Journal of Chromatography B (v.877, #10)

A sensitive high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for the determination of DNA adducts induced by nephrotoxic and carcinogenic aristolochic acid (AA) is presented. The DNA adduct of AAII (dA-AAII) was synthesized by in vitro incubation, purified by preparative HPLC, characterized using fluorescence spectroscopy and high-resolution mass spectrometry, and was used as the biomarker for AA exposure in rats. The developed HPLC-FLD method was validated and applied for the determination of dA-AAII in rat kidney tissues. The method provided a detection limit of 18.3 fmol, which allowed the detection of dA-AAII in the rat kidney tissue samples collected after a single oral dose of AA. dA-AAII was detected in the kidney DNA digestion extracts of the rats that were dosed with AA at 5 mg/kg and 30 mg/kg at concentrations of 6.2 ± 1.1 and 41.3 ± 8.0 dA-AAII per 109 normal dA, respectively.
Keywords: Aristolochic acid; DNA-AA adduct; Kidney tissue; High-performance liquid chromatography; Fluorescence detection;

A robust and rapid high-pressure liquid chromatography–electrochemical detection (HPLC–ECD) method was developed and validated for the accurate determination of ascorbic acid (AA) and uric acid (UA), in human plasma. Dehydroascorbic acid (DHAA) was indirectly measured by subtracting native ascorbic acid from total ascorbic acid concentrations; the latter was obtained after chemical reduction. A stable electrochemical active internal standard (homogentisic acid) was added for the accurate quantification of the analytes. The analyses were performed on a reverse-phase column with traditional HPLC and ultra-HPLC (UHPLC). The UHPLC method showed increased sensitivity with detection limit of 0.05 ng for both AA and UA, 2 times lower compared to conventional HPLC. UHPLC also reduced run times fourfold with less waste generation. Both assays showed good accuracy and precision, the intra- and inter-day CVs of AA and UA analysis are less than 7%.
Keywords: Ascorbic acid; Dehydroascorbic acid; Homogentisic acid; Internal standard; Ultra-HPLC; Electrochemical detection;

Analysis of BNP7787 thiol-disulfide exchange reactions in phosphate buffer and human plasma using microscale electrochemical high performance liquid chromatography by Dakshine Shanmugarajah; Daoyuan Ding; Quili Huang; Xinghai Chen; Harry Kochat; Pavankumar N. Petluru; Philippe Y. Ayala; Aulma R. Parker; Frederick H. Hausheer (857-866).
BNP7787 (disodium 2,2′-dithio-bis ethane sulfonate; Tavocept™) is a novel water-soluble investigational agent that is undergoing clinical development for prevention and mitigation of cisplatin-induced nephrotoxicity. BNP7787 is a disulfide that undergoes thiol-disulfide exchange reactions in vivo with physiological thiols. Mesna-disulfide heteroconjugates that form as a result of these exchange reactions may play a key role in the protection against cisplatin-induced nephrotoxicity. Although several analytical methods have been used to detect thiols and disulfides, they have notable limitations including (i) low sensitivity, (ii) interference by chemical modification by derivatization reagents, and (iii) cumbersome sample preparation. In this paper, a sensitive micro-HPLC-EC method is described that identifies BNP7787 and mesna in plasma and phosphate buffer across a broad concentration range from 500 nM to 100 μM. This method utilizes a dual electrochemical detector equipped with a wall-jet gold electrode. The approach described here facilitates the identification of BNP7787 and mesna down to nanomolar levels. Although we did not focus on optimizing the approach for other thiol and disulfide compounds, we believe this approach could be optimized and used in the identification of other thiols and disulfides in plasma. The assay requires significantly less sample preparation and does not involve the use of derivatizing agents (i.e., the thiol and disulfide species can be detected directly) and represents an important advance over previous methods. This method was used to detect and quantitate BNP7787 and to monitor and kinetically characterize the interactions of BNP7787 with glutathione, cysteine, cysteinyl-glycine, cysteinyl-glutamate and homocysteine.
Keywords: BNP7787; Electrochemical detection; HPLC-EC; Human plasma; Mesna; Mesna-cysteine; Mesna-cysteinyl-glycine; Mesna-cysteinyl-glutamate; Mesna-glutathione; Mesna-homocysteine; Thiol-disulfide exchange reactions;

A single LC–tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma by E.M. Hodel; B. Zanolari; T. Mercier; J. Biollaz; J. Keiser; P. Olliaro; B. Genton; L.A. Decosterd (867-886).
Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography–tandem mass spectrometry method (LC–MS/MS) requiring 200 μl of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20 mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20 mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1–12.6%) and sensitive (lower limits of quantification 0.15–3.0 and 0.75–5 ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC–MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 °C for up to 48 h before storage at −80 °C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.
Keywords: LC–MS/MS; Antimalarials; Pharmacokinetics; Plasma;

A CTAB based method for the preparation of total protein extract of wine spoilage microrganisms for proteomic analysis by Rita Polati; Giacomo Zapparoli; Paolo Giudici; Alessandra Bossi (887-891).
Mapping the proteome of microrganisms by 2D-electrophoresis is often a hard task, because many contaminants, e.g. polysaccharides of the cell wall and nucleic acid, can obstruct the pores of the IEF gel resulting in streaks and smears. A protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its salt-dependent solubility was developed. The cellulose-producing strain Gluconoacetobacter hansenii AAB0248 was resolved on 7 cm Minigels in over 500 protein spots (a hundred more than with protocols reported in literature). The method was further employed for mapping the proteome of some acid adapted, wine spoilage microrganisms e.g. acetic acid bacteria and a yeast.
Keywords: CTAB; Salt-dependent solubility; Wine spoilage; Acetic acid bacteria; Yeast; Proteome;

Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis by Maria Anderot; Mikael Nilsson; Ákos Végvári; Eva Horn Moeller; Marco van de Weert; Roland Isaksson (892-896).
In this manuscript we report the binding affinity between two model proteins, human serum albumin (HSA) and ribonuclease A (RNase A), and negatively charged polyelectrolytes, two different heparin fractions and dextran sulfate, by means of partial filling and affinity capillary electrophoresis. The apparent dissociation constants, K d, obtained by use of the partial-filling method, between HSA and heparin (17 kDa), heparin (3 kDa) and dextran sulfate (8 kDa) were 33 and 307 μM, respectively. A new method was developed to determine affinities that take in account different migration directions between the protein and the polyelectrolyte, which was required to study RNase A. By use of this affinity capillary electrophoresis two K d values were observed for the interaction between RNase A and heparin 17 kDa, yielding a high affinity binding with K d1 0.0075 μM, and a lower affinity binding with K d2 8.7 μM. For dextran sulfate 8 kDa these K d values were 0.027 and 10.4 μM, respectively. Heparin 3 kDa only showed a single K d value of 0.52 μM. The results show that the magnitude of the binding affinity depends on the type of polyelectrolyte and its molecular weight.
Keywords: Dissociation constant; Polyelectrolytes; Heparin; Dextran sulfate; Human serum albumin; Capillary electrophoresis; Ribonuclease A; RNase; Partial filling; Affinity; Binding;

To support the clinical pharmacokinetic trials of bencycloquidium bromide (BCQB), a specific and sensitive method based on weak cation-exchange solid phase extraction (WCX-SPE) and HPLC–ESI–MS techniques for the determination of BCQB in human plasma was developed and validated. BCQB and the internal standard 1-ethyl-bencycloquidium bromide were separated on a Zorbax Eclipse Plus C18 column (3.5 μm, 150 mm × 2.1 mm i.d.) with a mobile phase consisted of 20 mM ammonium acetate buffer solution containing 1% acetic acid (pH 3.6)–methanol (50:50, v/v), and the chromatographic run time for one sample was 8 min. The lower limit of quantitation (LLOQ) of the method is 5 pg/ml, which is critically important for the pharmacokinetic study of BCQB. The method possesses a reliable quantification range of 5–1000 pg/ml, the acceptable intra- and inter-batch precision of less than 8.9%, and the extraction recovery of more than 90.6%. The method was successfully applied for the single-dose pharmacokinetic study of BCQB nasal spray in healthy Chinese volunteers.
Keywords: Bencycloquidium bromide; SPE; WCX; LC–MS; Pharmacokinetics;

A selective and rapid method was developed and validated for determination of 6R-leucovorin (LV), 6S-leucovorin and 5-methyltetrahydrofolate (5-MeTHF) in human plasma using stereoselective liquid chromatography–tandem mass spectrometry. All analytes and the internal standard were extracted from plasma by solid phase extraction using Oasis® HLB C18 cartridges. A macrocyclic glycopeptide teicoplanin column was used for chiral separation of LV and 5-MeTHF isomers with NH4TFA or NH4OAc in methanol as mobile phase. Detection was performed on an API 4000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves were linear in the range of 0.050–20.0 μg/mL for 6R-LV and 6S-LV, and 0.025–10.0 μg/mL for 5-MeTHF. The intra- and inter-assay precision was 3.6–13.2%, 3.4–12.9% and 5.3–9.3% for 6R-LV, 6S-LV and 5-MeTHF, respectively. The accuracy was 99.4–102.4%, 95.3–96.8% and 93.0–110% for 6R-LV, 6S-LV and 5-MeTHF, respectively. The lower limit of quantification (LLOQ) was 0.050 μg/mL for each LV isomer and 0.025 μg/mL for 5-MeTHF. The method was successfully applied to a comparative pharmacokinetic study between leucovorin calcium and levoleucovorin calcium in 10 volunteers. No significant differences between levoleucovorin and leucovorin in pharmacokinetic parameters of 6S-LV and 5-MeTHF were found in volunteers.
Keywords: Stereoselective separation; Leucovorin; 5-Methyltetrahydrofolate; LC–MS/MS;

Thermodynamic study of the interaction between terbutaline and salbutamol with an immobilized β 2-adrenoceptor by high-performance liquid chromatography by Xinfeng Zhao; Xiaohui Zheng; Yinmao Wei; Liujiao Bian; Shixiang Wang; Jianbin Zheng; Youyi Zhang; Zijian Li; Weijin Zang (911-918).
Investigation of the interaction between drugs and receptors is very important in revealing the biologic basis and mechanism of the drug, and designing new bioactive compounds. The β 2-adrenoceptor (β 2-AR) was purified from rabbit lung tissues and immobilized on the surface of macro-pore silica gel through covalent bonds to prepare the stationary phase. Binding isotherms of terbutaline and salbutamol were determined by frontal analysis and the perturbation method, respectively. On the basis of the model of binding isotherm assumed, zonal elution was used to investigate the binding interaction of the receptor with terbutaline and salbutamol. The two drugs had one type of common binding site on immobilized β 2-AR. Salbutamol had at least one other major binding region. The association constant for terbutaline was (9.76 ± 0.67) × 104/M, and the concentration of the binding sites was (9.37 ± 1.32) × 10−6  M. Under identical conditions, association constants for salbutamol at the two types of binding site were (1.11 ± 0.08) × 104/M and (1.34 ± 0.13) × 103/M, and the concentration of the binding sites was (5.46 ± 0.35) × 10−6  M. Entropy increase was the main driving force for terbutaline and salbutamol to bind with β 2-AR. The associating reaction of terbutaline and β 2-AR was an exothermal process primarily from electrostatic interactions; hydrophobic force was the major factor contributing to the process for salbutamol.
Keywords: β 2-Adrenoceptor; Terbutaline; Salbutamol; Zonal elution; Thermodynamics;

AT1 receptor blockers are agents for the treatment of hypertension. Rapid and precise assay methods are needed to evaluate possible sub- or overdosage. A direct on-line solid phase extraction coupled to tandem mass spectrometry was developed and validated to determine valsartan (5–2000 ng/mL) or candesartan (1–200 ng/mL) in human plasma and urines. Both intra- and inter-assay accuracy and precision were in the range 89.2–111% and 0.9–16.9% RSD. Total run time was 4.5 min. This on-line clean-up–MS method was found to be robust for the analysis of a high number of samples with unvarying performance.
Keywords: On-line solid phase extraction; Mass spectrometry; High throughput; Valsartan; Candesartan;

Liquid chromatography–tandem mass spectrometry for the determination of methylprednisolone in rat plasma and liver after intravenous administration of its liver-targeted dextran prodrug by Shuang-Qing Zhang; Helen R. Thorsheim; Suman Penugonda; Venkateswaran C. Pillai; Quentin R. Smith; Reza Mehvar (927-932).
A specific and sensitive liquid chromatography (LC)–tandem mass spectrometric method for quantitative determination of methylprednisolone (MP) in rat plasma and liver was developed and validated using triamcinolone acetonide as an internal standard. Liquid–liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma and liver. The separation of MP was performed on a C18 column with a mobile phase of acetonitrile:0.5% formic acid aqueous solution (85:15, v/v) over 4 min. The assay was based on the selected reaction monitoring transitions at m/z 375 → 161 for MP in plasma, 375 → 357 for MP in liver, and 435 → 415 for internal standard in both plasma and liver. The lower limit of quantification was 20 ng/mL based on 100 μL of plasma or liver homogenate. Intra- and inter-day assay variations were ≤15%, and the accuracy values were between 85.8% and 118%. The extraction recoveries ranged from 76.8% to 79.2% for plasma and 76.8–80.8% for liver across the calibration curve range. The method was successfully applied to the measurement of low concentrations of regenerated MP in plasma and liver after intravenous administration of a single dose (5 mg/kg) of a liver-targeted dextran prodrug of MP to rats.
Keywords: Methylprednisolone; Dextran–methylprednisolone prodrug; Rat plasma; Rat liver; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics;

Peramivir is a novel influenza neuraminidase inhibitor. In this article, hydrophilic interaction chromatography coupled with tandem mass spectrometry was developed to determine peramivir in human plasma. The positive ion MRM mode was performed and the precursor to the product ion transitions of m/z 329 → 100 and 285 → 138 were used to measure peramivir and Ro 64-0802 (I.S.). Chromatographic separation was performed on an Amide-80 column with acetonitrile–water–formic acid (70:30:0.1, v/v/v, 0.5 mL/min). The method was linear over a concentration range of 10–10,000 ng/mL. The average inter-day/intra-day precision values were 3.7 ± 1.8% and 4.3 ± 1.8%, respectively, while the accuracy values were 97.0 ± 4.8%. This method has been successfully applied to Phase I of clinical research of peramivir.
Keywords: Hydrophilic interaction chromatography (HILIC); Tandem mass spectrometry; TSK gel Amide-80 column; Peramivir; Neuraminidase inhibitor; Avian influenza;

Synthesis of the flavonoid 3′,4′,5′-trimethoxyflavonol and its determination in plasma and tissues of mice by HPLC with fluorescence detection by Robert G. Britton; Isabel Fong; Shaban Saad; Karen Brown; William P. Steward; Andreas Gescher; Stewart Sale (939-942).
3′,4′,5′-Trimethoxyflavonol (TMFol) was synthesized as a potential colorectal cancer chemopreventive agent. An HPLC method for determination for TMFol in murine plasma and tissues was developed and validated using human plasma. Analyte was separated (C18 column; fluorescence detection 330 nm excitation, 440 nm emission) using 69% methanol and 0.1 M ammonium acetate buffer (pH 5.1) as mobile phase. The method was linear for 50–2500 ng/ml plasma and 0.05–10 μg/g tissue (r  > 0.99). TMFol was recovered from plasma or tissues using solid phase columns or organic solvent protein precipitation, respectively. Recovery at low, medium and high concentrations was 97.6–107.3%, with inter- and intra-day coefficients of variation of <10%. The lower limit of quantitation for plasma was 50 ng/ml. The method was applied to measure steady-state TMFol plasma and tissue levels in mice which received dietary TMFol (0.2%).
Keywords: 3′,4′,5′-Trimethoxyflavonol; High performance liquid chromatography; Cancer chemoprevention;

CVT-6883, a novel selective A2B adenosine receptor antagonist currently under clinical development, is highly lipophilic and exhibits high affinity for non-specific binding to container surfaces, resulting in very low recovery in urine assays. Our study showed the use of sodium dodecylbenzenesulfonate (SDBS), a low-cost additive, eliminated non-specific binding problems in the analysis of CVT-6883 in human urine without compromising sensitivity. A new sensitive and selective LC–MS/MS method for quantitation of CVT-6883 in the range of 0.200–80.0 ng/mL using SDBS additive was therefore developed and validated for the analysis of human urine samples. The recoveries during sample collection, handling and extraction for the analyte and internal standard (d 5-CVT-6883) were higher than 87%. CVT-6883 was found stable under the following conditions: in extract – at ambient temperature for 3 days, under refrigeration (5 °C) for 6 days; in human urine (containing 4 mM SDBS) – after three freeze/thaw cycles, at ambient temperature for 26 h, under refrigeration (5 °C) for 94 h, and in a freezer set to −20 °C for at least 2 months. The results demonstrated that the validated method is sufficiently sensitive, specific, and cost-effective for the analysis of CVT-6883 in human urine and will provide a powerful tool to support the clinical programs for CVT-6883.
Keywords: CVT-6883; A2B adenosin receptor antagonist; Human urine; Sodium dodecylbenzenesulfonate; SDBS; Adsorptive losses; Non-specific binding; LC–MS/MS; Method validation;

Coupling of a vented column with splitless nanoRPLC-ESI-MS for the improved separation and detection of brain natriuretic peptide-32 and its proteolytic peptides by Genna L. Andrews; Christopher M. Shuford; John C. Burnett; Adam M. Hawkridge; David C. Muddiman (948-954).
The circulating concentration of a biomarker for congestive heart failure, Brain (B-type) Natriuretic Peptide (BNP-32), is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of molecular specificity afforded by these assays has recently come into question as emerging studies indicate there are potentially multiple heterogeneous forms of BNP in circulation with immunoreactive capabilities. In order to better understand the molecular biology of BNP-32 as it relates to congestive heart failure, it would thus be advantageous to use a detection platform such as Fourier transform ion cyclotron resonance mass spectrometry. This high resolving power mass spectrometer can provide unparalleled molecular specificity and can facilitate identification and characterization of the various molecular forms across all disease states. Unfortunately, BNP circulates at low concentrations (as low as 3 fmol/mL). Thus, it will require a collaborative effort from a number of orthogonal front-end technologies to overcome the disconnect between the practical detection limits of this instrument platform and the physiological levels of BNP-32 and its alternative molecular forms. Herein, we begin optimization of these front-end techniques by first enhancing the conditions for online nanoLC-ESI-MS separations of BNP-32 and its proteolytic fragments. Through extensive analysis of various chromatographic parameters we determined that Michrom Magic C8 stationary phase used in conjunction with a continuous, vented column configuration provided advanced chromatographic performance for the nano-flow separations involving intact BNP-32 and its associated tryptic peptides. Furthermore, conditions for the tryptic digestion of BNP-32 were also studied. We demonstrate that the use of free cysteine as an alkylation quenching agent and a secondary digestion within the digestion scheme can provide targeted tryptic peptides with increased abundances. Combined, these data will serve to further augment the detection of BNP-32 by LC–MS.
Keywords: Vented column; BNP-32; Splitless nanoLC; Tryptic digestion;

Countercurrent chromatographic (CCC) separation and purification of various ribonucleases (RNases) was performed using the small-scale cross-axis coil planet centrifuge (X-axis CPC) with aqueous–aqueous polymer phase systems. RNases B and A were well resolved from each other with an aqueous–aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate (pH 9.2) as the mobile lower phase. The commercial RNase A samples obtained from three different companies were also highly purified using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system (pH 6.6) using the upper phase as the mobile phase. Recombinant RNase Po1, an RNase T1 family enzyme, was further successfully separated from the crude extract using the same solvent system with the lower phase used as the mobile phase. The RNase activities were well preserved during the CCC separation. The overall results demonstrate that the small-scale X-axis CPC is useful for a simple and rapid purification of various RNases in a preparative-scale.
Keywords: Countercurrent chromatography; Cross-axis coil planet centrifuge; Protein; Ribonuclease; Aqueous–aqueous polymer phase system;

Pharmacokinetics of fluoroquinolones in critical care patients: A bio-analytical HPLC method for the simultaneous quantification of ofloxacin, ciprofloxacin and moxifloxacin in human plasma by Julie De Smet; Koen Boussery; Kirsten Colpaert; Peter De Sutter; Peter De Paepe; Johan Decruyenaere; Jan Van Bocxlaer (961-967).
A high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed and validated for the simultaneous quantification of ofloxacin, ciprofloxacin and moxifloxacin in human plasma. Sarafloxacin was used as internal standard. Chromatography was carried out using a Waters XBridge™ C18 HPLC column and a gradient mobile phase consisting of CH3CN/MeOH/0.025 M TBA·Cl/TFA (eluent A at 75/25/899/1 (v/v); eluent B at 150/50/799/1 (v/v); both at pH 3.5). Excitation/emission wavelengths were 279/442 nm for ciprofloxacin and 290/500 nm for ofloxacin, moxifloxacin and internal standard. Prior to chromatography, plasma samples were treated with acetonitrile for protein precipitation, followed by evaporation of the liquid layer and reconstitution in eluent A. The method was validated for the three fluoroquinolones over the clinically relevant concentration range from 0.02 to 7.50 μg/ml. The method showed acceptable linearity with correlation coefficients, r 2  > 0.995, as well as high precision (RSD% <7% in each case), accuracy (90.4–105.4%) and selectivity. The limit of quantification for the three fluoroquinolones was established at 0.02 μg/ml. Ofloxacin, ciprofloxacin and moxifloxacin were extracted from plasma with a mean recovery of 95%, 86.4% and 94.2%, respectively. During validation, the concentration of the three fluoroquinolones was found to be stable after 3 freeze–thaw cycles and for at least 15 h after extraction. This bio-analytical method was finally applied to the analysis of samples which have been obtained from patients, participating in a pharmacokinetic study on moxifloxacin.
Keywords: Fluoroquinolones; Moxifloxacin; Ciprofloxacin; Ofloxacin; Plasma; HPLC; Fluorescence; Validation;

A rapid and reliable method was developed to quantitate tolterodine and its 5-hydroxymethyl metabolite in human plasma using liquid chromatography–electrospray tandem mass spectrometry. The assay was based on liquid–liquid extraction of the compounds from plasma with tert-butylmethylether and hydrophilic interaction chromatography performed on a silica column (30 mm × 4.6 mm, 3 μm particles), the mobile phase consisted of acetonitrile-20 mM ammonium acetate (70:30, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 326 → 147 for tolterodine, 342 → 223 for the 5-hydroxymethyl metabolite and 260 → 183 for the internal standard propranolol, respectively. The lower limit of quantitation was 49 and 46 pg/ml using 0.5 ml of plasma for the parent drug and its metabolite, respectively and linearity was observed up to 30 ng/ml. Within-day and between-day precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 7% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.
Keywords: Tolterodine; LC–MS/MS; HILIC; Pharmacokinetics;

A modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheat by Hetty C. van den Broeck; Antoine H.P. America; Marinus J.M. Smulders; Dirk Bosch; Rob J. Hamer; Ludovicus J.W.J. Gilissen; Ingrid M. van der Meer (975-982).
The detection, analysis, and quantification of individual celiac disease (CD) immune responsive gluten proteins in wheat and related cereals (barley, rye) require an adequate and reliable extraction protocol. Because different types of gluten proteins behave differently in terms of solubility, currently different extraction protocols exist. The performance of various documented gluten extraction protocols is evaluated for specificity and completeness by gel electrophoresis (SDS-PAGE), immunoblotting and RIDASCREEN® Gliadin competitive ELISA. Based on these results, an optimized, two-step extraction protocol has been developed.
Keywords: Celiac disease; Gluten protein extraction; Gliadins; Glutenins; Immunoblotting; Monoclonal antibodies; Wheat; R5;

Sphingosine-1-phosphate (S1P) is an important sphingolipid signaling molecule that regulates numerous cellular processes. In this paper we report a new method to quantify the levels of S1P in biological samples that relies on derivatization with naphthalene-2,3-dicarboxaldehyde (NDA) and quantification by reverse-phase high performance liquid chromatography (HPLC). The limit of detection (LOD) using S1P standards was 20.9 fmol (12.6 nM), and the limit of quantification (LOQ) was 69.6 fmol (41.7 nM). The recovery of S1P standards was up to 97.5%. The mean relative standard deviations (RSD) for the intra- and inter-day assay were 4.1% and 7.7%, respectively. To validate this procedure, we quantified the S1P levels in plasma from human, horse, and mouse (mean values of 0.45, 0.25, and 1.23 μM, respectively). We also used this technique to evaluate the S1P content in mouse tissues, as well as in rat neuronal cell cultures before and after sphingosine treatment. The results demonstrate that this new procedure can provide fast, sensitive, and reproducible S1P quantification, and offers several advantages over existing methods. The technique also may be used for determining the activity, as well as the inhibition, of sphingosine kinase. In the future it could be an important tool for investigators studying the role of S1P in signal transduction, cell growth and differentiation, and disease pathogenesis and treatment.
Keywords: Sphingosine-1-phosphate; Naphthalene-2,3-dicarboxaldehyde; HPLC; Biological samples;

An electroanalytical method for the determination of lincomycin in feeds, honey, milk and urine was demonstrated in this study. The procedure employed a solid-phase extraction for the isolation of lincomycin from real samples. The antibiotic residues were subsequently analyzed by high-performance liquid chromatography (HPLC) coupled with a disposable electrochemical sensor. The use of a disposable sensor together with the application of solid-phase extraction is attractive in practical application and should be useful in fast screening assay. The electroanalysis of lincomycin was first investigated using a preanodized screen-printed carbon electrode (SPCE*). Note that the SPCE* holds the advantages of low cost and easy to handle. The analytical parameters, such as, preanodization potential, preanodization time, solution pH, detection potential, cartridge, wash solution, elute solution and mobile phase, were further studied in detail. Under optimized conditions, the linear detection range for lincomycin is up to 1 mM (correlation coefficient = 0.999) with a detection limit of 0.08 μM (S/N = 3) and a quantification limit of 0.27 μM (S/N = 10). The applicability of the method was successfully demonstrated in real sample analysis.
Keywords: Lincomycin; Screen-printed carbon electrode; Solid-phase extraction; HPLC;

A sensitive and rapid method of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed and applied for the determination of aristolochic acid I (AAI) in rat urine and plasma. Prior to the HPLC analysis, the samples were derivatized to increase fluorescence character. The linear ranges of the calibration curves were 0.48–48 ng in urine and 0.23–69 ng in plasma. The intra- and inter-day precisions referred by relative standard deviation (RSD) were less than 3.2% and 4.0% for urine samples as well as less than 9.4% and 10.8% for plasma samples. The limits of quantification were 0.09 and 0.07 ng in urine and plasma, respectively. The developed method was successfully applied for the determination of AAI in rat urine and plasma samples collected after the oral administrations of AAI standard and Aristolochia contorta Bge. (Madouling) or Aristolochia manshuriensis (Guanmutong) herbal extracts. The ratios of the detected AAI amount in urine compared to the dosing amount of AAI were approximately constant. The concentrations of AAI in rat plasma were much lower than those in urine. The obtained results indicated that the metabolism of the AAI standard and AAI-containing herbs might be different, probably due to the complicated and multiple components in the herbs.
Keywords: Aristolochic acid; Pre-column derivatization; High-performance liquid chromatography; Fluorescence detection; Traditional Chinese medicine;

Preparative purification of antiamyloidogenic stilbenoids from Vitis vinifera (Chardonnay) stems by centrifugal partition chromatography by N. Zga; Y. Papastamoulis; A. Toribio; T. Richard; J.C. Delaunay; P. Jeandet; J.H. Renault; J.P. Monti; J.M. Mérillon; P. Waffo-Téguo (1000-1004).
Five stilbenoids, E-resveratrol, E-piceatannol, (+) E-(ɛ)-viniferin, (+)-ampelopsin A and vitisin C were isolated from methyl tert-butyl ether (MtBE) stem extract of Vitis vinifera (Chardonnay cv). Their purification on a preparative scale was obtained by centrifugal partition chromatography (CPC) using quaternary Arizona solvent systems composed of n-heptane/ethyl acetate/methanol/water. We tested 23 Arizona solvent systems to partition the extract and found that systems K and M (Hept/EtOAc/MeOH/water, 1:2:1:2 and 5:6:5:6, respectively; v/v) were the best to separate the stilbenes mentioned above. This support-free liquid–liquid chromatographic procedure made it possible to isolate ampelopsin A from V. vinifera for the first time. The antiamyloidogenic activity of the isolated stilbenes was evaluated versus β-amyloid fibrils. E-resveratrol and (+)-ampelopsin A were found to be the most active compounds with 63 and 46% inhibition at 10 μM, respectively. These findings suggest that E-resveratrol and (+)-ampelopsin A may function as attractive new candidates for protecting against brain cell dysfunction in vivo in AD by inhibiting the aggregation of Aβ.
Keywords: Stilbenoids; Grapevine; Centrifugal partition chromatography; Antiamyloidogenic activity; β-Amyloid fibrils; Alzheimer's disease (AD); Ampelopsin A;

Simultaneous measurement of diazolidinyl urea, urea, and allantoin in cosmetic samples by hydrophilic interaction chromatography by Takahiro Doi; Keiji Kajimura; Satoshi Takatori; Naoki Fukui; Shuzo Taguchi; Shozo Iwagami (1005-1010).
A new HPLC method for simultaneous measurement of diazolidinyl urea (DU), urea, and allantoin by hydrophilic interaction chromatography using a column packed with triazol-bonded silica particles is described. The calibration curves of DU, urea, and allantoin were linear over the ranges 2.5–125.0, 30–1250, and 0.25–18.75 mg/L, respectively. The recoveries of DU, urea, and allantoin from homemade cosmetic samples ranged from 92.84% to 101.69%, 98.19% to 103.22%, and 95.75% to 102.09%, respectively. The peak relative standard deviation (RSD) values for the recovery tests of 3 concentrations/5 replicates were 3.03% for all compounds. In day-to-day measurement of recovery tests from homemade lotions over 3 consecutive days, the RSDs were less than 2.5% in all cases. This method was well validated and would be helpful for quickly analyzing these compounds in cosmetic samples.
Keywords: Diazolidinyl urea; Allantoin; Urea; Hydrophilic interaction chromatography; Cosmetic;

A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with electrospray ionization in positive ion multiple reaction monitoring mode was developed for the quantification of mazindol (an anorectic agent) in human plasma. Fluoxetine was adopted as an internal standard (IS), and sample preparation involved one-step liquid/liquid extraction using ethyl acetate. The transition monitored were m/z 285 > 44 for mazindol and m/z 310 > 44 for IS. Chromatographic separation was achieved on a Capcell Pak MGII C18 column using an isocratic mobile phase, consisting of acetonitrile–20 mM ammonium formate in water (50:50, v/v, adjusted to pH 3.5 with formic acid) at a flow-rate of 0.2 mL/min. The retention times of mazindol and fluoxetine were 1.03 min and 1.45 min, respectively. The lower limit of quantitation (LLOQ) was 0.1 ng/mL using 200 μL of plasma, and no interferences were detected in chromatograms. The bench top stability of mazindol was evaluated in buffered and non-buffered plasma. The selectivity, linearity, precision, accuracy, recovery, and stability of the devised method were fully validated and absolute and relative matrix effects were evaluated. The described method provides a fast and sensitive analytical tool for determining mazindol levels in plasma, and was successfully applied to a pharmacokinetic study in 24 healthy human subjects after oral administration of 2 mg tablet formulation of mazindol under fasting conditions.
Keywords: Mazindol; LC/MS/MS; Human plasma; Pharmacokinetic study;