Journal of Chromatography B (v.796, #1)

News Section (N1-N2).

Procedure for separation of GM2 ganglioside species with different ceramide structures by a flash reversed-phase silica gel liquid chromatography by Laura Mauri; Manuela Valsecchi; Riccardo Casellato; Su-Chen Li; Yu-Teh Li; Sandro Sonnino (1-10).
GM2 ganglioside, β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)-]β-Gal-(1-4)-β-Glc-(1-1)-Cer, is the main ganglioside in the brain of Tay-Sachs patients. In this work, GM2 ganglioside was extracted from a Variant B Tay-Sachs human brain, purified to homogeneity of the oligosaccharide moiety by silica gel chromatography. It was further fractionated for the first time into the molecular species differing in the ceramide structures by reverse-phase flash chromatography. The GM2 ganglioside species were characterized by gas-chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. The major GM2 species contained the ceramides with d18:1–18:0 (40.5% of the total GM2 species), d20:1–18:0 (31%) and d18:1–20:0 (12%). We also found minor GM2 species with the ceramides with d18:1–24:1 (4%), d18:1–22:0 (2%) and d18:2–24:1 (1%), which have not been reported previously.
Keywords: GM2 ganglioside; Ceramide;

Determination of isoprostanes in urine samples from Alzheimer patients using porous graphitic carbon liquid chromatography–tandem mass spectrometry by Kristina Claeson Bohnstedt; Bo Karlberg; Lars-Olof Wahlund; Maria Eriksdotter Jönhagen; Hans Basun; Staffan Schmidt (11-19).
F2-isoprostanes (F2-iPs) comprise four classes of isomers produced non-enzymatically by free radical attack on arachidonic acid, a component of the cell membrane. This paper describes a new method for the quantification of F2-isoprostanes in urine samples from thoroughly diagnosed Alzheimer’s disease (AD) patients. The sample pretreatment consisted of liquid extraction of 900 μl urine with diethyl ether, its subsequent evaporation, and finally, reconstitution in 50 μl water. Of this, 20 μl was injected into a HPLC system with a 15  mm×1  mm porous graphitic carbon column coupled to a triple quadrupole mass spectrometer running in negative electrospray ionization mode. The F2-isoprostanes were separated in 15 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5. The average recovery obtained was approximately 75%. The limit of detection (3S/N) was calculated for iPF2α-III to be 0.7 pg injected on column, corresponding to 0.1 nM. The average level of iPF2α was 241±163 pg/mg creatinine in the urine samples from AD patients (average±standard deviation). The corresponding control values were 216±101 pg/mg creatinine, i.e. no statistically significant difference was noticed. No correlation pattern specific to Alzheimer’s disease was revealed by principal component analysis of the isoprostane peaks obtained either. The results from this study support earlier findings that levels of peripheral isoprostanes are not increased in patients with Alzheimer’s disease.
Keywords: Isoprostanes; Porous graphitic carbon;

A selective analytical method based on high-performance liquid chromatography (HPLC), combined with atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS), has been developed for simultaneous determination of B-trichothecenes and the major metabolites of deoxynivalenol. The method allows simultaneous analysis of nivalenol (NIV), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), fusarenon X (Fus-X) and de-epoxydeoxynivalenol (DOM-1). The method is based on one-step sample clean-up using a multifunctional MycoSep column. A linear gradient mobile phase system, consisting of water:acetonitrile:methanol (H2O:ACN:MeOH) at a flow-rate of 1 ml/min, and a Polar-RP C18 column, were utilised to obtain the best resolution of all tested compounds along with column and equilibrating within 30 min. Dexamethasone (Dex) was used as internal standard. The developed method shows good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r 2 ranged from 0.9936 to 0.9998). Average recoveries for tested compounds in both matrices have been determined ranging from 63.7 to 102.3% and limit of quantification (LOQ) ranged from 25 to 150 ng/g. The utility and practical impact of the method is demonstrated using contaminated pig urine and maize samples.
Keywords: B-Trichothecenes; Deoxynivalenol;

A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the enantiomers of a novel anticonvulsant agent, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine (AAP-Cl), in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation was accomplished by an HPLC system consisting of a Chirex chiral column (250  mm×4.6  mm i.d.) and a mobile phase of hexane:ethanol:tetrahydrofuran (280:20:40 (v/v)) containing trifluroacetic acid (0.3% (v/v)) and triethylamine (0.018% (v/v)) at a flow rate of 0.8 ml/min with UV detection. Male Sprague-Dawley rats were given (+)-AAP-Cl (10 and 20 mg/kg), (−)-AAP-Cl (10 mg/kg) or the racemic mixture (20 mg/kg) by i.v. bolus injection and serial blood samples were collected at different times after drug administration. (+)-AAP-Cl and (−)-AAP-Cl were separated with a resolution factor, R s, of at least 1.4, and a separation factor, α, greater than 1.09. Linear calibration curves were obtained over the concentration range of 0.5–30 μg/ml in plasma for both (+)-AAP-Cl and (−)-AAP-Cl (R 2≥0.996) with a limit of quantitation of 100 ng/ml and the recovery was greater than 80% for both enantiomers. The accuracy and precision for both enantiomers ranged from 96 to 102% (±0.2–7%) at upper and lower concentrations. The plasma concentration–time profiles of the enantiomers of AAP-Cl were best described by a two-compartment open model with a mean terminal half-life of about 5 h, volume of distribution at steady state of 3 l/kg and clearance of about 0.6 l/(h kg) in rats. There was no significant difference between the pharmacokinetic parameters of (+)-AAP-Cl and (−)-AAP-Cl, suggesting that the disposition of AAP-Cl in rats is not enantioselective. In addition, no chiral inversion of (+)-AAP-Cl to (−)-AAP-Cl or vice versa was observed. The results of this investigation have shed some light on the mechanism of action and disposition of AAP-Cl in rats.
Keywords: Enantiomer separation; Pharmacokinetics; N-(4-Chlorophenyl)-1-(4-pyridyl)ethylamine;

Enantioselective liquid chromatographic-electrospray mass spectrometric assay of β-adrenergic blockers: application to a pharmacokinetic study of sotalol in human plasma by Elena Badaloni; Ilaria D’Acquarica; Francesco Gasparrini; Silvana Lalli; Domenico Misiti; Franco Pazzucconi; Cesare R Sirtori (45-54).
An enantioselective high performance liquid chromatographic-electrospray ionization mass spectrometric (HPLC-ESI-MS) method for the direct determination of several β-adrenergic blockers was developed and validated. The method is based on the direct separation of the enantiomers of drugs on a laboratory-made chiral stationary phase (CSP) containing covalently bonded teicoplanin (TE) as chiral selector. Detection of the effluent was performed by electrospray ionization mass spectrometry, run in the selected-ion recording (SIR) mode. The method was applied to the pharmacokinetic monitoring of sotalol (STL) in the plasma of five young healthy volunteers, dosed with racemic drug. The limits of quantitation (LOQ) reached 4 ng/ml for both sotalol enantiomers. Such a method, fully validated, offers a novel, fast and very efficient tool for the direct determination of sotalol enantiomers in human plasma, and can be generally applied to the β-adrenergic blockers stereoselective pharmacokinetics.
Keywords: Enantiomer separation; Pharmacokinetics; Sotalol; β-Adrenergic blockers;

Identification of pheromones in mouse urine by head-space solid phase microextraction followed by gas chromatography–mass spectrometry by M.N. Kayali-Sayadi; José M. Bautista; L.M. Polo-Dı́ez; Ignacio Salazar (55-62).
Given the key role of pheromones in animal communication and behaviour, there is need to identify the different classes of these molecules under varying physiological conditions. However, the highly volatile nature of pheromones and the fact that they occur at very low concentrations in urine makes this task all the more difficult. Herein, we present a method of detecting and identifying the five main pheromones known: 2-sec-butyl-4,5-dihydrothiazole, geraniol, indole, trans-beta farnesene and trans-alpha farnesene in individual urine microsamples taken from male mice. Urine volumes as small as 20 μl were subjected to solid phase microextraction (SPME) followed by gas chromatography–mass spectrometry (GC–MS). This selective analytical method permits the rapid detection of these pheromones free from cross-contaminants as a clearly distinguishable spectral signals. Highest recovery rates of natural pheromones were achieved by extraction on a carboxen/polydimethylsiloxane (CAR/PDMS) fibre of 85 μm film thickness. This selective, sensitive and accurate method will help address the question of possible links between certain pheromone classes, and social and reproductive behaviour in mice.
Keywords: Pheromones;

A chromatographic method for determination of leucine aminopeptidase (LAP) activity in complex matrices is described. l-Leucine-β-naphthylamide was used as the substrate and its hydrolytic product, β-naphthylamine, was monitored by fluorescence at 280 nm excitation and 400 nm emission wavelengths. Under optimized conditions, the components in the incubation mixture were baseline separated and eluted out of a large-pore (300 Å) reversed-phase C4 column (RPC4) within 15 min with a non-linear gradient elution of methanol (0.05% (v/v) trifluoroacetic acid additive). The detection limit of the hydrolytic product reached 0.35 pmol at three time signal-to-noise (S/N) ratio with 5 μl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10 ng/ml to 80 μg/ml) and LAP (0.1–46.0 μg/ml) with correlation coefficient larger than 0.998 and reproducibility <3 and 10% R.S.D. (n=3), respectively. A fairly broad range of incubation time could be selected within 1 h. The LAP activities and concentrations in rabbit serum, tears, and mouse lens homogenates were determined to be 41.8 (0.3 mg/ml), 2.8 (40.0 μg/ml), and 1.6 pmol/(μl min) (17.5 μg/ml), respectively, with reproducibility of 2–9% R.S.D. (n=3) and intra- and inter-day variation for the retention time of the hydrolytic product being <1% R.S.D. (n=3). The results indicate that the present method is rapid and sensitive as compared to the conventional one.
Keywords: Enzymes; Leucine aminopeptidase;

In recent years, consumption of herbal supplements as an alternative to pharmaceutical drug therapy has increased. For example, with the health claims labeling which describes the link between soy-protein and a reduced risk of coronary heart disease (CHD), the consumption of soy and soy-derived phytoestrogens has increased dramatically. That being said, the oral bioavailability of only a few soy phytoestrogens such as Daidzein and Genestein have been previously estimated. In this paper, we present the calculated percent of rat oral bioavailability of five soy-derived phytoestrogens (Genistein, Daidzein, Biochanin A, Coumestrol, and Zearalenone) in male Sprague–Dawley rats. The plasma quantitation required for the bioavailability calculation is performed by using a rapid on-line plasma extraction procedure for the quantitative analysis. To further speed up the analysis the rats were dosed using the ‘n-in-one’ (cassette) protocol. The rapid on-line extraction/quantitation methodology coupled to the cassette dosing analysis of phytoestrogens is the key point of this paper. The limit of quantitation (LOQ) for each compound was 1–1000 ng/ml with each plasma sample analysis taking less than 2 min. In general the percent oral bioavailability was determined to be between 11 and 28%.
Keywords: Phytoestrogens; Plasma quantitation; Oral bioavailability;

High-performance liquid chromatographic assay of metabolites of thioguanine and mercaptopurine in capillary blood by Norbert Erb; Ulf Haverland; Dörthe O. Harms; Gabi Escherich; Gritta Janka-Schaub (87-94).
The main metabolites of the cytotoxic drugs thioguanine (6TG) and mercaptopurine (6MP) can be measured conveniently in red blood cells (RBC). Isolation of RBC, however, is laborious and requires some milliliters of blood. This HPLC assay allows measurements of thiopurine metabolites in very small blood samples obtained from the finger-tip. The metabolites, derivatives of 6TG and methylmercaptopurine (6MeMP), were extracted and hydrolized with perchloric acid to liberate the corresponding base. 6MeMP is completely transformed under these conditions to 4-amino-5-(methylthio)carbonyl imidazole. The chromatographic separation of 6TG and this imidazole was performed in a single run under isocratic conditions within 10 min using a 70 mm column. The quantification limit was 0.5 nmol/ml for 6TG and 3 nmol/ml blood for 6MeMP. The accuracy was 83% for 6TG (CV=3%) over the concentration range of 0.5–20 nmol/ml blood and 102% (CV=4%) for 6MeMP over the range of 3–150 nmol/ml blood. The intra-assay CV ranged from 5.4 to 7.4% for 6TG and from 6.2 to 10.6% for 6MeMP. The inter-assay CV was 7.5 and 9.5% in a pooled blood sample. The levels in RBC in whole blood were nearly coincident with those obtained in separated RBC, isolation of RBC therefore is not necessary for these measurements, if the drugs are given per os in the day before blood sampling. The concentration of 6MeMP nucleotides is more dependent on the given 6MP dose than the concentration of 6TG nucleotides. Intraindividual variations were small at unchanged drug doses, interindividual metabolite concentrations were highly variable.
Keywords: Thioguanine; Mercaptopurine;

There is considerable interest in quantifying morphine and its major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Available assays use gas chromatography–mass spectrometry or high-performance liquid chromatography (HPLC) with single or tandem mass spectrometry, ultraviolet, electrochemical, or fluorimetric detection. Nevertheless, few methods provide adequate sensitivity for all analytes, in a single injection, with the desired rate of sample throughput. A rapid and sensitive method for quantification of morphine, M3G and M6G from human plasma using HPLC with electrospray ionization mass spectrometry was developed using a Waters Oasis MCX 96-well plate for extracting both lipophilic morphine and its hydrophilic glucuronides, C18 separation using an isocratic mobile phase (methanol, acetonitrile and formic acid), and selected ion monitoring. Recoveries of morphine, M3G and M6G, respectively, were 81, 90 and 82% at the low (2, 25 and 2 ng/ml), 80, 77 and 75% at the medium (10, 250 and 10 ng/ml), and 74, 62 and 72% at the high (100, 1000 and 100 ng/ml) quality control samples. The limit of quantitation was 0.5 ng/ml morphine and M6G, and 5 ng/ml M3G. Analytes were validated over a linear range of 0.5–200 ng/ml morphine and M6G, and 5–2000 ng/ml M3G. This assay represents an improvement over existing methods through solid phase extraction with increased sample throughput (96-well plates), use of small samples (0.5 ml), and sub-nanogram detection.
Keywords: Morphine; Morphine glucuronides;

An improved reverse-phase high-performance liquid chromatographic method (RP-HPLC) for the determination of a novel iron chelator CP502 (1,6-dimethyl-3-hydroxy-4-(1H)-pyridinone-2-carboxy-(N-methyl)-amide hydrochloride) in rat plasma, urine and feces was developed and validated. The separation was performed on a polymeric column using a mobile phase composed of 1 mM ethylenediaminetetra-acetic acid disodium salt (EDTA), acetonitrile, methanol and methylene chloride. Separation of CP502 from plasma, urine or feces endogenous compounds was achieved by gradient elution. Retention times of CP502 and its major metabolite (glucuronide) were about 13 and 4 min, respectively. The method was validated in terms of limit of detection (LOD), limit of quantification (LOQ), selectivity (endogenous from plasma, urine or feces), linearity, extraction recovery, robustness (column selection, mobile phase composition, detection mode, internal standard (IS) selection, analyte stability), day-to-day reproducibility and system suitability (repeatability, peak symmetry and resolution). The method is applicable to bioavailability and pharmacokinetic studies of CP502 in rats.
Keywords: Pharmacokinetics; CP502; Iron chelator;

High-performance liquid chromatographic assay for the determination of Aloe Emodin in mouse plasma by M. Zaffaroni; C. Mucignat; T. Pecere; G. Zagotto; R. Frapolli; M. D’Incalci; M. Zucchetti (113-119).
An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3 ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol–water–acetic acid (65:35:0.2) and fluorescence detection at λ ex=410 nm and λ em=510 nm. The retention time of AE was 11.7 min. The assay was linear from 10 to 1000 ng/ml (r 2≥0.999), showed intra- and inter-day precision within 7.8 and 4.7%, and accuracy of 87.3–105.7%. Detection limit (LOD) and quantification limit (LOQ) were 4.5 and 5 ng/ml, respectively. The method was applied to determine for the first time the pharmacokinetic of AE in mice.
Keywords: Aloe Emodin;

A simple method for simultaneous determination of androstenediol 3-sulfate (Adiol-3S) and dehydroepiandrosterone sulfate (DHEA-S) in human serum using isotope diluted liquid chromatography–electrospray ionization-ion trap-mass spectrometry (LC–ESI-ion trap-MS) was developed. After addition of deuterated internal standards ([ 2 H 5 ]Adiol-3S and [ 2 H 4 ]DHEA-S), human serum (100 μl) was deproteinized with acetonitrile and then applied to a solid-phase extraction cartridge, Oasis HLB. The obtained steroid sulfates fraction was washed with hexane and then analyzed by LC–ESI-MS operated in the negative ion mode. The quantification ranges of Adiol-3S and DHEA-S were 10–400 ng/ml and 0.05–8 μg/ml, respectively. The method does not require the chemical or enzymatic hydrolysis of the conjugates and purification with high-performance liquid chromatography, and shows satisfactory reproducibility and accuracy. The concentrations of these sulfates in the sera of healthy male volunteers (n=14) were 19.2–245.3 mg/ml (Adiol-3S) and 0.175–5.16 μg/ml (DHEA-S), and those of patients with prostate cancer (n=19) were 15.3–182.7 ng/ml (Adiol-3S; four samples, not detectable) and 0.110–2.421 μg/ml (DHEA-S).
Keywords: Androstenediol 3-sulfate; Dehydroepiandrosterone sulfate;

The major urinary metabolites of 1,3-butadiene are monohydroxybutenyl-mercapturic acids (MHBMA) and dihydroxy-butyl-mercapturic acid (DHBMA). These metabolites can be used as biomarkers of exposure to this diene. In order to determine the smoking-related exposure to 1,3-butadiene, we have developed a rapid LC-MS/MS method for the determination of MHBMA and DHBMA in urine of humans and rats. The method requires 2–5 ml of urine which is solid phase extracted prior to LC-MS/MS analysis. Precision for MHBMA is ≤11.2% for human and ≤17% for rat urine. Corresponding values for DHBMA are ≤7.2 and ≤19%, respectively. Recovery rates are approximately 100% for both analytes in human urine and about 115% in rat urine. Limits of detection (LOD) are for humans 0.9 and 23 ng/ml and for rats 1.5 and 33 ng/ml for MHBMA and DHBMA, respectively. Application of the method to urine of humans and rats showed a significant effect of tobacco smoke exposure on the urinary excretion of MHBMA and the metabolic ratio DHBMA/(DHBMA + MHBMA).
Keywords: Mercapturic acids; 1,3-Butadiene;

Search for peptidic “middle molecules” in uremic sera: isolation and chemical identification of fibrinogen fragments by Batia Kaplan; Miriam Cojocaru; Edward Unsworth; Aaron Knecht; Brian M. Martin (141-153).
According to the “middle molecule” (MM) hypothesis, the uremic solutes ranging from 500 to 5000 Da are insufficiently eliminated by conventional hemodialysis and may act as uremic toxins. However, because of the methodological difficulties of MM purification, their chemical analysis is complicated and the precise structure of these molecules remains obscure. In the present study, a new micro-preparative procedure including SDS electrophoresis and liquid chromatography was applied for isolation of MM peptides from uremic sera. Microsequencing and MS/MS analyses of these peptides showed that most of the identified MM (22 out of 23) represented the N- and C-terminal fragments of the α- and β-chains of fibrinogen. The obtained data provide new information on the precise structure of fibrinogen fragments accumulating in uremic serum as MM.
Keywords: Middle molecules; Fibrinogen;

Linezolid is a new, promising antibacterial agent to treat severe infections. A rapid HPLC assay using UV detection for the determination in microdialysate and human plasma was developed. After sample preparation, using acetonitrile for plasma and water for microdialysate, 20 μl was injected and separated on a RP-18 column. Overall, the assay exhibited good precision and accuracy. The diffusion properties of linezolid investigated in in vitro microdialysis experiments revealed a mean relative recovery of 77.5% (CV: 5.4%; delivery and recovery experiments). Following characterization of linezolid in in vitro microdialysis, the setting is suitable for application in clinical studies.
Keywords: Microdialysis; Linezolid;

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid–liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol–acetonitrile–phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5–16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.
Keywords: Loratadine; Descarboethoxyloratadine;

Measurement of nicotine and cotinine in human milk by high-performance liquid chromatography with ultraviolet absorbance detection by Madhu Page-Sharp; Thomas W Hale; L.Peter Hackett; Judith H Kristensen; Kenneth F Ilett (173-180).
A high-performance liquid chromatographic (HPLC) assay for the determination of nicotine and cotinine in human milk was developed using an extraction by liquid–liquid partition combined with back extraction into acid, and followed by reverse-phase chromatography with UV detection of analytes. The assay was linear up to 500 μg/l for both nicotine and cotinine. Intra- and inter-day relative standard deviations (R.S.D.) were <10% (25–500 μg/l) for both nicotine and cotinine. Limits of quantitation (LOQ) were 10 and 12 μg/l for nicotine and cotinine, respectively, while the limits of detection (LOD) were 8 and 10 μg/l for nicotine and cotinine, respectively. The mean recoveries were 79–93% (range 25–500 μg/l) for nicotine and 78–89% (range 25–500 μg/l) for cotinine. The amount of fat in the milk did not affect the recovery. We found that this method was sensitive and reliable in measuring nicotine and cotinine concentrations in milk from a nursing mother who participated in a trial of the nicotine patch for smoking cessation.
Keywords: Nicotine; Cotinine;

Liquid-chromatographic determination of erlotinib (OSI-774), an epidermal growth factor receptor tyrosine kinase inhibitor by Erin R. Lepper; Sandra M. Swain; Antoinette R. Tan; William D. Figg; Alex Sparreboom (181-188).
A high-performance liquid-chromatographic (HPLC) assay with UV detection has been developed for the quantitative determination of erlotinib (OSI-774) in human plasma. Quantitative extraction was achieved by a single-solvent extraction involving a mixture of acetonitrile and n-butyl chloride (1:4, v/v). Erlotinib and the internal standard hydrochloride salt (OSI-597) were separated on a column packed with Nova-Pak C18 material and a mobile phase composed of acetonitrile and water, pH 2.0 (60:40, v/v). The column effluent was monitored with dual UV detection at wavelengths of 348 nm (erlotinib) and 383 nm (OSI-597). The calibration graph was linear in the range of 100–4500 ng/ml, with values for accuracy and precision ranging from 87.9 to 96.2% and 2.13 to 5.10%, respectively, for three different sets of quality control samples. The developed method was successfully applied to study the pharmacokinetics of erlotinib in a cancer patient at the recommended daily dose of 150 mg.
Keywords: Pharmacokinetics; Erlotinib; OSI-774;

Pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract by Fan Lei; Dong-Ming Xing; Lan Xiang; Yu-Nan Zhao; Wei Wang; Lu-Jun Zhang; Li-Jun Du (189-194).
Quantification of ellagic acid, the principal bioactive component of pomegranate leaf extract, in rats plasma following oral administration of pomegranate leaf extract was achieved by using a high-performance liquid chromatographic method. The calibration curve for ellagic acid was linear (r 2=0.9998) ver the concentration range 0.026–1.3 μg/ml. The intra- and inter-day assays of ellagic acid from rat plasma were less than 6.52% at concentration range from 26 to 1300 ng/ml and good overall recoveries (94.5–102.4%) were found on same concentrations. The concentration–time profile was fitted with an open two-compartment system with lag time and its max concentration of ellagic acid in plasma was 213 ng/ml only 0.55 h after oral administration extract 0.8 g/kg. The pharmacokinetic profile indicates that ellagic acid has poor absorption and rapid elimination after oral administration pomegranate leaf extract, and part of it was absorbed from stomach.
Keywords: Ellagic acid; Pharmacokinetics;

A simple, rapid, sensitive column-switching HPLC method is described for the analysis of the 10-hydroxycamptothecin (HCPT) in human serum. A pre-column containing restricted access media (RAM) is used for the sample clean-up and trace enrichment and is combined with a C18 column for the final separation. The analytical time is 8 min. The HCPT is monitored with fluorescence detector, excitation and emission wavelengths being 385 and 539 nm, respectively. There is a linear response range of 1–1000 ng/ml with correlation coefficient of 0.998 while the limit of quantification is 0.1 ng/ml. The intra-day and inter-day variations are less than 5%. This analytic procedure has been applied to a pharmacokinetic study of HCPT in clinical patients and the pharmacokinetic parameters of one-compartment model are calculated.
Keywords: Pharmacokinetics; Restricted access media; 10-Hydroxycamptothecin;

Improved procedure for the determination of malonaldehyde by gas-chromatography with electron-capture detection as 2,4,6-trichlorophenylhydrazine derivative by Lorenzo Sangalli; Luca Maria Chiesa; Elena Passerò; Ada Manzocchi; Giovanni Maffeo; Pier Antonio Biondi (201-207).
A previously described derivatization method using trichlorophenylhydrazine was developed for the estimation of malonaldehyde measured by gas-chromatography (GC) with electron-capture detection. The precision and reliability of the procedure are improved here by the use of methylmalonaldehyde as internal standard and by the introduction of a diverter valve at the end of the capillary column to protect the electron-capture detector, respectively.The method was applied to determine malonaldehyde content in bovine plasma samples.
Keywords: Derivatization; GC; Malonaldehyde;