Journal of Chromatography B (v.772, #1)

News Section (N1-N2).

Advanced fibre optical scanning in thin-layer chromatography for drug identification by Björn Ahrens; Dirk Blankenhorn; Bernd Spangenberg (11-18).
A systematic toxicological analysis procedure using high-performance thin layer chromatography in combination with fibre optical scanning densitometry for identification of drugs in biological samples is presented. Two examples illustrate the practicability of the technique. First, the identification of a multiple intake of analgesics: codeine, propyphenazone, tramadol, flupirtine and lidocaine, and second, the detection of the sedative diphenhydramine. In both cases, authentic urine specimens were used. The identifications were carried out by an automatic measurement and computer-based comparison of in situ UV spectra with data from a compiled library of reference spectra using the cross-correlation function. The technique allowed a parallel recording of chromatograms and in situ UV spectra in the range of 197–612 nm. Unlike the conventional densitometry, a dependency of UV spectra by concentration of substance in a range of 250–1000 ng/spot was not observed.

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2±0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm×75 μm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 μmol/l (median 6.71 μmol/l). The method was linear within the 50–200 μmol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.
Keywords: Amino acids; Peptides;

This paper reports on the selectivity behaviour of tryptic peptides on a Cu2+-loaded immobilised metal ion affinity chromatography (IMAC) support. Ovalbumin was chosen as a model protein for investigation of the selection and separation of histidine-containing peptides by IMAC off-line coupled with capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI–TOF). Two of five histidine-containing peptides in addition to some non-histidine-containing peptides from a tryptic digest of ovalbumin were captured by IMAC. To separate and purify the selected peptides, the IMAC sample was analysed by capillary zone electrophoresis (CZE). The sample was not separated by capillary zone electrophoresis, therefore, micellar electrokinetic chromatography (MEKC) using 10–75 mM SDS was used. Analysis of IMAC sample by MEKC, using low concentrations of SDS (10 mM) was characterised by MALDI–TOF. When using SDS at 75 mM, the migration times of reversed-phase fractions of the IMAC sample, were used to identify the peaks. One of the two selected histidine-containing peptides with two histidine residues was identified, analysing the sample by CZE or MEKC.
Keywords: Peptides; Histidine;

A new and in part automated headspace solid-phase microextraction method for quantitative determination of the highly toxic rodenticide fluoroacetic acid (FAA) in serum and other biological samples has been developed. FAA and deuterated acetic acid (internal standard) were extracted from acidified samples by a StableFlex divinylbenzene–Carboxen on polydimethylsiloxane fibre. The acids were derivatised on the fibre in-situ with 1-pyrenyldiazomethane and detected using gas chromatography–mass spectrometry with electron impact ionisation and selected ion monitoring. The calibration curve for FAA in serum was linear over the range from 0.02 to 5 μg/ml, with limits of detection and quantification of 0.02 and 0.07 μg/ml, respectively. The method was also tested with spiked whole blood, urine, stomach contents and kidney samples. It was sufficiently reliable, reproducible and sensitive for use in routine forensic toxicology applications.
Keywords: Fluoroacetic acid; 1-Pyrenyldiazomethane;

A selective, sensitive and accurate liquid chromatographic method with UV and fluorescence detection was developed, validated and applied for the determination of fluoroquinolones in human plasma. The effects of mobile phase composition, ion-pair and competing-base reagents, buffers, pH, and acetonitrile concentrations were investigated on the separation of six quinolones (cinoxacin, levofloxacin, ciprofloxacin, gatifloxacin, moxifloxacin and trovafloxacin). Sample preparation was carried out by adding internal standard and displacing agent and processing by ultrafiltration. This method uses ultraviolet and fluorescence detection and separation using a C18 column. The recovery, selectivity, linearity, precision, and accuracy of the method were evaluated from spiked human plasma samples. The method was successfully applied to patient plasma samples in support of a levofloxacin pharmacokinetic study.
Keywords: Levofloxacin; Ciprofloxacin; Gatifloxacin; Moxifloxacin; Trovafloxacin; Cinoxacin;

Determination of grepafloxacin and clinafloxacin by capillary zone electrophoresis by Alberto Navalón; Lilia Araujo; Avismelsi Prieto; José Luis Vı́lchez (65-72).
A simple and rapid capillary zone electrophoresis determination method with UV detection of grepafloxacin and clinafloxacin has been developed. The separation was performed in 35 mM borate–35 mM phosphate buffer solution (pH 8.6), containing 6% (v/v) of acetonitrile. Analyses were realised using fused-silica capillaries (57 cm length×75 μm I.D.) and the operating conditions were: 15 kV applied voltage, 30 °C and detection at 279 nm. Piromidic acid was used as an internal standard. The linear concentration range of application was 1.0–120.0 μg ml−1 for both compounds, with a detection limit of 0.2 μg ml−1 for grepafloxacin and 0.3 μg ml−1 for clinafloxacin. The analysis yielded good reproducibility (RSD between 3.37 and 1.74%). It was applied to the determination of grepafloxacin and clinafloxacin in human and rat urine samples. The method was validated using HPLC as a reference method. Recovery levels were between 94.5 and 103%.
Keywords: Grepafloxacin; Clinafloxacin;

An atmospheric pressure ionization liquid chromatographic–mass spectrometric assay was developed and validated for the determination of d-penicillamine2,5 enkephalin (DPDPE) in cerebrospinal fluid (CSF) from dog. DPDPE and internal standard (d-Ala2,d-Leu5 enkephalin=DADLE) were isolated from CSF by reversed-phase C18 solid-phase extraction with ZipTip micro-cartridges. Aliquots of extracted eluate were injected onto an Agilent Zorbax SB C18 column (30×2.2 mm; 3.5 μm) at a flow-rate of 0.4 ml/min. The isocratic mobile phase of methanol–10 mM ammonium formate (pH 3) (75:25, v/v) was then diverted to waste for 45 s after injection, after which time flow was directed to the single quadrupole mass spectrometer. DPDPE was detected by positive mode selected ion monitoring. Standard curves were linear (r 2≥0.991) over the concentration range 1–1000 ng/ml. The efficiency of extraction recovery was greater than 97%, and the intra-assay and inter-assay precisions were within 9% relative standard deviation. DPDPE and the internal standard were stable in the injection solvent at 4 °C for at least 48 h. The assay was applied to the pharmacokinetic study of intrathecal DPDPE administration in the dog animal model.
Keywords: d-Penicillamine-enkephalin (DPDPE); d-Ala; d-Leu-enkephalin (DADLE);

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight β-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 μM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 μM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035–0.065 pmol per 50 μl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight β-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic β-blockers through the corneal epithelial cell culture model.
Keywords: β-Blockers;

A generic isocratic HPLC–APCI-MS–MS method has been developed for the determination of plasma concentrations of bioactive compounds for the selection of potential new drug discovery candidates. A 4.6×50 mm cyano phase column eluted with an acetonitrile/water mobile phase containing 20 mM ammonium acetate and 0.4% TFA produces retention times of 1 min or less for a wide range of compounds. This is a great advantage in new drug discovery where many compounds are analyzed once and eliminated. No time is consumed developing chromatographic conditions for each new compound. The mass spectrometer can be optimized and the samples can be processed and analyzed, all in the same day. Multiple assays can be run consecutively without changing the column or mobile phase between assays.
Keywords: Drug discovery candidates;

A sensitive and automated method has been developed and validated to determine marker residue eprinomectin B1a in bovine milk. Extraction of eprinomectin B1a from milk is accomplished with acetonitrile after the addition of an internal standard. The extract containing the analytes is evaporated to dryness and reconstituted in a solution containing 30% 1-N-methylimidazole in acetonitrile. Online derivatization is carried out with trifluoroacetic anhydride. Determination of eprinomectin B1a and its internal standard is carried out by HPLC using a reversed-phase C18 column with a mobile phase consisting of methanol, acetonitrile, water, triethylamine and phosphoric acid. The overall extraction recovery of eprinomectin B1a is 94% with milk supplemented between 2 and 50 ng/ml eprinomectin B1a. Precision RSD averaged 3.0% in Laboratory 1 (n=25) compared to 4.3% in Laboratory 2 (n=35). The limit of quantitation is approximately 2 ng/ml eprinomectin B1a, the limit of detection is approximately 0.25 ng/ml using this method.
Keywords: Avermectin analogue; Eprinomectin;

All the available HPLC assays of cyclosporin A (CyA) use internal standards that are not commercially available. Our purpose was to develop an HPLC assay for measurements of CyA in rat blood and liver using a commercially available internal standard (I.S.). After the addition of tamoxifen (I.S.), blood (0.25 ml) or the liver homogenate (1 ml) samples were extracted into a mixture of ether:methanol (95:5). The residue after evaporation of the organic layer was dissolved in 200 μl of an injection solution and washed with 1 ml of hexane before analysis. The separation was achieved using an LC-1 column (70 °C) with a mobile phase of methanol–acetonitrile–0.01 M KH2PO4 (50:25:25, v/v) and a flow-rate of 1 ml/min. Detection was at 205 nm. Cyclosporin A and I.S. eluted at 5 and 7 min, respectively, free from endogenous peaks. Linear relationships (r>0.98) were observed between the CyA:I.S. peak area ratios and the CyA concentrations within the range of 0.2–10 μg/ml for blood and 0.1–4 μg/ml for the liver homogenates. The intra- and inter-run C.V.s and errors for both the blood and liver samples were <15%. The extraction efficiency (n=5) was close to 100% for both CyA and I.S. in both blood and liver homogenates. The lower limit of quantitation of the assay was 0.2 or 0.1 μg/ml based on 250 μl of blood or 1 ml of liver homogenate, respectively. The assay was capable of measuring blood and liver concentrations of CyA in a rat injected intravenously with a single 5-mg/kg dose of the drug.
Keywords: Cyclosporin A;

Development and validation of a liquid chromatographic method for Casiopeina IIIi® in rat plasma by Inés Fuentes-Noriega; Lena Ruiz-Ramı́rez; Araceli Tovar Tovar; Héctor Rico-Morales; Isabel Gracia-Mora (115-121).
A sensitive and specific liquid chromatographic method using extraction with zinc sulfate has been developed for the determination of Casiopeina IIIi and validated over the linear range 5–100 μg/ml in 1 ml of rat plasma. The analysis was performed on a Symmetry C18 (5 μm) column. The mobile phase was methanol: 0.01 M phosphate buffer pH 6.5 (40:60, v/v). The column effluent was monitored at 262 nm. The results showed that the assay is sensitive at 5 μg/ml. Maximum intra-day coefficient of variation was 10.6%. The recovery obtained in plasma was 87.2%. The method was used to perform protein binding studies by equilibrium dialysis in rat plasma and was found to be satisfactory.
Keywords: Casiopeina IIIi;

An HPLC system using a simple liquid–liquid extraction and HPLC with UV detection has been validated to determine tramadol concentration in human plasma. The method developed was selective and linear for concentrations ranging from 10 to 2000 ng/ml with average recovery of 98.63%. The limit of quantitation (LOQ) was 10 ng/ml and the percentage recovery of the internal standard phenacetin was 76.51%. The intra-day accuracy ranged from 87.55 to 105.99% and the inter-day accuracy, 93.44 to 98.43% for tramadol. Good precision (5.32 and 6.67% for intra- and inter-day, respectively) was obtained at LOQ. The method has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.
Keywords: Tramadol;

Cyanide (CN), a chemical asphyxiant, is a rapidly acting and powerful poison. We have developed a sensitive, rapid, simple, and fully automated method for measuring CN in whole blood. The assay is based on the use of gas chromatography (GC) with nitrogen–phosphorus detection and acetonitrile as an internal reference. Following the automated addition of phosphoric acid to the blood sample, the released hydrogen cyanide is analyzed using a fully automated headspace GC system. The assay, validated on human blood samples spiked with potassium cyanide and on clinical samples from fire victims who had smoke inhalation injury, can detect CN at a wide range of concentrations (30–6000 μg/l) in about 17 min (including incubation and GC run time, and <2 min for manual sample preparation). This automated, high-throughput, simple, and sensitive method is suitable for the rapid diagnosis of CN in clinical and forensic specimens.
Keywords: Cyanide;

Serum albumin is a mixture of mercaptalbumin (reduced form) and non-mercaptalbumin (oxidized form), i.e. a protein redox couple in serum. To investigate dynamic changes in the redox state of rat serum albumin (RSA), we developed a simple and sensitive high-performance liquid chromatographic (HPLC) system using an ion-exchange column with a linear gradient of ethanol concentration. Furthermore, we applied this HPLC system to examine dynamic changes in the redox state of RSA caused by severe oxidative stress such as exhaustive physical exercise. Using this system, we successfully separated RSA to rat mercaptalbumin (MAr) and rat non-mercaptalbumin (NAr), and also found the best conditions for the clear separation of RSA. In the experiments with exhaustive exercise, mean values for the MAr fraction in control and exercise groups were 76.2±1.8 and 69.0±3.5%, respectively. The MAr in the exercise group was significantly oxidized compared with that of the control group (P<0.01). These results suggested that RSA might act as one of the major scavengers in extracellular fluids under severe oxidative stress.
Keywords: Rat serum albumin;

Lewisite [dichloro(2-chlorovinyl)arsine] is a highly toxic chemical warfare agent with vesicant properties. The accidental exposure to lewisite or its intentional use as a chemical terrorism weapon are a public health threat and warrant investigations for the development of analytical methods to detect biomarkers of exposure to lewisite. Under aqueous conditions, lewisite rapidly hydrolyzes to the non-volatile 2-chlorovinylarsonous acid (CVAA). We have developed a sensitive, simple, and automated method for measuring CVAA in human urine. The assay is based on the use of solid-phase microextraction (SPME) and gas chromatography–mass spectrometry (GC–MS) after derivatization of the CVAA with 1,3-propanedithiol (PDT). The volatile CVAA-PDT is adsorbed onto a SPME fiber and analyzed by GC–MS. The assay was validated on human urine samples spiked with CVAA to determine the accuracy, precision, and limit of detection (LOD). The LOD was 7.4 pg in 1 ml of urine.
Keywords: 2-Chlorovinylarsonous acid;

The α3β4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) subtype was immobilized on a liquid chromatographic support and the resulting column used for the rapid and direct on-line screening for nAChR ligands. A multidimensional chromatographic system was developed consisting of the immobilized receptor column (NR column) connected via a switching valve to a C18 column that was, in turn, connected to a single quadrupole mass spectrometer. A mixture of 18 compounds, containing α3β4 nAChR (7) and compounds that are not α3β4 nAChR ligands (11), was injected onto the NR column. The mobile phase consisted of ammonium acetate (10 mM, pH 7.4)–methanol (95:5, v/v) and the flow-rate was 0.2 ml/min. For the first 8 min the eluent was directed to waste. At t=8 min, the switching valve was rotated and the NR column connected to the C18 column. The eluent from the NR column was directed to the C18 column for 12 min. At t=20 min, the switching valve was rotated and the NR column was disconnected from the C18 column. The compounds trapped on the C18 column were separated and eluted onto the mass spectrometer using a mobile phase of ammonium acetate (10 mM, pH 7.4)–methanol (40:60, v/v) at a flow-rate of 1.0 ml/min. Detection was accomplished using total ion monitoring. The multidimensional system correctly isolated six of the seven α3β4 nAChR ligands and only one of the 11 non-ligands was found with the α3β4 nAChR ligands. The results indicate that the multidimensional liquid chromatographic system can be used for the on-line screening of chemical mixtures for α3β4 nAChR ligands.
Keywords: Nicotinic receptor ligands;

Quinolones are a group of synthetic antibiotics that are widely used in veterinary medicine. Their residues may remain in tissues, milk, etc. intended for human consumption. The European Union fixes the maximum residue limits (MRLs) of veterinary medicinal products in foodstuffs of animal origin. Analytical methods are therefore needed to determine them in biological samples. In this study, we describe capillary isotachophoresis–capillary zone electrophoresis (ITP–CZE) to analyze three quinolones, enrofloxacin (ENR), ciprofloxacin (CPR) and flumequine (FLU), in pig plasma samples. We used solid-phase extraction with Oasis HLB cartridges as a sample pretreatment clean-up step. Capillary zone electrophoresis (CZE) requires low amounts of sample and is not as sensitive as one would wish. ITP–CZE is an easy way to increase the sample loadability and sensitivity. With this system sensitivity increases 40-fold. The detection limits for CPR, ENR and FLU were 70, 85 and 50 μg l−1, respectively, which were lower than their MRLs in different kinds of samples. This method is simple and sensitive, and is therefore an alternative tool to the existing HPLC methods for analyzing the residuals of these quinolones in biological samples.
Keywords: Ciprofloxacin; Enrofloxacin; Flumequine;

Liquid chromatography coupled with multi-channel electrochemical detection for the determination of daidzin in rat blood sampled by an automated blood sampling system by Feifei Tian; Yongxin Zhu; Hong Long; Meloney Cregor; Fuming Xie; Candice B Kissinger; Peter T Kissinger (173-177).
Daidzin, a soy-derived biologically active natural product, has been reported to inhibit mitochondrial aldehyde dehydrogenase and suppress ethanol intake. This paper describes a method for the determination of daidzin in rat blood. After administration of daidzin, blood samples were periodically collected from awake, freely moving animals by a Culex automated blood sampler. Daidzin was extracted from 50 μl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 12 min using a microbore C18 (100×1.0 mm) 3 μm column with a mobile phase containing 20 mM sodium acetate, 0.25 mM EDTA, pH 4.3, 4% methanol and 11% acetonitrile at a flow-rate of 90 μl/min. Detection was attained using a four-channel electrochemical detector with glassy carbon electrodes using oxidation potentials of +1100, 950, 850, 750 mV vs. Ag/AgCl. The limit of detection for daidzin in rat plasma was 5 ng/ml at a signal-to-noise ratio of 3:1. The extraction recovery of daidzin from rat plasma was over 74%. Linearity was obtained for the range of 25–1000 ng/ml. The intra- and inter-assay precisions were in the ranges of 2.7–6.6 and 1.9–3.7%, respectively. This method is suitable to routine in vivo monitoring of daidzin in rat plasma.
Keywords: Daidzin;

Determination of betaxolol in human aqueous humour by high-performance liquid chromatography with fluorescence detection by Berrak Dulger; Nursabah E. Basci; Ilgaz Sagdic-Yalvac; Aytekin Temizer (179-183).
A reversed-phase high-performance liquid chromatographic method is described for the determination of betaxolol in human aqueous humour. Betaxolol and the internal standard metoprolol were extracted with cyclohexane and separated on a reversed-phase column (Luna C18, 250×4.6 mm, 5 μm) with a mobile phase containing acetonitrile–phosphate buffer (40:60, v/v) at a flow-rate of 0.8 ml/min. The column effluent was monitored with a fluorescence detector at 227 nm (excitation) and 301 nm (emission). The retention times for metoprolol and betaxolol were 3.55 and 5.63 min, respectively. The recovery from aqueous humour was found to be 71.6% for betaxolol at 1.25 μg/ml. The within-day and day-to-day accuracy values were in the range of 96.17–105.2% for betaxolol at 0.1, 4 and 12 μg/ml (n=6), within-day and day-to-day precision values were less than 10% for betaxolol at the concentrations given above. The detection limit corresponding to the signal-to-noise ratio of 3:1 was 15 ng/ml. The presented method was suitable for measuring betaxolol levels in human aqueous humour samples obtained from patients after topical administration.
Keywords: Betaxolol; β-blocker;

A highly sensitive and specific method for the determination of norfloxacin in poultry tissues by LC–MS was developed and validated. An extract of the sample was separated on a C18 reversed-phase column and analyzed by LC–MS. The mobile phase was gradiently flowed with 2% acetic acid and acetonitrile. The limit of detection and limit of quantitation were 1 and 5 ng/g, respectively. Mean recoveries from various spiked tissues were 87.2% (ranging from 82.5 to 92.7%) for norfloxacin. The method has been successfully applied to determine norfloxacin in poultry muscle.
Keywords: Norfloxacin;

High-performance liquid chromatographic method for the quantification of unbound evernimicin in human plasma ultrafiltrate by Ruyun Zhong; Abraham Hernandez; Kevin B. Alton; Narendra S. Kishnani; James E. Patrick (191-195).
A rapid HPLC method was developed for quantification of unbound evernimicin in human plasma. Protein-free samples prepared by ultrafiltration were injected directly onto a polymeric reversed-phase column and the eluent monitored at 302 nm. Evernimicin that eluted within 3.5 min was well resolved from endogenous components. Linearity was established between peak height and evernimicin concentration from 25 to 2500 ng/ml. Assay precision (C.V.) was within 5% while bias was no greater than 3%. This method has been used for the ex vivo assessment of evernimicin protein binding in human plasma from safety and tolerance as well as liver dysfunction and renal insufficiency studies.
Keywords: Evernimicin;