Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method

We report a reversed-phase high-performance liquid chromatography method which resolves 13 identified carotenoids and nine unknown carotenoids from human plasma. A Nucleosil C₁₈ column and a Vydac C₁₈ column in series are used with an isocratic solvent system of acetonitrile–methanol containing 50 mM acetate ammonium–dichloromethane–water (70:15:10:5, v/v/v/v) as mobile phase at a flow-rate of 2 ml/min. The intra-day (4.5–8.3%) and inter-day (1.3–12.7%) coefficients of variation are suitable for routine clinical determinations.     

Determination of clozapine and its metabolite, N-desmethylclozapine, in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single solvent extraction step high-performance liquid chromatographic method is described for quantitating clozapine and its metabolite, N-desmethylclozapine, in rat serum microsamples (50 μl). The separation used a 2.1-mm I.D. reversed-phase Symmetry C₁₈ column with an isocratic mobile phase consisting of methanol–acetonitrile–28.6 mM sodium acetate buffer, pH 2.6 (10:20:70, v/v/v). The detection limit was 2.5 ng/ml for all the compounds using an ultraviolet detector operated at 230 nm. The method was used to study the pharmacokinetics of clozapine after an intravenous bolus dose (2.5 mg/kg).     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C₁₈ stationary phase and mobile phases of 20–40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4–5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the d- and l-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM β-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.     

Rapid and simple high-performance liquid chromatographic determination of nimesulide in human plasma

A high-performance liquid chromatographic method for the quantitation of nimesulide in human plasma is presented. The method is based on protein precipitation with methanol and reversed-phase chromatography with spectrophotometric detection at 404 nm. The separation was performed on a Nucleosil 120-5 C₁₈, 50×4-mm I.D. column and the mobile phase consisted of acetonitrile–methanol–15 mM potassium dihydrogenphosphate buffer, pH 7.3 (30:5:65, v/v). Only 250 μl of plasma are used for sample preparation and no internal standard is necessary. The limit of quantitation is 80 ng/ml and the calibration curve is linear up to 10 000 ng/ml. More than 20 samples can be analysed within 1 h. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Increasing NaCl and CaCl<Subscript>2</Subscript> concentrations in the growth medium of quince leaves: I. Effects on somatic embryo and root regeneration

Summary The effects of increasing concentrations of NaCl and CaCl₂ on quince (Cydonia oblonga Mill. BA 29 clone) somatic embryogenesis and adventitious root regeneration were investigated. Leaves collected from in vitro-grown shoots were used as explants and induced for 2d in liquid Murashige and Skoog medium containing 11.3 μM 2,4-dichlorophenoxyacetic acid. Explants were then cultured on semisolid Murashige and Skoog medium enriched with 4.7 μM kinetin and 0.5 μM naphthaleneacetic acid under red light for 25 d and under white light for another 25 d. Two experiments were performed: in the first, NaCl was used at 0,25, 50, 100, and 200 mM in factorial combination with CaCl₂ at 3, 9, and 27 mM; in the second, NaCl was applied at 0, 5, 10, 20, 40, and 80 mM in combination with CaCl₂ at 0.3, 1.0, and 3.0 mM. Quince leaves revealed the capacity to regenerate somatic embryos and/or adventitious roots. Quantitative and qualitative regeneration from leaves was affected by NaCl treatments: increasing NaCl concentrations, in combination with CaCl₂ at 1 mM, led to an increase in the proportion of leaves producing somatic embryos only, and to a decrease of both leaves regenerating roots only and leaves simultaneously producing somatic embryos and adventitious roots. This suggests a beneficial effect of salt stress on the embryogenic process. The regeneration response decreased with increasing salt concentrations and was almost totally inhibited above 50 mM NaCl and 9 mM CaCl₂. The presence of CaCl₂ in the culture medium apparently mitigated the effects of salt stress, but only when NaCl was applied at 40 mM. NaCl at 5 mM, in the presence of 0.3 or 1 mM CaCl₂, was favorable both to somatic embryo and root production. No value of the ratio Na⁺/Ca²⁺ was found to be optimal for the regeneration processes.     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.     

Sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its N-desethyl metabolite in human saliva or cerebrospinal fluid using solid-phase extraction

A sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its primary metabolite in human saliva or cerebrospinal fluid using solid-phase extraction is described. Samples mixed with internal standard and sodium phosphate buffer were applied to an activated C₁₈ solid-phase extraction column. The reconstituted eluate was injected onto a Zorbax RX C₈ column utilizing a mobile phase of 100 mM ammonium acetate (pH 4.0)–isopropyl alcohol–acetonitrile (55:20:25, v/v/v). Fluorescence detection was employed with excitation at 295 nm and emission at 456 nm. Quantitation was achieved using peak-height ratios. The detection response curve was linear from 2 to 850 nM for atevirdine in both human saliva and cerebrospinal fluid and from 2 to 250 nM for the metabolite in human saliva. The method was utilized to analyze cerebrospinal fluid and saliva samples from clinical studies.     

Microbore high-performance liquid chromatographic determination of cisapride in rat serum samples using column switching

For the determination of cisapride from serum samples, an automated microbore high-performance liquid chromatographic method with column switching has been developed. After serum samples (100 μl) were directly injected onto a Capcell Pak MF Ph-1 pre-column (10×4 mm I.D.), the deproteinization and concentration were carried out by acetonitrile–phosphate buffer (20 mM, pH 7.0) (2:8, v/v) at valve position A. At 2.6 min, the valve was switched to position B and the concentrated analytes were transferred from MF Ph-1 pre-column to a C₁₈ intermediate column (35×2 mm I.D.) using washing solvent. By valve switching to position A at 4.3 min, the analytes were separated on a Capcell Pak C₁₈ UG 120 column (250×1.5 mm I.D.) with acetonitrile–phosphate buffer (20 mM, pH 7.0) (5:5, v/v) at a flow-rate of 0.1 ml/min. Total analysis time per sample was 18 min. The linearity of response was good (r=0.999) over the concentration range of 5–200 ng/ml. The within-day and day-to-day precision (CV) and inaccuracy were less than 3.7% and 3.8%, respectively. The mean recovery was 96.5±2.4% with the detection limit of 2 ng/ml.     

Increasing NaCl and CaCl2 concentrations in the growth medium of quince leaves: II. Effects on shoot regeneration

Summary The effects of NaCl and CaCl₂ on shoot regeneration from quince (Cydonia oblonga BA L29 clone) leaves were investigated. Caulogenesis was induced on in vitro-grown leaves treated for 2d in liquid Murashige and Skoog (MS) medium with 11.3 μM 2,4-dichlorophenoxyacetic acid and cultured on MS gelled medium supplemented with 4.5 μM thidiazuron and 0.5 μM naphthaleneacetic acid. Three experiments were performed: in the first, we compared the effects of NaCl at 0, 25, 50, 100, and 200 mM in factorial combination with 3, 9, and 27 mM CaCl₂. In the second, NaCl was tested at 0, 5, 10, 20, 40, and 80 mM with CaCl₂ at 0.3, 1.0, and 3.0 mM. The third experiment was carried out with the same experimental design as the second one but replacing NaCl with Na₂SO₄. Shoot regeneration was evaluated after 50 d of culturing: 25 in darkness and 25 in white light. In the first experiment, shoot regeneration was very poor and was observed only at the lower salt concentrations. In the second experiment, the percentages of caulogenic leaves were much higher, but decreased with increasing NaCl concentration. The more pronounced negative effect of the highest NaCl concentrations appeared to be partly mitigated by CaCl₂ at 1 and 3 mM. The presence of 3 mM CaCl₂, in the experiment with Na₂SO₄, appeared to be even more effective in reducing the adverse effect of sodium stress on caulogenesis. This result was attributed to the lower Cl⁻ concentration in the growth medium, which resulted from replacing NaCl with Na₂SO₄. NaCl applied at low concentrations (5 and 10 mM) in combination with 3 mM CaCl₂ exerted a favorable effect on adventitious shoot regeneration. As regards the Na⁺ and Ca²⁺ interaction, when the Na⁺/Ca²⁺ ratio was below roughly 35 and 20, with NaCl and Na₂SO₄, respectively, at least 60% of leaves showed regenerating capacity, but optimal values of this ratio were not derived.     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC–ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100×1 mm I.D. C₈, 5 μm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 μl/min. For monoamines and metabolites we used a 150×1 mm I.D. C₁₈ 5 μm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 μM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-μm tissue slices. Tissue samples were homogenized in 50 or 100 μl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and γ-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

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.     

Application of reversed-phase liquid chromatography and prepacked C18 cartridges for the analysis of oxytetracycline and related compounds

The reversed-phase (RP) chromatographic separation of oxytetracycline (OTC) 4-epioxytetracycline (4-epiOTC), α-apooxytetracycline (α-apoOTC), and β-apooxytetracycline (β-apoOTC) has been accomplished on an Inertsil C₈ column at ambient temperature. Using the simplex method of solvent optimization, a 0.1 M ammonium acetate buffer (pH 3.0)-acetonitrile-tetrahydrofuran (72.5:12.5:15, v/v/v) mobile phase was found to give excellent separation of the compounds. OTC, 4-epiOTC, α-apoOTC and β-apoOTC were resolved in 35 min with calculated detection limits of 40, 20, 50 and 140 ng/ml, respectively. Solid-phase extraction (using RP C₁₈ cartridges) of OTC and OTC degradation compounds from distilled water and porcine muscle was tested at four concentration levels ranging from 200 to 2000 ng/ml (g); overall mean recovery of OTC from distilled water and porcine tissue was greater than 90% and 70%, respectively.     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

Excretion of peroxidase from horseradish hairy root in combination with ion supplementation

Summary The effect of ion-supplemented medium on peroxidase excretion from horseradish (Armoracia rusticana) hairy roots was studied. Supplementation of mannitol instead of ions revealed that the excretion was stimulated not by osmotic pressure in the medium but by ionic properties. Extracellular peroxidase activity per dry cell was proportionally correlated with the ionic strength of the cations. CaCl₂ or MgCl₂ was found to be the most effective agent for excretion among other combinations. CaCl₂ supplementation at the beginning of the culture caused higher peroxidase production in the medium without a significant loss of final cell mass compared with CaCl₂ addition during the culture. Repeated batch culture with 50 mM CaCl₂ supplementation allowed a continuous retention of cell viability over 149 days and produced a great amount of extracellular peroxidase, 12-fold higher than that achieved in a 40-day-old batch culture with 50 mM CaCl₂ supplementation. Correspondence to: T. Kobayashi     

Determination of a new non-narcotic analgesic, DA-5018, in plasma, urine and bile by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new non-narcotic analgesic, DA-5018 (I), in rat plasma, urine and bile samples, using propranolol for plasma samples and protriptyline for urine and bile samples as internal standards. The method involved extraction followed by injection of 100 μl of the aqueous layer onto a C₁₈ reversed-phase column. The mobile phases were 5 mM methanesulfonic acid with 10 mM NaH₂PO₄ (pH 2.5)-acetonitrile, 70:30 (v/v) for plasma samples and 75:25 (v/v) for urine and bile samples. The flow-rates were 1.0 ml/min for plasma samples and 1.2 ml/min for urine and bile samples. The column effluent was monitored by a fluorescence detector with an excitation wavelength of 270 nm and an emission wavelength of 330 nm. The retention time for I was 4.8 min in plasma samples and 10.0 min in urine and bile samples. The detection limits for I in rat plasma, urine and bile were 20, 100 and 100 ng/ml, respectively. There was no interference from endogenous substances.     

Ca2+ effects on ethylene, carbon dioxide and 1-aminocyclopropane-1-carboxylic acid synthase activity

The response of pericarp disks from ripening tomato (Lycopersicon esculentum Mill. cv. Traveler‘76) to CaCl₂, additions was studied to determine the effect of Ca²⁺ on ethylene and CO₂ production. Application of 5 mM CaCl₂ resulted in a 2, 20, 33, 39, and 50% increase in ethylene production in disks obtained from preclimacteric minimum, climacteric rise, climacteric peak, one, and two days postclimacteric fruit, respectively. CaCl₂ concentrations of 10 and 50 mM gave no additional stimulation of ethylene production; CO₂ production at 5 mM CaCl₂ was not different from controls, but is increased at 10 and 50mM CaCl₂. CaCl₂ also increased ethylene production in disks treated with 1-aminocyclopropane-1-carboxylic acid (ACC) or aminoethoxy-vinylglycine. Chloride salts of K⁺, Na⁺, Mg²⁺, Sr²⁺ and La³⁺ did not stimulate ethylene production. SrCl₂ stimulated ethylene production to a lesser degree than CaCl₂. Disks from potato (Solanum tuberosum L. cv. Katahdin) tubers produced greater quantities of ethylene and ACC when 5 mM CaCl₂ was included in the incubation medium (K. B. Evensen, 1983. Physiol. Plant. 60:125–128). Ca²⁺-treated disks had more than three times as much ACC synthase activity as control disks after 18 to 24 h incubation, when ethylene and ACC were maximal. The apparent K_m for S-adenosylmethionine was 13 μM at 29°C, pH 8.0 in extracts from both Ca²⁺-treated and control disks. Inclusion of 1 to 50 mM CaCl₂ in the assay medium did not significantly affect enzyme activity. ACC synthase extracted from control and Ca²⁺-treated disks had a pH optimum of 8.5 and an apparent molecular weight of 72 kdalton, estimated by gel filtration. It is likely that the presence of Ca²⁺ in the buffer allows greater synthesis of ACC synthase as part of the wound-healing response in potato, while in tomato the predominant effect is on membrane stabilization.