Journal of Chromatography B (v.821, #2)

A message to our readers, authors and reviewers regarding publication ethics by R. Bischoff; G. Hopfgartner; H.T. Karnes; W. Lindner; D.K. Lloyd (123).

Zirconia particles modified with N,N,N′,N′-ethylenediaminetetramethylenephosphonic acid (EDTPA), further referred to as r_PEZ, were studied as a support material for use in chromatography. Our previous studies have demonstrated the utility of r_PEZ in the separation of immunoglobulins from biological fluids. In the present study we sought to understand the underlying factors and identify the rate-limiting mechanisms that govern the transport of biomolecules in r_PEZ. Pulse injection techniques were used to elucidate the individual mass transfer parameters. Elution profiles obtained under retained and unretained conditions were approximated by the Gaussian equation and the corresponding HETP contributions were estimated. The dependence of the HETP values on incremental salt concentration in the mobile phase was determined. Resulting data in conjunction with the equations outlined in literature were used to estimate the theoretical number of transfer units for the chromatographic separation process. Our results indicate that surface diffusion probably plays a minor role; however pore diffusion was established to be the rate limiting mechanism for immunoglobulin G adsorption to r_PEZ. The HETP based methodology may be used to estimate the rate limiting mechanisms of mass transfer for any given chromatographic system under appropriate conditions.
Keywords: Zirconia; Pseudo-affinity matrix; Immunoglobulin; Pulse-injection; HETP;

Zalcitabine (ddC), lamivudine (3TC), didanosine (ddI), stavudine (d4T), carbovir (CBV), zidovudine (AZT), tenofovir (PMPA) and its administrated form (tenofovir diisoproxyl fumarate, TDF), are nucleosides currently approved in HIV therapy. To facilitate pharmacokinetics studies, a specific reversed-phase high-performance liquid chromatography (HPLC) method was developed for their analysis in rat plasma. The method involved a quantitative recovery of these drugs from rat plasma by solid-phase extraction on Oasis® HLB Waters cartridges followed by optimised HPLC separation on an Atlantis™ dC18 column with acetic acid–hydroxylamine buffer (ionic strength 5 mM, pH 7)-acetonitrile elution gradient. Quantitation was performed by HPLC/UV at 260 nm. Linear calibration curves were obtained within a 30–10,000 ng/mL plasma concentration range. Correlation coefficients (r 2) greater than 0.992 were obtained by least-squares regression and limits of quantification were in 30–90 ng/mL concentration range. Quantitative parameters (accuracy, intra-day repeatability and inter-day reproducibility) yielded satisfactory results. Finally, a new buffer, obtained with acetic acid and hydroxylamine, has been tested in HPLC/ESI-MS/MS and appears to be an efficient volatile buffer in the medium 5–7 pH range. Indeed, at pH 7 and low ionic strength (5 mM), its buffer capacity is one hundred times higher to that obtained for the usual acetic acid/ammonia buffer.
Keywords: HPLC; Hydroxylamine acetate buffer; Nucleoside; Antiretroviral; HIV plasma; Mass spectrometry;

In this study, we describe a simple liquid extraction (methanol/choloroform, 1:1, v/v) method for endogenous free cholesterol and administered sterols extracted from cultured Caco-2 cells. To quantify sterol contents in Caco-2 cells, a new HPLC–APCI-MS method was developed. All the sterols were baseline separated using reversed-phase column (C8, 2.1 mm × 150 mm, 3.5 μm) and isocratic conditions (90%, v/v, methanol–water mixture containing 0.2 mM ammonium acetate). The full scan mass spectra of sterols were measured by an ion trap mass spectrometer equipped with an APCI ion source. The intense fragment ions resulting from the loss of water [M + H–H2O]+ (m/z 369, 395, 397 and 399 for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively) were used for determinations. The absolute extraction recovery of sterols from the spiked cell samples were 109.7 ± 26.2, 105.7 ± 5.1, 109.8 ± 5.0 and 99.0 ± 7.0% for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively. Furthermore, no significant matrix effect was observed for the sterols in the cell samples. The sample assay was based on the internal standard method using stigmasterol as an internal standard. The method was linear over the concentration ranges of 0.45–9.0 μM (cholesterol) and 0.225–7.2 μM (sitosterol and sitostanol). The within- and between-day precision was less than 7% and accuracy ranged from 93.51 to 101.77%. The lowest limit of quantitation (LLOQ) was 0.225 μM for sitosterol and sitostanol, and 0.45 μM for cholesterol. The accuracy range was 95–106% and precision was lower than 9% for all LLOQ values.
Keywords: Cholesterol; Phytosterols; HPLC–APCI-MS; Caco-2 cells; Cellular uptake;

High-performance purification of gelsolin from plasma using anion-exchange porous hollow-fiber membrane by Kyohei Hagiwara; Shinji Yonedu; Kyoichi Saito; Tomoyuki Shiraishi; Takanobu Sugo; Tadashi Tojyo; Eisaku Katayama (153-158).
Gelsolin was purified from bovine plasma using an anion-exchange porous hollow-fiber membrane. The anion-change porous hollow-fiber membrane was prepared by radiation-induced graft polymerization of an epoxy-group-containing monomer, glycidyl methacrylate, and subsequent chemical modifications. Some of the epoxy groups of the polymer chain grafted onto the pore surface were converted into diethylamino groups, and the remaining epoxy groups were converted into 2-hydroxyethylamino groups. First, a gelsolin-containing dialyzed protein solution, prepared by pretreatments of ammonium sulfate precipitation and dialysis of plasma, was forced to permeate through the pores of an anion-exchange porous hollow-fiber membrane. Various proteins including gelsolin were adsorbed onto the anion-exchange polymer brush at a high rate with negligible diffusional mass-transfer resistance. Second, adsorbed gelsolin was specifically eluted by permeating 2 mM calcium chloride. The amount of recovered gelsolin was 0.1 mg per 1 mL of plasma. Third, the remaining adsorbed proteins were quantitatively eluted with 1 M sodium chloride, leading to a constant amount of recovered gelsolin during four cycles of purification. The total time required for gelsolin purification from 30 mL of bovine plasma was 11 h, during which the time for selective adsorption of various proteins and affinity elution of gelsolin using the anion-exchange porous hollow-fiber membrane was 20 min.
Keywords: Gelsolin; Plasma; Polymer brush; Anion exchange; Purification; Membrane chromatography; Calcium ion;

A sensitive method for quantitation of urinary 6beta-hydroxycortisol (6beta-HC) and cortisol using on-line SPE and LC–MS/MS was developed and validated. Human urine samples were injected directly onto an on-line solid phase extraction apparatus, Prospekt-2, followed by HPLC separation and electrospray triple quadrupole LC–MS/MS detection. The inter-day precision for the 6beta-HC:cortisol ratio was 7–9%. The lower limit of quantitation was 1 and 0.2 ng/mL for 6beta-HC and cortisol, respectively. Using the method we observed a diurnal variation on the 6beta-HC:cortisol ratio in healthy volunteers with the maximal ratio observed in the 2–10 pm urine collection period.
Keywords: 6beta-Hydroxycortisol; Cortisol; LC–MS/MS; CYP3A4; Human urine;

Simple sensitive and simultaneous high-performance liquid chromatography method of glucoconjugated and non-glucoconjugated porphyrins and chlorins using near infra-red fluorescence detection by Florentina Cañada-Cañada; Antonia Bautista-Sánchez; Myriam Taverna; Patrice Prognon; Philippe Maillard; David S. Grierson; Athena Kasselouri (166-172).
This paper reports, for the first time, a reversed-phase high performance liquid chromatographic method for the simultaneous determination of seven glucoconjugated and non-glucoconjugated porphyrins and chlorins, using near infra-red fluorescence detection. Chromatographic separation was performed on nucleosil-CN analytical column using an isocratic acetonitrile–0.1% (w/v) TFA at pH 1.8 (55:45, v/v) as mobile phase. Wavelength gradient was employed for sensitive detection, porphyrins derivates were monitored at λ exc  = 440 nm and λ emi  = 680 nm; and chlorins derivates at λ exc  = 420 nm, λ emi  = 650 nm. The method was validated and applied to monitor the biodegradation of a tri glucoconjugated chlorin derivative, TPC(glu)3, in spiked samples of human serum.
Keywords: Porphyrins; Chlorins; Glucoconjugated; Photodynamic therapy; HPLC;

Estimation of carboxylic acid metabolite of clopidogrel in Wistar rat plasma by HPLC and its application to a pharmacokinetic study by Sonu S. Singh; Kuldeep Sharma; Deepak Barot; P. Ram Mohan; Vidya B. Lohray (173-180).
A new HPLC method was developed for the estimation of carboxylic acid metabolite of clopidogrel bisulfate in rat plasma using atorvastatin as internal standard. Plasma samples were extracted with a mixture of ethyl acetate and di-chloro methane (80:20, v/v) followed by subsequent reconstitution in a mixture of water:methanol:acetonitrile (40:40:20, v/v). The chromatographic separation was achieved with gradient elution on Kromasil ODS, 250 mm × 4.6 mm i.d., 5 μm analytical column maintained at 30 °C. Carboxylic acid metabolite of clopidogrel as well as the internal standard were detected at a wavelength of 220 nm. The method was validated as per USFDA guidelines. Calibration curves were linear in the concentration range of 125.0–32,000 ng/ml and the correlation coefficient was better than 0.999. The extraction efficiency for the carboxylic acid metabolite of clopidogrel was more than 85.76%. The intra-day accuracy ranged from 98.9% to 101.5% with a precision of 1.30% to 6.06%. Similarly, the inter-day accuracy was between 96.2% and 101.1% with a precision of 3.47% to 4.30%. The drug containing plasma samples were stable at −70 °C for 48 days and at ambient temperature for 24 h. In the auto-sampler maintained at 15 °C, the processed and reconstituted samples were stable for 35 h. The drug containing frozen plasma samples were stable enough to with stand three freeze thaw cycles. The method was successfully applied to the pharmacokinetic study of the two different polymorphs of clopidogrel bisulfate in Wistar rat.
Keywords: Carboxylic acid metabolite of clopidogrel; Rat plasma; Validation and pharmacokinetics;

Since the NAD metabolite ADP-ribose (ADPR) has recently gained attention as a putative messenger, a method was established for the quantification of intracellular ADPR by reversed-phase HPLC. Cellular nucleotides were extracted with trichloroacetic acid, and crude cell extracts purified by solid phase extraction using a strong anion exchange matrix. After optimization of the extraction procedure, cellular ADPR levels were determined using two different reversed-phase columns (C18 versus C12), operated in ion pair mode. Intracellular ADPR concentrations in human Jurkat T-lymphocytes and murine BW5147 thymocytes were determined to be 44 ± 11 μM and 73 ± 11 μM, respectively.
Keywords: ADP-ribose; TRPM2; KCa channels; HPLC; Calcium signaling; Solid phase extraction;

Quantification by affinity perfusion chromatography of phosphorylated BRCAl and BRCA2 proteins from tumor cells after lycopene treatment by N. Chalabi; J.-C. Maurizis; L. Le Corre; L. Delort; Y.-J. Bignon; D.J. Bernard-Gallon (188-193).
A new procedure for the quantification of phosphorylated BRCA1 (P-BRCA1) and BRCA2 (P-BRCA2) proteins in breast cell lines after different treatments was carried out. Cells were cultivated with [35S]-methionine and extracts subjected to three perfusion chromatographies. First heparin affinity chromatography purified cellular DNA-binding proteins. Subsequent specific immunoprecipitation of BRCA1 and BRCA2 proteins was performed with antibodies raised against BRCA1 or BRCA2. The immune complexes were isolated by protein A affinity chromatography. Phosphorylated BRCA1 or BRCA2 proteins were then purified with a Poros 20 AL column where anti-phosphothreonine and anti-phosphoserine antibodies were previously bound. The percentage of phosphorylated BRCA1 or BRCA2 proteins was calculated as follows: 100 × dpm of P-BRCA1 or P-BRCA2 eluted from the POROS® 20AL column/total dpm eluted from POROS® 20AL column. Treatment with 10 μM lycopene increased P-BRCA1 and P-BRCA2 in the breast tumor cell line MCF7 but not in MDA-MB-231 or MCF-10a, breast tumor or fibrocystic cell lines, respectively.
Keywords: Breast cancer; BRCA1; BRCA2; Lycopene; Affinity chromatography;

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography–tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within ±15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with β-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.
Keywords: LC/MS/MS; Quercetin; Flavonoid; Analysis;

Simultaneous determination of nine fluoroquinolones in egg white and egg yolk by liquid chromatography with fluorescence detection by Zhenling Zeng; Aiguo Dong; Guixiang Yang; Zhangliu Chen; Xianhui Huang (202-209).
A liquid chromatographic (LC) method with fluorescence detection was developed for determination of nine fluoroquinolones (FQs) in egg white and yolk. Egg white samples were deproteinized with acidified ethanol (egg yolk samples with acetonitrile and acidified ethanol), followed by defatting with hexane once (white) or twice (yolk), and extracting FQs into acetonitrile. After acetonitrile was evaporated, the residue was dissolved in mobile phase, and FQs were detected in LC with a fluorescence detector. Recoveries for nine FQs from white and yolk were 74.7–85.6%, 79.1–91.2%, respectively, with excellent relative standard deviations. The limits of quantification were 5–20 ng g−1.
Keywords: Fluoroquinolones; LC; Eggs; Method;

Characterization of the urinary metabolites of dipetarudin by Mercedes López; Goetz Nowak (210-214).
Dipetarudin is a hybrid thrombin inhibitor composed of the N-terminal structure of dipetalogastin II and the exosite 1 blocking segment of hirudin. Pharmacokinetic studies demonstrated that it distributes in extravascular and intravascular spaces and is exclusively eliminated by the kidneys. Two active metabolites of dipetarudin with molecular masses of 6142 and 5395 Da, respectively, were isolated from rat urine. Analysis of their N-terminal sequences and molecular masses demonstrated that dipetarudin is cleaved in a first step at the peptide bond Phe55 ―Glu56 and then, at Gly3 ―Asn4. Nonmetabolized dipetarudin was not found in rat urine. Proteases localized in the proximal tubulus cells of kidneys might be responsible for its degradation.
Keywords: Dipetarudin; Renal metabolism; High performance liquid chromatography; Ecarin clotting time; Matrix assisted laser desorption/ionization time of flight;

Quantitation of niflumic acid in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection and its application to a bioequivalence study of talniflumate tablets by H.W. Lee; K.J. Won; S.H. Cho; Y.H. Ha; W.S. Park; H.T. Yim; M. Baek; J.H. Rew; S.H. Yoon; S.V. Yim; J.H. Chung; K.T. Lee (215-220).
A rapid and simple HPLC method with UV detection (288 nm) was developed and validated for quantitation of niflumic acid in human plasma, the active metabolite of talniflumate. After precipitation with 100% methanol containing the internal standard, indomethacin, the analysis of the niflumic acid level in the plasma samples was carried out using a reverse phase C18 CAPCELL PAK (5 μm, 4.6 mm × 250 mm) column. The chromatographic separation was accomplished with an isocratic mobile phase consisting of a mixture of 0.1 M sodium acetate in water and acetonitrile (37:63, v/v), adjusted to pH 6.4. This HPLC method was validated by examining its precision and accuracy for inter- and intra-day runs in a linear concentration range of 0.02–5.00 μg/mL. Stability of niflumic acid in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method was successfully applied to the bioequivalence study of talniflunate in healthy volunteers.
Keywords: Talniflumate; Niflumic acid; Validation; High-performance liquid chromatography (HPLC); Bioequivalence test;

Irinotecan (CPT-11) and its main metabolite SN-38 are potent anticancer derivatives of camptothecin (CPT), with active lactone and inactive carboxylate forms coexisting. A simple and sensitive HPLC method using the ion-pairing reagent tetrabutylammonium hydrogen sulfate (TBAHS) was developed to simultaneously determine all four analytes in rat plasma samples. Camptothecin (CPT) was used as internal standard. The mobile phase was 0.1 M potassium dihydrogen phosphate containing 0.01 M TBAHS (pH 6.4)–acetonitrile (75:25, v/v). Separation of the compounds was carried out on a Hypersil C18 column, monitored at 540 nm (excitation wavelength at 380 nm). All four compounds gave linear response as a function of concentration over 0.01–10 μM. The limit of quantitation in rat plasma was 0.01, 0.008, 0.005 and 0.005 μM for CPT-11 lactone, CPT-11 carboxylate, SN-38 lactone and SN-38 carboxylate, respectively. The method was successfully used in the study on the effect of coadministered thalidomide on the plasma pharmacokinetics of CPT-11 and SN-38 in rats. Coadministered thalidomide (100 mg/kg body weight by intraperitoneal injection) significantly increased the AUC0–10h values of CPT-11 lactone and CPT-11 carboxylate by 32.6% and 30.3 %, respectively, (P  < 0.01), but decreased the values by 19.2% and 32.4% for SN-38 lactone and carboxylate, respectively, (P  < 0.05). Accordingly, the value of total body clearance (CL) of CPT-11 lactone was significantly lower in combination group compared to the control (1.329 versus 1.837 L/h/kg, P  = 0.0002). Plasma t 1/2β values for SN-38 lactone and carboxylate were significantly (P  < 0.01) smaller in rats with coadministered thalidomide, as compared to rats receiving CPT-11 alone. Further studies are needed to explore the underlying mechanisms for the observed kinetic interaction between CPT-11 and thalidomide.
Keywords: HPLC; Irinotecan; SN-38; Lactone; Carboxylate;

The monitoring of uric acid (UA) and p-aminohippuric acid (PAH) levels in biological samples is routinely carried out in clinical laboratories as an indication of renal disease. With the aim of investigation of the correlation between the trace amounts of UA and PAH in human saliva or urine and renal diseases, we carried out the determination of UA and PAH in human saliva and urine by using capillary electrophoresis with electrochemical detection (CE-ED) in this work. Under the optimum conditions, UA, PAH and three coexisting analytes could be well separated within 21 min at the separation voltage of 14 kV in 80 mmol/L borax running buffer (pH 9.2). Good linear relationship was established between peak current and concentration of analytes over two orders of magnitude with detection limits (S/N  = 3) ranged from 5.01 × 10−7 to 2.00 × 10−6  mol/L for all analytes. The result shows that this proposed method could be successfully applied for the study on the correlation between the levels of UA and PAH in human saliva and urine and renal diseases, and provide an alternative and convenient method for the fast diagnosis of renal disease.
Keywords: Capillary electrophoresis; Electrochemical detection; Renal disease; Saliva; Urine; Uric acid; p-Aminohippuric acid;

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of epimedin C in rat plasma and applied to a pharmacokinetic study in rats after administration of Herba Epimedii extract. After addition of carbamazepine as an internal standard plasma samples were extracted with ethyl acetate. HPLC analysis of the extracts was performed on a Hypersil ODS2 analytical column using acetonitrile −0.4% acetic acid (25:75, v/v) as the mobile phase. The UV detector was set at 260 nm. The standard curve was linear over the range 0.05–4.0 μg/mL. The lower limit of quantification was 0.05 μg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of epimedin C in rat plasma after giving the animals Herba Epimedii extract.
Keywords: Epimedin C; Herba Epimedii; HPLC;

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid–liquid extraction with diethyl ether–dichloromethane–butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3–768 ng/ml) and ODT (1.5–384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.
Keywords: Tramadol; O-Desmethyltramadol; Sotalol;

Erratum to “Geometrical distortions in two-dimensional gels: applicable correction methods” by T. Aittokallio; J. Salmi; T.A. Nyman; O.S. Nevalainen (244).

Author Index (246-248).

Keyword Index (249-254).