Co-polymerization of MTPC (methylene tri p-cresol) and m-cresol using CiP (Coprinus cinereus peroxidase) to improve the dissolution characteristics of the enzyme-catalyzed polymer

MTPC (Methylene tri p-cresol) and m-cresol were copolymerized by Coprinus cinereus peroxidase in aqueous acetone. Although MTPC did not dissolve completely in the aqueous acetone, copolymerization was achieved owing to the radical transfer between solute and solid surface. Various polymerized products with different molecular weights and hydroxyl values were synthesized depending upon reaction compositions (ratio of MTPC to m-cresol and buffer to acetone). Poly(MTPC–m-cresol), a copolymer of MTPC and m-cresol, was mixed with a diazonaphthoquinone derivative to form a new type of photoresist, a thin film of which was formed on a silicon wafer and immersed in alkaline solution (tetramethylammonium hydroxide) to measure speed of dissolution. Poly(MTPC–m-cresol), with higher hydroxyl value (over 80%), showed remarkably improved dissolution characteristics (dark loss in alkaline solution decreased by almost half), which is prerequisite for sensitive photoresist polymer.     

Modelling on isoamyl isovalerate synthesis from Rhizomucor miehei lipasein organic media: optimization studies

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was employed in the esterification of isovaleric acid and isoamyl alcohol to synthesize isoamyl isovalerate in n-heptane. Response surface methodology (RSM) based on a five-level, five-variable central composite rotatable design (CCRD) was used to evaluate the effects of important variables: enzyme concentration (20–40% w/w of acid), acid concentration (0.2–1.0 M), incubation period (24–120 h), alcohol concentration (0.25–1.25 M) and temperature (30–70 °C) on the esterification yield of isoamyl isovalerate. Extent of conversion was found to be excellent at all acid and alcohol concentrations employed in the range of 0.2–1.25 M, even at low enzyme concentration (20% w/w). The optimum conditions arrived at are as follows: 35% (w/w) enzyme concentration, 1.0 M acid concentration, 1.25 M alcohol concentration and 120 h incubation period, at 35 °C. Under these conditions, the predicted value was 680 mM ester matched very well with an experimental value of 678 mM.     

Scanning electron microscopic studies of lipase-catalysed esterification catalysis for the synthesis of stearoyl lactate and p-cresyl laurate

The state of three lipases, two from Rhizomucor miehei and one from porcine pancreas, employed in the esterification reactions leading to the preparation of food additive esters were investigated by scanning electron microscopy (SEM). The lipases employed in the synthesis of stearoyl lactic acid and p-cresyl laurate in 10 ml solvent at 40–60 °C in shake-flask experiments and 150 ml in non-polar solvents at 50–60 °C in bench-scale level experiments were compared. All three lipases, which were subjected to high temperatures and non-polar solvents for a prolonged period of incubation of 72–120 h, showed decrease in the compactness when compared to unused lipase. The presence of buffer preserved the activity and compactness and the absence of the same reduced the amount of enzyme per unit area on the support. R. miehei lipase samples subjected to reaction in presence of 0.0004 ml of 0.1 M buffer/mg enzyme preparation at different pH values (4.0–9.0) showed a decrease in compactness of the enzyme on the surface which correlated to an increase in esterification activity. An increase in volume of buffer (0.0002–0.003 ml/mg enzyme preparation) in the reaction mixture at pH 7.0 showed a decrease in compactness and also a reduction in activity. The studies indicate that a compromise between pH and volume of buffer can lead to variation in the extent of adsorption, distribution and activity, enabling the achievement of maximum conversions in the esterification reactions.     

The multiple soluble malate dehydrogenase of Hoplias malabaricus(Erythrinidae, Characiformes)

To explain the six-banded pattern obtained upon electrophoresis of the soluble form of malate dehydrogenase (sMDH, EC 1.1.1.37) from the characiform Hoplias malabaricus, a recent locus duplication of its A isoform (sMDH-A*), in addition to its sMDH-B* isoform, was proposed. Klebe’s serial dilutions carried out using skeletal muscle, heart and liver extracts showed that the A1 and A2 subunits have the same visual end-points, indicating that these A-duplicated genes have a nondivergent pattern. Since there is no evidence of polyploidy in the Erythrinidae family, the MDH-A* loci have probably evolved from regional gene duplication. While these sMDH-A* loci encode nondivergent thermostable isoforms, the sMDH-B* encodes a thermolabile one. Thermostable sMDHs differ from the thermolabile sMDHs in that they have a higher K_m of oxaloacetate. Liver, muscle and heart unfractionated sMDH levels at three different temperature and two pH regimens were analysed and the results showed that, in the adaptative temperature range of Hoplias, the variation in K_m under conditions of constant pH (imidazole buffer) was less (approximately threefold) than that measured in the presence of temperature-dependent pH imidazole buffer (sevenfold). Estimation of the ratio of both isoforms in these tissues by Klebe’s method showed that, in unfractionated liver – where K_m values were the highest and the minimum K_m was obtained at 30^C (both for temperature-dependent pH and constant-pH imidazole buffer) – the duplicate A (thermostable, A1 and A2) and B (thermolabile) subunits were detected in a ratio of 2:1. On the other hand, in muscle extracts – in which the lowest K_m values were measured, with the minimum K_m at 10–20^C (temperature-dependent pH and constant-pH imidazole buffer, respectively) – a ratio of two thermolabile to one thermostable subunits was observed.     

Noncompetitive Activation of the Peroxidase-Catalyzed Oxidation of o-Phenylenediamine by Melamine

Peroxidase-catalyzed oxidation of o-phenylenediamine (PDA) is greatly activated with melamine (MA) in 15 mM phosphate–citrate buffer at pH 6.0–7.4 in a noncompetitive manner: k _cat and K _m increase in direct proportion to the MA concentration. An extent of the activation is quantitatively characterized with a coefficient (in M^–1), which essentially increases along with the rise in pH from 6.0 to 7.4. MA acts as a nucleophilic catalyst in the oxidation process: it most likely affects the peroxidase active site from the distal position of heme. MA noncompetitively inhibits the peroxidase oxidation of PDA at pH 4.3, since it completely loses its nucleophilic properties in acidic medium. A rapid, highly accurate, and simple analytical test system based on the kinetics of melamine-activated oxidation of PDA is proposed for the quantitative determination of melamine within the concentration range of 10^–4–10^–3 M. This test system uses the spectrophotometric determination of the PDA oxidation product at 455 nm.     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Inhibition of hepatic lipase by m-aminophenylboronate application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases

Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K₁ value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h⁻¹ mg⁻¹ protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h⁻¹ mg⁻¹ The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

Quantification of nicotine, chlorpyrifos and their metabolites in rat plasma and urine using high-performance liquid chromatography

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1–85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2±7.9, 74.9±8.5, 81.7±6.9, 73.1±7.8, 74.3±8.3, 80.8±6.6, 81.6±7.3 and 81.4±6.5, and from urine 79.4±6.9, 77.8±8.4, 83.3±6.6, 72.8±9.0, 76.3±7.7, 83.4±7.9, 81.6±7.9 and 81.8±6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.     

Enzyme inhibition by p-cresol and lacticacid in lipase-mediated synthesis of p-cresyl acetate and stearoyl lactic acid: a kinetic study

A detailed kinetic study was carried out to investigate the porcine pancreatic lipase-catalysed esterification reactions of p-cresol–acetic acid and lactic acid–stearic acid. The kinetic data were in agreement with a Ping Pong Bi–Bi mechanism being followed by the enzyme, where inhibition is indicated in the presence of p-cresol and lactic acid in the respective reactions. Mathematical analyses of experimentally observed initial rates yielded various kinetic parameters, K _m(p-cresol) = 0.1, K _{m(acetic acid)} = 0.54, K _{m(lactic acid)} = 0.059 M, K _{m(stearic acid)} = 0.04 M, V _{max(p-cresol–acetic acid)} = 13.2(h^–1), V _{max(lactic acid–stearic acid)} = 0.00163 M/h, K _i(p-cresol) = 0.59 and K _{i(lactic acid)} = 0.079 M. The K _m and K _i values of p-cresol and lactic acid observed in the respective reactions showed both the competitive nature of binding between the substrates p-cresol and acetic acid on the one hand and lactic acid and stearic acid on the other and the inhibitory nature of p-cresol and lactic acid.     

Sensitivity variations of Listeria strains to the bacteriocins, lactocin 705, enterocin CRL35 and nisin

Five strains of Listeria monocytogenes, four strains of Listeria innocua and a strain of Listeria seeligeri showed different sensitivities to lactocin 705 (17 000 AU ml^–1), enterocin CRL35 (8500 AU ml^–1) and nisin (2500 IU ml^–1) at different pHs (5, 6 and 7). The susceptibility of Listeria strains to bacteriocins at each pH was strain dependent, and it was enhanced at the low pH. L. monocytogenes had enhanced nisin tolerance while the non-nisin bacteriocins were more inhibitory with viability losses of 3–3.4 in contrast with 1.5–1.8 log cycles, respectively. Lower viability loss values were obtained with L. innocua strains with all three bacteriocins while L. seeligeri was more sensitive to nisin than to lactocin 705 or enterocin CRL35.     

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography–mass spectrometry

We report here the development and validation of an LC–MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C₁₈ reversed-phase column using an acetonitrile–water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS–MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477→266), DMLOP (m/z 463→252) and A-LOP (m/z 519→266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3±1.50% (range: 70.7–73.7%) and 79.4±12.8% (64.9–88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.     

The effect of temperature on growth and cellulase (β-1,4-endoglucanase) production in the compost fungusChaetomium thermophile var.dissitum

Chaetomium thermophile var.dissitum, isolated from an experimental urban refuse compost, had the following growth characteristics: Minimum temperature, 27±1°C; optimum, 45–50°C; maximum, 57±1°C; pH optimum 5.5–6.0.A number of carbohydrates could be used for growth, but cellulase formation measured with carboxymethylcellulose as substrate was initiated only on cellulose or xylan. With cellulose as the carbon source, cellulase accumulation in the culture filtrate followed closely that of growth, when the temperature was varied. pH optimum for the cellulase system was 5.0.The optimum temperature for cellulase activity with carboxymethylcellulose as substrate varied between 77°C with 1/2 h incubation time and 58°C with 10 h incubation time.With cotton as substrate, the optimum temperature was 58°C regardless of incubation time. Carboxymethylcellulose had a higher stabilizing effect on the enzyme than cotton. The temperature stability of the cellulase was highest at pH 6.0.     

A study of factors influencing 2-methoxy-3-isopropylpyrazine production by Pseudomonas perolens using acid trap and UV spectroscopy

Summary A method employing acid-trapping of air-purged volatiles followed by UV absorption was used to determine factors influencing the production of 2-methoxy-3-isopropylpyrazine (MIPP) by Pseudomonas perolens. MIPP in 6 abetm HCl was found to have absorption maxima at 220 nm (A + 9290 cm^–1m^–1) and 307 nm (A = 9370 cm^–1m^–1). In liquid media, the amount of recoverable MIPP was dependent on the pH of the buffer system. It varied from 50% recovery at pH 6.0 to 83% recovery at pH 9.0. From solid media, about 54% of the MIPP added was recovered by the air purge and acid trap device. Exogenous addition of valine, glycine, and methionine did not increase MIPP production suggesting that precursors are endogenous to the cell. The amount of MIPP production was influenced by the initial cell density. The highest amount of MIPP was observed at a cell density of 10⁶ cfu/ml or less. Higher cell densities resulted in lower production. Offprint requests to: G. A. Reineccius     

Thermophilic H2 production from a cellulose-containing wastewater

Cellulose in wastewater was converted into H₂ by a mixed culture in batch experiments at 55°C with various wastewaters pH (5.5–8.5) and cellulose concentrations (10–40 g l^–1). At the optimal pH of 6.5, the maximum H₂ yield was 102 ml g^–1 cellulose and the maximum production rate was 287 ml d^–1 for each gram of volatile suspended solids (VSS). Analysis of 16S rDNA sequences showed that the cellulose-degrading mixed culture was composed of microbes closely affiliated to genus Thermoanaerobacterium.     

Kinetic studies of acetate fermentation by Methanosarcina sp. MSTA-1

Summary The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55° C, and optimum pH was 6.5–7.5, giving a minimum generation time of 12.6–13.9 h (µ_max = 0.050–0.055 h^–1) and a maximum value of the specific acetate consumption rate (q _infs ^supps ) of 14–20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55° C and at constant pH 7 resulted in a K _m value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively. Offprint requests to: R. Moletta     

Stomatal responses in isolated epidermis of the crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr.

The optimal conditions for opening of stomata in detached epidermis of the Crassulacean Acid Metabolism (CAM) plant Kalanchoe daigremontiana were determined. Stomatal opening in CO₂–free air was unaffected by light so subsequently all epidermal strips were incubated in the dark and in CO₂–free air. Apertures were maximal after 3 h incubation and were significantly greater at 15° C than 25° C. Thus stomata in isolated epidermis of this species can respond directly to temperature. Stomatal opening was greatest when the incubating buffer contained 17.6 mol m^–3 K⁺, but decreased linearly with increasing K⁺ concentrations between 17.6 and 300 mol m^–3; the decrease in aperture was shown to be associated with increasing osmotic potentials of the solutions. Reasons for this behaviour, which differs from that of many C₃ and C₄ species, are discussed. Stomatal apertures declined linearly upon incubation of epidermis on buffer solutions containing between 10^–11 and 10^–5 mol m^–3 abscisic acid (ABA). Hence stomata on isolated epidermis of K. daigremontiana respond to lower concentrations of ABA than those of any species reported previously.     

Sensitive isocratic liquid chromatographic assay for the determination of 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C₁₈ column (70×4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5–2000 and 10–1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.