Journal of Chromatography B (v.820, #2)

A group of 18-mers of the same base sequence, but with differing alkyl modifications is used to investigate effects of these modifications on retention of oligonucleotides using ion-pairing reversed-phase liquid chromatography (IP-RPLC). It is shown that IP-RPLC is able to distinguish between oligonucleotides differing only by a single alkyl group. The identity of the nucleobase and position and length of the alkyl adduct affect retention of the oligonucleotide. These separation phenomena result from changes in charge and hydrophobicity upon alkylation. As demonstrated in this paper; chromatographic selectivity, unique to IP-RPLC, greatly facilitates the purification process of modified oligonucleotides.
Keywords: IP-RPLC; Oligonucleotides; Adducts; Purification; HPLC; Methylation;

A repeatable and highly sensitive analytical method using gas chromatography–mass spectrometry (GC–MS) in the selected ion monitoring mode (SIM) is developed for the simultaneous detection of ketamine (KT), norketamine (NK), and newly introduced dehydronorketamine (DHNK) in urine. The test specimen along with the deuterium analogues as internal standards (IS): d4-KT for KT and d4-NK for NK/DHNK, was extracted on an automatic solid-phase extraction (SPE) apparatus. The extracted eluate then was dried and derivatized with N-methyl-bis(trifluoroacetamide) (CF3CONCH3COCF3, MBTFA). Finally, the cooled derivatized solution was directly injected into the GC–MS system for analysis. The proposed process achieves high sensitivity for the detection of KT, NK, and DHNK. Correlation coefficients derived from typical calibration curves in the range of 20–2000 ng/mL are 1.000 for KT and NK, 0.999 for DHNK. The limits of detection (LODs) and limits of quantitation (LOQs) are 0.5–1.0 and 1.5–3.0, respectively. The overall method recoveries of KT, NK, and DHNK are 82.2–93.4. The intra- and inter-day run deviations are smaller than 5.0%. The analytical scheme was also applied to the determination of KT, NK, and DHNK in 20 KT suspected urine specimens, and the results reconfirm that DHNK is a main metabolite of KT.
Keywords: Ketamine; Norketamine; Dehydronorketamine; GC–MS; MBTFA;

A selective and sensitive method has been developed and validated for simultaneous quantification of methylephedrine and noscapine in human plasma. Analytes were extracted from human plasma samples by liquid–liquid extraction, separated on a Diamonsil C18 column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization (APCI) interface. Diphenhydramine was used as the internal standard (I.S.). The method was found to be precise and accurate within the linear range 0.1–100 ng/ml for each analyte. The intra- and inter-day relative standard deviations (R.S.D.s) were below 5.2% for methylephedrine and 6.7% for noscapine. The inter-day relative error (RE) as determined from quality control samples (QCs) was less than 3.0% for each analyte. The assay was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation containing 20 mg dl-methylephedrine hydrochloride, 16 mg noscapine, 300 mg paracetamol and 1 mg of chlorpheniramine maleate.
Keywords: Methylephedrine; Noscapine;

Liquid chromatographic/tandem mass spectrometric methods using pneumatically assisted electrospray ionisation (LC–ESI-MS/MS) was developed for determination of 18 mycotoxins and metabolites—ochratoxin A, zearalenone, α-zearalenol, β-zearalenol, α-zearalanol (zeranol), β-zearalanol (taleranol), fumonisin B1, fumonisin B2, T-2 toxin, HT-2 toxin, T-2 triol, diacetoxyscirpenol (DAS), 15-monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), deepoxy-deoxynivalenol (DOM-1) and aflatoxin M1—in milk. The mycotoxins were extracted and cleaned up simultaneously. Extraction and removal of lipophilic compounds was performed at pH 2 using a two-phase mixture of acetonitrile and hexane. The acetonitrile concentration of the aqueous phase was reduced and the pH was adjusted to 8.5 before clean up by solid phase extraction (SPE) on Oasis HLB. The toxins DON, DOM-1, 3-AcDON, 15-AcDON, ochratoxin A, zearalenone, α-zearalenol, β-zearalenol, α-zearalanol and β-zearalanol were detected in negative ion mode after separation on a Hypersil ENV analytical column, while the toxins T-2 toxin, HT-2 toxin, T-2 triol, DAS, MAS, fumonisin B1, fumonisin B2 and aflatoxin M1 were detected in positive ion mode after separation on a Luna C18 column. Two transition products were monitored for each compound. The extraction and SPE conditions were optimised to obtain maximum recovery and minimum signal suppression/enhancement. The detection capabilities related to the transition products of lowest abundance were in the range 0.020–0.15 μg/l. The mean true recoveries were in the range 76–108% at levels of 0.2–10 μg/l.
Keywords: Tandem mass spectrometry; Milk; Mycotoxins; Ochratoxin A; Trichothecenes; Zearalenone; α-Zearalenol; β-Zearalenol; α-Zearalanol; Zeranol; β-Zearalanol; Taleranol; Fumonisin B1; Fumonisin B2; T-2 toxin; HT-2 toxin; T-2 triol; Diacetoxyscirpenol; DAS; Monoacetoxyscirpenol; MAS; Deoxynivalenol; DON; Vomitoxin; 3-Acetyldeoxynivalenol; 3-AcDON; 15-Acetyldeoxynivalenol; 15-AcDON; Deepoxy-deoxynivalenol; DOM-1; Aflatoxin M1;

The synthesis and initial characterization of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic stationary phase by Ruin Moaddel; Gerry B. Price; Jean-Marc Juteau; Michael Leffak; Irving W. Wainer (197-203).
The DNA unwinding element binding protein (DUE-B) plays a key role in DNA replication. The DUE-B protein has been immobilized on a liquid chromatography support and the resulting immobilized protein column was used for the on-line screening of a series of steroids. The DUE-B protein was expressed with an added C-terminal sequence of six adjacent histidine residues, a His6-tag and immobilized on a chiral ligand exchange support, the CLC-L column, using Ni2+ as the coordinating metal ion. The chromatographic retentions of 12 steroids were determined on the DUE-B/CLC-L column. The magnitudes of the steroid-immobilized DUE-B interactions, reflected by the observed retention times, correlated to the effect of the steroids in the cell-free replication system, i.e. the longer the retention, the greater the increase in DNA replication. The coefficient of determination for the %DNA activities linear relation to retention time was 0.9694. The data suggest that the DUE-B/CLC-L phase can be used for on-line pharmacological studies. The results also indicated that His-tagged proteins can be directly immobilized on the CLC-L stationary phase and the resulting columns used as rapid screens for the isolation and identification of small molecule or protein ligands from complex biological or chemical mixtures.
Keywords: Affinity chromatography; Immobilized protein; DNA replication; Metal complexation chromatography; On-line screening;

One-step purification of metallothionein extracted from two different sources by Rubens T. Honda; Roziete Mendes Araújo; Bruno Brasil Horta; Adalberto L. Val; Marilene Demasi (205-210).
We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS–PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.
Keywords: Metallothionein; Environmental sciences; Ni and Cd binding; Metal-chellating affinity chromatography;

Capillary zone electrophoresis (CZE) with fluorescence detection was applied to the simultaneous determination of histamine and polyamines including spermine, spermidine, diaminopropane, putrescine, cadaverine, diaminohexane with 4-fluor-7-nitro-2,1,3-benzoxadiazole (NBD-F) as the fluorescent derivatization reagent. The seven NBD-F labeled amines was separated within 200 s using 85 mM phosphate running buffer at pH 3.0. The concentration limits of these amines ranged from 5.1 × 10−8  M for spermine to 2.1 × 10−8  M for histamine. The relative standard deviations for migration time and peak height were less than 1.5% and 6.0%, respectively. The method was successfully applied to the analysis of biogenic amines in the lysate of tobacco mesophyll protoplasts, and spermidine and putrescine were detected in the lysate with satisfying recovery.
Keywords: Capillary electrophoresis; 4-Fluor-7-nitro-2,1,3-benzoxadiazole; Histamine; Polyamines; Tobacco protoplast;

A rapid, sensitive and selective LC–MS–MS method for the simultaneous quantitation of picroside-I and kutkoside (active constituents of herbal hepatoprotectant picroliv) was developed and validated in rabbit plasma. The analytes and internal standard (Amarogentin) were extracted using Oasis® HLB solid phase extraction cartridges. Analysis was performed on Spheri RP-18 column (10 μm, 100 mm × 4.6 mm i.d.) coupled with guard column using acetonitrile:MilliQ water (50:50, %v/v) as mobile phase at a flow rate of 1 ml/min with a retention time of 1.39, 1.33 and 1.42 min for picroside-I, kutkoside and amarogentin, respectively. The quantitation was carried out using an API-4000 LC–MS–MS with negative electro spray ionization in multiple reaction monitoring (MRM) mode. The precursor to product ion transitions for picroside-I, kutkoside and amarogentin were m/z 491 > 147, 199; 511 > 167, 235; 585 > 227, respectively. The method was validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy and precision (within- and between-assay variation), freeze–thaw (f–t), auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56–400 ng/ml with a limit of detection (LOD) of 0.5 ng/ml for both analytes. The recoveries from spiked control samples were >60 and >70% for picroside-I and kutkoside, respectively. Accuracy and precision of the validated method were within the acceptable limits of <20% at low and <15% at other concentrations. The analytes were stable after three freeze–thaw cycles and their dry residues were stable at −60 °C for 15 days. The method was successfully applied to determine concentrations of picroside-I and kutkoside post i.v. bolus administration of picroliv in rabbit.
Keywords: LC–MS–MS; Hepatoprotectant; Picroside-I; Kutkoside; MRM;

Quantification of phenolic metabolites of environmental chemicals in human urine using gas chromatography–tandem mass spectrometry and isotope dilution quantification by Roberto Bravo; Lisa M. Caltabiano; Carolina Fernandez; Kimberly D. Smith; Maribel Gallegos; Ralph D. Whitehead; Gayanga Weerasekera; Paula Restrepo; Amanda M. Bishop; José J. Perez; Larry L. Needham; Dana B. Barr (229-236).
We have developed a method to measure 12 urinary phenolic metabolites of pesticides or related chemicals. The target chemicals for our method are 2-isopropoxyphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; carbofuranphenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; 3,5,6-trichloro-2-pyridinol; para-nitrophenol, ortho-phenylphenol, pentachlorophenol, 1-naphthol and 2-naphthol. The sample preparation involves enzyme hydrolysis, isolation of the target chemicals using solid phase extraction cartridges, a phase-transfer catalyzed derivatization, cleanup using sorbent-immobilized liquid/liquid extraction cartridges, and concentration of the sample. Derivatized samples are analyzed by capillary gas chromatography–tandem mass spectroscopy using isotope dilution calibration for quantification. The limits of detection are in the mid ng/L range and the average coefficient of variation was below 15% for most of the analytes. Using our method, we measured concentrations of the target chemicals in urine samples from the general population.
Keywords: Phenolic metabolites; Human urine; GC–MS/MS;

A sensitive LC–MS–MS assay for the quantitative determination of bromocriptine has been developed and validated and is described in this work. The assay involved the extraction of the analyte from 1 ml of human plasma using a solid phase extraction on Oasis MCX cartridges. Chromatography was performed on a Symmetry C18 (2.1 mm × 100 mm, 3.5 μm) column using a mobile phase consisting of 25:75:01 acetonitrile–water–formic acid with a flow rate of 250 μl/min. The linearity was within the concentration range of 2–500 pg/ml. The lower limit of quantification was 2 pg/ml. This method has been demonstrated to be an improvement over existing methods due to its greater sensitivity and specificity.
Keywords: Bromocriptine; Mass spectrometry; Liquid chromatography;

A high-performance liquid chromatography (HPLC)–mass spectrometry (MS) method has been developed for the analysis of 9-β-d-arabinofuranosyl-2-fluoroadenine 5′-triphosphate (F-ara-ATP) from biological samples. Quantification is carried out by selected ion monitoring of the parent ion. Baseline separation of the monophosphate (F-ara-AMP) and diphosphate (F-ara-ADP) is achieved using the volatile ion-pairing reagent dimethylhexylamine. This method is selective and sensitive with an on-column detection limit of ∼50 fmol. It also permits simultaneous monitoring of endogenous adenosine phosphates. The utility of the assay has been demonstrated by the analysis of F-ara-ATP in human leukemic cells after incubation with 9-β-d-arabinosyl-2-fluoroadenine (F-ara-A) at clinically relevant concentrations.
Keywords: LC–MS; Fludarabine triphosphate; Leukemia;

Liquid chromatography–mass spectrometry assay for quantitation of ifosfamide and its N-deschloroethylated metabolites in rat microsomal medium by Thomas Storme; Lionel Mercier; Alain Deroussent; Micheline Re; Thierry Martens; Jacques Royer; Philippe Bourget; Gilles Vassal; Angelo Paci (251-259).
A specific and sensitive quantitative assay has been developed using high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) for the simultaneous quantitation of the antitumor drug ifosfamide (IFM) and its two metabolites, N 2-deschloroethylifosfamide (N 2-DCE-IFM) and N 3-deschloroethylifosfamide (N 3-DCE-IFM) in microsomal medium. The analytes and the internal standard (cyclophosphamide) were isolated by ethylacetate extraction from rat liver microsomes. They were analysed on a Nucleosil® C18 HD column (125 mm × 4 mm, 5 μm) using a step gradient with the mobile phase (2 mM ammonium formate and methanol). The HPLC–ESI-MS method used selected ion monitoring of ions m/z 199.1 Th and m/z 261.1 Th and was validated in the concentrations ranges of 100–5000 ng/mL for IFM and 50–2500 ng/mL for its N-deschloroethylated metabolites (DCE-IFM) with good accuracy and precision (CV less than 15%). The low limits of quantitation (LLOQ) were found at 50 ng/mL for N-deschloroethylated metabolites and at 100 ng/mL for the parent drug (IFM). The method was applied for the determination of ifosfamide and its N-deschloroethylated metabolites in rat microsomal incubations.
Keywords: Ifosfamide; Cyclophosphamide; Microsomal metabolism; HPLC–ESI-MS; Neurotoxicity;

A method is described which permits the quantitative detection of the chemical coccidiostats halofuginone, robenidine, diclazuril, nicarbazin and dimetridazole and its main metabolite 2-hydroxydimetridazole in poultry eggs and feed. Sample preparations were kept very simple and are based upon extraction with an organic solvent. Sample extracts were injected into the liquid chromatography tandem mass spectrometry (LC–MS/MS) system on a C18 column and a gradient elution was performed. Dimetridazole-D3 and diclazuril-bis, a structural analogue of diclazuril, were used as internal standards. Detection was performed on a triple quadrupole mass spectrometer in the selected reaction monitoring mode after ionisation in the positive or negative electrospray ionisation mode. Argon was applied as collision gas for collision induced dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC [Official Journal of the European Communities L221 (2002) 8].
Keywords: Coccidiostats; Eggs; Feed; Residues; LC–MS/MS;

Thymidine phosphorylase (TP) catalyses the conversion of thymidine into thymine. A non-radiochemical assay procedure for TP was developed in which thymine was detected at 265 nm after separation with reversed-phase HPLC. A complete separation of thymidine and thymine was achieved in 6 min and the minimum amount of thymine that could be detected was 0.8 pmol. The assay was linear with reaction times, up to at least 4 h, and protein concentrations up to at least 65 μg/ml. Population analysis showed no differences in TP activity between man and women or with increasing age.
Keywords: Thymidine phosphorylase; MNGIE; HPLC;

A fast and sensitive high-performance liquid chromatographic method for determination of azithromycin in human serum using fluorescence detection was developed. The drug and an internal standard (clarithromycin) were extracted from serum using n-hexan and subjected to pre-column derivatization with 9-fluorenylmethyl chloroformate as labeling agent. Analysis was performed on a phenyl packing material column with sodium phosphate buffer containing 2 ml/l triethylamine (pH 5.9) and methanol (29:71, v/v) as the mobile phase. The standard curve was linear over the range of 10–500 ng/ml of azithromycin in human serum. The means between-days precision were from 13.3% (for 10 ng/ml) to 2% (500 ng/ml) and the within-day precision from 11.9 to 1.7% determined on spiked samples. The accuracy of the method was 100.7–107.2% (between days) and 100.3–107.8% (within day). The limit of quantification was 10 ng/ml. This method was applied in a bioequivalence study of four different azithromycin preparations in 12 healthy volunteers.
Keywords: Reverse phase chromatography; HPLC; Azithromycin; Serum; Bioequivalence study; Macrolide antibiotics;

Huperzine A–human serum albumin association: Chromatographic and thermodynamic approach by François Darrouzain; Claire André; Lhassane Ismaili; Myriam Matoga; Yves Claude Guillaume (283-288).
The synthesis of six new huperzine analogues was reported. Each product presents an amidification of the free amine on huperzine A. The synthesis strategy of these new huperzine A derivatives is based on a condensation with an acyl anhydride. The binding on HSA of two molecule series (huperzine and benzodiazepine, respectively) was investigated with high performance liquid affinity chromatography (HPLAC) using an HSA column. A thermodynamic approach showed that binding huperzine A on HSA involved hydrophobic and Van der Waals interactions. A comparative thermodynamic study with benzodiazepine molecules was carried out to determine the potential binding site of huperzine derivatives on HSA.
Keywords: Thermodynamic; Huperzine; HPLC; Human serum albumin;

Author Index (289-291).

Keyword Index (293-299).