Use of enzymatic reactors in the high performance liquid chromatographic determination of ethanol and methanol with electrochemical detection

Despite numerous applications both in industry and in analytical chemistry, immobilized enzyme technology has encountered only limited success in modern liquid chromatography. The strict requirements of enzymatic reactors for high performance liquid chromatography (HPLC) (i.e., mechanical resistance, limited void volumes, high contact surface, high binding capacity, stable enzyme-to-matrix bond without loss of enzyme activity) are not yet fully met by commercially available products. In our laboratory, the development of post-column reactors with immobilized alcohol oxidase on-line with an HPLC/EC system has been undertaken. Three home-made reactors were produced: in the first alcohol oxidase from Candida boidinii was quasi-immobilized onto an HPLC-sized anion exchange support, in the second the enzyme was bound to a silica-based support via Schiff's base formation, and in the third a covalent linkage to an epoxy-activated hydroxyalkyl methacrylate material was chosen. Chemical immobilization proved superior to the ionic binding. The importance of limiting the post-column band spreading due to the reactor void volume, by using HPLC-size supports, was confirmed.     

Direct injection high-performance liquid chromatographic method with electrochemical detection for the determination of ethanol and methanol in plasma using an alcohol oxidase reactor

A highly sensitive reversed-phase high-performance liquid chromatographic assay for ethanol and methanol in plasma, using a post-column enzymic reactor with electrochemical detection, has been developed. The alcohols, separated on the column, were converted by immobilized alcohol oxidase into their respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, detected via oxidation at a platinum electrode. As the chromatographic column, two glass cartridges (150 mm x 3 mm I.D.) in series, packed with 10 microns HEMA-S 1000 packing, were used. Alcohol oxidase from Candida boidinii was immobilized onto HEMA-BIO 1000 VS-L (10 microns), packed in a 30 mm x 3 mm I.D. glass cartridge. The reaction product, hydrogen peroxide, was detected with an amperometric detector with a platinum electrode, operated at +500 mV vs. an Ag/AgCl reference electrode. A 20-microliters volume of ten-fold diluted plasma was injected without any pre-treatment. Under the described conditions, methanol and ethanol were well resolved from each other and from the "front" of the chromatogram. The limit of detection was ca. 2.5 nmol for ethanol and 0.6 nmol for methanol in plasma, at a signal-to-noise ratio of 3. Excellent linearity was observed for ethanol, in the range 0.125-4 micrograms injected (r = 0.9999). In contrast, the response for methanol was markedly non-linear above 500 micrograms injected, presumably owing to progressive saturation of the reactor. The precision and accuracy of the assay were satisfactory, as was the reactor life (one month).     

Flow-rate dependence of post-column reaction chromatographic detectors and optimization of reactor length for slow chemical reactions

In high-performance liquid chromatography, use of any post-column reactor invariably involves a compromise between the conditions needed to obtain complete reaction and avoidance of excessive dispersion by band broadening in the reactor. Flow rate and the reactor geometry interact to establish the final chromatographic performance. Based on the flow-rate dependence of the peak area and peak height, post-column detectors constitute a distinct class of detectors which differ from mass-flow and concentrations-sensitive detectors such as the flame ionization and absorbance detector, respectively. The concept of reactor length optimization is developed for first-order chemical reactions in a post-column detector. The findings are applicable to both chromatographic and flow-injection systems.     

Determination of the antibiotic fludalanine in plasma and urine by high-performance liquid chromatography using a packed-bed, post-column reactor with o-phthalaldehyde and 2-mercaptoethanol

Fludalanine is a novel anti-bacterial agent active against gram-negative and gram-positive bacteria. A high-performance liquid chromatographic assay has been developed using ion-pair chromatography to resolve fludalanine and the internal standard 3,3-difluoro-D-alanine from plasma and urine background. The mobile phase contains sodium dodecyl sulfonate and methanol in a phosphate buffer. Fludalanine is derivatized post-column with o-phthalaldehyde via a packed-bed chemical reactor. The adduct is detected fluorometrically. The plasma and urine assays are sensitive to 0.25 and 0.5 micrograms/ml, respectively.     

Specific detection of acetyl-coenzyme A by reversed-phase ion-pair high-performance liquid chromatography with an immobilized enzyme reactor

A selective chromatographic detection system for the determination of acetyl-coenzyme A (CoA) is reported. The short-chain acyl-CoA thioesters were separated by reversed-phase ion-pair high-performance liquid chromatography (HPLC), and then acetyl-CoA was selectively detected on-line with an immobilized enzyme reactor (IMER) as a post-column reactor. Thio-CoA liberated enzymatically from acetyl-CoA was determined spectrophotometrically after reaction with Ellman's reagent in the reagent stream. The IMER with phosphotransacetylase had a substrate specificity sufficient to determine acetyl-CoA and was active and stable in the mobile phase containing methanol and the ion-pair reagent. The calibration graph was linear between 0.2 and 10 nmol, with a detection limit of 0.05 nmol. This HPLC system with detection by IMER allows the selective identification and determination of acetyl-CoA in a mixture of acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA, which are difficult to separate with ion-pair HPLC.     

Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20alpha-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.     

Enhancing the response of alkyl methylphosphonic acids in negative electrospray ionization liquid chromatography tandem mass spectrometry by post-column addition of organic solvents

A method to enhance the signal intensity and signal-to-noise of several alkyl methylphosphonic acids in negative electrospray ionization liquid chromatography tandem mass spectrometry (ESI LC-MS/MS) is presented. This class of compound represents the initial metabolites and environmental degradants of the nerve agents: VX, rVX (Russian VX), GB (Sarin), GF (Cyclosarin), and GD (Soman). Compared with the post-column addition of the mobile phase, the post-column addition of aprotic solvents and longer chain alcohols enhance the signal intensity and signal-to-noise ratio (S/N) of the chromatographic peaks by factors of up to 60 and 19, respectively. The post-column addition of water, methanol, and ethanol resulted in little or no relative signal enhancement. It is proposed that the post-column addition of these solvents do not result in the same enhancements due to stabilization of analyte solvation through hydrogen bonding.     

Glassy carbon pre-column for direct determination of acetylcholine and choline in biological samples using liquid chromatography with electrochemical detection.

The determination of acetylcholine and choline has been quite successfully accomplished using liquid chromatography with electrochemical detection following the original reports of Potter et al. [J. Neurochem., 41 (1984) 188]. A post-column reactor containing acetylcholinesterase and choline oxidase allows conversion of the desired species into hydrogen peroxide, an electrochemically active substance. However, the direct injection of tissue homogenates and other biological samples into such a system exhibits quite large solvent fronts and unidentified peaks. Using a pre-column packed with glassy carbon particles, we were able to dramatically decrease the size of the solvent front for such injections and tentatively identify the unknown peaks to be caused, at least in part, by common catecholamines. The glassy carbon pre-column, in addition to increasing the selectivity of the results, allowed the required chromatographic time per sample to be decreased from 20 to 10 min.     

Determination of 2-mercaptopropionylglycine and its metabolite, 2-mercaptopropionic acid, in plasma by ion-pair reversed-phase high-performance liquid chromatography with post-column derivatization.

A simple and fast high-performance liquid chromatographic method was developed for the simultaneous measurement of 2-mercaptopropionylglycine (Tiopronine) and its metabolite (2-mercaptopropionic acid) in human plasma after the administration of a pharmaceutical dosage form (Acadione). The sample treatment before high-performance liquid chromatographic analysis consisted of the reduction of the corresponding disulphides by tri-n-butylphosphine and protein precipitation with ethanol. Separation was achieved by ion-pair high-performance liquid chromatography on a reversed-phase column (LiChrospher RP 18e) with cetrimonium bromide as counter ion and detection by fluorimetry after post-column derivatization with a selective thiol reagent, i.e. pyrenemaleimide. The high frequency of the analyzed samples and validation results make the method suitable for pharmacokinetic studies, and this was demonstrated by the first results obtained after the administration of an oral dose of 500 mg of Tiopronine to two healthy subjects.     

Effect-directed analysis by high-performance liquid chromatography with gas-segmented enzyme inhibition

A reversed-phase high-performance liquid chromatography system with UV-detector was equipped with an on-line acetylcholinesterase inhibition assay to achieve effect-directed analysis of potentially toxic samples. The enzyme activity was detected colorimetrically using Ellman's reagent. The inhibition and substrate conversion took place in glass capillaries at a 100 microL/min flow rate. Extra-column band spreading in the reaction coils reduces the sensitivity and separation power of biochemical detectors severely. Knitted reactors exhibited no reduction of longitudinal dispersion in the tested flow range. The implementation of air-segmentation allowed an extended inhibition and substrate conversion time without a significant loss of chromatographic resolution. The limit of detection of two model compounds carbofuran (carbamate) and paraoxon-ethyl (organophosphate) was determined to be 13 ng (injected mass) and 7.4 ng, respectively, applying an isocratic chromatography method. A mixture of five insecticides was separated by a gradient elution and the inhibitory effect on the enzyme activity could be detected with high resolution. The band width at half height of the enzyme inhibition detector signal after a reaction time of about 8 min or 4.2 m of capillary, respectively, increased only by a factor of 1.4 compared to the UV-detector signal.     

Rapid separation and measurement of rat urinary kallikrein by high-performance liquid chromatography with a continuous flow enzyme detector

Rat urinary kallikrein was separated by high-performance liquid chromatography (HPLC) using an ion-exchange or gel-permeation column. Kallikrein activity was monitored continuously with peptidase or esterase activity using a post-reactor system directly adapted to HPLC. A PTFE helically coiled tube served as the enzyme reactor vessel. Four and three peaks with peptidase and esterase activity, respectively, were detected on application of normal rat urine.     

The use of immobilized alcohol oxidase in the continuous flow determination of ethanol with an oxygen electrode

Immobilized alcohol oxidase was used in the determination of blood alcohol. The alcohol oxidase catalyzed the aerobic oxidation of ethanol and the oxygen concentration was monitored with an oxygen membrane electrode in a flow cell. The enzyme was immobilized either by covalent attachment via glutaraldehyde to the inside walls of nylon tubing, or by adsorption onto three separate controlled-pore glass support materials: TiO₂, SiO₂, or AL₂O₃. The supports were packed into 10 cm lengths of 3 mm i.d. glass tubing or 30 cm lengths of 5 mm i.d. nylon tubing. The five methods of immobilization were compared for stability and activity toward ethanol. Immobilization on silanized glass beads results in the highest activity and greatest stability of the reactor.     

High-performance liquid chromatographic assay of L-alpha-glycerophosphorylcholine using a two-step enzymic conversion.

A high-performance liquid chromatographic method using an enzymic reactor for determination of L-alpha-glycerophosphorylcholine in pharmaceutical forms is described. The procedure includes incubation of L-alpha-glycerophosphorylcholine with glycerophosphorylcholine phosphodiesterase (EC 3.1.4.2), giving choline and glycerophosphate, and subsequent chromatography of choline with a post-column enzymic reactor and electrochemical detection. The results obtained show a close linearity of the whole assay from 2 to 150 nmol/ml L-alpha-glycerophosphorylcholine, the sensitivity being 2 pmol per 20 microliters of injected sample. The precision of the method in the analysis of L-alpha-glycerophosphorylcholine in pharmaceutical forms, ampoules and capsules, was 1.34 and 1.21%, respectively.     

Novel quartz flow-cell as a post-column photochemical reactor for high-performance liquid chromatography

The construction of a new post-column photochemical reactor with quartz flow cells in series for high-performance liquid chromatography (HPLC) is described. The performance of the new reactor was compared with a conventional open tubular PTFE coil reactor. The sensitivity, accuracy and precision obtained with both reactors are comparable. The new reactor has the obvious advantages of smaller cell volume as well as inertness and resistance to not only light and heat produced by the UV lamp, but also to organic solvents in the mobile phases, which results in greatly improved durability, reduced peak broadening and shorter chromatographic run times. Application of the new reactor to the fluorescence detection of DU-6859a, a new fluoroquinolone antimicrobial agent, in human serum is reported.     

Determination of extracolumn effects in recycle gas chromatography with capillary columns

Extracolumn band broadening in multidimensional systems utilizing flow switching is clearly undesirable. In certain cases, i.e. capillary recycle gas chromatography, the success of an experiment is contingent on the minimization of pre- and post-column dispersion of the peaks. Knowledge of these sources of peak distortion is necessary to optimize the experimental design. A system that extracts statistical parameters from real chromatographic peaks is discussed and used to evaluate band broadening in a capillary recycle experiment.     

Improved method for the determination of the cortisol production rate using high-performance liquid chromatography and liquid scintillation counting

Two new methods for the determination of the cortisol production rate using reversed-phase high-performance liquid chromatography are described. One uses ultraviolet detection at 205 nm, the other on-line post-column derivatization with benzamidine, followed by fluorimetric detection. The specific activity of tetrahydrocortisol and tetrahydrocortisone in urine from patients who had received tritium-labelled cortisol was determined by the indicated methods, followed by fraction collection and liquid scintillation counting. The post-column reaction detection procedure was superior to ultraviolet detection, both in selectivity and analysis time. Intra- and inter-assay variance of the post-column reaction detection procedure were 3.7 and 4.7%, respectively. A good correlation (r = 0.99) was obtained between values determined by this procedure and by a thin-layer chromatographic procedure.     

Determination of plasma tranexamic acid using cation-exchange high-performance liquid chromatography with fluorescence detection

A procedure is described for the determination of plasma tranexamic acid concentrations using cation exchange high-performance liquid chromatography with fluorescence detection following post-column derivatisation with omicron-phthalaldehyde. The chromatographic conditions were optimised with respect to detector performance and the method applied to measuring the plasma tranexamic acid levels of patients in a double-blind trial.     

Determination of cyclodextrins in biological fluids by high-performance liquid chromatography with negative colorimetric detection using post-column complexation with phenolphthalein

A rapid and sensitive high-performance liquid chromatographic method for the analysis of beta- and gamma-cyclodextrin in aqueous biological fluids such as plasma, urine, or tissue homogenate is described. The chromatographic system consists of a microBondapak Phenyl column as stationary phase and a mobile phase of water with 10% methanol. After post-column addition of an alkaline solution of phenolphthalein, negative colorimetric detection is used. The elution solvent and post-column reagent were mixed in a capillary tubing of 1.5 m (1.0 mm I.D.). Two methods of sample treatment are given, one for large (1.0 ml) and one for small (0.1 ml) sample volumes. Both methods were shown to be linear and reproducible. The detection limit for beta-cyclodextrin was 1.0 microgram/ml (0.77 nmol/ml). The method was used in the determination of some pharmacokinetic parameters of beta-cyclodextrin in rats after intravenous injection.     

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.     

High-performance liquid chromatographic assays for protoporphyrinogen oxidase and ferrochelatase in human leucocytes

Rapid, sensitive and specific high-performance liquid chromatographic assays are described for protoporphyrinogen oxidase and ferrochelatase in human leucocytes. The enzyme reaction products were separated and quantitated by reversed-phase high-performance liquid chromatography with fluorescence detection. The optimal pH for the protoporphyrinogen oxidase assay was 8.6 and the Michaelis constant for protoporphyrinogen IX was 9.78 +/- 0.96 microM (mean +/- S.D.). The mean (+/- S.D.) activity of protoporphyrinogen oxidase in fourteen apparently healthy subjects was 0.146 +/- 0.023 nmol protoporphyrin IX per min per mg protein. In one patient with variegate porphyria, the activity was 0.028 nmol protoporphyrin IX per min per mg protein. The optimal pH for ferrochelatase was 7.4 and with protoporphyrin and Zn2+ as substrates, the Michaelis constants were 1.49 and 8.33 microM, respectively. The mean activity of ferrochelatase in ten control subjects was 0.24 nM Zn-protoporphyrin or 2.05 nM Zn-mesoporphyrin formed per h per mg protein.