Search

Dual counter-current chromatography (dual CCC)-tandem mass spectrometry (MS/MS) was successfully performed with a newly designed spiral column for dual CCC. The small column capacity required for directly coupling with electrospray MS/MS was accomplished by forming a rectangular spiral groove on a plastic disk and sealing it with a PTFE sheet. This novel dual CCC-MS/MS technique was successfully applied for the rapid determination of methomyl, fenobucarb and carbaryl pesticides in food. A two-phase solvent system of n-hexane-acetonitrile-0.1% formic acid (45:45:10) was suitable for both good dual CCC separation and sufficient ionization of pesticides. Recoveries of these three pesticides from mandarin orange and spinach samples fortified at 0.05 mg/kg were in the range of 93-107% with relative standard deviations of 2.4-3.8%.

Dual counter-current chromatography (dual CCC) has been successfully applied to rapid sample preparation for the simultaneous determination of residual carbaryl, fenobucarb and methomyl in vegetable oil and citrus fruit. The citrus fruit samples were extracted with n-hexane solution containing stable isotopically labeled internal standards (methomyl-d3, fenobucarb-d3 and carbaryl-d9), and applied to dual CCC using a two-phase solvent system of n-hexane-acetonitrile to purify the carbamate pesticides from aliphatic sample matrix. The coiled column was rotated at 420 rpm, the lower mobile phase was introduced through the head toward the tail, and the upper mobile phase in the opposite direction. Due to the high partition efficiency of dual CCC, the lower phase fraction collected from 2 to 5 min after injection could be subjected to flow-injection tandem mass spectrometry directly after concentration. Repetitive sample injection can be performed at high reproducibility without a risk of contamination from the compounds retained in the column.

Quinoline yellow (Color Index No. 47005) consists of multiple components that show a large difference in their partition coefficients ( K), ranging from 0.03 to 3.3 in the solvent system tert.-butyl methyl ether (MTBE)-1-butanol-acetonitrile- aqueous 0.1 M trifluoroacetic acid (TFA). Consequently, it requires an excessively long elution time for the simultaneous separation of all components by the standard high-speed counter-current chromatography technique, which uses a constant flow-rate of the mobile phase. In order to overcome this problem, we increased the flow-rate of the mobile phase stepwise from 0.1 to 2.0 mL / min. Using this new procedure, six components (0.2-6.1 mg) were successfully isolated from 25 mg of a commercial quinoline yellow preparation in a single run using a two-phase solvent system composed of MTBE-1-butanol- acetonitrile-aqueous 0.1 M TFA (1:3:1:5, v / v). The purified components were analyzed by high-performance liquid chromatography, electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy.  2003 Elsevier Science B.V. All rights reserved.

The WAP-8294A complex was isolated from the fermentation broth of Lysobacter sp. WAP-8294, whose major component, WAP-8294A2, showed a strong activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci in vitro, and also exhibited a potent activity against MRSA in vivo. The previous separation procedure using the conventional chromatographic methods was laborious and time-consuming, and the recovery of the desired compound was often unsatisfactory. In the present study, high-speed counter-current chromatography (HSCCC) was applied to the separation of the main components of the WAP-8294A complex. Due to the high polarity of the target compounds, we selected a hydrophilic two-phase solvent system composed of n-butanol-ethyl acetate-aqueous 0.005 M trifluoroacetic acid (1.25:3.75:5, v/v/v) which provided a suitable range of partition coefficient values for these compounds. Although the settling time of this solvent system was much longer than the optimum range, suggesting a low retention of the stationary phase under the standard experimental conditions, the separation was successfully performed at the low flow-rate of 0.5 ml/min. A sample size of 25 mg yielded pure fractions of three components (1-6 mg). The identification of each component was carried out by HPLC and fast atom bombardment mass spectrometry. The method will contribute to the clinical development of WAP-8294A2 as an anti-MRSA agent.

A multiresidue analytical method was developed for the confirmation of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in bovine tissues using electrospray ionization liquid chromatography-tandem mass spectrometry (LC-ESI-MS-MS) with a product ion scan mode. All penicillins gave [M-H]-, [M-H-CO2]- and [M-H-141]- as the product ion, when [M-H]- was selected as the precursor ion. Combination of an ion-exchange cartridge clean-up and the LC-ESI-MS-MS method can reliably identify all of these penicillins fortified at a concentration of 0.05 mg/kg in bovine tissues, including liver, kidney and muscle. The limits of confirmation satisfy the maximum residue limits for each of the penicillins established by the World Health Organization, US Food and Drug Administration, European Union and Japan.

High-speed counter-current chromatography was successfully applied to the separation of the main components of spiramycin. A 25-mg quantity of sample was separated using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:6:5:5), and the fractions were analyzed by high-performance liquid chromatography and fast atom bombardment mass spectrometry. The separation yielded 13.4, 0.7 and 1.7 mg of spiramycins I, II, and III with purities of 98.2, 92.3 and 97.4%, respectively.

A simple method has been developed for the simultaneous determination of triclabendazole and its metabolites (sulphoxide and sulphone) in bovine milk by reversed-phase high-performance liquid chromatography (HPLC). A milk sample was homogenized with sodium sulfate anhydrous and acetonitrile, and centrifuged. The supernatant was isolated, rinsed with n-hexane saturated with acetonitrile, and evaporated. The residue was dissolved with 0.1 M potassium dihydrogenphosphate, and 0.1 M sodium hydrogencarbonate, and then cleaned up on a Bond Elut C18 cartridge. The three compounds were separated on a Capcell Pak C18 UG 120 (5 microm, 150x4.6 mm I.D.) column and determined by UV detection at 295 nm. The mobile phase was acetonitrile-0.05 M ammonium acetate (50:50), and the flow-rate was 0.8 ml/min at 40 degrees C. The mean recoveries (n=4) were 89.1-95.0% with a relative standard deviation of 1.1-2.6%. The detection limits were 0.004-0.006 microg/g in milk. The proposed method was used to monitor raw milk samples for the market, and applied to the analysis of milk samples from 10 cows which had been administered with triclabendazole to control the liver fluke. The confirmation of the triclabendazole and its metabolites in the above milk sample has been achieved by electrospray LC-MS for the first time.

A simple, rapid, and reliable method for the determination of residual sulphonamide antibacterials (SAs) (sulfadiazine, sulfamerazine, sulfadimidine, sulfamethoxypiridazine, sulfisozole, sulfamonomethoxine, sulfamethoxazole, sulfisoxazole, sulfadimethoxine, and sulfaquinoxaline) in animal liver and kidney was developed using a combination of clean-up on a Bond Elut PSA cartridge and HPLC with UV detection. The SAs were extracted with ethyl acetate and then dissolved in 5 ml of 50 v/v% ethyl acetate-n-hexane after being evaporated to dryness. For clean-up of the crude sample, the resuspended extract was applied to a Bond Elut PAS (primary/secondary amine cartridge), and then SAs were eluted from the cartridge using 5 ml of 20 v/v% acetonitrile-0.05 M ammonium formate before being analysed by HPLC. Recoveries of the SAs at the levels of 0.5 and 0.1 microg/g were 70.8-98.2%, the rerative standard deviation were less than 7.0%, and the detection limits were 0.03 microg/g. The present analysis method of SAs in animal kidney and liver using HPLC with a clean-up procedure was demonstrated to be highly applicable to the direct LC-MS-MS analysis without any modification.

A multiresidue analytical method was developed for the quantification of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in bovine tissues using liquid chromatography– electrospray ionization tandem mass spectrometry (LC–ESI MS/MS) with a multiple reaction monitoring technique. Using the deuterated PCG and NFPC as internal standard was effective for improvement of repeatability and accuracy. We chose [M − H-141] − as a monitor ion of MRM analysis and [M − H] − as a precursor ion for each penicillin. Combination of an ion-exchange cartridge clean-up and ion-pair LC enable us to determine the residual penicillins using the standard curves made from standard solutions without the influence of sample matrix on the MS. The average recoveries of PCG, PCV, MPIPC, MCIPC, NFPC and MDIPC from bovine liver, kidney and muscle at the same concentrations as the tolerance levels of PCG (50 μg/kg) ranged from 77 to 101% with the coefficients of variation ranging from 0.7 to 4.2% ( n =5). The limits of quantification for the six penicillins were 2–10 μg/kg in bovine muscle, liver and kidney (S/N ratio >10).

We will review recent developments in mass spectrometric analysis of tetracycline antibiotics (TCs) in foods. The mass spectrometric techniques discussed are as follows: the collision-activated decomposition mass-analysed ion kinetic energy spectrometry (CAD MIKES), thin-layer chromatography (TLC)- fast atom bombardment (FAB) mass spectrometry (MS), particle beam (PB) liquid chromatography (LC)-MS, LC-fit FAB MS, thermospray (TSP) LC-MS, atmospheric chemical ionization (APCI) LC-MS and tandem electrospray (ESI) LC-MS. Their advantages and limitations are described in the confirmation of TCs in foods: CAD MIKES can confirm TCs with high sensitivity; however, its practical application is questionable because of uncommon instrumentation. TSP has a problem in reproducibility of the mass spectrum. Although TLC-FAB-MS can be applied to any kind of samples, it cannot be used for the quantitative analysis. LC-frit FAB-MS is a useful technique for the confirmation of TCs in honey, but it cannot be applied to animal tissues because of a lack of sensitivity. PB negative chemical ionization, APCI, and ESI-MS-MS can reliably confirm TCs in foods with good reproducibility.

In order to facilitate the application of the foam counter-current chromatography (CCC) technique to various natural products, we have introduced two parameters, i.e., “foaming power” and “foam stability”, which reliably indicate applicability of the sample to foam CCC. In each test, the sample was delivered into a graduated cylinder with a ground stopper followed by vigorous shaking. The foaming power was expressed by the volume ratio of the resulting foam to the remaining solution, and the foam stability by the duration of the foam. A set of data obtained from various samples including bacitracin, gardenia yellow, phloxine B, rose bengale and senega methanol extract revealed a significant correlation between these parameters and the degree of foam enrichment by foam CCC. The overall results indicated that a sample with the following properties may be effectively enriched by foam CCC: the foaming power is greater than 1.0 and the foam stability is over 250 min. We applied these experimental results to the enrichment of microcystins in a cyanobacteria bloom sample extract using foam CCC.

High-speed counter-current chromatography (HSCCC) has been successfully applied to the separation of the ivermectin components. A 25-mg quantity of the sample was separated using a two-phase solvent system of n -hexane-ethyl acetate-methanol-water (19:1:10:10, v/v). The fractions were analyzed by high-performance liquid chromatography, nuclear magnetic resonance, and fast-atom bombardment mass spectrometry. The separation yielded 18.7 mg of 99.9% pure ivermectin B1a, 1.0 mg of 96.0% pure ivermectin B1b, and 0.3 mg of 98.0% pure avermectin Bla (precursor of ivermectin).

A thin-layer chromatographic-fast atom bombardment mass spectrometric (TLC-FAB-MS) method incorporating an analyte condensation technique was established for the identification of the 27 food dyes consisting the twelve dyes permitted for use in foods and the fifteen unlawful dyes in Japan. The use of magic bullet [1,4-dithiothreitol-1,4-dithioerythritol (3:1)] as a matrix allowed the measurement of the FAB mass spectra of the food dyes except for Food Blue No. 2 (Indigo Carmine). The separation was performed on a C18-modified silica gel TLC plate using the following two solvent systems: methanol-acetonitrile-5% aqueous sodium sulphate solution (3:3:10) and methyl ethyl ketone-methanol-5% aqueous sodium sulphate solution (1:1:1). The condensation technique for concentration of a diffuse sample spot on the TLC plate improved the detection limit 4-20-fold with good reproducibility. The method was successfully applied to the identification of unlawful dyes in imported foods.

A simultaneous determination of three polyether antibiotics, salinomycin, monensin and lasalocid, was established by silica gel and RP-18 high-performance thin-layer chromatography (HPTLC) using 18,19-dihydrosalinomycin and 18,19-dihydro-20-ketosalinomycin as internal standards. Fluorescent derivatives of the polyethers were prepared by reaction of their sodium salts with 1-bromoacetylpyrene and Kryptofix 222 in acetonitrile. A silica gel HPTLC plate with carbon tetrachloride-ethyl acetate-acetonitrile (50:5:10) as a solvent system and a RP-18 HPTLC plate with dichloromethane-ethyl acetate-acetone-acetonitrile (15:2:1:55) gave satisfactory separations of the five pyrenacyl esters of salinomycin, monensin, lasalocid, 18,19-dihydrosalinomycin and 18,19-dihydro-20-ketosalinomycin. Under these conditions the internal standard 18,19-dihydrosalinomycin was found to be suitable for the simultaneous determination of salinomycin and monensin, and 18,19-dihydro-20-ketosalinomycin for lasalocid. A linear relationship was obtained between the fluorescence intensity and the amount of each antibiotic in the range 2–14 ng. The detection limit of the three antibiotics was 100 pg.

The major components of bacitracin were separated and purified using high-speed counter-current chromatography (HSCCC). A systematic search for optimum two-phase solvent systems resulted in two systems: chloroform—ethanol—methanol—water (5:3:3:4) and chloroform—ethanol—water (5:4:3). These were selected based on the determination of the partition coefficients of all the components and the setting time of the phase. HSCCC with these solvent systems separated two components, bacitracins A and F. Improvements in the flow-cell arrangement eliminated noise in detection, making in-line monitoring possible. A tandem mass spectrometric technique was used to characterize the isolated components.

We have introduced two series of two-phase solvent systems which facilitate the systematic search for the solvent systems suitable for high-speed counter-current chromatography. The n-hexane-ethyl acetate-n-butanol-methanol-water systems provide a broad range of hydrophobicity, while the chloroform-methanol-water systems are extremely useful for separations of various natural products with moderate hydrophobicity. The practical use of these solvent series was demonstrated with several test samples which include dinitrophenyl amino acids, S-triazine herbicides, indole auxins, and non-ionic organic solvents.

The semipreparative capability of the newly developed high-speed counter-current chromatograph equipped with a set of three multilayer coils has been demonstrated in separations of a variety of biological samples including triterpenoic acids, indole auxins, bacitracin, flavonoids and tetracycline derivatives, each with a suitable two-phase solvent system. The sample quantities ranging from 50 to 500 mg were efficiently separated within a few hours. The separation of tetracycline derivatives was remarkably improved by adding ammonium acetate to the solvent system.

A technique for the simultaneous determination of sorbic acid, benzoic acid, dehydroacetic acid, p-hydroxybenzoic acid esters (ethyl, isopropyl, n-propyl, isobutyl and n-butyl p-hydroxybenzoate) and saccharin sodium using ion-pair reversed-phase high-performance liquid chromatography is described. The nine food additives were separated on a Nucleosil 3C18 (3 microns) column (75 x 4.6 mm I.D.) using methanol-acetonitrile-0.05 M aqueous acetonic acid solution (pH 4.5) (1.5:1:3.1) containing 2.5 mM cetyltrimethylammonium chloride as the mobile phase at a flow-rate of 1.0 ml/min, and detected at 233 nm. The food additives were separated within 18 min and their calibration graphs were linear between 2 and 200 ng.

A simple, rapid and reliable chemical analysis method for microcystins (cyanoginosins) has been studied. Three different mobile phases for high-performance liquid chromatography were selected and optimized. Also the adsorptive powers of three commercially available C18 cartridges were compared and the results successfully applied to the clean up of three of the toxins. Finally a total system for the analysis and isolation of microcystins was established.

A novel design of the cross-axis synchronous flow-through coil planet centrifuge is introduced. The apparatus holds a pair of large coil holders symmetrically, one on each side of the rotary frame, at a lateral position 12.5 cm from the center of the holder shaft held 10 cm from the centrifuge axis. Mathematical analysis of acceleration generated by the planetary motion of the apparatus revealed a unique centrfugal force field which promises high retention of the stationary phase in the multilayer coil to perform efficient preparative-scale counter-current chromatography.

Foam counter-current chromatography has been successfuly applied to continuous removal and concentration of hydrophobic bacitrcin (BC) components from a large volume solution using nitrogen gas and distilled water free of surfactants or other additives. The experiment was initiated by introducing nitrogen at the head inlet of the coil rotated at 500 rpm. Then, a 2.5-1 volume of the sample solution containing BC at 50 ppm was continuously introduced into the middle portion of the coil at 1.5 ml/min. The hydrophobic components produced a thick foam which was carried with the gas phase and collected from the tail end of the coil while the other components stayed in the liquid stream and eluted from the head outlet of the coil. High-performance liquid chromatographic analysis of the foam fraction revealed that the degree of enrichment increased with hydrophobicity of the BC components. BC-A and -F were enriched 1400 and 2260 times, respectively, compared with the original concentration in the sample solution. These results clearly indicate that the present method will be quite effective for detection and/or isolation of a small amount of natural products present in a large volume of aqueous solution.

A simple and rapid method for the determination of residual pyridonecar☐ylic acid antibacterials (PCAs) (oxolinic acid, nalidixic acid and piromidic acid) in fish was developed using a combination of high-performance liquid chromatography (HPLC) and clean-up with an amino-type prepacked cartridge. PCAs were extracted with n -hexane—ethyl acetate (1:3) and the extract was applied to a Baker 10 amino cartridge. PCAs were eluted from the cartridge with acetonitrile—methanol—0.01 M aqueous oxalic acid solution (pH 3.0) (3:1:6) and were determined by HPLC. The separations were performed on Nucleosil 3C 18 (3 μm, 75 × 4.6 mm I.D.) using a mobile phase containing oxalic acid. The recoveries of PCAs from various fishes fortified at the level of 1.0 ppm were 77.1–95.5%, and the detection limits were 0.05 ppm. The analytical time per sample was less than 30 min.

An isocratic high-performance liquid chromatographic method for the determination of tetracyclines is described using a mobile phase containing oxalic acid and C8- and C18-modified silica gel columns. For good separations of tetracyclines, oxalic acid concentrations of above 0.01 and 0.2 M respectively for parent tetracyclines (group I) and impurities in tetracycline (group II) are required. The optimum pH of the aqueous oxalic acid solution in the mobile phase is 2.0. The combinations of the C8-modified silica gel column with methanol—acetonitrile—0.01 M aqueous oxalic acid solution pH 2.0 (1:1.5:5) and the C18-modified silica gel column with methanol—acetonitrile—0.2 M aqueous oxalic acid solution pH 2.0 (1:1:3.5) gave satisfactory results for groups I and II, respectively.

A technique for the determination of tetracyclines using reversed-phase thin-layer chromatographic (TLC) plates is described. A solvent system containing 0.5 M aqueous oxalic acid solution makes posible the determination of tetracyclines on an unreated reversed-phase TLC plate. The separation is dependent on the pH of the aqueous acidic solution in the solvent system, the optimum pH being 2.0. The combinations of a C 8 TLC plate with methanol—acetonitrile—0.5 M aqueous oxalic acid solution (pH 2.0) (1:1:4) as the solvent system, and a C 18 TLC plate with methanol—acetonitrile—0.5 M aqueous oxalic acid solution (pH 2.0) (1:1:2) as the solvent system gave satisfactory results for parent tetracyclines and impurities in tetracycline, respectively. Depending on the purpose, good separations among tetracyclines can be obtained by changing the TLC system.

A compact desktop model of a high-speed counter-current chromatograph holds three identical multilayer coils in the symmetrical positions around the rotary frame to maintain perfect balance of the centrifuge system without the use of a counterweight. These multilayer coils are connected in series to make up a total capacity of 400 ml while the unique gear arrangement on the rotary frame establishes a twist-free mechanism of the flow tubes so that continuous elution can be performed without the use of rotary seal. The high performance of the present system was successfully demonstrated in separations of 10–250 mg of 2,4-dinitrophenyl amino acid mixtures in a two-phase solvent system composed of chloroform—acetic acid—0.1 M hydrochloric acid (2:2:1, v/v/v).

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.

A simple, rapid and precise analytical method for the residual tetracyclines in honey has been established using a tandem cartridge clean-up system (prepacked reversed-phase and ion-exchange cartridges) followed by high-performance liquid chromatography. The recoveries of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC) from honey spiked at a level of 1.0 ppm are 87.1, 85.3, 98.0 and 99.0%, respectively, with coefficients of variation of 1.1–3.9%. The detection limits in honey are 0.02 ppm for OTC and TC, and 0.05 ppm for CTC and DC, respectively. The time required for the analysis of four samples isonly 1 h.

In order to examine the suitability of our four established methods for the analysis of tetracyclines (high-performance liquid chromatography, reversed-phase thin-layer chromatographic densiometry, reversed-phase thin-layer chromatographic spray reagents and silica gel high-performance thin-layer chromatographic densitometry) for the determination of impurities in tetracycline pharmaceutical preparations, sixteen samples available on the Japanese market were analysed. Beacuse these methods did not always give satisfactory results, their analytical conditions were modified. The comparative studies indicated these methods were accurate and reliable. Therefore, we recommend a combination of these methods for the analysis of impurities, in which the spray reagents are used first as a screening method, and then if the results exceed the limits, a precise determination is carried out using either high-performance liquid chromatography or reversed-phase thin-layer chromatographic densitometry.