Production of α-tocopherol by sequential heterotrophic–photoautotrophic cultivation of Euglena gracilis

Photoautotrophic cultivation of Euglena gracilis results in cells with high α-tocopherol content but the final cell concentration is usually very low due to the difficulty of supplying light efficiently to the photobioreactor. On the other hand, Euglena grows heterotrophically to high cell concentrations, using various organic carbon sources, but the α-tocopherol contents of heterotrophically grown cells are usually very low. Sequential heterotrophic/photoautotrophic cultivation, by which cells are grown heterotrophically to high cell concentrations and then transferred to photoautotrophic culture for accumulation of α-tocopherol was therefore investigated for efficient α-tocopherol production. In batch culture, using glucose as the organic carbon source, the cellular α-tocopherol content increased from 120 μg g⁻¹ at the end of heterotrophic phase to more than 400 μg g⁻¹ at the end of the photoautotrophic phase. By using ethanol as the organic carbon source during the heterotrophic phase, adding corn steep liquor as a nitrogen source and optimizing light supply during the photoautotrophic phase, the α-tocopherol content of the cells at the end of the photoautotrophic phase increased to 1700 μg g⁻¹. A system consisting of a mini-jar fermentor (for the heterotrophic phase) and an internally illuminated photobioreactor (for the photoautotrophic phase) was then constructed for continuous sequential heterotrophic/photoautotrophic cultivation. The cells were continuously cultivated heterotrophically in the mini-jar fermentor and the effluent was continuously passed through the photobioreactor for α-tocopherol accumulation. In this way, it was possible to produce 7 g l⁻¹ cells containing about 1100 μg α-tocopherol per g-cell continuously for more than 420 h. The continuous process resulted in α-tocopherol productivity of 100 μg l⁻¹ h⁻¹ which is about 9.5 and 4.6 times higher than those obtained in batch photoautotrophic culture and batch heterotrophic cultures, respectively.     

Linoleic and α-linolenic fatty acids affect biomass and secondary metabolite production and nutritive properties of Panax ginseng adventitious roots cultured in bioreactors

The effect of elicitation with linoleic (C18:2) and α-linolenic (C18:3) fatty acids (LLA and α-LNA) was investigated in Panax ginseng C.A. Meyer adventitious roots cultured in 5 l balloon-type bioreactors. Fatty acids were added in culture medium at 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μmol l⁻¹ at day 40, at the end of exponential growth phase. Roots were harvested and assayed at day 47. Elicitation with both LLA and α-LNA enhanced accumulation of total polyphenolics and flavonoids in roots compared with control without elicitation. The highest accumulation of flavonoids was observed at 5.0 μmol l⁻¹ for both elicitors. Total phenolics production reached its highest value of about 4.0 mg g⁻¹ DW under the elicitation with 5.0 μmol l⁻¹ LLA and 5.0–20.0 μmol l⁻¹ α-LNA. Meanwhile, α-LNA was more effective than LLA for increasing biomass and ginsenoside production. The biomass of 11.1 g DW l⁻¹ and maximal total ginsenoside content of 7.9 mg g⁻¹ DW were achieved at 5 μmol l⁻¹ α-linolenic acid. The essential polyunsaturated linoleic (C18:2) and α-linolenic (C18:3) fatty acids were accumulated in roots in response to elicitation while content of palmitic (C16:0) and oleic (C18:1) acids declined. The activities of SOD, G-POD and CAT were enhanced by two elicitors to similar extent while APX activity was preferably stimulated by α-LNA. Our results demonstrate that elicitation with α-linolenic acid stimulates production of biomass and secondary metabolites in bioreactor-cultured P. ginseng adventitious roots.     

5α,6α-Epoxy-cholestan-3β-ol (cholesterol α-oxide): A specific substrate for rat liver glutathione transferase B

A semi-micro assay was developed for the conjugation of 5α,6α-epoxy-cholestan-3β-ol (cholesterol α-oxide) with glutathione. The soluble supernatant of rat liver homogenate catalysed the reaction at a rate of 0.2–0.5 pmol.min⁻¹ .mg protein⁻¹ with 4μM cholesterol α-oxide, while the reaction in the presence of GSH alone was barely detectable. Enzymic activity in the soluble supernatant was due equally to the two forms of glutathione transferase B (100 pmol.min⁻.mg protein⁻¹), glutathione transferases AA, A, C and E being unreactive. The activity of purified glutathione transferase B was about 5-times that expected from the activity of the soluble supernatant. Complex enzyme kinetics were obtained suggestive of substrate inhibition.     

Determination of phytic acid by gas chromatography–mass spectroscopy: application to biological samples

A GC–MS method is reported for the determination of phytic acid based on purification by anion-exchange chromatography, enzymatic hydrolysis of phytic acid to myo-inositol and derivation to trimethylsilyl derivative, with scyllo-inositol as an internal standard. Analytical features of the method are: limit of detection 9 μg l⁻¹ phytic acid, linear working range 18–500 μg l⁻¹ phytic acid, and coefficient of variation 1.9%. The method has been successfully applied to a variety of biological samples: various rat organs (kidney, liver, brain and bone), human plasma and urine and kidney stones. A comparative study of sample treatments, including deproteization, lipid extraction and the presence of a chelator, is also reported. Phytic acid amounts found in rat organs ranged from 1.07 g kg⁻¹ for bone to 32.0 g kg⁻¹ for brain. Phytic acid in human plasma was of the order of 0.14 mg l⁻¹. In kidney stones, phytic acid was found in calcium containing stones.     

Development and Application of an Immunoaffinity Column Enzyme Immunoassay for Mycotoxin Zearalenone in Complicated Samples

The zearalenone (ZEA) monoclonal antibody (mAb) 2D3, one of the highest sensitivity antibodies, was developed. Based on this mAb, it was established of an immunoaffinity column (IAC) coupled with an indirect competitive enzyme-linked immunosorbent assay (icELISA). After optimization, the icELISA allowed an IC₅₀ against ZEA of 0.02 µg L⁻¹. The mAb 2D3 exhibited a high recognition of ZEA (100%) and β-zearalenol (β-ZOL, 88.2%). Its cross-reactivity with α-zearalenol (α-ZOL) and β-zearalanol (β-ZAL) were found to be 4.4% and 4.6%, respectively. The IAC-icELISA method was employed to analyze ZEA contamination in food samples, compared with high-performance liquid chromatography (HPLC). The spiked assay for ZEA demonstrated the considerable recoveries for IAC-icELISA (83–93%) and HPLC (94–108%) methods. Results showed that the mAb 2D3 and IAC-icELISA method posed potential applications in sensitively determination of ZEA in maize.     

Determination of clindamycin in human plasma by liquid chromatography–electrospray tandem mass spectrometry: application to the bioequivalence study of clindamycin

A simple, sensitive and specific liquid chromatography–electrospray tandem mass spectrometry (LC–MS–MS) method for the determination of clindamycin (I) was developed. Both I and verapamil (II, internal standard) were analyzed using a C₁₈ column with a mobile phase of 80% acetonitrile–0.01% trifluoroacetic acid. Column eluents were monitored by electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to daughter combinations of m/z 425→126 and 455→165 was used to quantitate I. A limit of quantitation of 0.0500 μg/ml was found. The assay exhibited a linear dynamic range of 0.0500–20.0 μg/ml and gave a correlation coefficient (r²) of 0.998 or better. The chromatographic run time was approximately 2 min. The intra-batch precision and accuracy of the quality controls (QCs, 0.0500, 0.150, 1.50, 15.0 and 20.0 μg/ml) were characterized by coefficients of variation (CVs) of 5.13 to 13.7% and relative errors (REs) of −4.34 to 4.58%, respectively. The inter-batch precision and accuracy of the QCs were characterized by CVs of 4.35 to 8.32% and REs of −10.8 to −4.17%, respectively. The method has successfully been applied to the analysis of samples taken up to 12 h after oral administration of 300 mg of I in healthy volunteers.     

Binding Rather Than Metabolism May Explain the Interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative ά-Zearalenol

The interaction between two Fusarium mycotoxins, zearalenone (ZEN) and its derivative ¯α-zearalenol (¯α-ZOL), with two food-grade strains of Lactobacillus was investigated. The mycotoxins (2 μg ml⁻¹) were incubated with either Lactobacillus rhamnosus strain GG or L. rhamnosus strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and ¯α-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or ¯α-ZOL by lyophilized L. rhamnosus GG and L. rhamnosus LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with ¯α-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds.     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

High-performance liquid chromatographic determination of tianeptine in plasma applied to pharmacokinetic studies

An improved analytical method for the quantitative measurement of tianeptine and its main metabolite MC₅ in human plasma was designed. Extraction involved ion-paired liquid–liquid extraction of the compounds from 1.0 ml of human plasma adjusted to pH 7.0. HPLC separation was performed using a Nucleosil C₁₈, 5 μm column (150×4.6 mm I.D.) and a mixture of acetonitrile and pH 3, 2.7 g l⁻¹ solution of sodium heptanesulfonate in distilled water (40:60, v/v) as mobile phase. UV detection was performed using a diode array detector in the 200–400 nm passband, and quantification of the analytes was made at 220 nm. For both tianeptine and MC₅ metabolite, the limit of quantitation was 5 μg l⁻¹ and the calibration curves were linear from 5 to 500 μg l⁻¹. Intra- and inter-assay precision and accuracy fulfilled the international requirements. The recovery of tianeptine and its metabolite from plasma was, respectively, 71.5 and 74.3% at 20 μg l⁻¹, 71.2 and 70.8% at 400 μg l⁻¹. The selectivity of the method was checked by verifying the absence of chromatographic interference from pure solutions of the most commonly associated therapeutic drugs. This method, validated according to the criteria established by the Journal of Chromatography B, was applied to the determination of tianeptine and MC₅-metabolite in human plasma in pharmacokinetic studies.     

Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography–tandem mass spectrometry

A column-switching liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 μl) were injected onto a C₁₈-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0–acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C₁₈ column, with a mixture of 20 mM ammonium acetate–acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M−H]⁻ were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M−H]⁻ in MS–MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M−H−Glu]⁻, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025–100 μg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78–103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.     

3β-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β,17β-diol and dehydroepiandrosterone as substrates

3β-Hydroxysteroid dehydrogenase (3β-HSD)/Δ^5→4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3β-HSD/Δ^5→4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3β-HSD activity. For this purpose, we compared the efficiencies of a 3β-hydroxy-5-ene steroid (DHEA) and a 3β-hydroxy-5α-reduced steroid (5α-androstane-3β,17β-diol, 5α-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K_m) for 5α-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent K_m of placental 3β-HSD for 5α-A-diol was in the range of 18 to 40 μmol/l (n = 3) vs 0.45 to 4 μmol/l for DHEA (n = 3); for the liver enzyme, 17 μmol/l for 5α-A-diol and 0.60 μmol/l for DHEA, and for the skin enzyme 14 and 0.18 μmol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5α-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to K_ms and rates of product formation was obtained by use of purified placental 3β-HSD with DHEA, pregnenolone, and 3β-hydroxy-5α-androstan-17-one (epiandrosterone) as substrates: the K_m of 3β-HSD for DHEA was 2.8 μmol/l, for pregnenolone 1.9 μmol/l, and for epiandrosterone 25 μmol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein·min and, with epiandrosterone, 127 nmol/mg protein·min. With placental homogenate as the source of 3β-HSD, DHEA at a constant level of 5 μmol/l behaved as a competitive inhibitor when the radiolabeled substrate, [³H]5α-A-diol, was present in concentrations of 20 to 60 μmol/l, but a lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [³H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5α-A-diol (40 μmol/l). These findings are indicative that both steroids bind to a common site on the enzyme, however, the binding affinity for these steroids appear to differ markedly as suggested by the respective K_ms. Studies of inactivation of purified placental 3β-HSD/Δ^5→4-isomerase by an irreversible inhibitor, viz 5,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroidal substrate has a 5α-configuration, e.g. epiandrosterone, or a C-5,C-6-double bond e.g. DHEA or pregnenolone. The lower rates of product formation obtained with placenta and fetal tissues by use of 3β-hydroxy-5-ene steroids as substrates when compared with those obtained with 3β-hydroxy-5α-reduced steroids may be explained by a combination of factors, including: (i) inhibition of 3β-HSD activity by end products of metabolism of 3β-hydroxy-5-ene steroids, e.g. 4-androstene-3,17-dione formed with DHEA as substrate; (ii) higher binding affinity of the enzyme for 3β-hydroxy-5-ene steroids—and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for the isomerization step with 5α-reduced steroids as substrates, and (iv) the possible presence in fetal tissues of an enzyme with 3β-HSD activity only (i.e. no Δ^5→4-isomerase).     

Inversion of sucrose with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min⁻¹ g⁻¹. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Biological monitoring of dinitrotoluene by gas chromatographic–mass spectrometric analysis of 2,4-dinitrobenzoic acid in human urine

The method of analysis described permits the determination of 2,4-dinitrobenzoic acid down to the lower μg l⁻¹ range in the urine of persons exposed to dinitrotoluene. 2,4-Dinitrobenzoic acid is the main metabolite of 2,4-dinitrotoluene and technical dinitrotoluene. After acidic hydrolysis, which served to release the conjugated part of the 2,4-dinitrobenzoic acid, the analyte was selectively separated from the urine matrix via various extraction steps and then derivatised to the methyl ester. Quantitative analysis was carried out using capillary gas chromatography and mass selective detection. 3,5-Dinitrobenzoic acid was used as an internal standard. The detection limit was 1 μg l⁻¹ urine. The relative standard deviations of within-series imprecision were between 5 and 6%. The relative recoveries were between 91 and 110% depending on the concentration. The analytical method developed as part of this study was used to investigate a collective consisting of 82 urine samples from persons working in the area of explosives disposal. The concentrations of 2,4-dinitrobenzoic acid determined ranged from the detection limit to 95 μg l⁻¹ urine. The method allowed the quantification of low-level internal exposure to dinitrotoluene.     

Parallel solid-phase synthesis of 3β-peptido-3α-hydroxy-5α-androstan-17-one derivatives for inhibition of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Δ⁴-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3β-peptido-3α-hydroxy-5α-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23–58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17β-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3β-(N-heptanoyl- -phenylalanine- -leucine-aminomethyl)-3α-hydroxy-5α-androstan-17-one (42) inhibited the enzyme with an IC₅₀ value of 227 nM, which is twice as potent as the natural substrate Δ⁴-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR⁺) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 μM (less than previously reported type 3 17β-HSD inhibitors) and, interestingly, no proliferation at 0.1 μM.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of the heavy metal tolerant and hyperaccumulator <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> (Brassicaceae)

Plants were regenerated from root explants of Arabidopsis halleri (L.) O’Kane and Al-Shehbaz via a three-step procedure callus induction, induction of somatic embryos and shoot development. Callus was induced from root segments, leaflets and petiole segments after incubation for 2 weeks in Murashige and Skoog medium (MS) supplemented with 0.5 mg/l⁻¹ (2.26 μM) 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.05 mg/l⁻¹ (0.23 μM) kinetin. Only calli developed from root segments continued to grow when transferred to a regeneration medium containing 2.0 mg/l⁻¹ (9.8 μM) 6-γ-γ-(dimethylallylamino)-purine (2ip) and 0.05 mg/l⁻¹ (2.68 μM) α-naphthalenacetic acid (NAA) and eventually 40 of them developed embryogenic structures. On the same medium 38 of these calli regenerated shoots. Rooting was achieved for 50 of the shoots subcultured in MS medium without hormones. The regeneration ability of callus derived from root cuttings, observed in this study, makes this technique useful for genetic transformation experiments and in vitro culture studies.     

Determination of benzimidazole residues using liquid chromatography and tandem mass spectrometry

The determination of residues of benzimidazole using liquid chromatography and tandem mass spectrometry (LC–MS–MS) with ion spray ionization is described. Swine muscle tissue was spiked with a mixture of fifteen benzimidazoles, including metabolites of fenbendazole and albendazole. As clean-up procedure, an ethyl acetate extraction followed by solid-phase extraction on styrol-divinyl-benzene cartridge was used. The evaluation was performed by selecting the characteristic product ions for the benzimidazoles and using multiple reaction mode. 2-n-Butylmercaptobenzimidazole was used as internal standard. Blank muscle samples were fortified in the concentration range of 1–22 μg/kg. The limits of detection were below 6 μg/kg and the limits of quantification for most benzimidazoles were below 10 μg/kg. The matrix effect was checked using spiked muscle tissues of cattle and sheep as well as liver of cattle. Practical application will be shown by incurred egg material from laying hens treated with flubendazole. The recovery of the clean-up was mostly above 50% in muscle tissue and 70% in egg yolk.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of reserpine in FVB/N mouse plasma using a “chelating” agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid–liquid extraction

A sensitive, specific, accurate and reproducible analytical method employing a divalent cation chelating agent (disodium EDTA) for sample treatment was developed to quantitate reserpine in FVB/N mouse plasma. Samples pretreated with 40 μl of 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid–liquid extraction (LLE) procedure to isolate reserpine and a structural analog internal standard (I.S.), rescinnamine, from mouse plasma. The extracts were analyzed by turbo ionspray liquid chromatography–tandem mass spectrometry (LC–MS–MS) in the positive ion mode. Sample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The assay demonstrated a lower limit of quantitation of 0.02 ng/ml using 0.1-ml plasma sample aliquots. The calibration curves were linear from 0.02 to 10 ng/ml for reserpine. The intra- and inter-assay precision of quality control (QC) samples ranged from 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from −8.17 to 8.61%. Reserpine and the I.S. were found to be highly bound to FVB/N mouse plasma protein. This is the first report of disodium EDTA employed as a special protein-bound release agent to recover protein-bound analytes from plasma. These matrix effects and the effects of pH in the HPLC mobile phase on the sensitivities of LC–MS–MS are discussed in this paper.     

Determination of l-α-acetylmethadol, l-α-noracetylmethadol and l-α-dinoracetylmethadol in plasma by gas chromatography—mass spectrometry

A method is described for the simultaneous determination of l-α-acetylmethadol (LAAM) and its N-demethylated metabolites, l-α-noracetylmethadol (norLAAM) and l-α-dinoracetylmethadol (dinorLAAM), in plasma by gas chromatography—chemical ionization mass spectrometry. Deuterated internal standards for each analyte serve as carriers and control for recovery during sample purification on a solid-phase extraction column (C₁₈), and subsequent separation and analysis on a DB-17 capillary column. With this method, we have determined levels of LAAM, norLAAM, and dinorLAAM in small volumes of plasma (100 μl). The limit of quantitation for all analytes was approximately 1.0 ng/g plasma and the limit of detection was approximately 0.5 ng/g plasma. An experimental application is also described where these analytes are quantitated in plasma obtained from rats before, during, and after chronic administration of LAAM-HCl. Since this technique affords a selective and sensitive means of detection of LAAM and its active, N-demethylated metabolites in small samples of blood, it may enable patient compliance to be more easily assessed by allowing samples to be collected by a simple finger-prick technique.