Journal of Chromatography B (v.779, #2)

News Section (N1-N2).

A non-competitive immunoassay based on micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence (LIF) detection has been developed for the determination of alpha-fetoprotein (AFP). The anti-AFP antibody was labeled with fluorescein isothiocyanate (FITC) and the product was used as a fluorescent tracer, then AFP was mixed with the labeled antibody. After incubation, the immune AFP–antibody complex was separated from labeled free antibody by MECC. The parameters affecting separation such as the concentration of sodium dodecyl sulfate (SDS), the buffer pH and separation voltage were investigated and the following conditions were selected: 20 mM tetraborate containing 100 mM SDS at pH 9.50, and 20 kV separation voltage. The detection limit of this assay was 0.1 ng/ml with a linear range spanning two orders of magnitude. This method was applied to determine AFP in human serum.
Keywords: α-Fetoprotein;

Electrophoresis using ultra-high voltages by Maribel Vazquez; Gareth McKinley; Luba Mitnik; Samantha Desmarais; Paul Matsudaira; Daniel Ehrlich (163-171).
Optimization of electrophoretic techniques is becoming an increasingly important area of research as microdevices are now routinely adapted for numerous biology and engineering applications. The present work seeks to optimize electrophoresis within microdevices by utilizing ultra-high voltages to increase sample concentration prior to separation. By imaging fluorescently-tagged DNA samples, the effects of both conventional and atypical voltage protocols on DNA migration and separation are readily observed. Experiments illustrate that short periods of high voltage during electrophoretic injection do not destroy the quality of DNA separations, and in fact can enhance sample concentration five-fold. This study presents data that illustrate increases in average resolution, and resolution of longer fragments, obtained from electrophoretic injections utilizing voltages between 85 and 850 V/cm.
Keywords: DNA;

A sensitive and selective HPLC–MS–MS method was developed for the determination of trimebutine maleate (TM) and its major metabolites N-monodemethyltrimebutine (TM-MPB), N-didemethyltrimebutine (APB) and 3,4,5-trimethoxybenzoic acid (TMBA) in human plasma. The analytes were extracted from plasma samples by liquid–liquid extraction and chromatographed on a YMC J’sphere C18 column. The mobile phase consisted of 2 mM ammonium acetate buffer (pH 6.5)–methanol (20:80, v/v), and at a flow-rate of 0.2 ml/min. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive–negative switching electrospray ionization (ESI). The method was validated over the concentration range of 1–100 ng/ml for trimebutine maleate and APB, 1–500 ng/ml for MPB, and 50–10 000 ng/ml for TMBA. Inter- and intra-day precision (RSD%) for trimebutine maleate and its three metabolites were all within ±15% and the accuracy was within 85–115%. The limit of quantitation was 1 ng/ml for trimebutine maleate, TM-MPB and APB, and 50 ng/ml for TMBA. The extraction recovery was on average 58.2% for trimebutine maleate, 69.6% for MPB, 51.2% for APB and 62.5% for TMBA. The method was applied to the pharmacokinetic study of trimebutine maleate and its metabolites in healthy Chinese volunteers.
Keywords: Trimebutine maleate; N-Monodemethyltrimebutine; N-Didemethyltrimebutine; 3,4,5-Trimethoxybenzoic acid;

A sensitive and specific isotope dilution liquid chromatography–electrospray tandem mass spectrometry method was developed for the determination of the 3-nitrotyrosine residue levels in rat plasma proteins. The assay is based on the cleavage of proteins with concentrated hydrochloric acid to release both 3-nitrotyrosine and tyrosine. To control the potential artifactual nitration of tyrosine residues during the proteolysis, samples are spiked with 13C9-labeled tyrosine and the level of 13C9-labeled 3-nitrotyrosine is measured. The clean-up process entails hydrolysate fortification with 2,5,6-d 3-3-nitrotyrosine, followed by solid-phase extraction on octadecylsilyl (to isolate tyrosine) and aminopropylsilyl (to isolate 3-nitrotyrosine) cartridges. Tyrosine and 3-nitrotyrosine fractions are mixed in an appropriate ratio prior to the analysis. The method was applied to animals exposed to ferric nitrilotriacetate to induce oxidative stress.
Keywords: Nitrotyrosine; Tyrosine; Proteins;

Measurements of nitrite (NO2 ) and nitrate (NO3 ) in biological fluids are proposed as indices of cellular nitric oxide (NO) production. Determination of NO2 and NO3 in standard solutions is not difficult, however, determinations which reflect accurately cellular NO synthesis represent a considerable analytical challenge. Problems are often encountered arising from background NO2 /NO3 contamination in experimental solutions and laboratory hardware, and with methods for sample extraction. We investigated potential procedures for the extraction and determination of NO2 and NO3 in biological samples. Consequently, a protocol was devised which yielded acceptable results regarding extraction efficiency, assay reproducibility, sample throughput and contaminant minimisation. It entailed rigorous washing of all equipment with water of low NO2 and NO3 content, sample deproteinisation by centrifugal ultrafiltration through a 3K filter and analysis by high-performance anion-exchange liquid chromatography with UV detection. Retention times for NO2 and NO3 in standards and plasma were 4.4 and 5.6 min, respectively. Assay linearity for standards ranged between 31 nM and 1 mM. The limit of detection for NO2 and NO3 in standards was 3 pmol. Recoveries of NO2 and NO3 from spiked plasma (1–100 μM KNO2/KNO3) and from extracted standards (1–250 μM) were approximately 100%. Intra-assay and inter-assay RSDs for NO2 and NO3 in spiked and unspiked plasma were 10.6% or less. Assays on washed platelet supernatants demonstrated collagen-induced platelet generation of NO products and analysis of murine and rat cardiac perfusates was achieved. Our procedure may be suitable for routine determination of NO2 and NO3 in various biological fluids, e.g., plasma.
Keywords: Nitrite; Nitrate;

Metallothionein (MT) isoforms, MT-1 and MT-2, in biological specimens are clearly separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated capillary. The effectiveness of CZE analysis in the study of MT isoforms in biological specimens is discussed. We did two experiments to determine the MT-1/MT-2 ratio in biological specimens. The ratio of MT-1/MT-2 can be determined by CZE under a neutral pH without any detergents. One of these studies is time-dependent changes of the MT-1/MT-2 ratio in the cytosol of the pancreas and liver in mice after Zn or Cd injection. In the pancreas, both isoforms were detected in the control mice and the ratio of MT-1/MT-2 was below 1.0. When Zn was injected, the maximum peak areas of both isoforms were obtained at 24 h, and the ratios increased over a value of 1.0 at 3 h and peaked at 10 h. However, in the Cd-injected mice, the peak areas of both isoforms increased up to 72 h, and the ratios were below 1.0 up to 72 h. On the contrary, neither isoform was detected in the livers of control mice. The ratios of Zn-injected mice liver were near the value 1.0 between 6 and 72 h, although the areas of both isoforms showed peaks at 48 h. The ratios of Cd-injected mice livers were detected to be over 1.0 from 10 h, but there were no significant difference between 10 and 72 h, and the areas of both isoforms showed peaks at 24 h. The other experiment investigated the ratio in each fraction of cell fractionation. Cell fractionation was done in the livers of Zn-treated mice. Twenty-four hours after the injection, the ratio of MT-1/MT-2 was 0.80±0.12 and 1.19±0.21 (mean±SD) in nuclear and cytosol fractions, respectively. Neither isoform was detected in mitochondrial or microsomal fraction. From the present results, CZE analysis is a suitable method for observation of the ratio of MT-1/MT-2 in biological specimens, and dynamic changes in both isoforms can be detected.
Keywords: Metallothionein;

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25–35 μg/ml (r 2=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 μg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1–5), LHRH (1–4), LHRH (1–3) and LHRH (3–4).
Keywords: Luteinizing hormone releasing hormone;

A capillary electrophoresis (CE) and a high-performance liquid chromatography (HPLC) method to analyze biogenic amines in food were compared. An automated precolumn derivatization with o-phthaldialdehyde (OPA) allows for the determination of aliphatic amines and amino acids by HPLC. In contrast, for the measurement of histamine and tyramine by CE, no laborious sample pretreatment was necessary. The biogenic amines were separated by CE or HPLC in less than 9 or 20 min, respectively. The calibration curves were linear to at least 100 mg/kg (r=0.999) and 1000 mg/kg for HPLC and CE, respectively, with detection limits for histamine of 0.5 mg/kg (fluorescence detector) or 1 mg/kg (diode array detector) with HPLC and 2 mg/kg with CE. The detection limits for tyramine were 1.5 mg/kg with HPLC and 6 mg/kg with CE and for further amines (e.g., putrescine, spermidine, cadaverine, agmatine) ranging from 1.0 to 8.5 mg/kg with HPLC. There was a good correlation between CE and HPLC (correlation coefficient for histamine: 0.994).
Keywords: Biogenic amines; Tyramine;

Ribavirin is a purine nucleoside analog with broad spectrum activity against a spectrum of DNA and RNA viruses. To facilitate pharmacokinetics studies, a LC–MS–MS method for the analysis of ribavirin in rat and monkey plasma was developed and validated. The method involved the addition of acyclovir as an internal standard and protein precipitation with acetonitrile followed by separation by an Intertsil Silica column and quantification by a MS–MS equipped with a positive electrospray ionization in the multiple reaction monitoring mode. The MS–MS reaction was selected to monitor the 245→113 and 226→152 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 10–5000 ng/ml. The lower limit of quantitation was 10 ng/ml, the coefficient of variation (CV) was 8–11%, and the bias was 1–3%. Intra-day and inter-day analysis of QC samples at 30, 1500 and 3500 ng/ml indicate that the method was precise (CV<18%) and accurate (bias<13%). Ribavirin in rat and monkey plasma was stable at 5 °C for at least 24 h, 0 °C for at least 4 h, and after three freeze–thaw cycles. This specific, accurate and precise assay is useful in the study of the pharmacokinetics of this compound.
Keywords: Ribavirin;

Methods employing monolithic HPLC columns for the determination of the cyclooxygenase II inhibitors rofecoxib (I) and 3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5′-dimethyl-5H-furan-2-one (DFP, III) in human plasma are described. Each analyte, together with an internal standard was extracted from the plasma matrix using solid-phase extraction in the 96-well format. The analytes were chromatographed on a Chromolith Speed Rod monolithic HPLC column (4.6×50 mm). Analyte detection for rofecoxib was via fluorescence following post-column photochemical derivatization. Detection for III was based on the native fluorescence of the compound. The precision, accuracy, and linearity of the methods were found to be comparable to those obtained using methods employing conventional packed HPLC columns. Use of the monolithic column permitted mobile phase flow-rates of up to 6.5 ml/min to be employed in the assays. The use of elevated flow-rates enabled the per sample analysis time to be reduced by up to a factor of 5 compared with assays based on packed HPLC columns. The results of experiments aimed at evaluating the ruggedness and reproducibility of monolithic columns employed in bioanalytical methods are presented.
Keywords: Cyclooxygenase II inhibitors;

A high-performance liquid chromatographic method was developed for the quantification of doxorubicin derived from PEGylated liposomal doxorubicin (Doxil) and its major metabolite in human plasma. This method utilizes Triton X-100 to disperse the liposome, followed by a protein precipitation step with 5-sulfosalicylic acid. Analytes in the resultant supernatant are separated on a Discovery RP amide C16 column (250×3 mm I.D., 5 μm) using an isocratic elution with a mobile phase consisting of 0.05 M sodium acetate (pH 4.0) and acetonitrile (72:28). The retention times for doxorubicin and the internal standard daunorubicin were 4.8 and 10.1 min, respectively. The column eluate was monitored by UV–visible detection at 487 nm. The determination of doxorubicin was found to be linear in the range of 1.0 ng/mL to 25 μg/mL, with intra-day and inter-day coefficients of variation and percent error ≤10%. The recovery of doxorubicin from plasma was >69.3%, with a liposomal dispersion efficiency of >95.7%. Our analytical method for free and PEGylated doxorubicin in human plasma is rapid, avoids organic extractions, and maintains sensitivity for the parent compound and its major metabolite, doxorubicinol.
Keywords: Doxorubicin; Doxorubicinol;

Determination of N,N-dimethyltryptamine and β-carboline alkaloids in human plasma following oral administration of Ayahuasca by Mercedes Yritia; Jordi Riba; Jordi Ortuño; Ariel Ramirez; Araceli Castillo; Yolanda Alfaro; Rafael de la Torre; Manel J Barbanoj (271-281).
Ayahuasca is a South American psychotropic beverage prepared from plants native to the Amazon River Basin. It combines the hallucinogenic agent and 5-HT2A/2C agonist N,N-dimethyltryptamine (DMT) with β-carboline alkaloids showing monoamine oxidase-inhibiting properties. In the present paper, an analytical methodology for the plasma quantification of the four main alkaloids present in ayahuasca plus two major metabolites is described. DMT was extracted by liquid–liquid extraction with n-pentane and quantified by gas chromatography with nitrogen–phosphorus detection. Recovery was 74%, and precision and accuracy were better than 9.9%. The limit of quantification (LOQ) was 1.6 ng/ml. Harmine, harmaline, and tetrahydroharmine (THH), the three main β-carbolines present in ayahuasca, and harmol and harmalol (O-demethylation metabolites of harmine and harmaline, respectively) were measured in plasma by means of high-performance liquid chromatography (HPLC) with fluorescence detection. Sample preparation was accomplished by solid-phase extraction, which facilitated the automation of the process. All five β-carbolines were measured using a single detector by switching wavelengths. Separation of harmol and harmalol required only slight changes in the chromatographic conditions. Method validation demonstrated good recoveries, above 87%, and accuracy and precision better than 13.4%. The LOQ was 0.5 ng/ml for harmine, 0.3 ng/ml for harmaline, 1.0 ng/ml for THH, and 0.3 ng/ml for harmol and harmalol. Good linearity was observed in the concentration ranges evaluated for DMT (2.5–50 ng/ml) and the β-carbolines (0.3–100 ng/ml). The gas chromatography and HPLC methods described allowed adequate characterization of the pharmacokinetics of the four main alkaloids present in ayahuasca, and also of two major β-carboline metabolites not previously described in the literature.
Keywords: N,N-Dimethyltryptamine; β-Carboline alkaloids;

Determination of the enantiomerization energy barrier of some 3-hydroxy-1,4-benzodiazepine drugs by supercritical fluid chromatography by Peter Oswald; Koen Desmet; Pat Sandra; Jan Krupčı́k; Pavol Májek; Daniel W Armstrong (283-295).
The first-order kinetic equation for irreversible reactions was used to determine the enantiomerization barrier of some of 3-hydroxy-1,4-benzodiazepine enantiomers by supercritical fluid chromatography (SFC). The racemates of lorazepam, oxazepam and temazepam were separated by SFC on chiral (R,R)-Whelk-O1 column with supercritical carbon dioxide containing 12.5% methanol and 0.5% diethylamine as a mobile phase. Peak areas of enantiomers prior to (A A0, A B0) and after the separation (AA , AB ), used for calculation of the enantiomerization barrier, were determined by computer-assisted peak deconvolution of peak clusters from the chromatograms. It was demonstrated for the first time that using a model for a four-peak cluster produces height precise results, and most closely approximates the published results. The kinetic equation for irreversible reactions was used to determine apparent enantiomerization rate constants. The dependence of the apparent enatiomerization barrier (ΔG app AB , ΔG app BA ) on temperature was used to determine apparent activation enthalpy (ΔH app RS , ΔH app SR ) and entropy (ΔS app RS , ΔS app SR ) for all studied benzodiazepines.
Keywords: Lorazepam; Temazepam; Oxazepam;

A fast and robust liquid chromatography–mass spectrometry (LC–MS–MS) method has been developed for simultaneous quantitation of the angiotensin-converting enzyme (ACE) inhibitor, ramipril and its metabolite ramiprilat in human plasma. The method involves a solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables a detection limit at sub-nanogram levels. The proposed method has been validated with a linear range of 0.5–250 ng/ml for both ramipril and ramiprilat. The overall recoveries for ramipril and ramiprilat were 88.7 and 101.8%, respectively.
Keywords: Ramipril; Ramiprilat;

A sensitive high-performance liquid chromatography (HPLC) method using UV detection for the determination of gabapentin in human plasma has been developed. In this method, gabapentin was extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge followed by derivatization with phenylisothiocyanate. Analysis was achieved by using a HPLC system that was equipped with a UV detector. The quantitation limit of gabapentin in human plasma was 0.03 μg/ml. The method is sensitive with excellent selectivity and reproducibility and it has been applied to a bioequivalence clinical study with great success.
Keywords: Gabapentin;

There is a need to measure dietary transfer of gossypol and its metabolite, gossypolone in aquatic animals because of common use of cottonseed meal as a feed ingredient and fertilizer. The analytical method for gossypol and gossypolone enantiomers, therefore, becomes important. HPLC techniques have been developed by using mainly UV detection. We simultaneously used both UV and electrochemical (EC) detectors, and found that each individual detector has its own advantage which can increase accuracy and ease of identification. EC detection (2.5 and 50 ng/ml) exhibited a significantly lower detection level for both gossypol and gossypolone enantiomers than the UV detection (40 and 300 ng/ml) in the rainbow trout tissues, while UV detectors showed more stable detection than EC. We were able to detect a very low concentration of each gossypol enantiomer by EC but not UV detection especially in seminal plasma. For the first time gossypolone enantiomers were quantified in fish tissues by HPLC and its method was described. The technique, simultaneous adoption of both UV and EC detectors, could be helpful for a very low concentration of gossypol and/or gossypolone enantiomers in tissues of other animals and humans.
Keywords: Gossypol; Gossypolone;

Simultaneous, quantitative determination of opiates, amphetamines, cocaine and benzoylecgonine in oral fluid by liquid chromatography quadrupole-time-of-flight mass spectrometry by Kjell A Mortier; Kristof E Maudens; Willy E Lambert; Karine M Clauwaert; Jan F Van Bocxlaer; Dieter L Deforce; Carlos H Van Peteghem; André P De Leenheer (321-330).
A method using liquid chromatography coupled to tandem mass spectrometry is described for the determination of drugs of abuse in oral fluid. The method is able to simultaneously quantify amphetamines (amphetamine, methamphetamine, MDA, MDMA and MDEA), opiates (morphine and codeine), cocaine and benzoylecgonine. Only 200 μl of oral fluid is spent for analysis. The sample preparation is easy and consists of mixed mode phase solid-phase extraction. Reversed-phase chromatography is carried out on a narrow bore phenyl type column at a flow-rate of 0.2 ml/min. A gradient is applied ranging from 6 to 67.6% methanol with ammonium formate (10 mM, pH 5.0) added to the mobile phase. The column effluent was directed into a quadrupole-time-of-flight instrument by electrospray ionization, without the use of a splitter. A validation study was carried out. Recovery ranged from 52.3 to 98.8%, within-day and between-day precision expressed by relative standard deviation were less than 11.9 and 16.8%, respectively, and inaccuracy did not exceed 11.6%. The limit of quantification was 2 ng/ml (0.66×10−5–1.48×10−5 M) for all compounds. Internal standards were used to generate quadratic calibration curves (r 2>0.999). The method was applied to real samples obtained from suspected drug users. An interference was observed from the device used to sample the oral fluid, consequently this was excluded from the method which was validated on oral fluid obtained by spitting in a test-tube.
Keywords: Opiates; Amphetamines; Cocaine; Benzoylecgonine;

Immobilized liposome chromatography (ILC) was used to screen and analyze permeable compounds in traditional Chinese medicines (TCMs), testing extracts from Radix Angelica Sinensis. More than 10 peaks were resolved based on their interactions with the ILC stationary phase, a system which mimics biomembranes; this means that more than 10 components in Radix Angelica Sinensis extract have significant retention on an ILC column. Two of them, ligustilide and ferulic acid, were identified from their MS spectrum and with standard samples. A possible molecular structure of another component retained on ILC was also preliminarily identified as 3-butylidene-4,5-dihydro-2(1,3H)-1-isobenzofuranol according to its MS spectrum, hydrophobicity and 1H NMR spectrum. Of all detected components, ligustilide had the best penetration ability through the biomembrane. The effects of pH, column temperature, and ionic strength on the chromatography of methanolic extracts of Radix Angelica Sinensis were also investigated. It was found that the separation selectivity on ILC is strongly affected by the eluent pH, but only slightly by the column temperature and ionic strength.
Keywords: Permeable compounds;

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) assay for a new structurally unique carbapenem antibiotic, ertapenem, in urine has been improved for selectivity and automated using a Packard MultiPROBE® II EX pipetting station. The method uses column-switching for on-line extraction of the urine sample. The extraction column, analytical column, mobile phase, and timing of the column-switching valve have been changed to enhance selectivity for the analyte over endogenous background material. Sample transfer and dilution prior to direct-injection into the HPLC system have been accomplished using a Packard MultiPROBE® II EX robotic liquid handling system.
Keywords: Ertapenem;

Capillary zone electrophoresis was employed for the analysis of histamine in single rat peritoneal mast cells using an amperometric detector with a carbon fiber microdisk bundle electrode. In this method, individual mast cells and then 0.02 mol/l NaOH as a lysing solution are injected into the front end of the separation capillary by electromigration with an aid of a inverted microscope. A cell injector was constructed. Using it, the cell suspension was static, when a voltage for injecting single cells was applied. Histamine in single rat peritoneal mast cells have been identified. Quantitation has been accomplished through the use of calibration curves. The mean amount of histamine for nine cells is 95.8 fmol, which is consistent with the literature value.
Keywords: Histamine;

An improved sample work-up and derivatisation procedure for the quantitative determination of paroxetine in human plasma by gas chromatography–negative ion chemical ionisation mass spectrometry is presented. Solvent extraction from plasma samples at alkaline pH was combined with derivatisation to the pentafluorobenzyl carbamate derivative in one step and subsequently analysed without any further purification. Thus, lengthy and time-consuming solvent evaporation steps are avoided to assure high-throughput analysis. Complete validation data are presented. The method is rugged, rapid and robust and has been applied to the batch analysis of paroxetine during pharmacokinetic profiling of the drug.
Keywords: Paroxetine;

A rapid, shorter, isocratic high-performance liquid chromatographic method is described for the determination of plasma total homocysteine. In this method the sample preparation was modified for reduction of the time of the thiolic reduction from 30 at room temperature to 10 min at 37 °C with tris-(2-carboxyethyl) phosphine (TCEP), reduction the time of derivatization from 60 to 10 min at 60 °C and elution of the SBD-thiols derivative by a shorter HPLC-column which is commercially available. The SBD–homocysteine derivative was eluted at 3.7 min. The method was equally precise and faster for quantification of tHcy in plasma as other previously described method and should be very useful for epidemiological studies in which large numbers of samples have to be analyzed
Keywords: Homocysteine;