Gas chromatographic-mass spectrometric identification and quantitation of ethylene glycol in serum after derivatization with perfluorooctanoyl chloride: a novel derivative

Ethylene glycol poisoning is a common clinical problem and identification as well as quantitation of ethylene glycol in serum is important for medical and legal purposes. Most investigators described determination of ethylene glycol by gas chromatography without derivatization or derivatives forming a molecular ion <200. We describe a novel derivatization technique of ethylene glycol using perfluorooctanoyl chloride, after extraction from serum using acetone. This derivative has a molecular mass of 854 and produces a base peak at m/z 441 and other diagnostic strong peaks for unambiguous identification. Moreover, this derivative is less volatile and is free from interferences from endogenous serum components. Quantitation can be achieved by using 1,4-butanediol as an internal standard. The assay showed within-run and between-run precision of 7.2% and 8.0%, respectively, and linearity over the serum ethylene glycol concentration range 70–2240 μg/ml with a detection limit of 5 μg/ml.     

High-performance liquid chromatographic procedure for the measurement of nitrobenzodiazepines and their 7-amino metabolites in blood

A simple and sensitive HPLC method is described for the determination of the nitrobenzodiazepines, nitrazepam, flunitrazepam and clonazepam and their respective 7-amino metabolites in post-mortem blood. Using a single-step extraction the nitrobenzodiazepines were recovered from 0.5 ml of blood using butyl chloride. The solvent was evaporated to dryness and the reconstituted residue injected onto an HPLC system. Separation was achieved using a phenyl-bonded reversed-phase column with an acetonitrile-phosphate buffer mobile phase. Ultraviolet detection (240 nm) was used for quantitation. A linear response was observed between 0.01 and 0.60 mg/l. Assay precision between and within assays was less than 20% for all analytes. The limits of detection ranged from 0.001 to 0.008 mg/l with a limit of quantitation of 0.01 mg/l for all analytes.     

Glycolic acid production using ethylene glycol-oxidizing microorganisms.

Screening for microorganisms oxidizing ethylene glycol to glycolic acid was carried out. Among stock cultures, several yeasts and acetic acid bacteria showed high glycolic acid producing activity. Pichia naganishii AKU 4267 formed the highest concentration of glycolic acid, 35.3 g/l, from 10% (v/v) ethylene glycol (molar conversion yield, 26.0%). Among soil isolates, Rhodotorula sp. 3Pr-126, isolated using propylene glycol as a sole carbon source, formed the highest concentration of glycolic acid, 25.1 g/l, from 10% (v/v) ethylene glycol (molar conversion yield, 18.5%). Rhodotorula sp. 3Pr-126 showed higher activity toward 20% (v/v) ethylene glycol than P. naganishii AKU 4267. Optimization of the conditions for glycolic acid production was investigated using P. naganishii AKU 4267 and Rhodotorula sp. 3Pr-126. Under the optimized conditions, P. naganishii AKU 4267 and Rhodotorula sp. 3Pr-126 formed 105 and 110 g/l of glycolic acid (corrected molar conversion yields, 88.0 and 92.2%) during 120 h of reaction, respectively.     

The kinetics of glycol destruction by a Pseudomonas putida BS-2 strain]

Destruction of ethylene glycol and diethylene glycol by Pseudomonas putida BS-2 culture under conditions of its batch cultivation has been studied for its physiological regularities. The specific rate of the biomass growth in the region of limiting substrate concentrations depends on the diethylene glycol concentration in the medium and follows the Mono equation. A semisaturation constant for diethylene glycol is 209 +/- 17 mg/d. The specific rate of the culture growth is independent of the ethylene glycol concentration in the medium within a wide range from 0.08 to 10 g/l. Kinetics of the bacteria growth inhibition by excess of substrates is a complex character and obeys none of the known models of the substrate inhibition.     

Simultaneous high-performance liquid chromatographic analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization with o-phtaldialdehyde and fluorescence detection

A rapid, simple and robust method is presented for the simultaneous determination of the gamma-amino-n-butyric acid (GABA) derivatives pregabalin (PGB), gabapentin (GBP) and vigabatrin (VGB) in human serum by high-performance liquid chromatography (HPLC). Serum is deproteinized with trichloroacetic acid and aliquots of the supernatant are precolumn derivatized with o-phtaldialdehyde (OPA) and 3-mercaptopropionic acid. Separation is achieved on a Alltima 3C18 column using isocratic elution; the drugs are monitored using fluorescence detection. Norvaline is used as an internal standard. Within-day precision (COV; n = 10) is 1.2% for PGB (serum concentration 10.0 mg/l), 1.1% for GBP (serum concentration 15.8 mg/l) and 0.3% for VGB (serum concentration 15.5 mg/l). The method is linear up to at least 63 mg/l for PGB, 40 mg/l for GBP and 62 mg/l for VGB. Lower limits of quantitation (LOQ) are 0.13 mg/l for PGB, 0.53 mg/l for GBP and 0.06 mg/l for VGB. No interferences were found from commonly coadministered antiepileptic drugs (AEDs) and from endogenous amino acids. Experimental design in combination with statistical evaluation (ANOVA) was used to study the robustness of chromatography and sample preparation. The method is very suitable for routine therapeutic drug monitoring and for pharmacokinetic studies.     

High-performance liquid chromatographic determination of fluconazole in plasma

A high-performance liquid chromatographic method with ultraviolet absorbance detection at 260 nm was developed for the analysis of fluconazole in plasma. The method involves sample clean-up by liquid-liquid extraction. The proposed technique is reproducible, selective, reliable and sensitive. Calibration standards were prepared in the range 1.25-20 mg/l. The limit of quantitation was 0.4 mg/l. The coefficients of variation were 5% between measurements of a single extract injected in duplicate, and 7% between two extractions of spiked samples at the same concentrations. The separation between fluconazole and endogenous substances was satisfactory. This method was designed in order to minimise the risk of interference from substances that could be co-administered to critically ill patients undergoing hemodiafiltration. With a run time below 5 min, the present method is rapid and easy to use for later clinical studies, as well as for routine monitoring.     

Rapid determination of ethylene glycol at toxic levels in serum and urine

The rapid gas chromatographic detection and determination of ethylene glycol in biological fluids is described. Phenylboronic acid in acetone was used for the esterification of glycol. The phenylboronates of ethylene glycol and 1,2-propylene glycol are not separated on a packed column of medium polarity (OV-17), but they can be separated on a non-polar column (OV-101). In both instances 1,3-propylene glycol can be used as an internal standard. The method requires only 100 μl of serum or urine and is suitable for trace analysis in an emergency toxicological laboratory. The utility of the method is demonstrated on two cases of human intoxication with ethylene glycol.     

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid-liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025-10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.     

Effect of methyl alcohol and ethylene glycol on the work of activated sludge.

The effect of methyl alcohol and ethylene glycol on the work of activated sludge grown in synthetic wastewater was investigated. Wastewater carrying these compounds could be purified by the activated sludge method, providing the concentration of methyl alcohol and glycol did not exceed 5,000 mg/l and 1,000 mg/l, respectively. At these values reduced purification efficiency, increased volumetric index of the sludge and changes in the structure of the activated sludge flocs could be observed.     

Effects of small neutral osmolytes on the supercoiling free energy and intrinsic twist of p30delta DNA

Both theory and experiments are employed to investigate the effects of small neutral osmolytes on the average intrinsic twist (l0), the torsion and bending elastic constants, and the twist energy parameter (ET) that governs the supercoiling free energy. The experimental data for ethylene glycol and acetamide at 37 degrees C suggest, and are interpreted in terms of, a model wherein the DNA exhibits an equilibrium between two distinct conformational states that possess different numbers of bound water molecules and exhibit different intrinsic twists and torsion and bending elastic constants. Expressions are derived to relate the effective ET and l0 to the equilibrium constant, water activity (aw), and number (n) of bound water molecules released per cooperative domain undergoing the two-state transition. The variations of l0 and ET with -ln(aw) are similar for acetamide and ethylene glycol at 37 degrees C. Fitting the theory to those data yields the range n = 103-125 for ethylene glycol and n = 71-113 for acetamide, depending on the assumed value of ET for the dehydrated state. The cooperative domain size of the two-state transition is estimated to exceed about 25-30 base pairs (bp). Between 0 and 19.4 w/v % ethylene glycol, the torsion elastic constant, measured by time-resolved fluorescence polarization anisotropy (FPA), increases by 1.37-fold, whereas the measured ET decreases by 1.15-fold over that same range. The implied decrease in bending rigidity over that range is by a factor of about 0.7. The variations of l0 and ET with increasing -ln(aw) due to added ethylene glycol at 37 degrees C are far smaller than the corresponding variations observed previously at 14 and 15 degrees C. However, at 21 degrees C, upon adding either ethylene glycol or acetamide, l0 and ET initially decline steeply with increasing -ln(aw), with slopes possibly comparable to those seen at 14 and 15 degrees C, but then flatten out and follow curves similar to those at 37 degrees C. Possible origins of such mixed behavior are discussed. The effects of betaine at both 37 and 21 degrees C differ qualitatively and quantitatively in various respects from those of ethylene glycol and acetamide. Upon adding sucrose, l0 initially jumps to higher plateaus at both 37 and 21 degrees C, but its effects on ET cannot be reliably assessed, due to the limited range of -ln(aw).     

The effect of ethylene glycol on the phytovolatilization of 1,4-dioxane

Phytoremediation at contaminated sites is often complicated by the presence of more than one chemical However, the effects of common co-contaminants such as ethylene glycol on the phytoremediation of other chemicals, e.g., 1,4-dioxane, is not well understood. Field studies with DN34 poplar trees revealed a 28% decline in growth rate in response to 10 g/L ethylene glycol in the groundwater, thus indicating a significant and deleterious effect on tree viability, and likely, the plants' utility for phytoremediation. Thorough investigations using Arabidopsis thaliana, with its small size and rapid life cycle, indicated significant growth reduction at 10 g/L and complete inhibition of germination at 40 g/L ethylene glycol Ethylene glycol was almost as severe a stressor as the well characterized osmotic inhibitor, sorbitoL Watering potted trees with 10 g/L ethylene glycol reduced their growth by more than 50%, and similar results were observed in hydroponically grown poplar and willow trees. Under hydroponic conditions, 60 g/L ethylene glycol inhibited the phytovolatilization of l,4-dioxane by more than 80%, and all trees evapo-transpired 1,4-dioxane less efficiently than water. In fact, this efficiency differed between trees and the difference became more pronounced in the presence of ethylene glycol.     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

Validated gas chromatographic–mass spectrometric assay for determination of the antifreezes ethylene glycol and diethylene glycol in human plasma after microwave-assisted pivalylation

A gas chromatographic–mass spectrometric assay is described for identification and quantification of the antifreezes ethylene glycol (EG) and diethylene glycol (DEG) in plasma for early diagnosis of a glycol intoxication. After addition of 1,3-propanediol as internal standard, the plasma sample was deproteinized by acetone and an aliquot of the supernatant was evaporated followed by microwave-assisted pivalylation. After gas chromatographic separation, the glycols were first identified by comparison of the full mass spectra with reference spectra and then quantified. The quantification has been validated according to the criteria established by the Journal of Chromatography B. The assay was found to be selective. The calibration curves for EG and DEG were linear from 0.1 g/l to 1.0 g/l. The limit of detection for EG and DEG was 0.01 g/l and the limit of quantification for both was 0.1 g/l. The absolute recoveries were 50 and 65% for the low quality control samples and 51 and 73% for the high quality control samples of EG and DEG, respectively. Intra- and inter-day accuracy and precision were inside the required limits. The glycols in frozen plasma samples were stable for more than 6 months. The method was successfully applied to several authentic plasma samples from patients intoxicated with glycols. It has also been suitable for analysis of EG and DEG in urine.     

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.     

Development of a sensitive radiorespiration method for detecting microbial activity at subzero temperatures

We have developed a simple and sensitive method to detect microbial respiration at subzero temperatures. Microbial activity was detected by measuring (14)CO(2) evolved during the microbial-mediated mineralization of [1-(14)C] acetic acid or [2-(14)C] glucose in microcosm assays using modified (14)CO(2) traps. Various (14)CO(2) traps, designed to withstand freezing at subzero temperatures, were tested for their quench characteristics during liquid scintillation spectrometry and their ability to trap (14)CO(2). Solutions consisting of 1 M KOH supplemented with 20% or 30% v/v ethylene glycol did not freeze at temperatures above -20 degrees C and had a minor quenching effect on liquid scintillation spectrometry. Addition of ethylene glycol did have an effect on the efficiency of (14)CO(2) trapping, as the cumulative recovery of (14)CO(2) was reduced by 14% and 32% in the 1 M KOH+20% ethylene glycol and 1 M KOH+30% ethylene glycol solutions, respectively. Using the modified (14)CO(2) traps, microbial activity in representative Canadian high Arctic environmental samples was detected at temperatures as low as -15 degrees C. This simple method allows for sensitive, specific, and reliable detection of microbial activity occurring at subzero temperatures and is readily adaptable for studies in other cryoenvironments.     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

Methods for the determination of seven selective serotonin reuptake inhibitors and three active metabolites in human serum using high-performance liquid chromatography and gas chromatography

This paper describes a set of simple and sensitive multiresidue methods for the determination of the specific serotonin reuptake inhibitors (SSRIs) used as antidepressant drugs, and some of their respective active metabolites in human serum. It involves liquid–liquid extraction procedures followed by gas chromatography coupled to nitrogen phosphorus detection or isocratic reversed-phase high-performance liquid chromatography combined with fluorescence detection (HPLC–FL), depending on the analytes. Extraction recoveries were between 71 and 96% for the eight SSRIs and their metabolites analysed by GC and between 41 and 77% for the two of them analysed by HPLC. Limits of detection (LODs) and limits of quantitation (LOQs) ranged, respectively, from 2.5 to 5 μg/l and from 10 to 20 μg/l. Intra-assay and inter-assay precision was studied at three and four concentration levels, respectively, and was less than 19% for all compounds. Accuracy was also satisfactory for all. An excellent linearity was observed from the LOQs up to 1000 μg/l for milnacipram and paroxetine and from each LOQ up to 400 mg/l for the other compounds. The performance of the methods described thus allows the therapeutic drug monitoring of the currently commercialised SSRIs.     

Determination of BAY x 7195, a novel leukotriene D4 antagonist,in human plasma by high-performance liquid chromatography with post-column photo derivatisation and fluorescence detection

Methods to determine plasma concentrations of the leukotriene D₄ antagonist BAY x 7195 by HPLC with post-column photo derivatisation and fluorescence detection are described. Following dilution and centrifugation plasma supernatant is injected onto the HPLC system allowing the selective determination of the drug with a limit of quantitation (LOQ) of 10 μg/l (method A). Sensitivity was further enhanced to a LOQ of 0.6 μg/l by employing solid-phase extraction whereby the analyte concentration in the injection solution was increased (method B). Data on recovery, accuracy and precision of both methods throughout the working range are presented. BAY x 7195 is stable in plasma after repeated freeze-thaw cycles and upon storage at −20°C for at least 13 months. Method A was applied to a clinical study with oral administration of 250 mg BAY x 7195 where ca. 1% of the maximum plasma concentrations still could be accurately and precisely quantified. Method B was employed to determine the drug in plasma after administration of 1 mg as aerosol.     

Cryoprotectants for Crithidia fasciculata Stored at -20 C, with Notes on Trypanosoma gambiense and T. conorhini

SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log-phase or end-of-log-phase cultures were frozen for 24–48 hr at ∼ -20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2'-dioxyethanol (diethylene glycol); 1,2,4-butanetriol; 1,4-cyclohexanediol; dimethylsulfoxide; propylene glycol; and N -acetylethanolamine. Several sugars were active, e.g., D-arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood-enriched media + 2-2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3-week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.     

Toxic effects of some alcohol and ethylene glycol derivatives on Cladosporium resinae.

Eleven commercially available alcohol and ethylene glycol derivatives were tested for their toxicity toward a problem organism in jet fuel, Cladosporium resinae. In the presence of glucose, 20% (vol/vol) ethylene glycol monomethyl ether prevented spore germination and mycelial growth, and 10% (vol/vol) 2-ethoxybutanol, 10% 2-isopropoxyethanol, 10% 3-methoxybutanol, 5% 2-butyloxyethanol, 5% ethylene glycol dibutyl ether, and 5% diethylene glycol monobutyl ether were found to have similar effects. In a biphasic kerosene-water system, 3-methoxybutanol, 2-butyloxyethanol, and diethylene glycol monobutyl ether were again found to be more toxic than ethylene glycol monomethyl ether. Considerable potassium efflux, protein leakage, and inhibition of endogenous respiration were observed in the presence of the more toxic compounds. 2-Butyloxyethanol also caused loss of sterols from cells.