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A liquid chromatographic (HPLC) method has been developed for the quantitative determination of different forms of vitamin E (alpha-, gamma-, and delta-tocopherol) in aquatic organisms. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with fluorescence detection. The efficiency of the extraction method was equivalent or superior to that of more complex approaches for the isolation of tocopherols. Linearity has been achieved over the range of 0.02 to 3 micrograms/ml for alpha-tocopherol and within-run and between-run coefficients of variation at three levels were 0.7-2.9 and 1.2-3.7%, respectively. The recovery at three concentrations ranged from 73.8 to 96.6% and the minimal quantity that could be detected was 0.6 ng. Comparable figures were obtained for gamma- and delta-tocopherol. This method has been routinely applied to determine vitamin E in Artemia, rotifers, turbot and sea bass larvae, and shrimp postlarvae.

In a previous paper (H.J. Nelis, P. Léger, P. Sorgeloos, and A. P. De Leenheer, Antimicrob. Agents Chemother. 35:2486-2489, 1991) it was reported that two selected antibacterial agents, i.e., trimethoprim and sulfamethoxazole, can be efficiently bioencapsulated in nauplii of the brine shrimp Artemia franciscana for administration to fish. This follow-up study showed that larvae of the sea bass and the turbot as well as postlarvae of the white shrimp accumulate the therapeutic agents in high quantities when fed medicated A. franciscana. To monitor their levels as a function of time, the liquid chromatographic method originally developed for the analysis of A. franciscana was modified with respect to chromatography, internal standardization, and sample pretreatment. The levels of trimethoprim ranged from 1 to 7 micrograms/g (sea bass), 1 to 13 micrograms/g (turbot), and 4 to 38 micrograms/g (white shrimp). The corresponding values for sulfamethoxazole were 0.3 to 4 micrograms/g (sea bass), 1 to 42 micrograms/g (turbot), and 4 to 35 micrograms/g (white shrimp). Only the two fish species, unlike the shrimp, metabolized the latter to N-acetylsulfamethoxazole (concentration range, 1 to 10 micrograms/g). These data suggest the potential of the bioencapsulation of therapeutic agents in live food as a tool to control infectious diseases in aquaculture. A preliminary challenge test also confirmed the in vivo efficacy of this approach.

Volume 61, no. 12, p. 4505, Introduction, column 1, line 13, and column 2, line 5: reference 19, which refers to a toxicity test based on the inhibition of (beta)-glucuronidase, not (beta)-galactosidase, in E. coli, should not be cited. [This corrects the article on p. 4505 in vol. 61.].

As part of ongoing studies in the biosynthesis of the lantibiotic nisin, we investigated the proteolytic cleavage of a synthetic Fmoc-labeled decapeptide mimicking a key amino acid sequence of the precursor prenisin in extracts of a Lactococcus lactis subsp. lactis strain. Reverse-phase high-performance liquid chromatography with photodiode array detection was used to trace and purify potential enzymatic conversion products. Of the three newly appearing chromatographic peaks, one was identified by means of electrospray mass spectrometry, amino acid analysis, and amino acid sequencing as an Fmoc-labeled hexapeptide derived from cleavage at the Arg-1-Ile+1 bond. This assay will be useful to monitor the purification of the endoproteinase that reportedly cleaves prenisin at the same Arg-1-Ile+1 site present in the model substrate.

We developed a chemiluminometric assay of beta-galactosidase in coliform bacteria, using a phenylgalactose-substituted 1,2-dioxetane derivative as a substrate. Permeabilization of cells is required to ensure the efficient cellular uptake of this compound. By this method, one coliform seeded in 100 ml of sterile water can be detected after a 6- to 9-h propagation phase followed by a 45-min enzyme assay in the presence of polymyxin B. Compared with fluorometry and colorimetry, chemiluminometry afforded 4- and 1,000-fold increases in sensitivity and 1- and 6-h increases in the speed of detection, respectively.

A liquid chromatographic method for the determination of oxytetracycline in bovine plasma was developed. Contrary to most current tetracycline assays involving solid-phase extraction, oxytetracycline and the internal standard tetracycline were isolated from buffered plasma by double-phase extraction with ethyl acetate-isopropyl alcohol. The latter component was found to be essential to ensure reproducible partitioning of both tetracyclines into the organic layer. Chromatography was conducted on a Lichrosorb RP8 column, compounds were eluted with 0.01 M oxalic acid-methanol-acetonitrile, and detection was at 357 nm. Linearity was observed over the 0-8.5-micrograms/mL range, and the detection limit approximated 5 ng/mL. The extraction yield was 80.4 +/- 3.4% (n = 12), and the coefficients of variation for repetitive within-run and between-run analyses at two concentration levels (0.25 and 2 micrograms/mL) were 1.0-4.3 and 2.4-7.2%, respectively. The method was applied to a pharmacokinetic study of oxytetracycline in three nonlactating cows after a single intramuscular administration of a long-acting preparation.

We report a means of determining amoxicillin in bovine plasma by liquid chromatography with UV detection at 235 nm. Purification and concentration of extracts were accomplished by a tandem solid-phase extraction procedure with two reversed-phase columns. Separation of amoxicillin from interferences was improved by the incorporation of a crown ether in the solvent systems used both for the solid-phase extraction and the final high-pressure liquid chromatography. Cefadroxil was added as an internal standard. The average recovery of amoxicillin from plasma (n = 23) was 78.2 +/- 3.0%, and the within-run and between-run coefficients of variation ranged from 1.8 to 7.0%. The detection limit was estimated at 0.1 microgram/ml. This method was used to determine amoxicillin in bovine plasma after intramuscular administration of the drug.

The determination of ampicillin in plasma and serum by reversed-phase high-performance liquid chromatography with UV detection suffers from poor selectivity and sensitivity. Currently, the most common approach to overcoming these problems consists of improving the compound's detectability via pre- or postcolumn derivatization. In the method that we describe, however, enhanced selectivity is afforded by sample purification by a tandem solid-phase extraction method (ion-exchange and reversed-phase). This approach permits detection at wavelengths of as low as 210 nm, which results in enhanced sensitivity (detection limit, 0.01 microgram/ml). A second factor that affects selectivity is the addition to the chromatographic eluent of a crown ether to optimize the separation between ampicillin and polar endogenous plasma constituents. This combination of improved sample pretreatment and a more selective chromatographic system in conjunction with internal standardization forms the basis of a new assay for the quantitation of ampicillin in plasma. The overall recovery of ampicillin was 76.4% +/- 4.9% (n = 24), and the within-run and between-run coefficients of variation ranged from 1.6 to 7.2%. The method was applied to pharmacokinetic studies in cows and dogs after intramuscular or oral administration of the drug.

The brine shrimp Artemia, an excellent live food source in aquaculture, has been studied as a carrier to deliver selected chemotherapeutic agents to fish for prophylactic treatment of infectious diseases. To monitor the efficiency of incorporation of trimethoprim and sulfamethoxazole in Artemia franciscana, a sensitive and specific analytical method was developed. It is based on homogenization of Artemia nauplii in methanol, extraction of lipids with hexane, solid-phase cleanup on C18 cartridges, and reversed-phase liquid chromatography with detection at 210 nm. The method is sensitive (detection limit, on the order of 3 micrograms/g with a sample quantity of 30 mg [dry weight]) and reproducible (coefficients of variation, 2.2 and 1.8% for trimethoprim and sulfamethoxazole at levels of 79.6 and 257 micrograms/g of body weight, respectively). Preliminary quantitative data indicated excellent uptake and persistence of both therapeutic agents in A. franciscana, with levels of 115 micrograms/g for trimethoprim and 277 micrograms/g for sulfamethoxazole.

The stability of Azure B and Eosin Y in stock solutions of the individual compounds as well as in mixtures of the two dyes was studied. The purpose of the study of these two essential constituents of the Romanowsky Giemsa stain, commonly used in cytology and histology, was to select a stable mixture as a definitive stock solution. Two specific high performance liquid chromatographic methods were used to monitor qualitative and quantitative changes in solutions. Several parameters influencing the stability of Azure B were examined e.g. the type of counter ion, the presence of Eosin Y and the type of solvent used. The second part focused on the stability of Eosin Y in mixtures with different cationic dyes submitted to high temperatures. In conclusion, an Azure B SCN-Eosin Y acid mixture in dimethylsulphoxide (concentrations 0.75% and 0.12%, respectively) was selected as being the most appropriate composition of a stock solution for the Romanowsky Giemsa stain.

Quality control of albumin solutions for human use is of utmost importance because the presence of impurities can provoke adverse reactions in treated patients. Three different techniques were used to detect the presence of albumin oligomers and polymers as well as foreign proteins in commercial solutions. The relative concentrations of the former two were estimated using size-exclusion high-performance liquid chromatography. Isoelectric focusing and two-dimensional immunoelectrophoresis allowed detection of other contaminants of proteinaceous nature. The application of this combination of techniques supersedes the traditional approaches (gel filtration on polydextran gels, electrophoresis) in specificity and speed. Analysis of 34 lots of commercial albumin solutions from 22 manufacturers revealed the superior quality of preparations of placental rather than plasmatic origin.

Urinary 2-thioxothiazolidine-4-carboxylic acid is a useful indicator to assess the degree of occupational exposure to carbon disulfide. A new procedure is described for the isolation of this compound from urine prior to reversed-phase high-performance liquid chromatography. It is based on liquid-liquid extraction with methyl tert-butyl ether, followed by affinity chromatography on organomercurial agarose gel. 5-Carboxythiouracil is used as internal standard. The superior selectivity of affinity chromatography for the isolation of 2-thioxothiazolidine-4-carboxylic acid from urine permits an isocratic high-performance liquid chromatographic analysis. The total recovery of 2.5 mg of 2-thioxothiazolidine-4-carboxylic acid/g of creatinine in spiked urine by liquid-liquid extraction combined with affinity chromatography was 48.0% (SD 2.0%, n = 8). Within-run and within-day relative standard deviations averaged 4.0% (means = 2.48 mg/g of urinary creatinine, n = 9) and 6.5% (means = 1.19 mg/g of urinary creatinine, n = 15), respectively. The detection limit of the method was estimated at 0.05 mg of 2-thioxothiazolidine-4-carboxylic acid/g of urinary creatinine. The identity and spectral purity of 2-thioxothiazolidine-4-carboxylic acid detected in the urine extracts were confirmed by diode-array UV-vis detection. Typical 2-thioxothiazolidine-4-carboxylic acid levels in individual workers exposed to carbon disulfide ranged from nondetectable to 11 mg/g of urinary creatinine, several of which exceeded the generally accepted biological exposure index of 5 mg/g of urinary creatinine.

Different high performance liquid chromatographic systems were applied to the investigation of vitamin A metabolism in subjects undergoing haemodialysis. Plasma levels of retinol, retinyl esters and retinoic acid were measured. There was a significant elevation of plasma retinol and dialysis failed to normalise this level. No correlation with plasma concentrations of creatinine or urea was found. No differences in retinyl ester and retinoic acid levels were observed between healthy subjects and haemodialysis patients. These results suggest that retinol accumulation is not caused by a deficiency in its oxidative metabolism.

A new assay for retinol in human serum, based on nonaqueous reversed-phase liquid chromatography, is presented. Sample preparation includes addition of the internal standard, retinyl propionate, deproteinization of 100 microL of serum with acetonitrile, and extraction with hexane. The standard curve is linear up to 2 mg/L. The assay is characterized by excellent sensitivity (detection limit, 15 micrograms/L) and good within-run and between-run precision (CVs of 2.6 and 2.7%, respectively), and results compare favorably with those by fluorimetry. We assayed 135 samples from hospitalized patients by both methods. Although the two sets of values correlated well (r = 0.955) the fluorimetric method occasionally suffers from interferences. In practice, fluorimetry proves valuable as a routine method, while liquid chromatography meets the criteria of a potential reference method.

The significance of carotenoid accumulation in crustacean eggs remains obscure, particularly because neither eggs nor female animals have been found to display specific pigment patterns in relation to reproduction. We report here the first example of carotenoids found exclusively in the ovaries, the eggs, and the hemolymph, but not in the carcass of a female, reproductively active crustacean, i.e. the brine shrimp Artemia. These pigments are virtually absent in males and in immature animals and disappear very rapidly in growing nauplii following hatching of encysted embryos. Within the cysts, they are preferably localized in the yolk platelets. We have identified them as mono-cis- canthaxanthins on the basis of their mass and absorption spectra and by comparison with synthetic components. Carotenoids with the unusual cis-configuration have never been isolated from animals, nor are there reports on the occurrence of carotenoid pigments at specific sites. Our findings may thus provide a clue to a precise function for carotenoids in Artemia and, possibly, related Crustacea.

A review is presented of current gas and liquid chromatographic methods for the determination of the fat-soluble vitamins A, D, E and K and the provitamin A beta-carotene in biological samples of human origin. For each vitamin, the discussion successively focuses on procedures for sample preparation, gas and liquid chromatographic systems and principles of detection. The emphasis is on liquid chromatography, which is gradually becoming a standard technique in fat-soluble vitamin assays. New trends in the liquid chromatography of these compounds include the use of smaller particles and shorter columns, to improve speed, and the advance of electrochemical detection as an alternative to absorbance and fluorescence detection. Bonded phases, both normal and reversed phase, tend to be preferred over underivatized silica as column supports. Gas chromatography remains of particular value in combination with mass spectrometry, a technique which may form the basis of reference methods. In general, despite the availability of well established analytical methods for fat-soluble vitamins, the wealth of recent literature in this area indicates that there continues to be a need for new assays with enhanced speed, specificity and sensitivity.

A rapid, specific, and sensitive liquid chromatographic assay for minocycline in human serum is described. The drug and an internal standard (oxytetracycline) were extracted into ethyl acetate from 0.5 ml of buffered serum (pH 6.5). Chromatographic separation was achieved on a 10-micrometer Lichrospher 100 CH 8 column with acetonitrile--citric acid (0.1 M) as the eluent. The column effluent was monitored at 352 nm. The assay was linear up to 3 micrograms/ml with a mean coefficient of variation of 3.3% (n = 6). An extraction recovery of 89.4 +/- 3.2% (mean +/- SD, n = 17) was obtained over the 0.5--2.6 micrograms/ml range. The detection limit averaged 50 ng/ml. A serum concentration-time profile in humans after oral intake is presented.

A rapid, sensitive and specific liquid chromatographic method is described for the determination of doxycycline in human tissues. The procedure involves mechanical homogenization of tissue samples in hydrochloric acid followed by extraction of the drug and an internal standard into ethyl acetate. Chromatographic separation is performed on a reversed phase column and allows quantitation of tissue levels as low as 0.68 nmol/g using a 200-400 mg sample. Application of the assay to tissue samples obtained from 36 patients confirmed the excellent penetration of doxycycline in organs. The method supersedes the classical microbiological assays in specificity and speed.

The hydrocarbon-utilizing Brevibacterium sp. strain KY-4313 was reevaluated for its potential to produce canthaxanthin, a carotenoid pigment of strong commercial interest. Three approaches were used to optimize the canthaxanthin yield from this organism, i.e., the preparation of mutants, the addition of supposedly carotenogenic chemicals to the growth medium, and growth promotion. Following treatment of the parent strain with N-nitrosomethylurea, a presumed mutant was isolated which showed a 32% increase in cellular canthaxanthin content. No effective carotenogenic chemicals were found in connection with hydrocarbon fermentations, in which mainly growth promotion through periodic medium renewal proved conducive to enhanced pigment production. Carotenogenesis could be stimulated in brain heart infusion broth by adding alcohols or retinol. Improved growth in this medium was generally not associated with higher canthaxanthin yields. Both superior growth and pigment levels were obtained in a newly designed medium based on fumaric acid-molasses. The maximum yields of canthaxanthin in shake flasks were (in milligrams per liter) 4.2 (brain heart infusion broth plus propanol-zinc sulfate), 3.6 (hydrocarbon medium), and 9.3 (fumaric acid-molasses), which represent a significant improvement over the originally reported optimal result (1 mg/liter). The corresponding yields of echinenone, the direct precursor of canthaxanthin, were 1.2, 1.6, and 2.3 mg/liter, respectively. Two-liter hydrocarbon batch fermentations involving medium renewal maximally produced 7.2 mg of canthaxanthin and 3.7 mg of echinenone per liter.

Metabolites of tetracycline antibiotics have not previously been isolated from human excreta. It is now demonstrated that minocycline is the first tetracycline to be metabolized in man. A two-step liquid chromatographic procedure was used to isolate three metabolites from human urine. The principal metabolite was tentatively identified as 9-hydroxyminocycline by mass spectral and spectrophotometric analysis and comparison with a synthetic compound. The latter was prepared by incubation of minocycline in a modified Udenfriend system, simulating an enzymatic oxygenase. Two other major metabolites are probably mono-N-demethylated derivatives. Substantial amounts of 4-epiminocycline, which probably resulted from minocycline epimerization rather than biotransformation, were also present.

An analytical method for the profiling and quantitative determination of carotenoids in bacteria is described. Exhaustive extraction of the pigments from four selected bacterial strains required treatment of the cells with potassium hydroxide or liquefied phenol or both before the addition of the extracting solvent (methanol or diethyl ether). The carotenoids in the extracts were separated by nonaqueous reversed-phase liquid chromatography in conjunction with photodiode array absorption detection. The identity of a peak was considered definitive only when both its retention time and absorption spectrum, before and after chemical reactions, matched those of a reference component. In the absence of the latter, most peaks could be tentatively identified. Two examples illustrate how in the analysis of pigmented bacteria errors may result from using nonchromatographic procedures or liquid chromatographic methods lacking sufficient criteria for peak identification. Carotenoids of interest were determined quantitatively when the authentic reference substance was available or, alternatively, were determined semiquantitatively.

A reversed-phase high-performance liquid chromatographic method for quantitative doxycycline determination in human serum and urine is described. The drug was extracted from buffered (pH 6.1) serum or urine into ethyl acetate. A structural analog, demeclocycline, was added as the internal standard. A 10-cm X 2-mm i.d., 5-micrometers Lichrosorb RP8 column with acetonitrile-0.1 M citric acid as the eluent was used. The effluent was monitored at 350 nm. The extraction recovery from spiked serum was 87-8 +/- 4.3% (mean +/- SD, n = 11); for urine, a value of 92.2 +/-2.0% (mean +/- SD, n = 10) was found. Within-run and within-day relative standard deviations averaged (x = 2.5 micrograms/ml, n = 10) and 4.75% (x = 2.6 micrograms/ml, n = 9), respectively. The detection limit was estimated at 50 ng/ml of serum. No significant extra peaks were observed in chromatograms obtained on serum or urine extracts, suggesting the probable absence of metabolic processes in vivo.

A liquid chromatographic procedure for the quantitative determination of the thiazine dye azure B, the principal constituent of Romanowsky stains, is presented. Unlike previous methods relying on peak area normalization, the present approach involves real quantitation through calibration with the reference standard azure B tetrafluoroborate. The method has been used for the quality control of commercial azure B preparations and to study their stability in stock and staining solutions, either or not in the presence of eosin Y. Results suggest that highly pure azure B perchlorate meets the requirements of a reference material, useful for standardization of Romanowsky-Giemsa staining in haematology.

Chromatographic procedures have been developed for determination of the stabilizers N-acetyl-DL-tryptophan and octanoic acid in human albumin solutions. N-Acetyl-DL-tryptophan and the internal standard, N-formyl-DL-tryptophan, were separated by liquid chromatography on a reversed-phase column with UV detection at 280 nm. Deproteinization and extraction were carried out with methanol. The extraction recovery at the level of 4.9 mM was 92.5 +/- 2.5% (S.D.) (n = 10), and the average coefficient of variation (C.V.) for replicate analyses of albumin solutions (mean = 2.57, 10.44 and 17.10 mM) was 1.10% (n = 27). Octanoic acid was determined gas chromatographically as its methyl ester, with nonanoic acid as the internal standard. The sample pretreatment included acidification, extraction with hexane and derivatization with methanol-sulphuric acid. The relative recovery from albumin solutions was 89.7 +/- 5.8% (S.D.) (n = 6), and replicate determinations of the compound yielded a C.V. of 5.5% (mean = 14.82 mM, n = 9).

Several conflicting observations in the literature raised considerable doubt about the metabolic fate of doxycycline, which, like other tetracyclines, has been claimed to be metabolically inert. A double liquid chromatographic approach was used in an attempt to demonstrate the polar metabolites and/or conjugates in excreta of human volunteers who ingested the drug. Both ion-exchange and reversed-phase chromatography failed to reveal significant by-products in urine and feces, except for minor amounts of 4-epidoxycycline. In addition, enzymatic hydrolysis procedures did not present any evidence of the conjugates. Thus, the different excretion behavior of doxycycline, compared to other analogs, cannot be explained in terms of increased metabolism.

In order to study the biological fate of all-trans- and cis-canthaxanthin in the brine shrimp Artemia, a quantitative method was developed for the determination of both carotenoids and their metabolic precursors in encysted embryos (cysts), nauplii, whole animals, organs, and subcellular fractions. This method is based on nonaqueous reversed-phase chromatography, two new exhaustive extraction procedures, and the determination of proteins in the extracted residue. Hydration of Artemia cysts caused a reversible conversion of part of the all-trans- to cis-canthaxanthin. During further pre-emergence embryonic development, there was little change in the levels of both isomers. After hatching of cysts, cis-canthaxanthin was progressively isomerized to the all-trans form, while the total (all-trans + cis) canthaxanthin to protein ratio tended to remain constant. Cis-canthaxanthin rapidly became undetectable in animals fed on algae and reappeared in females at an advanced stage of the reproductive cycle. All-trans-canthaxanthin remained present during the whole Artemia life cycle in addition to its metabolic precursors echinenone and beta-carotene. The carotenoid distribution in organs and subcellular fractions indicated high affinity of cis-canthaxanthin for the female reproductive system, oocytes in general, and yolk in particular. A role for cis-canthaxanthin is suggested at an early developmental stage, i.e., in cysts, before hatching.