethroid insecticides in 6 crops ranged from 83.8 to 112.8%, with a coefficient of variation (CV) of 2.00 to 12.09% for the narrow-bore capillary GC (n = 6) and from 82.8 to 106.4%, CV = 2.93-12.19%, for the wide-bore capillary GC (n = 6). The minimum detectable levels of 0.004-0.028 mg/kg (for fruits and vegetables) or 0.01-0.08 mg/kg (for grains) for the 8 pyrethroids are easy to detect.

Simultaneous determination of ethylene glycol, propylene glycol, 1,3-butylene glycol and 2,3-butylene glycol in human serum and urine by wide-bore column gas chromatography

A method has been developed for the separation and measurement of ethylene glycol and three other glycols (propylene glycol, 1,3-butylene glycol and 2,3-butylene glycol) in biological samples by wide-bore column gas chromatography with a flame ionization detector. The method used 1,3-propylene glycol (1,3-propanediol) as an internal standard. The method was linear at least from 2 to 1000 μg/ml, with a detection limit of 1 μg/ml. Analytical recoveries were 89–98% for the different concentrations. Precision studies showed coefficients of variation of 1.5–7.7% for the different concentrations. The assay was applied to the analysis of biological samples from two patients who had ingested ethylene glycol and/or other glycols in a suicide attempt.

Chemistry, occurrence and biological effects of saturated pyrrolizidine alkaloids associated with endophyte-grass interactions

Saturated amino pyrrolizidine alkaloids with an oxygen bridge between C-2 and C-7 have been isolated from grasses and the chemistry and biology of these substances are reviewed in this paper. These substances are commonly referred to as loline alkaloids as they are derivatives of loline and originally were isolated from Lolium spp. The saturated amino pyrrolizidine alkaloids are not hepatotoxic and must be distinguished from the 1,2-unsaturated pyrrolizidine alkaloids which are very significant animal and human toxins and carcinogens of plant origin. Chemical synthesis of loline has been achieved based on starting materials of a nitrone and methyl 4-hydroxycrotonate. N-formylloline is made by addition of ethyl formate to loline and removal of excess ethyl formate and ethanol formed. N-acetylloline is synthesized by addition of acetyl chloride to loline in chlorofirm. N-acetylloline is partitioned into a dilute acid, then from a dilute base with CHCl 3, and the CHCl 3 removed to yield N-acetylloline. N-methylloline, norloline, N-formynorloline, N-acetylloline, and N-propionylnorloline are also readily formed from loline. Little is known about the biosynthesis of the loline alkaloids but from biosynthesis of other pyrrolizidine alkaloids a proposed biosynthesis is ornithine → putrescine → spermidine → → dialdehyde → → norloline → → loline. Loline was first isolated from Lolium temulentum L. ( Lolium cuneatum Nevski) and later several derivatives were identified in tall fescue ( Festuca arundinacea Schreb.). Chromatographic methodologies have been used to separate the pyrrolizidine alkaloids but 50-m capillary columns (0.32 mm i.d.) or 10-m wide-bore columns (0.53 mm i.d.) provide the greatest resolution of the many loline derivatives. Loline alkaloids have been found primarily in plants known to be infected witn Acremonium coenophialum Morgan-Jones and Gams. Lolines have been found in Lolium perenne L., F. arundinacea, and Poa autumnalus Muhl. ex Ell. infected with A. coenophialum and in Festuca gigantea (L.) Vill. infected with an unidentified Acremonium spp. Loline alkaloids were present in infected tall fescue in greatest amounts in seed, followed in decreasing amounts in the rachis, stem, leaf sheath, and leaf blade. Site of synthesis, plant and/or endophyte, is not known nor is the translocation tissue known. During the main part of the growing season there is little change in the accumulation of the loline alkaloids in leaf blade. Alkaloid levels increased with plant age and increased in regrowth tissue. The concentration of endophyte mycelium in leaf sheaths is positively associated with loline alkaloid accumulation. N-formylloline is toxic to several different insects from ingestion, topical and injected applications. Larger animal bioassay have not been conducted with pure compounds but the observations suggest some pharmacological activity in these systems.

Rapid toxicological screening of barbiturates in plasma by wide-bore capillary gas chromatography and nitrogen-phosphorus detection.

A rapid screening technique for the identification and quantitation of some barbiturates in plasma is reported. The method employs a one-step extraction at pH 5.8 into an organic phase, separation of compounds by gas chromatography with a wide-bore (0.75-mm i.d.) fused-silica capillary column connected to a direct injection kit, and nitrogen-phosphorus detection. Identification of each barbiturate is by retention time relative to brallobarbital, confirmed using another wide-bore column of slightly different polarity, connected to a flame-ionization detector. Rapid quantitation may be obtained with extracted standards used for drug screening. Detection threshold (0.5 micrograms/mL in a 0.5-mL serum sample and 0.5-microL injection volume), precision (CV < or = 11%), and linearity (r > 0.99) were satisfactory for routine toxicological applications.

Taguchi design experiments for optimizing the gas chromatographic analysis of residual solvents in bulk pharmaceuticals

Taguchi design experiments were conducted in order to identify and optimize the parameters that yield a maximum separation of 26 solvents commonly found in bulk pharmaceuticals. Wide-bore columns with chemically cross-linked stationary phases of different polarities were retained owing to their sensitivity and specificity in optimizing the analysis via a simple response function based on information theory.

Determination of three metabolites of a new angiotensin-converting enzyme inhibitor, imidapril, in plasma and urine by gas chromatography—mass spectrometry using multiple ion detection

A specific and sensitive gas chromatographic—mass spectrometric method for the determination of three metabolites of the angiotensin-converting enzyme inhibitor, imidapril, in plasma and urine was developed. The metabolites were isolated from plasma and urine using a Bond Elut C 18 solid-phase extraction cartridge. The isolated metabolites were converted to sensitive derivatives by pentafluorobenzyl bromide and heptafluoro- n-butyric acid anhydride. Following derivatization, the sample solutions were analysed by wide-bore column gas chromatography—mass spectrometry with multiple ion detection. The detection limits of the three metabolites were each 1 ng/ml in plasma and 5 ng/ml in urine. Analysis of the spiked plasma and urine samples demonstrated the good accuracy and precision of the method. This method was very useful for use in pharmacokinetic and bioavailability studies of the three metabolites of imidapril in humans.

Multiresidue Method for Quantitation of Organophosphorus Pesticides in Vegetable and Animal Foods

Abstract A critical review of multiresidue analytical methods was performed to develop an easy procedure that can be applied with slight modifications to vegetable and animal food samples. The multiresidue method described Is applicable to the quantitative determination of 28 organophosphorus pesticides In fatty as well as nonfatty foods. The foods are divided Into 4 groups according to extraction process (acetone and acetone-water for samples with moisture content &amp;lt;45%) and cleanup (on active carbon- Cellte, on disposable mlnicolumns of Kieselguhr- type material, and on disposable mlnicolumns of bonded-phase silica, according to fat and pigment content). A further fractionation on silica-gel microcolumns can be Included In the procedure. A wide-bore column (SPB-5) with flame photometric detection operated in phosphorus mode (FPD-P) was chosen for gas chromatographic determination. Recoveries from apples, olive oil, whole milk, pasta, and eggs are reported. Two packed columns (4% OV-101 and SP2250- SP2401) with FPD-P were tested as confirmation columns. The method is suitable for a total diet study.

Determination of isradipine and its pyridine metabolite in serum by capillary column gas chromatography with nitrogen-selective detection

A relatively simple, sensitive and precise gas chromatographic method for the determination of isradipine, a calcium antagonist of the dihydropyridine type, and its main metabolite in serum is described. Using a one-step extraction procedure, a wide-bore column and a nitrogen—phosphorus detector, a limit of quantitation of 0.5 and 2.0 n M for isradipine and the metabolite was found. No interferences from several drugs were observed. The method was successfully used in a pharmacokinetic study in hypertensive women during pregnancy.

Determination of urinary 2,5-hexanedione concentration by an improved analytical method as an index of exposure to n-hexane.

2,5-Hexanedione is a main metabolite of n-hexane and is considered as the cause of n-hexane polyneuropathy. Therefore, it is useful to measure 2,5-hexanedione for biological monitoring of exposure to n-hexane. The analytical methods existing for n-hexane metabolites, however, were controversial and not established enough. Hence, a simple and precise method for determination of urinary 2,5-hexanedione has been developed. Five ml of urine was acidified to pH 0.5 with concentrated hydrochloric acid and heated for 30 minutes at 90-100 degrees C. After cooling in water, sodium chloride and dichloromethane containing internal standard were added. The sample was shaken and centrifuged. 2,5-Hexanedione concentration in an aliquot of dichloromethane extract was quantified by gas chromatography using a widebore column (DB-1701). Urinary concentration of 2,5-hexanedione showed a good correlation with exposure to n-hexane (n = 50, r = 0.973, p less than 0.001). This method is simple and precise for analysis of urinary 2,5-hexanedione as an index of exposure to n-hexane.

Determination of peroxyacetyl nitrate, peroxypropionyl nitrate and alkyl nitrates of atmospheric importance using capillary columns

Three capillary columns were studied for the determination of peroxyacetyl nitrate, peroxypropionyl nitrate and some alkyl nitrates of atmospheric importance. Two non-polar HP-1 100% dimethyl polysiloxane columns, of 0.53 and 0.32 mm I.D., yielded the best resolution of all compounds studied. The intermediate polarity HP-17 50% methyl-50% phenyl polysiloxane wide-bore column could not resolve peroxypropionyl nitrate from 2-butyl nitrate. Compared with capillary columns with polar Carbowax satisfactory phases, the columns studied gave a better resolution of peroxyacetyl nitrate and peroxypropionyl nitrate, and there was no interfering water peak. Alkyl nitrates could be determined simultaneously. Detection limits were in the low ppb range.