Journal of Chromatography B (v.787, #2)

News Section (N1-N2).

An analytical method was developed for the determination, in blood plasma, of a novel peroxisome proliferator-activated receptor (PPAR) agonist drug, tesaglitazar. The drug and the isotope labelled internal standard were isolated by solid-phase extraction (SPE) on hexylsilica, separated by reversed-phase liquid chromatography and quantified by tandem mass spectrometry. Factorial design and a robotic sample processor were employed in the exploration and optimisation of the SPE procedure in the 96-well format. This allowed rapid development of the method, notably limiting the process to four experiments before validation. The detectability was greatly improved by utilising the formation of sodium adducts in atmospheric pressure positive ionisation mass spectrometry. Absolute recovery was more than 95% with a coefficient of variation of 5% at a level of 8.7 nM. The accuracy and precision of the automated SPE method presented here matched the excellence of the previously used method based on manual liquid–liquid extraction. Furthermore, the method resulted in an increased sample throughput.
Keywords: Tesaglitazar;

A rapid and simple method for the determination of morphine (M), normorphine (NM), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in plasma by high-performance liquid chromatographic separation with mass spectrometric detection (HPLC–MS) has been developed. Samples (40 μl) were cleaned-up by protein precipitation with two volumes (80 μl) of acetonitrile and reconstituted in formic acid 0.1% in water. Naloxone was used as internal standard. Analytes were separated on a phenyl–hexyl column using a step-gradient (1 ml/min) of acetonitrile and formic acid in water. Acetonitrile was added post-column (0.3 ml/min). Quantification of morphine and its metabolites was achieved with an Agilent 1100 series HPLC–MS system equipped with electrospray interface set to selected ion-monitoring (SIM) mode. Calibration curves covered a wide range of concentrations (2.44–10 000 nM) and were best fitted with a weighed quadratic equation. The limits of quantification achieved with this method were 2.44 nM for M and 4.88 nM for NM, M3G and M6G. The method proved accurate (85–98%), precise (C.V.<10%) and was successfully applied to a wide range of in vitro and in vivo pharmacokinetic studies in rodents.
Keywords: Morphine; Normorphine; Morphine glucuronides;

Cathepsin K is a cysteine proteinase, primarily expressed in osteoclasts, which has a strong collagenolytic activity and plays an essential role involved in bone matrix degradation. Its inhibition could provide a novel approach to the treatment and prevention of osteoporosis. One structural class of lead compounds in our cathepsin K inhibitors program is based on an arylaminoethyl amide scaffold, which has potential metabolic weak points that might be stabilized by appropriate chemical modification(s). For the identification of potential metabolic “soft spots” and the rational design of improved derivatives, early biotransformation of a potent arylaminoethyl amide cathepsin K inhibitor (NVP-AAV490-NX) was investigated in plasma, urine and liver homogenates of rats after intravenous bolus administration of 10 mg/kg. The detection and identification of metabolites was achieved by high-resolution mass spectrometry (time-of-flight MS) and multi-dimensional mass spectrometry (ion trap MS). Both mass spectrometers were combined with reversed-phase capillary high-performance liquid chromatography columns. It was demonstrated that both mass analyzers complement each other and that, even in the sub-nanogram range, the resulting set of MS data can be successfully used to elucidate most of the metabolic changes unambiguously, solely by mass spectrometric techniques. The proposed metabolite structures were additionally corroborated by exact mass measurement of the protonated molecular ions to confirm the predicted elemental composition, by determination of the number of the exchangeable hydrogen atoms replacing water against deuterium oxide as mobile phase and, in one case, by an MS3 product ion experiment in order to elucidate the site of conjugation.
Keywords: Cathepsin K inhibitor; NVP-AAV490-NX;

Clinical use of polyethylene glycols as marker substances and determination in urine by liquid chromatography by Gisela Gauchel; Bernd Huppertz; Heribert Feiertag; Ruprecht Keller (271-279).
Adulteration of samples is a serious problem in the analysis of drugs of abuse. One of the most frequent methods is substitution of urines by “clean” urines to gain false-negative results in laboratory tests for drugs of abuse. One way to approach this problem may be to label the patient’s urine with marker substances which are given orally prior to the delivery of urine. This concept is based on methods for determining malabsorption in pediatric medicine. We report a protocol for evaluating low-molecular-mass polyethylene glycols as enteral labelling marker substances. For monitoring renal excretion of the ingested polyethylene glycols we have developed and optimised an isocratic reversed-phase high-performance liquid chromatographic method with automatic sample cleanup by column switching in the back-flush technique and with RI detection. The chromatographic procedure is simple, reliable and rapid, allowing a high sample throughput for routine screening.
Keywords: Poly(ethylene glycol);

Liquid chromatographic assay for the simultaneous determination of indole-3-carbinol and its acid condensation products in plasma by Mark J Anderton; Rebekah Jukes; John H Lamb; Margaret M Manson; Andreas Gescher; William P Steward; Marion L Williams (281-291).
A high-performance liquid chromatographic method was developed for the simultaneous determination of indole-3-carbinol (I3C), 3,3′-diindolylmethane (DIM), [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr1), and indolo[3,2b]carbazole (ICZ). Compounds were extracted from mouse plasma using tert.-butyl methyl ether, incorporating 4-methoxy-indole as internal standard. Chromatographic separation utilized a Waters Symmetry RP18 in tandem with a Thermoquest BDS C18 column, an acetonitrile–water gradient and UV (280 nm) in series with fluorescence (ex. 335 nm; em. 415 nm) detection. Calibration curves were linear (r 2>0.99) between 50 and 15,000 ng/ml for I3C; 150 and 15,000 ng/ml for LTr1; and 0.15 and 37.5 ng/ml for ICZ and the method was reproducible and precise (within-day and between-day coefficients of variation below 9.7 and 13%, respectively). The method described is suitable for comprehensive pharmacokinetic studies with indole-3-carbinol.
Keywords: Indole-3-carbinol; 3,3′-Diindolylmethane;

Generalized least squares regression with variance function estimation was used to derive the calibration function for measurement of methotrexate plasma concentration and its results were compared with weighted least squares regression by usual weight factors and also with that of ordinary least squares method. In the calibration curve range of 0.05 to 100 μM, both heteroscedasticity and non-linearity were present therefore ordinary least squares linear regression methods could result in large errors in the calculation of methotrexate concentration. Generalized least squares regression with variance function estimation worked better than both the weighted regression with the usual weight factors and ordinary least squares regression and gave better estimates for methotrexate concentration.
Keywords: Methotrexate;

Simple immunoaffinity method to purify recombinant hepatitis B surface antigen secreted by transfected mammalian cells by Sergio da Silva e Mouta Junior; Carlos Otávio Alves Vianna; Ilka Ennes; Selma de Andrade Gomes; Marcos da Silva Freire; Edimilson Domingos da Silva; Salvatore Giovanni De Simone; Marcia Terezinha Baroni de Moraes (303-311).
Purification of recombinant hepatitis B surface antigen (recHBsAg) produced in a stable Chinese hamster ovary (CHO) cell line was evaluated using Linx Affinity Purification System (Invitrogen, USA). To purify HBsAg secreted by this cell line, a murine monoclonal antibody (MAbAH1) raised against native HBsAg was used. The purified AH1MAb was conjugated with phenyldiboronic acid (PDBA) and immobilized on the immunoaffinity chromatographic support. Using an optimized protocol the affinity column was able to purify recHBsAg from supernatant of mammalian cells cultures with more than 80% purity. This method showed to be simple and quicker than the current ultracentrifugation methods. The method is also efficient and economical in obtaining purified recHBsAg.
Keywords: Recombinant hepatitis B surface antigen;

Development of a liquid chromatography–mass spectrometric method for measuring the binding of memantine to different melanins by Martin J Koeberle; Patrick M Hughes; Clive G Wilson; Graham G Skellern (313-322).
A sensitive and selective liquid chromatography–mass spectrometric method was validated for the determination of free memantine in melanin binding studies. The sources of melanin studied were sepia, synthetic and bovine melanin. Memantine was chromatographed on a reversed-phase column (Prodigy 5 μm, ODS(3), 100 Å, 100×4.6 mm) using gradient elution with mobile phases of 0.1% formic acid in deionised water and 0.1% formic acid in methanol at a flow-rate of 0.8 ml/min. The mode of ionisation was atmospheric pressure–electrospray and detection by single ion monitoring of the memantine ion m/z 180. Validation of the method showed that the assay was linear from 0.1 to 1200 nM and 0.5 to 1200 nM memantine in deionised water and phosphate-buffered saline (PBS), respectively. Accuracy for sample preparations in deionised water was between 80 and 108% and between 80 and 123% for PBS. For both media, intra- and inter-day precision was below 1% for retention time and below 5% for analyte peak area. At the LLOQ, the variation of peak area was less than 17%. Binding of memantine to melanin was measured indirectly by determining the unbound fraction of memantine. After incubation of melanin with memantine, the sample was centrifuged and filtered to separate the memantine–melanin complex effectively from suspension. The filtrate was then assayed for free memantine from which the extent of binding was then calculated.
Keywords: Memantine; Melanins;

On-line determination of fluconazole in blood and dermal rat microdialysates by microbore high-performance liquid chromatography by François-Xavier Mathy; Benoı̂t Vroman; Denis Ntivunwa; Ann J De Winne; Roger K Verbeeck; Véronique Préat (323-331).
To study the distribution of fluconazole in the dermis of the rat, on-line microdialysis using double-site sampling coupled with a microbore HPLC system was developed. The chromatographic conditions consisted of a mobile phase of 20 mM diammonium phosphate–acetonitrile (75:25, v/v, pH 7.0) pumped through a microbore C18 column at 40 μl/min. The eluent was monitored with UV detector with UZ flow cell (30 mm path length) at 210 nm. A microbore 10-port pneumatic valve fitted with two loops of 1 μl was used to collect and directly inject microdialysates from jugular and dermal probes. The retention time was 5.8 min for fluconazole and 10.1 min for its fluorinated analog, UK-54373 used as a retrodialysis marker. The assay was precise, with inter- and intra-assay relative standard deviation values of 0.64 and 0.71%, respectively, and with a good linearity (r=0.999) in the range of 0.15–20 μg/ml with only 1 μl injected onto the column. The LOD and LOQ values for fluconazole were 0.100 and 0.150 μg/ml, respectively. The applicability of the method was demonstrated by studying the disposition of fluconazole in blood and dermis following i.v. bolus at a dose of 10 mg/kg.
Keywords: Fluconazole;

A sensitive and specific liquid chromatography–tandem mass spectrometry assay was developed to quantify the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma. The analytes (I, II) and their stable isotope-labeled analogues as internal standards were extracted on a C18 solid-phase extraction cartridge using a Zymark RapidTrace™ automation system. The chromatographic separation was carried out on a narrow-bore reversed-phase Zorbax XDB-C8 HPLC column with a mobile phase of acetonitrile/water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). The analytes were ionized using negative-to-positive switch electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 415→163 and m/z 431→337 was used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10–2500 ng/ml of plasma for both I and II. The lower limit of quantification was 10 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A throughput of 80 human plasma standards and samples per run was achieved with run time of 5 min for each injection. The assay has been successfully used in analyses of human plasma samples to support clinical studies.
Keywords: Eplerenone;

Determination of Chinese hamster ovary cell-derived recombinant thyrotropin by reversed-phase liquid chromatography by João Ezequiel de Oliveira; Fernanda de Mendonça; Cibele N Peroni; Paolo Bartolini; Maria Teresa C.P Ribela (345-355).
A reversed-phase high-performance liquid chromatography (RP-HPLC) methodology for the qualitative and quantitative analysis of human thyrotropin (hTSH) in CHO cell conditioned medium and in purified preparations has been set up and validated for accuracy, precision and sensitivity. A recovery test indicated a bias of less than 2% and intra-day and inter-day quantitative determinations presented relative standard deviations (RSD) always <7%, while sensitivity was 0.2 μg (RSD=5.6%). The novel methodology was applied to the study of the best cultivation conditions and was able to detect a significant difference in retention time (t R) between pituitary and recombinant hTSH, probably reflecting the influence of the heterogeneity of the carbohydrate moiety on the hydrophobic properties of the molecule.
Keywords: Thyrotropin;

Purification strategy for recombinant Phl p 6 is applicable to the natural allergen and yields biochemically and immunologically comparable preparations by Roland Suck; Bernhard Weber; Brigitte Schäffer; Ellen Diedrich; Timo Kamionka; Helmut Fiebig; Oliver Cromwell (357-368).
The recombinant major grass pollen allergen Phl p 6 has been expressed with a N-terminal 6×His-tag sequence and subsequently purified using nickel-chelating Sepharose. After cleavage of the tag-sequence, a second pass over the affinity chromatography revealed that even untagged rPhl p 6 bound tightly. In order to determine if that property is typical for Phl p 6, the natural allergen was purified in the same way starting with a grass pollen extract. Indeed, nPhl p 6 could be highly enriched in one step using nickel-chelating Sepharose. In addition to this new powerful purification method, the results provide further information in that the recombinant and natural allergens share a lot of properties, since biochemical characteristics are reflected in the purification strategies. The preparations of natural and recombinant Phl p 6 were used for comparative electrophoretic, chromatographic and immunological analysis which demonstrated high similarity.
Keywords: Recombinant allergen; Phl p 6;

A quantitative liquid chromatography mass spectrometry (LC–MS) methodology with online sample clean up by column switching is described for the simultaneous determination of the hydroxycinnamates, caffeic acid and chlorogenic acid, and of the catechins, epicatechin and catechin in human urine samples. Enzymatically treated urine samples were directly injected onto the LC–MS system, where sample clean up was performed by a reversed-phase Zorbax 300SB C3 column and selective elution of the target compounds onto a Zorbax SB C18 column resulted in final separation prior to detection by atmospheric pressure chemical ionization (APCI) MS using single ion monitoring (SIM) in negative mode. Linear calibration graphs were achieved in the dynamic range of 10–1000 ng/ml urine. The inter- and intraassay coefficients of variation (C.V.%) for the analysis of the four compounds in quality control urine samples were between 7.8 and 10.9, n=17 (reproducibility), and the repeatability of the assay was between 2.5 and 5.0% (n=12). Analyses of urine samples from a human dietary intervention study with intake of 600 g of fruits and vegetables were demonstrated. To our knowledge, this is the first method described that allows simultaneous determination of both hydroxycinnamates and catechins in biological samples.
Keywords: Hydroxycinnamates; Catechins;

The paper describes the development of a method for the determination of 15 nucleotides in cultured mononuclear blood and umbilical vein endothelial cell lysates by solvent generated ion-pair chromatography. The phase system is generated via a mobile phase of 100 mM phosphoric acid adjusted to pH 6.2 with triethylamine. Nucleotides are eluted by applying a linear magnesium ion gradient. The method is robust, highly reproducible and easily adaptable to other cell lysates and allows the separation and quantitation of the nucleotides with detection limits in the range from 17 (ADP) to 126 (CDP) pmol in 20-μl aliquots.
Keywords: Nucleotides;

Determination of protease inhibitors using liquid chromatography–tandem mass spectrometry by Valerie A Frerichs; Robin DiFrancesco; Gene D Morse (393-403).
A method for the analysis of six protease inhibitors and one metabolite has been developed and validated. Amprenavir, ritonavir, saquinavir, lopinavir, indinavir, nelfinavir, and an active metabolite of nelfinavir (M8) are quantitated using reversed-phase liquid chromatography coupled to tandem mass spectrometry, equipped with an electrospray ionization source (ESI-LC–MS–MS). The validation data presented here shows that the method allows the rugged analysis of these species from one aliquot. The evolution of complex drug interactions assessments and the clinical use of therapeutic drug monitoring for these antiretrovirals will be a potential immediate application of this method.
Keywords: Protease inhibitors;

Glutathione S-transferase of Ochrobactrum anthropi (OaGST), a bacterium isolated from soils contaminated by xenobiotic pollutants, was recently purified, cloned and characterised in our laboratories. The recombinant OaGST (rOaGST), highly expressed in Escherichia coli, when purified by glutathione-affinity chromatography and then analysed by electrospray ionisation mass spectrometry (ESI-MS), has evidenced a disulphide bond with glutathione (S-glutathiolation), which was removable by reduction with 2-mercaptoethanol. Enzymatic digestion of rOaGST with endoproteinase Glu-C, followed by liquid chromatography (LC)–ESI-MS analyses of the peptide mixtures under both reducing and not reducing conditions, have shown that glutathione was covalently bound to the Cys10 residue of rOaGST. Furthermore, LC–ESI-MS analyses of overexpressed rOaGST in Escherichia coli crude extracts, with and without incubation with glutathione, have not shown any S-glutathiolation of the recombinant enzyme.
Keywords: Cysteine; Glutathione S-transferase;

Nile Red fluorescent marker is widely-used for different purposes, such as staining cell structures and for the visualization and localization of colloidal drug carriers. However, when fluorescence-dependent imaging or quantification is performed, the risk of inexact results is increased due to photobleaching. The proposed, simple quantification method of using an HPLC–UV–Vis system allows the determination of Nile Red even in photobleached samples. The intra- and inter-assay accuracies for all analytes were found to be within 94.9 and 100.8%, respectively, of target values. When samples underwent photobleaching by laser, UV–Vis detection varied at around 99±5%, whereas fluorescence decreased down to 86%. Such results show this method to be interesting for approaches where quantification should be performed after analysis such as fluorescent imaging.
Keywords: Nile Red;

A sensitive, simple, and accurate method for determination of spinosin in rat plasma with sulfamethoxazole (SMZ) as internal standard was developed using RP-HPLC with UV detection. Sample preparations were carried out by protein precipitation with acetonitrile, followed by the evaporation of the acetonitrile to dryness. The resultant residue was then reconstituted in mobile phase and injected onto a Hypersil C18 (200×4.6 mm I.D., 5 μm) analytical column. The mobile phase consisted of acetonitrile–water (15:85, v/v) with 1% glacial acetic acid. The assay was shown to be linear over the range of 18.07–903.5 ng/ml (R 2=0.995). Mean recovery was determined as 93.6%. Within- and between-day precisions were ≤8.9% RSD. The limit of quantitation was 18.07 ng/ml. The HPLC method developed has been applied to determine the pharmacokinetics of spinosin in rat plasma after having taken Suanzaoren decoction.
Keywords: Spinosin;