Journal of Chromatography B (v.805, #2)

News Section (N1-N2).

OFC (OFC).

This study developed an acid hydrolysis method instead of using enzyme extraction, equipped with column-switching system for the pretreatment of samples, in the determination of 1-hydroxypyrene in the urine from children and pyrene in airborne particulates. We collected both types of samples from areas near a petrochemical industry and rural areas as reference. Samples were first treated with acid hydrolysis and followed by solvent extraction prior to being injected into the separation system for the determination with high performance liquid chromatography and fluorescence. A column-switching system was on-line with a C18 separation column to remove matrix interference and obtain a stable baseline of the chromatogram. The eluent used to separate the 1-hydroxypyrene was 60% (v/v) aqueous acetonitrile solution. A fluorescence detector was used to monitor 1-hydroxypyrene at λ ex =348  nm and λ em =388  nm , and pyrene at λ ex =331  nm and λ em =390  nm . Both calibration graphs were linear with very good correlation coefficients (r>0.999) and the detection limits were ca. 2 pg (5 ng/l). Results showed that there was a significant association between 1-hydroxypyrene levels in urine specimens and pyrene levels in airborne particulate samples (r=0.68, P<0.05). The average levels of pyrene in the particulates (0.18 versus 0.09 ng/m3) and of 1-hydroxypyrene in urine specimens (155.9 versus 110.2 ng/g creatinine) were higher for the petrochemical area than for the rural area. This method is stable and sensitive for measuring polycyclic aromatic hydrocarbons in environmental samples.
Keywords: 1-Hydroxypyrene;

Analysis of PEG 400 and 4000 in urine for gut permeability assessment using solid phase extraction and gel permeation chromatography with refractometric detection by S. Loret; G. Nollevaux; R. Remacle; M. Klimek; I. Barakat; P. Deloyer; C. Grandfils; G. Dandrifosse (195-202).
We developed a treatment of urine samples allowing the analysis of two intestinal permeability markers: polyethylene glycol (PEG) 400 (highly diffusible; basal permeability indicator) and PEG 4000 (poorly diffusible; indicator of an abnormal increase of permeability) by a unique gel permeation chromatography (GPC) with refractometric detection. Urinary PEG were extracted using a mixed-bed resin composed of C2 and C18 layers. Permeability mean values determined in 11 human healthy subjects were 24.20±9.30% and 0.12±0.08% for, respectively, PEG 400 and 4000. The percentage of the PEG 4000 permeability value to the one of PEG 400 corresponded to an intestinal permeability index (IPI) of 0.52±0.35 expressing a low diffusion of this poorly permeability marker.
Keywords: Poly(ethylene glycol);

A sensitive, rapid and selective liquid chromatography–positive electrospray ionization tandem mass spectrometry (LC–(ESI+)-MS–MS) method has been developed and validated for the simultaneous quantification of beclomethasone dipropionate (BDP) and its active metabolite, beclomethasone 17-monopropionate (17-BMP) in rat plasma and different tissues using fluticasone propionate (FP) as the internal standard. The method was validated over a linear range from 0.05 to 5 ng/ml for both analytes. A solid-phase extraction procedure was used for plasma samples and a liquid–liquid extraction procedure for tissues samples (lung, liver and kidney). The between-day and within-day coefficients of variation for all compounds were ≤20% at the concentrations of lower limit of quantification (LLOQ) and ≤15% at other quality control concentrations. The same method was also partially validated for human plasma. The utility of this assay was demonstrated by monitoring BDP and 17-BMP plasma and tissue concentrations in an animal study designed for evaluation of pulmonary targeting after intratracheal administration of BDP dry powder.
Keywords: Beclomethasone propionates;

Terbutaline is a β-adrenergic receptor antagonist that acts as a bronchodilator in the treatment of asthma and chronic bronchitis. In the present work, a column-switching high-performance liquid chromatographic method was developed to monitor terbutaline sulphate in dog plasma. The system consists of a C2 pre-column (PC) and a C18 analytical column connected in series via a switching valve. Atenolol was used as the internal standard. Good linearity was achieved in the range of 5–800 ng/ml plasma. The mean intra- and inter-assay variation coefficients for this analysis were 2.3 and 4.7%, respectively. The average recovery for terbutaline was 87.4% from plasma. The mean concentration after three freeze-thaw cycles was 99.4% of the normal value. The analytical sensitivity and accuracy of this assay is adequate for characterisation of the pharmacokinetics of oral administration of terbutaline to dogs and has been successfully used to provide pharmacokinetic data using pulsatile and immediate-release tablets.
Keywords: Terbutaline;

In this study immunosuppressants, i.e. cyclosporin A (CyA), tacrolimus (TRL), sirolimus (SRL) and everolimus (RAD) were quantified in whole blood samples from immunosuppressant treated transplant recipients by an integrated on-line solid phase extraction–high performance liquid chromatography–tandem mass spectrometry (SPE–HPLC–MS/MS) system. This method has been developed to improve the following characteristics: speed, robust analysis, simultaneous determination and low cost. This can be achieved by the use of a perfusion column as an extraction cartridge in combination with a short HPLC column and highly selective and sensitive atmospheric pressure ionisation tandem mass spectrometry (API–MS/MS) in the multiple reaction monitoring (MRM) detection mode. This high throughput technique is perfectly appropriate for routine therapeutic drug monitoring (TDM) of organ transplanted patients.
Keywords: Cyclosporin A; Tacrolimus; Sirolimus; Everolimus;

Toxicological examination of fatal aviation accident victims routinely includes analysis of ethanol levels. However, distinguishing between antemortem ingestion and postmortem microbial formation complicates all positive ethanol results. Development of a single analytical approach to determine concentrations of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA), two well-known metabolites of serotonin, has provided a convenient, rapid and reliable solution to this problem. Antemortem ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio for 11–19 h after acute ingestion. The liquid–liquid extracts of postmortem urine samples were subjected to liquid chromatography–mass spectrometry (LC–MS) for the simultaneous quantitation of these two analytes, yielding detection limits of 0.1 ng/ml for each. Examination of the 5-HTOL/5-HIAA ratio was undertaken for 44 urine samples known to be antemortem ethanol-positive or antemortem ethanol-negative. Recent ethanol ingestion was conveniently and accurately separated using a 5-HTOL/5-HIAA ratio of 15 pmol/nmol, a value previously suggested using human volunteers. All 21 ethanol-negative postmortem samples were below this cutoff, while all 23 ethanol-positive postmortem samples were above this cutoff. Thus, we recommend the employment of this cutoff value, established using this straightforward LC–MS procedure, to confirm or deny recent antemortem ethanol ingestion in postmortem urine samples.
Keywords: Postmortem urine; Ethanol; Serotonin;

Acetone is an important volatile disease marker. Due to its nature of activity and volatility, it is a difficult task to measure the concentration of acetone in biological samples with accuracy. In this paper, we developed a novel method for determination of trace amount acetone in human plasma by solid-phase microextraction technique with on-fiber derivatization. In this method, the poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber was used and O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) was first loaded on the fiber. Acetone in plasma sample was agitated into headspace and extracted by solid-phase microextraction (SPME) fiber and subsequently derivatized with PFBHA on the fiber. Acetone oxime was analyzed by gas chromatography–mass spectrometry (GC–MS). Quantitative analysis of acetone in plasma was carried out by using external standard method. The SPME conditions (extraction temperature and time) and the method validation were studied. The present method was tested by determination of acetone in diabetes plasma and normal plasma. Acetone concentration in diabetes plasma was found to be higher than 1.8 mM, while in normal plasma was lower than 0.017 mM. The results show that the present method is a potential tool for diagnosis of diabetes.
Keywords: Derivatization; GC; Acetone;

An accurate, sensitive, and specific reverse-phase high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of HIV-protease inhibitors (PIs) (indinavir, IDV; amprenavir, APV; saquinavir, SQV; nelfinavir, NFV; ritonavir, RTV; and lopinavir, LPV) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) (nevirapine, NVP; delavirdine, DLV; and efavirenz, EFV) in human blood plasma is described. The method provides excellent resolution and peak shape for nine analytes through a linear gradient (36–86%) of 25% phosphate buffer (pH 4.5), 60% acetonitrile, 15% methanol, and 0.75 ml TFA, with a gradient mobile phase flow rate (0.9–1.1 ml) over 30 min run time. The optimized solid phase extraction (SPE) extraction method using (1.0 ml, 100 mg BOND ELUT-C18 Varian) column provides a clean base line and high extraction efficiency using a 550 μl plasma sample. The method was validated over the range of 10–10,000 ng/ml for NVP, IDV, and SQV; 10–5000 ng/ml for EFV; 25–10000 ng/ml for APV; and 25–5000 ng/ml for DLV, NFV, RTV, and LPV. This method is accurate (average accuracies of three different concentrations ranged from 91 to 112%), and precise (within- and between-day precision measures ranged from 0.2 to 5.7% and 0.1 to 5.4%, respectively). This method is suitable for use in clinical pharmacokinetic studies as well as in therapeutic drug monitoring (TDM).
Keywords: HIV protease inhibitors; Non-nucleoside reverse transcriptase inhibitors;

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (λ=224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 μg/ml for each enantiomer of esmolol and 0.07–8.0 μg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.
Keywords: Enantiomer separation; Esmolol;

The method for separation and determination of dexamethasone sodium phosphate (DexP) in cochlear perilymph fluid (CPF) of cavy was developed using HPLC with ultraviolet (UV) monitoring and electrospray ionization/mass spectrometry (ESI/MS) identification. The quantitative determination of DexP in CPF was achieved by HPLC with UV detection at 245 nm. The separation was carried out on a Phenomenex ODS(3) column (250  mm×4.6  mm i.d., 5 μm) with the mobile phase of acetonitrile–5 mmol/l ammonium acetate (23:77 (v/v)) at a flow rate of 1.0 ml/min. DexP was baseline separated from the matrices of CPF blanks within 15 min. The linearity ranged from 0.5 to 50 μg/ml. The limit of detection was 0.10 μg/ml. The recovery ranged from 98.5 to 100.8%. The relative standard deviations (R.S.D.s) of intra- and inter-day peak area were between 0.7–1.3 and 1.2–3.5%, respectively. Both full scan MS and MS2 of DexP with positive and negative polarity were obtained and elucidated. The specific ions were chosen to characterize DexP in the CPF sample. Using the proposed HPLC-UV-ESI/MS method, the concentration of DexP in CPF samples after both vein and middle ear injections were determined, and the relationships between concentration and time were obtained. This method offered reference data for clinical investigation of DexP to cure ear diseases.
Keywords: Dexamethasone sodium phosphate;

Determination of A 3,4-diaminopyridine in plasma by liquid chromatography with electrochemical detection using solid-phase extraction by S Goulay-Dufaÿ; B Do; M.D Le Hoang; J.A Raust; H Graffard; F Guyon; D Pradeau (261-266).
In order to quantify a small amount of a drug, 3,4-diaminopyridine (3,4-DAP), in animal plasma samples, an analytical method was developed. It involved an extraction of 3,4-DAP and phenylephrine, used as internal standard (IS), from plasma with solid-phase extraction (SPE) on C18 cartridges. This analytical method is a hyphenated technique based on high-performance liquid chromatography with electrochemical detection (HPLC–EC) whose purpose is to obtain first a sensitive method and second a satisfying separation between 3,4-DAP and phenylephrine. The analytical method is accurate, specific, and linear between 10 and 500 μg of 3,4-DAP per litre. The recovery of 3,4-DAP is estimated at 70.8% with a 95% confidence interval of (66.0–75.6%). Intermediate precision was evaluated on three quality control samples; the intra-day precision was estimated at 13.5, 9.1, 7.8% and the inter-day precision at 17.9, 8.4, 9.3%. The limit of quantification of the method was evaluated at 10 μg l−1. First toxicokinetic parameters determined on dogs plasma samples after one 3,4-DAP oral administration of 1 mg kg−1 were: C max=395.7 μg l−1; T max=15 min; t 1/2=113.6 min; Clearance/F=16.8 ml kg−1  min−1 and Vd/F=2.7 l kg−1.
Keywords: 3,4-Diaminopyridine;

A rapid and sensitive HPLC method for the simultaneous quantification of albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in sheep blood plasma has been developed. Plasma samples were extracted with ethyl acetate under alkaline conditions. Separation was achieved on a C18 reversed-phase analytical column, in the presence of positively- (tetra-n-butylammonium hydrogen sulphate) and negatively-charged (octanesulphonate sodium) pairing ions, while detection was performed fluorometrically. Excitation and emission wavelengths were 290 and 320 nm, respectively. Limits of quantification were defined at 39 ng/ml for ABZ-SO, 4.95 ng/ml for ABZ-SO2 and 4 ng/ml for ABZ-SO2NH2. Accuracy data, in terms of recovery efficiency showed overall values (±S.E.M.) of 85.6±1.0% for ABZ-SO, 100.0±1.0% for ABZ-SO2 and 89.1±0.6% for ABZ-SO2NH2. The method was successfully applied to quantitatively determine the three albendazole metabolites in plasma samples collected from sheep that had been orally administered albendazole.
Keywords: Albendazole sulphoxide; Albendazole sulphone; Albendazole 2-aminosulphone;

A new method for the determination of tranexamic acid (TA) in human plasma using high performance liquid chromatography with tandem mass spectrometric detection was described. TA and the internal standard, methyldopa, was extracted from a 200 μl plasma sample by a one-step deproteination using perchloric acid. Chromatographic separation was performed on an Xtrra™ MS C18 Column (2.1  mm×100  mm, 3.5 μm) with the mobile phase consisting of 10% acetonitrile in 2 mM ammonium acetate buffer (pH 3.5) at a flow rate of 0.15 ml/min. The total run time was 5 min for each sample. Detection and quantitation was performed by the mass spectrometer using the multiple reaction monitoring of the precursor-product ion pair m/z 158 → 95 for TA and m/z 212 → 166 for methyldopa, respectively. The method was linear over the concentration range of 0.02–10.00 μg/ml with lower limit of quantification of 0.02 μg/ml for TA. The intra- and inter-day precision was less than 11% and accuracy ranged –10.88 to 11.35% at the TA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around time. The method has been successfully applied to a clinical pharmacokinetic study of TA in 12 healthy subjects.
Keywords: Tranexamic acid;

A method for simultaneous determination of polyamines and catecholamines in cell extracts by micellar electrokinetic capillary chromatography with UV detection at 254 nm was established at the first time. The polyamines (putrescine, spermidine and spermine) and catecholamines (dopamine, serotonin, norepinephrine and epinephrine) were extracted from PC-12 cells and were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Different derivatization conditions such as temperature, ratio of derivatization reagents and incubation time were investigated to find the best reaction condition which gave the highest detection sensitivity for polyamines and catecholamines. The influence of running buffer and additives on the separation such as pH, sodium dodecyl sulfate (SDS) concentrations and various additives was also investigated. Separation was achieved within 20 min with good repeatability in a 100 mM boric acid buffer containing 10 mM SDS and 10 mM 18-crown-6 at a pH of 9.5. The detection limit ranged from 1.0×10−7 to 9.0×10−7  M, which is sufficient for determination of polyamines and catecholamines in many cell extracts. This technique can be easily applied to polyamine-related anticancer drug studies or clinical follow-ups after each dosage of these anticancer drugs, since these drugs not only have great inhibition on polyamine levels in blood, but also have a large influence on catecholamine levels in blood.
Keywords: PC-12 cell extracts; Catecholamines; Polyamines;

In the present study, a simple and rapid reversed-phase HPLC procedure has been developed for the simultaneous determination of eight fat-soluble vitamins (retinol, menadione, menaquinone, δ-tocopherol, cholecalciferol, α-tocopherol, α-tocopherol acetate and phylloquinone) in biological fluids: blood serum and urine. The analytical column, Phenomenex Luna C18 (150  mm×4.6  mm) 3 μm, was operating at ambient temperature. Mobile phase consisted of a mixture of CH3OH–CH3CN delivered using a linear gradient, starting with a composition of 50–50% v/v and ending at 30–70% at a flow rate of 1.3 ml/min. Xanthophyll was used as internal standard (2 ng/μl). Detection and identification was performed using a photodiode array detector. Eluent monitoring was achieved at 280 nm for vitamins and 450 nm for the internal standard. However, quantitation was performed at maximum wavelength for each vitamin. Detection limits were found in the range of 1.4–6.6 ng per 20-μl injected samples, while linearity held up to 25 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. The biological fluids were treated using solid-phase extraction cartridges, to remove all endogenous interferences from sample matrix. The solid-phase extraction protocol was optimized in terms of retention and elution. High extraction recoveries from biological matrices: blood serum and urine, (average recovery ranging between 95 and 97.6% for blood serum and between 94.2 and 95.8% for urine) were achieved for the eight fat-soluble vitamins, using Cyclohexyl J.T. Baker SPE cartridges with methanol as eluent, requiring small volumes, 100 μl of blood serum and 100 μl of urine.
Keywords: Vitamins;

Analysis of coenzyme Q10 in human plasma by column-switching liquid chromatography by Ping Jiang; Meihui Wu; Yufang Zheng; Chang Wang; Yonghang Li; Jian Xin; Guowang Xu (297-301).
A new method of determining coenzyme Q10 in human plasma was developed based on column-switching high performance liquid chromatography (HPLC). CoQ10 was quantitatively extracted into 1-propanol with a fast one-step extraction procedure, after centrifugation, the supernatant was cleaned on an octadecyl-bonded silica column and then transferred to reversed-phase column by a column-switching valve. Determination of CoQ10 was performed on a reversed-phase analytical column with ultraviolet detection at 275 nm and the mobile phase containing 10% (v/v) isopropanol in methanol at a flow-rate of 1.5 ml/min. The sensitivity of this method allows the detection of 0.1 μg/ml CoQ10 in plasma (S/N=3). The linearity between the concentration and peak height is from 0.05 to 20 mg/l. The reproducibility (R.S.D.%) of the method is less than 2% (within day) and less than 3% (between day), the average recovery is 100.9+2.1%, it takes only 30 min to complete an analysis procedure, suitable for the determination of CoQ10 in human plasma especially for batch analysis in clinical laboratories. Finally, the method was applied to determine the plasma CoQ10 levels in healthy subjects, hyperthyroid and hypothyroid patients.
Keywords: Coenzyme Q10;

A relatively rapid and specific method for the determination of chloralose in animal tissues by LCMSMS was developed. Isocratic reverse phase HPLC was used to introduce samples for electrospray negative ionisation tandem mass spectrometry. Methanol extracts were diluted to approximate the mobile phase composition, then filtered prior to analysis. Residues were identified by monitoring the multiple reaction monitoring (MRM) transitions of precursor ions mass:charge (m/z) 309 and 307 to a common m/z 161 product ion. Qualitative and quantitative confirmation data were acquired simultaneously by monitoring alternative MRM transitions. Calibration was linear over a working range of 0.025–1.3 μg/ml, and the limit of quantitation (LOQ) was 0.28 mg/kg for liver. The mean recovery was 88.5% from chicken muscle tissue fortified at 198–237 mg/kg, and ranged from 81.3 to 94.3% from liver tissue fortified at 1–52 mg/kg. The method is compared to a gas chromatography (GC) procedure previously employed.
Keywords: Chloralose;

Determination of benidipine in human plasma using liquid chromatography–tandem mass spectrometry by Wonku Kang; Hwi-Yeol Yun; Kwang-Hyeon Liu; Kwang-il Kwon; Jae-Gook Shin (311-314).
We developed a method for determining benidipine, a dihydropyridine analogue calcium-channel blocker, in plasma using liquid chromatography–tandem mass spectrometry (LC–MS–MS). Benidipine and benidipine-d5, an internal standard, were extracted from plasma using diethyl ether in the presence of 5 M NaOH. After drying the organic layer, the residue was reconstituted in acetonitrile and injected onto a reversed-phase C18 column. The isocratic mobile phase (acetonitrile–5 mM ammonium acetate, 90:10, v/v) was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 506–174 for benidipine and m/z 511–179 for the internal standard. The coefficient of variation of the assay precision was less than 13%, and the accuracy exceeded 92%, except at the limit of quantification, 0.05 ng/ml with 1 ml of plasma, when it was 85%. This method was used to measure the benidipine concentration in plasma from healthy subjects after a single 4-mg oral dose of benidipine. This method is a very simple, sensitive, and accurate way to determine the plasma benidipine concentration.
Keywords: Benidipine;

Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption–elution cycles.
Keywords: Surface polarity; Lysozymes; Dye-ligands;

A new HPLC assay for the detection of l-arginine, N G,N G-dimethyl-l-arginine (ADMA) and N G,N G′-dimethyl-l-arginine (SDMA) in plasma using the derivatisation reagent AccQ-Fluor™ (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) is described. The fluorescent derivatives produced are extremely stable enabling routine processing of large numbers of samples. Arginine and its metabolites are extracted from plasma on strong cation exchange (SCX) cartridges with N G-monomethyl-l-arginine (NMMA) as internal standard, derivatised and separated on a C18 column with acetonitrile in 0.1 M sodium acetate buffer pH 6. Separation of the stereoisomers ADMA and SDMA was excellent and improvements to the solid phase extraction (SPE) procedure enabled good recovery (>80%) of arginine, ADMA and SDMA. The utility of the method is exemplified by comparison of plasma concentrations of ADMA, SDMA and arginine in healthy volunteers and diabetic/ischaemic patients.
Keywords: Derivatisation, LC; l-Arginine; Dimethyl-l-arginine;

A simple method for the separation of the four major neutral glycosphingolipids, present in all human tissue, was developed. This gradient normal phase-HPLC method utilises a polyvinyl alcohol bonded stationary phase and an evaporative light-scattering detection (ELSD). Screening pure solvents in a binary gradient elution mode allowed, in a first step, to assess the behaviour of the studied solutes and to select the solvents for further mobile phase optimisation. The proportion of the remaining solvents was defined to reach a maximal resolution. The reduction of the analysis time and the enhancement of the signal were obtained by optimising the gradient slope and the flow-rate. Optimal levels of triethylamine and formic acid (TEA-FA) for the enhancement of the evaporative light scattering detector response were established at 0.1% (v/v). Thus, the optimal conditions for the separation of the four glycosphingolipids was obtained with a gradient elution from a 100% chloroform to a 100% acetone:methanol (90:10 (v/v)) mobile phase at 0.2 ml min−1, using a 10% min−1 gradient slope. Finally, this method was applied to detect the excess of one of the neutral sphingolipids, namely globotriaosylceramide (Gb3) in the urine of patients affected with Fabry disease. A liquid–liquid extraction of the sediments obtained from an aliquot of only ten ml of urine proved sufficient to detect the excess of Gb3 present in both hemizygote and heterozygote patients. In all, the ability of our method to detect abnormal amounts of Gb3 in urinary sediments could allow the diagnosis of weakly symptomatic Fabry patients in large screening programs
Keywords: Glycosphingolipids; Globotriaosylceramide;

A non-radioactive procedure to measure the deoxycytidine kinase (dCK) activity in crude cell free homogenates was developed. 2-Chlorodeoxyadenosine (CdA) was used as the substrate for dCK and was separated from its product 2-chlorodeoxyadenosine-5′-monophosphate (CdAMP) by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 30 min. The minimum amount of CdAMP that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3 h. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 μg/ml in the assay. An amount of 8×103 cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. CdA was not only converted to CdAMP but also to 2-chloroadenine and, surprisingly, also to 2-chlorodeoxyinosine, in MOLT-3 cells. The deamination of CdA was completely inhibited by deoxycoformycin, which clearly demonstrates that CdA is a substrate for adenosine deaminase.
Keywords: Deoxycytidine kinase; Enzymes; 2-Chlorodeoxyadenosine;

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of d-(−) and l-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and dl-(±)-lactic acids in calf feces and dl-(±)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 μm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-l-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2–14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for d- and l-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for dl-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.
Keywords: Enantiomer separation; Lactic acid; Pyruvic acid; Acetic acid;

Liquid chromatographic assay for dicloxacillin in plasma by Oscar Alderete; Dinora F González-Esquivel; L Misael Del Rivero; Nelly Castro Torres (353-356).
A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH=4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 μm) column, using methanol–0.05 M phosphate buffer, pH=4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 μg/ml. The response is linear in the range of 0.5–10 μg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at −20 °C for 2 months, processed samples were stable at least for 24 h and also after two freeze–thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans.
Keywords: Dicloxacillin;

A simple method for determining the concentration of astilbin, a flavanone, in rabbit plasma has been developed. After liquid–liquid extraction, the flavanone was detected by HPLC on a 4.6-μm octadecylsilica column (Nova-Pak C-18) at 291 nm. Linear calibration graphs for astilbin were constructed from 0.44 to 22.17 μM. The limit of quantitation was 0.44 μM in plasma. The method has been applied to pharmacokinetic studies after a single i.v. and an oral administration of the compound to rabbits.
Keywords: Astilbin;

Combinatorial phage display was used to discover peptides that selectively bind to the α-cobratoxin (neurotoxin) component of the multi-component venom of the Thai cobra, Naja kaouthia. Peptide sequences determined in this way were synthesized chemically and were covalently attached to agarose through the α-amino terminus. Such affinity chromatography supports selectively bound the α-cobratoxin component from crude venom, while passage of the crude venom over the support selectively depleted the venom of this component. The selective binding of α-cobratoxin to peptide-based solid-phase supports suggests that a limitless variety of peptides similarly obtained by combinatorial phage display can be used to craft specific analytical and preparative tools.
Keywords: α-Cobratoxin; Combinatorial phage display peptides;

Determination of paraldehyde by gas chromatography in whole blood from children by Isaiah M. Githiga; Simon N. Muchohi; Bernhards R. Ogutu; Charles R.J.C. Newton; Godfrey O. Otieno; Evelyn N. Gitau; Gilbert O. Kokwaro (365-369).
A rapid, sensitive and selective gas chromatographic method with flame ionization detection was developed for the determination of paraldehyde in small blood samples taken from children. Whole blood samples (300 μl) collected in a 3 ml Wheaton® glass sample vial were spiked with acetone (internal standard: 15 ng) followed by addition of concentrated hydrochloric acid. The mixture was heated in the sealed airtight sample vial in a water bath (96 °C; 5 min) to depolymerize paraldehyde to acetaldehyde. A 2 ml aliquot of the headspace was analyzed by gas chromatography with flame ionization detector using a stainless steel column (3  m×4  mm i.d.) packed with 10% Carbowax® 20M/2% KOH on 80/100 Chromosorb® WAW. Calibration curves were linear from 1.0–20 μg (r 2>0.99). The limit of detection was 1.5 μg/ml, while relative mean recoveries at 2 and 18 μg were 105.6±8.4 and 101.2±5.9%, respectively (n=10 for each level). Intra- and inter-assay relative standard deviations at 2, 10 and 18 μg were <15%. There was no interference from other drugs concurrently used in children with severe malaria, such as anticonvulsants (diazepam, phenytoin, phenobarbitone), antipyretics/analgesics (paracetamol and salicylate), antibiotics (gentamicin, chloramphenicol, benzyl penicillin) and antimalarials (chloroquine, quinine, proguanil, cycloguanil, pyrimethamine and sulfadoxine). The method was successfully applied for pharmacokinetic studies of paraldehyde in children with convulsions associated with severe malaria.
Keywords: Paraldehyde;