Journal of Chromatography B (v.819, #2)

Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells by Alain Beck; Marie-Claire Bussat; Nathalie Zorn; Virginie Robillard; Christine Klinguer-Hamour; Stéphane Chenu; Liliane Goetsch; Nathalie Corvaïa; Alain Van Dorsselaer; Jean-François Haeuw (203-218).
7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI–TOF, ES–TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 149 297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 152 878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 154 459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing.
Keywords: CHO cells; NS0 cells; Peptide mapping; Glycopeptide mapping; Recombinant monoclonal antibodies; Insulin-like growth factor-1 receptor;

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography by J. Randall Slemmon; Cory L. Painter; Sashi Nadanaciva; Florentina Catana; Karen Kaup; Rachel Scherrer; Virginia Casadas; Youyang Zhao; Marcia I. Heron (219-228).
Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.
Keywords: Peptides; Proteomics; Biomarkers; Chromatography; SPE; Automation; Heart; Matrix metalloprotease;

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5–2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.
Keywords: Purines; Uric acid; Allopurinol; Methylxanthines; Urolithiasis; Urinary calculi; Inborn errors of purine metabolism; Xanthinuria; Dihydroxyadeninuria; High-performance liquid chromatography;

Determination of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine and plasma by gas chromatography–mass spectrometry by Brian A. Logue; Nicholas P. Kirschten; Ilona Petrikovics; Matthew A. Moser; Gary A. Rockwood; Steven I. Baskin (237-244).
The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography–mass spectrometry (GC–MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d2), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra- and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females.
Keywords: Cyanide; 2-Aminothiazoline-4-carboxylic acid; GC–MS; Chemical warfare agent;

Flurogestone (FGA) is a synthetic progesterone, with a progestational action higher than that of progesterone itself. It is intended for vaginal use in large animals to induce oestrus synchronization. A quantitative method for the analysis of flurogestone acetate (FGA) in ovine plasma by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) has been developed. After the incorporation of megestrol acetate (MGA) as internal standard (IS) and followed by a liquid–liquid extraction from plasma, FGA and MGA were chromatographed using a reverse-phase HPLC column and detected by tandem mass spectrometry with a TurboIonSpray® source. Multiple reaction-monitoring (MRM) mode was used for the quantitative determination of FGA in ovine plasma. The precursor ions [M  + H]+ at m/z 407.2 and 385.1 for FGA and MGA, respectively, produced product ions at m/z 267.1/285.1 for FGA and m/z 267.1/224.0 for MGA. The validated concentration range was 0.2–5.0 ng/ml based on 500 μl plasma aliquots. The lower limit of quantitation was 0.2 ng/ml. Fully validated selectivity, accuracy, precision and reproducibility criteria for routine use in pharmacokinetic studies were demonstrated.
Keywords: Oestrus synchronization; Flurogestone acetate;

Determination and occurrence of polybrominated diphenyl ethers in maternal adipose tissue from inhabitants of Singapore by Qing Qing Li; Annamalai Loganath; Yap Seng Chong; Jeffrey Philip Obbard (253-257).
Polybrominated diphenyl ethers (PBDEs), as a specific group of brominated flame retardants (BFR), are used in a variety of consumer products including electronics and household furnishings. In recent years, a marked increase in the levels of PBDEs in human biological tissues and fluids, especially breast milk, has been reported in several countries. However, few data are available from countries in the Asia-pacific region, including Singapore. This study presents a validated method procedure and the first available data of the concentrations of PBDE congeners: PBDE-47 (2,2,4,4-Tetrabromodiphenyl ether), PBDE-99 (2,2′,4,4′,5-Pentabromodiphenyl ether), PBDE-100 (2,2′,4,4′,6-Pentabromodiphenyl ether), PBDE-153 (2,2′,4,4′,5,5′-Hexabromodiphenyl ether), PBDE-154 (2,2′,4,4′,5,6′-Hexabromodiphenyl ether) in maternal adipose tissue collected from inhabitants of Singapore. Microwave-assisted extraction (MAE) of PBDEs spiked adipose tissues coupled with GC–MS analysis achieved comparable recoveries to a conventional Soxhlet Extraction (SE) procedure of between 70 and 130%. MAE also yielded comparable precision data (variance less than 13%) relative to the SE procedure. Spiked Carbon-13 PBDE congeners were also used as surrogates for MAE quality assurance and confirmed the efficiency of the procedure. PBDE congeners were detected in all of 16 maternal adipose tissues collected in Singapore, where levels were comparable to available data from Belgium.
Keywords: Flame retardants; Polybrominated diphenyl ethers; Soxhlet extraction; Microwave-assisted extraction; Adipose tissue; Women; Singapore;

A sensitive and accurate liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the intracellular determination of nine antiretroviral drugs in human peripheral blood mononuclear cells (PBMCs) is proposed. PBMCs are isolated by density gradient centrifugation using Vacutainer™ CPT tubes and cell count is performed with a Coulter® instrument. Single-step extraction of drugs from PBMCs pellets is performed with MeOH 50% (with clozapine added as internal standard, I.S.) and the supernatant is injected onto a 2.1 mm × 30 mm SymmetryShield™ 3.5 μm-RP18 column equipped with a 2.1 × 10 mm guard column. Chromatographic separations are performed using a gradient program with a mixture of 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electro-spray ionisation–triple quadrupole mass spectrometry using the selected reaction monitoring (SRM) detection mode. The positive mode is used for the HIV protease inhibitors (PIs) indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, and the negative mode is applied for efavirenz. The calibration curves are prepared using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.5 to 100 ng/ml of cell extracts and fitted to a quadratic regression model weighted by 1/(concentration)2. The lower limit of quantification is less than 0.5 ng/ml. The mean extraction recovery for all PIs/NNRTIs is always above 88%. The method is precise, with mean inter-day CV% within 0.6–10.2%, and accurate (range of inter-day deviation from nominal values −7.2 to +8.3%). This analytical method can be conveniently used in clinical research for the assessment of intracellular levels of all PIs/NNRTIs commercially available at present using a simple one-step cell extraction of PBMCs followed by liquid chromatography coupled with tandem triple quadripole mass detection.
Keywords: Protease inhibitors; Non-nucleoside reverse transcriptase inhibitors; PBMCs; Peripheral blood mononuclear cells; HPLC–MS/MS; Indinavir; Amprenavir; Saquinavir; Ritonavir; Nelfinavir; Lopinavir; Atazanavir; Efavirenz; Nevirapine;

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed to determine cefixime ((6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(carboxymethoxyimino)acetamido]-8-oxo- 3-vinyl-5-thia-1-azabicyclo-[4,2,0]-oct-2-ene-2-carboxylic acid) in human plasma. After a simple protein precipitation using acetonitrile, the post-treatment samples were analyzed on a C8 column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water–formic acid (40:60:0.5, v/v/v). The analyte and internal standard cefetamet were both detected by use of selected reaction monitoring mode. The method was linear in the concentration range of 0.05–8.0 μg/ml. The lower limit of quantification was 0.05 μg/ml. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.7%. The accuracy determined at three concentrations (0.05, 0.80 and 7.2 μg/ml for cefixime) was within ±2.0% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of cefixime capsule in 24 healthy volunteers.
Keywords: Cefixime;

Characterization of lipophilicity and antiproliferative activity of E-2-arylmethylene-1-tetralones and their heteroanalogues by B. Hallgas; Zs. Dobos; E. Ősz; F. Hollósy; R.E. Schwab; E.Z. Szabó; D. Erős; M. Idei; Gy. Kéri; T. Lóránd (283-291).
A molecular library based on E-2-arylmethylene-1-tetralone has been designed and synthesized. A reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed and applied to separate them and to characterize their lipophilicity. The chromatographic method applied here was suitable to separate the structural (ortho and para) isomers of compounds and was sensitive enough to differentiate their lipophilicities. The measured (k′) and computer calculated (CLOGP) lipophilicity values has been compared. Good linear correlation has been found in the case of these structurally related molecules. In vitro biological assay has been performed with Methylene blue dying to investigate the antiproliferative potency of the compounds synthesized in this work. The measured (k′) and calculated (CLOGP) lipophilicities of the compounds were compared with the antiproliferative activities and an optimum value of lipophilicity has been found for these compounds.
Keywords: Tetralones; Molecular library; Lipophilicity; Retention factor and CLOGP calculation; Antiproliferative activity;

I, 2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid is an α peroxisome proliferator-activated receptor (PPAR) agonist with some γ activity being investigated for potential use in the treatment of Type II diabetes mellitus and dyslipidemia. Two automated liquid–liquid extraction methods were developed and validated for the determination of I in human plasma. Concentrations of I were determined over a wide range of clinical doses. For Method A, plasma was acidified and extracted with ethyl acetate using a fully automated procedure. Analysis was performed by LC-MS/MS with a turbo ionspray source in negative ion mode. For Method B, a larger volume of plasma was extracted and a heated nebulizer source was used on the mass spectrometer. Method A was linear from 0.05 to 50 ng/mL and Method B from 0.2 to 1000 ng/mL. Validation procedures showed that both methods were robust, specific and reproducible.
Keywords: Insulin sensitizer; PPAR; LC-MS/MS; Human plasma; Automated liquid–liquid extraction;

Microporous polyamide membranes were activated by bisoxirane and subsequently bound with chitosan (CS) to amplify reactive groups. Then polylysine (PLL) as ligand was immobilized onto the CS-coated nylon membranes. The contents of CS and PLL of PLL-attached membranes were 93.2 and 90.4 mg/g nylon membrane, respectively. Such PLL-attached membranes were used to adsorb bilirubin from the bilirubin–phosphate solution and bilirubin–albumin solution. The adsorption mechanism of bilirubin and the effects of temperature, initial concentration of bilirubin, albumin concentration and ionic strength on adsorption were investigated by batch experiments. The results showed that the adsorption capacity increased with increasing the temperature while decreased with increasing the NaCl concentration and albumin concentration, and the adsorption isotherm fitted the Freundlich model well. The result of dynamic experiment showed PLL-attached membranes can well remove the bilirubin from the bilirubin–albumin solution.
Keywords: Chitosan; Bilirubin; Affinity membrane; Poly-l-lysine; Adsorption;

Novel and simple high-performance liquid chromatographic method for determination of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity by Michele Buffalini; Raffaella Pierleoni; Chiara Guidi; Paola Ceccaroli; Roberta Saltarelli; Luciana Vallorani; Sabrina Zeppa; Vilberto Stocchi (307-313).
We present here a high-performance liquid chromatographic method for the evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. The automated method was applied to fungal and mouse liver extracts and validated by the addition of mevastatin to the reaction mixture and by several intra- and inter-day assays. This method offers important advantages over those previously reported because no radiolabeled substrates or expensive techniques such as mass spectrometry are required, and the time of analysis is relatively short. Moreover, the method can be successfully applied to different biological samples; hence, it should be very useful in evaluating potential inhibitors of the HMG-CoA enzyme and investigating cholesterol metabolism, cell growth and differentiation processes.
Keywords: Mevalonolactone; Cholesterol; Mevastatin; HPLC analysis;

To prove the intake of recently controlled designer drugs, N-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a simple, sensitive and reliable method which allows us to simultaneously detect BZP, TFMPP and their major metabolite in human urine has been established by coupling gas chromatography–mass spectrometry (GC–MS) and high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS). GC–MS accompanied by trifluoroacetyl (TFA) derivatization and LC–MS analyses were performed after the enzymatic hydrolysis and the solid phase extraction with OASIS HLB, and BZP, TFMPP and their major metabolites, 4′-hydroxy-BZP (p-OH-BZP), 3′-hydroxy-BZP (m-OH-BZP) and 4′-hydroxy-TFMPP (p-OH-TFMPP), have found to be satisfactorily separated on a semi-micro SCX column with acetonitrile–40 mM ammonium acetate buffer (pH 4) (75:25, v/v) as the eluent. The detection limits produced by GC–MS were estimated to be from 50 ng/ml to 1 μg/ml in the scan mode, and from 200 to 500 ng/ml in the selected ion monitoring (SIM) mode. Upon applying the LC–ESI-MS technique, the linear calibration curves were obtained by using the SIM mode for all analytes in the concentration range from 10 ng/ml to 10 μg/ml. The detection limits ranged from 5 to 40 ng/ml in the scan mode, and from 0.2 to 1 ng/ml in the SIM mode. These results indicate the high reliability and sensitivity of the present procedure, and this procedure will be applicable for proof of intake of BZP and TFMPP in forensic toxicology.
Keywords: N-Benzylpiperazine; BZP; Hydroxy-N-benzylpiperazine; 1-(3-Trifluoromethylphenyl)piperazine; TFMPP; Hydroxy-1-(3-trifluoromethylphenyl)piperazine; GC–MS; LC–ESI-MS;

Enantioselective analysis is used as a valuable tool for determining the biological origin of chiral derivatives of arachidonic, 11,14-eicosadienoic and linoleic acid in psoriatic skin scales and for clarifying their role in pathogenesis. This paper reports on a simple and rapid enantioselective determination (without any derivatization) of the fatty acid derivatives 13(R,S)-hydroxyoctadecadienoic acid [13(R,S)-HODE], 9(R,S)-hydroxyoctadecadienoic acid [9(R,S)-HODE] and 12(R,S)-hydroxyeicosatetraenoic acid [12(R,S)-HETE], using high-performance liquid chromatography (HPLC) with Chiralpak® AD as the chiral selector and electrospray ionisation mass spectrometry (ESI-MS). The enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE in psoriatic skin scales of untreated patients (untreated during the last 4 weeks before sampling) was evaluated in comparison to psoriatic skin scales of patients underlying systemic treatment. The enantiomeric distribution of 12-HETE and 9-HODE showed no remarkable differences, whilst samples of patients under systemic treatment exhibited a lower predominance of 13(S)-HODE than samples of untreated patients. Furthermore, the effect of UVB phototherapy on the enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE was studied and a semiquantitation of these compounds in psoriatic skin scales performed. The detected amounts of 9-HODE in samples of untreated patients were remarkably lower than those in samples of patients underlying systemic treatment. In the case of UVB phototherapy, no influence on the enantiomeric distribution could be observed.
Keywords: Enantioselective analysis; Psoriasis; Polyunsaturated hydroxy fatty acids; Chiralpak® AD; High-performance liquid chromatography; UVB; Systemic treatment;

Quantitative determination of perfluorooctanoic acid in serum and plasma by liquid chromatography tandem mass spectrometry by John M. Flaherty; Paul D. Connolly; Emily R. Decker; S. Mark Kennedy; Mark E. Ellefson; William K. Reagen; Bogdan Szostek (329-338).
A selective and sensitive method for analysis of perfluorooctanoic acid (PFOA) in human serum and plasma, utilizing liquid chromatography tandem mass spectrometry (LC–MS/MS), has been developed and thoroughly validated to satisfy strict FDA guidelines for bioanalytical methods. A simple, automated sample preparation procedure, involving extraction of the target analyte with acetonitrile on protein precipitation media in a 96-well plate format was developed, allowing efficient handling of large numbers of samples. The proposed method uses the calibration standards prepared in a surrogate matrix (rabbit serum or plasma) and 13C-labeled PFOA as the internal standard to account for matrix effects, instrument drift, and extraction efficiency. Human serum and plasma could not be used for matrix matching of calibration standards as endogenous levels of PFOA observed in the control human serum and plasma significantly exceeded the targeted lower limit of quantitation (LLOQ) of the method. Precision and accuracy of the method were demonstrated by analysis of rabbit serum and plasma control samples fortified at 0.5, 5, and 40 ng/mL PFOA and human serum and plasma fortified at 1.0, 5.0, 40 ng/mL PFOA. The LLOQ of 0.5 ng/mL PFOA was experimentally demonstrated for rabbit and human serum and plasma. Within-day precision and accuracy, short-term stability, freeze–thaw stability, equivalence of response between PFOA and APFO (the ammonium salt of PFOA), and dilution of concentrated samples were also investigated. The results of the validation experiments comply with the precision and accuracy limits defined by the FDA guidance document: “Guidance for Industry, Bioanalytical Method Validation”, May 2001.
Keywords: Perfluorooctanoic acid; LC–MS/MS; Serum; Plasma;

Development of a sensitive and specific technique for the quantitation of drug metabolites without the use of synthetic analytical standards or radiolabel would represent a major advance in preliminary route of metabolism screening in drug discovery. In this study, the ability of evaporative light-scattering detection (ELSD) to quantify metabolites of 7-ethoxycoumarin (EC) was evaluated. Because ELSD operates as a mass detector, the complex nature of in vitro-derived samples from hepatocyte incubations resulted in an inability to detect the analytes of interest in this matrix using ELSD. Additionally, the gradient nature of the analysis required to temporally separate ethoxycoumarin from its metabolites and matrix components interfered with the ELSD response. Furthermore, using less-complex contrived mixtures, ELSD demonstrated insufficient sensitivity (limit of detection of 1000–10,000 ng/mL) and an inconsistent inter-analyte response. Together, the limitations outlined in these experiments demonstrate that ELSD is at present an inadequate technique for generating semi-quantitative data on metabolites in drug discovery.
Keywords: Evaporative light-scattering detector; Metabolism; 7-Ethoxycoumarin; Metabolite identification; HPLC;

Author Index (347-350).

Keyword Index (351-358).