Journal of Chromatography B (v.801, #2)

OFC: Update (OFC).

News Section (N1-N2).

An analytical process generally involves four main steps: (1) sample preparation; (2) analytical separation; (3) detection; and (4) data handling. In the bioanalytical field, sample preparation is often considered as the time-limiting step. Indeed, the extraction techniques commonly used for biological matrices such as liquid–liquid extraction (LLE) and solid-phase extraction (SPE) are achieved in the off-line mode. In order to perform a high throughput analysis, efforts have been engaged in developing a faster sample purification process. Among different strategies, the introduction of special extraction sorbents, such as the restricted access media (RAM) and large particle supports (LPS), allowing the direct and repetitive injection of complex biological matrices, represents a very attractive approach. Integrated in a liquid chromatography (LC) system, these extraction supports lead to the automation, simplification and speeding up of the sample preparation process. In this paper, RAM and LPS are reviewed and particular attention is given to commercially available supports. Applications of these extraction supports, are presented in single column and column-switching configurations, for the direct analysis of compounds in various biological fluids.
Keywords: Reviews; Restricted access materials; Large particle supports; Direct injection;

DE-310 is a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, DX-8951, which is conjugated to a water-soluble polymer via a peptide spacer. Assay methods have been developed for the determination of a polymer-bonded DX-8951 conjugate, DX-8951, and Glycyl-DX-8951 (G-DX-8951) in mouse plasma. Free DX-8951 and Glycyl-DX-8951 were extracted from plasma by protein precipitation and analyzed by HPLC (Method I). Conjugated DX-8951 was extracted by protein precipitation and digested by using a thermolysin. The productive compound was analyzed by HPLC (Method II). The lower limits of quantitation of DX-8951, Glycyl-DX-8951, and Conjugated DX-8951 were 0.60, and 0.77 ng/ml and 3.45 μg/ml (as DX-8951 equivalent). These two methods showed satisfactory sensitivity, precision, accuracy, recovery, and selectivity.
Keywords: DE-310; Camptothecin;

This study describes a high-performance liquid chromatographic (HPLC) method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF) and cortisol in human urine using either methylprednisolone or beclomethasone as internal standard. Separation was achieved on a reversed-phase phenyl column by a gradient elution of 0.05 M KH2PO4–0.01 M CH3COOH (pH 3.77) and 0.05 M KH2PO4–0.01 M CH3COOH with acetonitrile (4:6, v/v). 6β-Hydroxycortisol and cortisol were monitored by UV absorption at 239 nm. The lower quantitation limits of the present HPLC method were 21.5 ng/ml for 6β-OHF and 5.0 ng/ml for cortisol in urine. The within-day reproducibilities in the amounts of 6β-OHF and cortisol determined were in good agreement with the actual amounts added, the relative error being less than 1.59%. The inter-assay precisions (R.S.D. values) were less than 7.91% for 6β-OHF and cortisol. The method was compared with the GC/MS method by measuring 6β-OHF in the same urine samples. A good correlation was found between the amounts determined by the two methods. The regression equations for the HPLC (y) and GC/MS (x) methods were: y=1.0701x+17.389 (r=0.9772) for methylprednisolone as internal standard and y=1.0827x+6.1364 (r=0.9794) for beclomethasone as internal standard.
Keywords: Phenotyping; Cortisol; 6β-Hydroxycortisol; CYP3A activity;

Quantification and confirmation of flunixin in equine plasma by liquid chromatography–quadrupole time-of-flight tandem mass spectrometry by Yi Luo; Jeffrey A Rudy; Cornelius E Uboh; Lawrence R Soma; Fuyu Guan; James M Enright; Deborah S Tsang (173-184).
The method describes quantification and confirmation of flunixin in equine plasma by liquid chromatography–quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS/MS). Samples were screened by enzyme-linked immunosorbent assay (ELISA) and only those samples presumptively declared positive were subjected to quantification and confirmation for the presence of flunixin by this method. The method is also readily adaptable to instrumental screening for the analyte. Flunixin was recovered from plasma by liquid–liquid extraction (LLE). The sample was diluted with 2 ml saturated phosphate buffer (pH 3.10) prior to LLE. The dried extract was reconstituted in acetonitrile:water:formic acid (50:50:0.1, v/v/v) and subsequently analyzed on a Q-TOF tandem mass spectrometer (Micromass) operated under electrospray ionization positive ion mode. The concentration of flunixin was determined by the internal standard (IS) calibration method using the peak area ratio with clonixin as the IS. The limits of detection (LOD) and quantification (LOQ) for flunixin in equine plasma were 0.1 and 1 ng/ml, respectively, whereas the limit of confirmation (LOC) was 2.5 ng/ml. The qualifying ions for the identification of flunixin were m/z 297 [M+H]+, 279 (BP), 264, 259, 239 and those for clonixin (IS) were m/z 263 [M+H]+, 245 (BP) and 210. The measurement uncertainty about the result was 8.7%. The method is simple, sensitive, robust and reliably fast in the quantification and confirmation of flunixin in equine plasma. Application of this method will assist racing authorities in the enforcement of tolerance plasma concentration of flunixin in the racehorse on race day.
Keywords: Flunixin;

Biological action and activity reflect an aspect of the fundamental physicochemical properties of the bioactive compounds. As an alternative to classical QSAR studies, in this work different quantitative retention–activity relationships (QRAR) models are proposed, which are able to describe the role of hydrophobicity on the binding affinity to different brain monoamine receptors (H1-histamine, α1-noradrenergic and 5-HT2-serotonergic) of different families of psychotherapeutic drugs. The retention of compounds is measured in a biopartitioning micellar chromatography (BMC) system using Brij-35 mobile phases. The adequacy of the QRAR models developed is due to the fact that both the retention of compounds in BMC and the drug–receptor interaction are described by the same hydrophobic, electronic and steric properties of compounds. The obtained results indicate that, for structurally related compounds that present the same molecular features as the basic pharmacophore, there is a retention range in which compounds present the highest affinity to all of monoamine receptors.
Keywords: Hydrophobicity; Quantitative retention–activity relationships; Biopartitioning micellar chromatography; Monoamine receptor;

An improved and simplified high-performance liquid chromatographic (HPLC) method at UV detection 265 nm is presented for the determination of d4T in rat plasma. The mobile phase consists of methanol–distilled water–acetic acid in the 23:77:0.2 (v/v) ratio. Quantification is achieved by the peak-area ratio method with reference to the internal standard. This paper presents linearity, accuracy, precision, limit of quantification and limit of detection, specificity–selectivity and sample stability data. Based on the intra and inter-day validation, all coefficients of variation (CV) were found less than 15%. The assay is sufficiently rapid and sensitive and was applied in a pharmacokinetic study in rats.
Keywords: Stavudine;

An assay was developed to quantify norepinephrine (NE) and its metabolites (MHPG and DHPG) by high-performance liquid chromatography with electrochemical detection method (HPLC-ECD) in brain tissue and plasma of rats treated by LiCl. Separation on C18 column was obtained by a mobile phase consisting of 4.5% methanol in buffer (0.1 M sodium acetate, 0.2 M citric acid) containing 0.2 mM ethylenediaminetetraacetic acid disodium salt (EDTA Na2) and 0.4 mM sodium octylsulfate, operated at a flow rate of 0.8 ml/min. A potential of +0.78 V was applied across the working and reference electrodes of the detector. The precision was in the range 2.88–4.35% for NE, 5.94–11.0% for MHPG and 1.97–4.40% for DHPG. Accuracy was 98.8–99.3% for NE, 97.4–100% for MHPG and 96.1–101% for DHPG. The limit of detection was 0.6 ng/ml for NE, 0.5 ng/ml for MHPG and 0.2 ng/ml for DHPG. The linearity is over the range 20–60 ng/ml for NE, 7–23 ng/ml for MHPG and 6–20 ng/ml for DHPG. The assay has been applied successfully to measure simultaneously cortex and plasmas concentrations of these three catecholamines in rats.
Keywords: Norepinephrine; 3-Methoxy-4-hydroxyphenylglycol; 3,4-Dihydroxyphenylglycol; Lithium chloride;

Supported liquid membrane (SLM) technique for sample work-up and enrichment was used for determination of tricyclic antidepressant drugs in urine by high-performance liquid chromatography (HPLC) with UV detection. The studied antidepressant drugs were amitriptyline, opipramol, noxiptyline and additionally diethazine was used as possible internal standard. Alkaline phosphoric buffer with urine sample, as the donor solution, was passed over the liquid membrane into which investigated substances were extracted. On the other side of the membrane, analyzed compounds were trapped due to creating non-extractable form in acidic acceptor solution. Enriched and cleaned up drugs were then injected into a HPLC system with ultraviolet detection to analyze of their concentration in acceptor solution. Optimum extraction efficiency was determined by changing acceptor and donor solutions pH, application of different flow rates of donor solution and by using different solvents in the membrane. Also, donor solution volume, extraction time and concentration of analytes were varied to check the linearity of extraction process. The highest extraction efficiency: 43% for opipramol, 56% for noxiptyline, 43% for amitriptyline and 42% for diethazine (R.S.D. values were <6% and n=3) was achieved when 0.05 M phosphate buffer pH 4.0 and 9.5 were used as donor and acceptor solutions, respectively, n-undecane with 5% tri-n-octylphosphine oxide (TOPO) was used as liquid membrane. Limit of quantification (LOQ) for tricyclic antidepressants after enrichment of 100 ml of urine sample was about 1 ng/ml.
Keywords: Amitriptyline; Opipramol; Noxiptyline; Diethazine;

In this paper, the anti-coagulant rodenticide-human serum albumin (HSA) binding was investigated using a perturbation method to calculate the solute distribution isotherms. It was shown that rodenticide can bound either on the benzodiazepine HSA site with low affinity (site I) or on the warfarin HSA site with high affinity (site II). The thermodynamic parameters of this association were calculated for the two HSA binding sites. For the site II, the rodenticide-HSA association was governed enthalpically whereas for the site I, this one was driven entropically. Moreover, the role of the magnesium (Mg2+) and calcium (Ca2+) on this association was carried out. It was clearly demonstrated that the rodenticide affinity for the site I was not affected by modifying the bulk solvent surface tension whereas for the site II the association constant increased strongly with the Mg2+ or the Ca2+ concentration in the bulk solvent. These results showed that the rodenticide-HSA affinity and thus the rodenticide toxicological effect depends on the Mg2+ or Ca2+ concentration.
Keywords: Human serum albumin; Rodenticide;

Comparison of measured and calculated lipophilicity of substituted aurones and related compounds by B Hallgas; T Patonay; A Kiss-Szikszai; Zs Dobos; F Hollósy; D Erős; L Őrfi; Gy Kéri; M Idei (229-235).
A molecule library containing 55 aurone- and thioaurone-type structures has been designed and synthesised. Reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed to separate these compounds and to characterise their lipophilicity by experimental method (k′). The experimental lipophilicity data have been compared with the computer calculated lipophilicity parameters (CLOGPs) of the same molecules. In general, good correlations between the measured and calculated lipophilicities have been found with the exception of structure isomers and compounds capable for hydrogen bonding. The chromatographic method was suitable to separate the structure (ortho and para) isomers of aurone and thioaurones and was sensitive enough to differentiate their lipophilicities. Our findings suggest the usefulness of the chromatographic method in fast characterisation of the lipophilicity of structurally closely related molecules.
Keywords: Lipophilicity; Aurones;

Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells by Annie Costa; Jean-Paul Paı̈s de Barros; Gérard Keith; Wlodzimierz Baranowski; Jean Desgrès (237-247).
Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNAAsn and tRNAHis, mannosyl-queuosine (manQ) in tRNAAsp, and galactosyl-queuosine (galQ) in tRNATyr. An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs from various mammalian hepatic cells. Our results show that the four Q-tRNAs are fully modified in liver tissues from adult mammals, regardless of the mammal species. However, a lack in the Q-modification level was observed in Q-tRNAs from newborn rat liver, as well in Q-tRNAs from normal rat liver cell cultures growing in a low queuine content medium, and from a rat hepatoma cell line. It is noteworthy that in all cases of Q-tRNA hypomodification, our analytical procedure showed that tRNAAsp is always the least affected by the hypomodification. The biological significance of this phenomenon is discussed.
Keywords: Queuosine; tRNA;

Galactonate determination in urine by stable isotope dilution gas chromatography–mass spectrometry by Peter Schadewaldt; Hans-Werner Hammen; Sara Stolpmann; Loganathan Kamalanathan; Udo Wendel (249-255).
A stable isotope dilution assay was developed for the sensitive determination of d-galactonic acid. d-[U- 13 C 6 ]galactono-1,4-lactone was prepared as internal standard. Unlabelled and U- 13 C -labelled d-galactonic acid species were converted to the N-(1-butyl)galactonamide pentaacetate derivatives and assessed by gas chromatography–mass spectrometry (GC–MS). Positive chemical ionisation and monitoring of the [MH-60]+-ions in the galactonate chromatographic peak at m/z 402 and m/z 408 were used for quantification. The procedure was applied to study the variability of d-galactonate excretion in healthy subjects and galactosemic patients and to monitor the d-galactonate–d-galactitol ratio in human urine.
Keywords: Galactonate;

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to determine the enantiomers of the muscle relaxant baclofen in human plasma and cerebrospinal fluid (CSF) has been developed. A commercially available ultrafiltration membrane is used to prepare the sample. A chiral CROWNPAK CR(+)® stationary phase column is then used to perform complete resolution of the S(+)- and R(−)-enantiomers of baclofen. This method was used to analyze human plasma and CSF spiked with baclofen, and the calibration curves for both biologic samples were linear over a concentration range of 0.15–150 ng enantiomer/ml. The lower limit of quantification was 0.15 ng enantiomer/ml in both fluids. Finally, the method was tested with an artificial CSF as an alternative to authentic human CSF. The results showed that no matrix effects and no interfering peaks were observed using this artificial CSF.
Keywords: Enantiomer separation; Baclofen;

Glipizide and rosiglitazone are widely used to treat Type 2 diabetes. In order to investigate drug–drug protein binding interaction between glipizide and rosiglitazone, a method was developed and validated for simultaneously determining the free (unbound) fraction of glipizide and rosiglitazone in plasma employing equilibrium dialysis for the separation of free drug and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for quantitation. Post-dialysis human plasma or buffer samples of 0.2 ml were extracted using a liquid–liquid extraction procedure and analyzed by a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Zorbax SB-Phenyl column, ionized using an atmospheric pressure electrospray ionization source and analyzed in positive ion mode with multiple reaction monitoring. The ion transitions monitored were m/z 446→321 for glipizide, m/z 358→135 for rosiglitazone, and m/z 271→155 for tolbutamide (internal standard, IS). The chromatographic run time was 5 min per injection, with retention times of 2.3, 3.4 and 2.3 min for glipizide, rosiglitazone and IS, respectively. The calibration curves of glipizide and rosiglitazone were over the range of 1–2000 ng/ml (r 2>0.9969) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v). The inter-assay precision and accuracy of the quality control samples were <10.9% of coefficient of variability and >93.5% and 94.5% of nominal concentration for glipizide and rosiglitazone, respectively. The lower limit of quantitation of both glipizide and rosiglitazone was 1.0 ng/ml. Both glipizide and rosiglitazone bound to plasma protein extensively (>99% bound). Glipizide and rosiglitazone free fraction averaged 0.678±0.071 and 0.389±0.061%, respectively, at plasma concentration of 1000 ng/ml. This developed method proves reproducible and sensitive and its application to clinical samples is also reported.
Keywords: Equilibrium dialysis; Glipizide; Rosiglitazone;

The origin, i.e. natural occurrence or illegal treatment, of findings of 17α-boldenone (α-Bol) and 17β-boldenone (β-Bol) in urine and faeces of cattle is under debate within the European Union. A liquid chromatographic positive ion electrospray tandem mass spectrometric method is presented for the confirmatory analysis of 17β-boldenone, 17α-boldenone and an important metabolite/precursor androsta-1,4-diene-3,17-dione (ADD), using deuterium-labelled 17β-boldenone (β-Bol-d3) as internal standard. Detailed sample preparation procedures were developed for a variety of sample matrices such as bovine urine, faeces, feed and skin swab samples. The method was validated as a quantitative confirmatory method according to the latest EU guidelines and shows good precision, linearity and accuracy data, and CCα and CCβ values of 0.1–0.3 and 0.4–1.0 ng/ml, respectively. Currently, the method has been successfully applied to suspect urine samples for more than a year, and occasionally to faeces, feed and swab samples as well. Results obtained from untreated and treated animals are given and their impact on the debate about the origin of residues of 17β-boldenone is critically discussed. Finally, preliminary data about the degree of conjugation of boldenone residues are presented and a simple procedure for discrimination between residues from abuse versus natural origin is proposed.
Keywords: Boldenone; Androsta-1,4-diene-3,17-dione;

Astragaloside IV (AGS-IV) is an active constituent of Radix Astragali used in many Traditional Chinese Medicines. This paper describes a sensitive and specific assay for the quantitation of AGS-IV in rat plasma. After solid phase extraction (SPE), samples were analyzed by liquid chromatography electrospray ionization mass spectrometry using a reversed-phase C18 column. The assay was linear in the range 1–500 ng/ml with a limit of detection of 0.5 ng/ml. The recovery was 92.5% and within-day and between-day precision were 3.7–6.0 and 2.8–9.8%, respectively. The assay was applied to a pharmacokinetic study in rat after a single oral dose. The drug was rapidly absorbed and subsequently eliminated according to a biphasic concentration–time curve.
Keywords: Astragaloside IV;

An increasing number of synthetic drugs are appearing on the illicit market and on the scene of drug use by youngsters. Official figures are underestimated. In addition, immunochemical tests are blind to many of these drugs and appropriate analytical procedures for routine clinical and epidemiological purposes are lacking. Therefore, the perceived increasing abuse of recreational drugs has not been proved yet. In a previous paper, we proposed a procedure for the preliminary screening of several recreational substances in hair and other biological matrices. Unfortunately, this procedure cannot apply to cocaine. Consequently, we performed a new headspace solid-phase microextraction and gas chromatography–mass spectrometry (HS-SPME–GC–MS) procedure for the simultaneous detection of cocaine, amphetamine (A), methamphetamine (MA), methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE), N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), ketamine, and methadone in human hair. Hair was washed with water and acetone in an ultrasonic bath. A short acid extraction with 1 M hydrochloric acid was needed; the fiber was exposed to a 5 min absorption at 90 °C and thermal desorption was performed at 250 °C for 3 min. The procedure was simple, rapid, required small quantities of sample and no derivatization. Good linearity was obtained over the 0.1–20.0 ng/mg range for the target compounds. Sensitivity was good enough: limits of detection (LOD) were 0.7 ng/mg of hair for the majority of substances. The intra-day precision ranged between 7 and 20%. This paper deals with the analytical performance of this procedure and its preliminary application to hair samples obtained on a voluntary basis from 183 young people (138 males and 45 females) in the Rome area.
Keywords: Headspace analysis; Hair analysis; Amphetamine-like drugs; Cocaine;

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC–MS n . This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R 2=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.
Keywords: Verapamil; Molecularly imprinted polymers;

We developed a new 3-D HPLC method for on-line clean-up and simultaneous quantification of two important naphthalene metabolites, 1-naphthol and 2-naphthol, in human urine. Except an enzymatic hydrolysis no further sample pre-treatment is necessary. The metabolites are stripped from urinary matrix by on-line extraction on a restricted access material pre-column (RAM RP-8), transferred in backflush mode onto a silica-based CN-(cyano)phase column for further purification from interfering substances. By another successive column switching step both analytes are transferred with a minimum of overlapping interferences onto a C12 bonded reversed phase column with trimethylsilyl endcapping where the final separation is carried out. The entire arrangement is software controlled. Eluting analytes are quantified by fluorescence detection (227/430 nm) after an external calibration. Within a total run time of 40 min we can selectively quantify both naphthols with detection limits in the lower ppb range (1.5 and 0.5 μg/l for 1- and 2-naphthol, respectively) with excellent reliability (ensured by precision, accuracy, matrix-independency and FIOH quality assurance program participation). First results on a collective of 53 occupationally non exposed subjects showed mean levels of 11.0 μg/l (1-naphthol) and 12.9 μg/l (2-naphthol). Among smokers (n=21) a significantly elevated mean level of urinary naphthols was determined (1-naphthol: 19.2 μg/l and 2-naphthol: 23.7 μg/l) in comparison to non smokers (n=32; 1-naphthol: 5.6 μg/l, 2-naphthol: 5.6 μg/l).
Keywords: Naphthol;

A new technique for sample preparation on-line with LC and GC–MS assays was developed. Microextraction in a packed syringe (MEPS) is a new miniaturised, solid-phase extraction technique that can be connected on-line to GC or LC without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100–250 μl) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising. It is very easy to use, fully automated, of low cost and rapid in comparison with previously used methods. This paper presents the development and validation of a method for microextraction in packed syringe MEPS on-line with GC–MS. Local anaesthetics in plasma samples were used as model substances. The method was validated and the standard curves were evaluated by the means of quadratic regression and weighted by inverse of the concentration: 1/x for the calibration range 5–2000 nM. The applied polymer could be used more than 100 times before the syringe was discarded. The extraction recovery was between 60 and 90%. The results showed close correlation coefficients (R>0.99) for all analytes in the calibration range studied. The accuracy of MEPS–GC–MS was between 99 and 115% and the inter-day precision (n=3 days), expressed as the relative standard deviation (R.S.D.%), was 3–10%.
Keywords: Microextraction in packed syringe; Anaesthetics;

Capillary porous graphitic carbon (PGC) columns have been utilized for separation of several catecholamines and related compounds (i.e. l-tyrosine, l-DOPA, 3-O-methyl-DOPA, dopamine, 3,4-dihydroxy-phenyl-acetic acid (DOPAC), homovanillic acid, noradrenaline, vanillomandelic acid and adrenaline) on-line with electrospray ionization tandem mass spectrometry (ESI–MS/MS). The use of a mobile phase without ion-pairing agents and with high content of organic modifier facilitated the coupling to the selective and sensitive mass spectrometric detection. Minimum detectable sample concentration (MDC sample) for noradrenaline, dopamine and l-tyrosine in a standard solution was estimated to 3, 10 and 30 nM, respectively (3 S/N corresponds to MDQ for l-tyrosine of approximately 8×10−14  mol). The developed strategy was applied for analysis of brain tissue, i.e. a substantia nigra (ns) sample.
Keywords: Catecholamines; Porous graphitic carbon; l-DOPA; Dopamine; Polar analytes;

Application of short monolithic columns for fast purification of plasmid DNA by Karmen Branovic; Dubravko Forcic; Jelena Ivancic; Ales Strancar; Milos Barut; Tanja Kosutic Gulija; Renata Zgorelec; Renata Mazuran (331-337).
Anion-exchange chromatography is one of the most important methods in downstream processing of plasmid DNA, both as a process and as an analytical technique. Separation of plasmid DNA on traditional particle-based anion-exchange supports is usually slow. Moreover, such supports have a low capacity for plasmid DNA due to the steric exclusion effects. In this work, the separation of plasmid DNA using short monolithic columns, Convective Interaction Media, will be presented. It will be demonstrated that plasmid DNA can be purified from bacterial cells using alkaline lysis followed by chromatography on a very short weak anion-exchange chromatographic columns—disks—with good purity and quality within a short time. Furthermore, the separation of plasmid DNA from cell RNA can be carried out without the need of adding RNAse. Fast and efficient method for in-process control of the purified plasmid will be described as well.
Keywords: Purification; Monolithic columns; Plasmid DNA;

A simple, rapid method for the simultaneous determination of cardiovascular drugs: celiprolol, bisoprolol and irbesartan in human plasma is described. The two main features of the proposed method deal first, with a simultaneous solid phase extraction of weakly basic beta-blockers derivatives and irbesartan which exhibit weak acidic properties; second with an absorbance monitoring using diode array detection in order to insure an improved selectivity. The separation is performed on a C18 Kromasil® 4.6  mm×150  mm column using a linear gradient to achieve an entire separation of the four species in less than 20 min. The full analytical validation is performed according to guidance for industry for bioanalytical method validation. Linearity of the response was demonstrated for each drug for a range fulfilling the reported plasma levels, that is 10–500, 5–250 and 20–1000 ng l−1 for celiprolol, bisoprolol and irbesartan respectively. Intra- and inter-day relative standard deviations for all compounds were, in any case, lower than 11% and the method exhibits a convenient accuracy (percentage of relative error lower than 6% for each drug). In each case, the LOD were sufficient to detect post dose trough concentrations for checking patient’s observance. Moreover, selectivity towards either endogenous species or co-administered drugs was demonstrated by combination of the use of the solid phase extraction process, gradient elution and diode array detection facilities, making thus, the proposed technique especially suitable for routine drug monitoring of resistant hypertensive patients.
Keywords: Celiprolol; Bisoprolol; Irbesartan;

The analysis of corticosterone in mouse blood serum (metabolic-stress experiment) and 17-hydroxycorticosterone in human urine (exercise-stress experiment) samples by means of capillary electrophoresis/UV absorbance in conjunction with online sample concentration techniques is described. The use of normal MEKC had an analyte detection limit of 7 μg/ml (S/N=3); whereas when online sample concentration methods, including sweeping-micellar electrokinetic chromatography (Sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used, the detection limits could be improved to 3 and 5 ng/ml, respectively. In the analysis of actual samples from animal metabolic-stress experiments (39 mouse), chronically stressed animals showed a higher level (552±152 ng/ml) and acute stressed animals showed an intermediate level (375±105 ng/ml). In comparison, normal animals show a lower concentration level of corticosterone (153±109 ng/ml). In addition, based on a human exercise-stress experiment (seven volunteers), the acute stressed humans (after exercise, 800 m of running) show a higher concentration of 17-hydroxycorticosterone (113±55 ng/ml for males; 128±25 for females) and the non-stressed humans (before exercise) show a lower concentration (63±37 ng/ml for male; 60±20 for female), respectively.
Keywords: Corticosterone; 17-Hydroxycorticosterone;

Quantitative analysis of human salivary glucose by gas chromatography–mass spectrometry by Maria Luisa Di Gioia; Antonella Leggio; Adolfo Le Pera; Angelo Liguori; Anna Napoli; Carlo Siciliano; Giovanni Sindona (355-358).
A reference analytic methodology was developed for the determination of human salivary glucose concentration. The technique involves the glucose derivatization with acetic anhydride and subsequent analysis of glucose penta-acetylated by gas chromatography combined with mass spectrometry. Glucose concentration in the biological fluid depends on the physiological status of the donor.
Keywords: Glucose;

Determination of 3-amino-5-mercapto-1,2,4-triazole in serum by G.J. Depree; P.D. Siegel (359-362).
A high performance liquid chromatography (HPLC) method using fluorescence detection to determine 3-amino-5-mercapto-1,2,4-triazole (AMT) levels in serum has been developed. Sample preparation involved treatment with tributylphosphine (TBP) to reduce disulfides formed during storage, precipitation of proteins with acetonitrile (ACN), and precolumn derivatization using the thiol reactive fluorescent probe monobromobimane (MBB). The conjugate (AMT-MBB) was resolved by gradient elution from a C18 reversed-phase column. The assay method was linear over a concentration range of 0.78–50 μg/ml and had a limit of detection (LOD) of 0.05 μg/ml AMT (10 μl injection). This method provides a sensitive and specific tool for the determination of AMT in serum and may have potential industrial hygiene application.
Keywords: 3-Amino-5-mercapto-1,2,4-triazole;

Naringenin and hesperetin, the aglycones of the flavanone glucosides naringin and hesperidin occur naturally in citrus fruits. They exert a variety of pharmacological effects such as antioxidant, blood lipid-lowering, anticarcinogenic and inhibit selected cytochrome P-450 enzymes resulting in drug interactions. A specific, sensitive, precise, and accurate solid-phase extraction high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of naringenin and hesperetin in human plasma was developed and validated. After addition of 7-ethoxycoumarin as internal standard, plasma samples were incubated with β-glucuronidase/sulphatase, and the analytes were isolated from plasma by solid-phase extraction using C18 cartridges and separated on a C8 reversed phase column with methanol/water/acetic acid (40:58:2, v/v/v) as the eluent at 45 °C. The method was linear in the 10–300 ng/ml concentration range for both naringenin and hesperetin (r>0.999). Recovery for naringenin, hesperetin and internal standard was greater than 76.7%. Intra- and inter-day precision for naringenin ranged from 1.4 to 4.2% and from 1.9 to 5.2%, respectively, and for hesperetin ranged from 1.3 to 4.1% and from 1.7 to 5.1%, respectively. Accuracy was better than 91.5 and 91.3% for naringenin and hesperetin, respectively.
Keywords: Naringenin; Hesperetin;