Journal of Chromatography B (v.857, #1)

A selective and sensitive LC/MS/MS assay was developed for the quantification of d2-nicotine and d2-cotinine in plasma of current and past smokers administered d2-nicotine. After solid phase extraction and liquid–liquid extraction, HPLC separation was achieved on a capillary hydrophilic interaction chromatography phase column. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated for d2-nicotine (0.03–6.0 ng/ml plasma) and d2-cotinine (0.15–25 ng/ml plasma). The lower limits of quantitation were 0.15 ng/ml and 0.25 ng/ml for d2-nicotine and d2-cotinine, respectively. The coefficient of variation was 3.7% for d2-nicotine and 2.5% for d2-cotinine. The method was applied to two ongoing studies of d2-nicotine metabolism in prior and current smokers. Preliminary analysis of a subset of subjects from these studies detected a significantly lower rate of nicotine conversion to cotinine by past smokers compared to current smokers.
Keywords: Nicotine metabolism; d2-nicotine; HILIC;

Quantitative determination of tilmicosin in canine serum by high performance liquid chromatography–tandem mass spectrometry by Michael Herrera; Haiqing Ding; Robert McClanahan; Jane G. Owens; Robert P. Hunter (9-14).
A highly sensitive and quantitative LC/MS/MS assay for the determination of tilmicosin in serum has been developed and validated. For sample preparation, 0.2 mL of canine serum was extracted with 3 mL of methyl tert-butyl ether. The organic layer was transferred to a new vessel and dried under nitrogen. The sample was then reconstituted for analysis by high performance liquid chromatography–tandem mass spectrometry. A Phenomenex Luna C8(2) analytical column was used for the chromatographic separation. The eluent was subsequently introduced to the mass spectrometer by electrospray ionization. A single range was validated for 50–5000 ng/mL for support of toxicokinetic studies. The inter-day relative error (inaccuracy) for the LLOQ samples ranged from −5.5% to 0.3%. The inter-day relative standard deviations (imprecision) at the respective LLOQ levels were ≤10.1%.
Keywords: Tilmicosin; Canine; Liquid chromatography/mass spectrometry;

An HPLC-MS/MS method was developed for the determination of MK-0518 (raltegravir), an HIV integrase inhibitor, in human plasma over the concentration range of 2–1000 ng/mL. Stable isotope labeled 13C6-MK-0518 was used as an internal standard. The sample preparation procedure utilized liquid–liquid extraction with hexane:methylene chloride in the 96-well format with a 200 μL plasma sample size. The compounds were chromatographed on an Ace C18 (50 × 3.0 mm, 3 μm, titanium frits) column with 42.5/57.5 (v/v %) 0.1 mM EDTA in 0.1% formic acid/methanol mobile phase at a flow rate of 0.5 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for MK-0518 (m/z 445 → 109) and 13C6-MK-0518 (m/z 451 → 367) on an Applied Biosystem API 4000 HPLC-MS/MS was used for quantitation. Intraday precision of standard curve concentrations in five different lots of control plasma was within 3.2%, while accuracy ranged from 94.8 to 106.8%. The mean extraction recovery of spiked plasma samples was 87%. Quality control (QC) samples were stored at −20 °C. Initial within day analysis showed QC accuracy within 7.5% of nominal with precision of 3.1% or less. The plasma QC samples were demonstrated to be stable for up to 23 months at −20 °C. The method described has been used to support over 18 clinical studies during Phase I through III of clinical development.
Keywords: Integrase inhibitor; Raltegravir; MK-0518; Plasma; Bioanalytical; HPLC–MS/MS; Liquid–liquid extraction;

Interaction of lysozyme with negatively charged flexible chain polymers by Diana Romanini; Mauricio Braia; Rodrigo Giatte Angarten; Watson Loh; Guillermo Picó (25-31).
The complex formation between the basic protein lysozyme and anionic polyelectrolytes: poly acrylic acid and poly vinyl sulfonic acid was studied by turbidimetric and isothermal calorimetric titrations. The thermodynamic stability of the protein in the presence of these polymers was also studied by differential scanning calorimetry. The lysozyme–polymer complex was insoluble at pH lower than 6, with a stoichiometric ratio (polymer per protein mol) of 0.025–0.060 for lysozyme–poly vinyl sulfonic acid and around 0.003–0.001 for the lysozyme–poly acrylic acid. NaCl 0.1 M inhibited the complex precipitation in agreement with the proposed coulombic mechanism of complex formation. Enthalpic and entropic changes associated to the complex formation showed highly negative values in accordance with a coulombic interaction mechanism. The protein tertiary structure and its thermodynamic stability were not affected by the presence of polyelectrolyte.
Keywords: Lysozyme; Poly vinyl sulfonate; Poly acrylic acid; Protein–polyelectrolyte complex;

A sensitive and specific method was developed and validated for the determination of paeoniflorin in rat brain with liquid chromatography–tandem mass spectrometry. Sample pretreatment involved protein precipitation following solid-phase extraction. Paeoniflorin and geniposide (internal standard) were separated isocratically on a Waters Symmetry C18 column (150 mm × 2.1 mm i.d., 5 μm), using a mobile phase of methanol/water with 0.1% formic acid (50:50, v/v) at a flow-rate of 200–300 μL/min in 4 min. A Finngan LTQ tandem mass spectrometer equipped with electrospray ionization source was operated in the positive ion mode. Selective reaction monitoring was performed to quantify paeoniflorin and the internal standard at m/z transitions of 503 → 381 and 411 → 231, respectively. A good linearity was found in the range of 2–500 ng/mL (R 2  = 0.9939). The intra- and inter-batch assay precisions (coefficient of variation, CV) at 5, 50 and 400 ng/mL (n  = 5) ranged from 6.3% to 9.7% and 1.2% to 7.2%, respectively, and the accuracies were from 95.9% to 101.6% and 99.4% to 102.9%, respectively. The mean recoveries of paeoniflorin were 81.2%, 80.9% and 82.3% at 5, 50 and 400 ng/mL (n  = 5), respectively, and the mean recovery of the internal standard was 76.7% with a concentration of 50 ng/mL (n  = 5). Stability studies showed that paeoniflorin was stable in different conditions. Finally, the method was successfully applied to the pharmacokinetic study of paeoniflorin in rat brain following a single subcutaneous administration (10 mg/kg) to rats.
Keywords: LC–MS/MS; Paeoniflorin; Brain distribution; Matrix effects; Pharmacokinetics;

A rapid, simple and robust method is presented for the simultaneous determination of seven antiepileptic drugs (AEDs), including primidone, phenobarbital, phenytoin, carbamazepine with its two major metabolites carbamazepine-10,11-epoxide and carbamazepine-10,11-(trans)-dihydrodiol and the new AEDs lamotrigine, hydroxycarbazepine (active metabolite of oxcarbazepine) and zonisamide in serum by high performance liquid chromatography (HPLC)-diode array detector (DAD). After solid-phase extraction, separation is achieved on an Alltima 3C18 analytical column using isocratic elution with a mixture of acetonitrile, methanol and phosphate buffer at 45 °C. The method is exhaustively validated, including experimental design in combination with statistical evaluation (ANOVA) to study the robustness of chromatography and sample preparation. Commonly co-administered antiepileptic drugs do not interfere with the method. Intra-day precision (RSD < 1.9%), linearity, lower limit of quantitation (LOQ < 0.065 mg/l) and robustness make the method suitable for daily therapeutic drug monitoring and pharmacokinetic studies.
Keywords: Anticonvulsants; Zonisamide; Carbamazepine; Oxcarbazepine; Lamotrigine; Phenytoin; Primidone; Phenobarbital; Drug analysis; HPLC; Validation; Design of experiments;

The prenyl-flavones, icaritin and desmethylicaritin, are bioactive compounds from the traditional Chinese medicinal herb, Epimedium, extracts of which can enhance bone health in animal models. In order to examine their bioavailability in humans, we have developed and validated a sensitive method to quantify icaritin and desmethylicaritin in human sera, using gas chromatography–mass spectrometry. The serum samples were extracted with ethyl acetate and then derivatized with BSTFA in pyridine (4:1). With genistein as internal standard, calibration curves with good linearity (R 2  > 0.99) within the concentration range of 0.15–10 nM in the selective ion monitoring mode were obtained. The limits of detection and quantization were 11 and 33 pM for icaritin, and 23 and 70 pM for desmethylicaritin, respectively; inter- and intra-assay variabilities were <15%, and accuracies were between 89 and 110%. Icaritin, but not desmethylicaritin, was detected from 1 h, increasing to a peak at 8 h (1.51 ± 1.6 nM) in sera of human volunteers after ingestion of an aqueous decoction of Epimedium. This sensitive method can be used to quantify serum levels of icaritin and desmethylicaritin for pharmacokinetic studies.
Keywords: Icaritin; Desmethylicaritin; Prenyl-flavones; Gas chromatography–mass spectrometry (GC–MS); Selective ion monitoring (SIM) mode; Human serum;

A rapid and sensitive RP-HPLC method with fluorescence detection has been developed for the quantitative analysis of trace amounts of monofluoroacetate (MFA) in biological samples as serum, food and meat. 9-Chloromethylanthracene (9-CMA) is used as the fluorescence labeling reagent. Samples were extracted and reacted with 9-chloromethylanthracene together with tetrabutylammonium bromide as catalyst at 80 °C for 50 min to give a new fluorescent derivative as 9-methyleneanthracene monofluoroacetate (MA-MFA). The resulting MA-MFA was characterized with IR, 1H NMR, 13C NMR and MS. Chromatography separation is performed on an Agilent Hypersil ODS column with a fluorescent detector employed with the excitation and emission wavelengths as 256 nm and 412 nm, respectively. Optimal conditions for derivatization, fluorescence detection and chromatographic separation have been established. The novel method yields a good linear relationship when the MFA concentration in serum within 1 and 250 ng/mL (r  = 0.9988). The detection limit (signal-to-noise ratio = 3 with 2 μL injected) was 0.25 ng/mL. The practical applicability of this method was demonstrated by quantitative determination of MFA-Na in a blood sample from a person who had ingested the poison.
Keywords: Monofluoroacetate (MFA); 9-Chloromethylanthracene (9-CMA); 9-Methyleneanthracene monofluoroacetate (MA-MFA); High-performance liquid chromatography;

Determination of non-steroidal anti-inflammatory drugs in pharmaceuticals and human serum by dual-mode gradient HPLC and fluorescence detection by Hany Ibrahim; Alexandre Boyer; Jalloul Bouajila; François Couderc; Françoise Nepveu (59-66).
Non-steroidal anti-inflammatory drugs (NSAIDs) such as piroxicam and mefenamic acid are commonly prescribed to treat inflammation, pain and fever. Similarly acetylsalicylic acid is used to prevent strokes and heart attacks. A rapid and selective method was developed for the simultaneous assay of three NSAIDs and salicylic acid via HPLC with fluorescence detection. The separation was performed using a “dual-mode” gradient (acetonitrile–0.1% aqueous orthophosphoric acid) and the analysis was completed within 7 min using an ACE® column C18, 5 μm, 150 mm × 4.6 mm. Naproxen was used as internal standard. The proposed method is simple, selective as well as with a good sensitivity reaching LOD lower than 2 pmol (0.05 μM) and was applied for quantitative analysis in pharmaceuticals and in human serum samples. The mean recovery was more than 95% and the within-day and between-days precisions were found to be satisfactory having RSD within the acceptable limits (<10%).
Keywords: NSAIDs; Acetylsalicylic acid; Mefenamic acid; Piroxicam; Salicylic acid; “Dual-mode” gradient; Fluorescence;

A sensitive, stereoselective assay using solid phase extraction and LC–MS–MS was developed and validated for the analysis of (R)- and (S)-bupropion and its major metabolite (R,R)- and (S,S)-hydroxybupropion in human plasma and urine. Plasma or glucuronidase-hydrolyzed urine was acidified, then extracted using a Waters Oasis MCX solid phase 96-well plate. HPLC separation used an α1-acid glycoprotein column, a gradient mobile phase of methanol and aqueous ammonium formate, and analytes were detected by electrospray ionization and multiple reaction monitoring with an API 4000 Qtrap. The assay was linear in plasma from 0.5 to 200 ng/ml and 2.5 to 1000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. The assay was linear in urine from 5 to 2000 ng/ml and 25 to 10,000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. Intra- and inter-day accuracy was >98% and intra- and inter-day coefficients of variations were less than 10% for all analytes and concentrations. The assay was applied to a subject dosed with racemic bupropion. The predominant enantiomers in both urine and plasma were (R)-bupropion and (R,R)-hydroxybupropion. This is the first LC–MS/MS assay to analyze the enantiomers of both bupropion and hydroxybupropion in plasma and urine.
Keywords: Bupropion; Hydroxybupropion; Mass spectrometry; Chiral analysis; LC–MS–MS;

A rapid and sensitive HPLC–MS method for the detection of plasma and cellular rifampicin by Ruben C. Hartkoorn; Saye Khoo; David J. Back; John F. Tjia; Catriona J. Waitt; Masautso Chaponda; Geraint Davies; Alison Ardrey; Samantha Ashleigh; Stephen A. Ward (76-82).
Rifampicin is active against both intracellular and extracellular Mycobacterium tuberculosis. The ability to measure rifampicin drug concentrations in both plasma and in cells may be useful in evaluating the suitability of dosage regimens for populations and individuals. Here a novel simple, precise and accurate method for the quantification of rifampicin in both cells and plasma is reported. Sample proteins were precipitated with acetonitrile containing the internal standard and then diluted with water. Aliquots of supernatant were then injected into the HPLC–MS system for chromatographic separation and detection. Rifampicin calibration curves encompassed concentrations from 100 to 12,800 ng/mL. Intra- and inter-assay precision and accuracy were determined using low, medium and high concentration quality control samples and was found to be within 10% in all cases. Rifampicin concentrations were found to be unaffected by freeze–thaw cycles, but were significantly affected by heat-inactivation (58 °C, 40 min). This assay was successfully utilised to determine the pharmacokinetic profile of rifampicin in plasma and peripheral blood mononuclear cells (PBMC) in 8 tuberculosis patients receiving rifampicin over an 8 h period.
Keywords: Rifampicin; HPLC–MS; Plasma; Cellular;

Method to determine stability and recovery of carboprost and misoprostol in infusion preparations by Kai On Chu; Chi Chiu Wang; Chi Pui Pang; Michael Scott Rogers (83-91).
The two synthetic prostaglandin analogues, carboprost and misoprostol, are used extensively in obstetric and gynaecological practice. Our recent research of these compounds’ use for intra-umbilical injection to treat adherent placenta necessitated their storage in solution for 3–4 days. This raised concerns over the stability and applied dosage in the in-house infusion preparations. It requires various pharmacological preparations before administration in clinical practice. We used LCMS to develop a simultaneous, valid, fast and simple method to assess the stability and recovery of their in-house preparations in different conditions. The linearity between 0–40 μg/ml was above 0.995. The reproducibility (CV) was within 5.2%. The limit of quantitation of the method for both compounds is about 2 μg/ml. The accuracy of both compounds from 0.4–40 μg/ml is 96.4–104.3% while the precision is 0.4–7.4%. The recoveries of carboprost in the infusion were from 100.3 ± 4.0 to 102.4 ± 1.6% and that of misoprostol in Cytotec tablet was from 44.9 ± 3.5 to 50.0 ± 5.0% in water and saline at 4 °C and room temperature. No interference was found from the matrix and between the tested compounds. The compounds were basically stable for 6 days in water and in saline, whether they were stored at 4 °C or at room temperature. However, only half of the dosage of misoprostol was recovered in the solution. Therefore, misoprostol dosage should be adjusted before clinical application.
Keywords: Carboprost; Misoprostol; Stability; Recovery;

Quantification of cyclosporine A in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry using a column-switching approach by Nicolas Ansermot; Marc Fathi; Jean-Luc Veuthey; Jules Desmeules; Denis Hochstrasser; Serge Rudaz (92-99).
As a potential alternative to cyclosporine A (CsA) monitoring in whole blood, a sensitive and selective method was developed for quantifying this immunosuppressive drug in human peripheral blood mononuclear cells (PBMCs) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). PBMCs were isolated from whole blood by density gradient centrifugation. After purification, cell counts were performed to express CsA amounts per single cell. The pelleted cells were then lysed and CsA was extracted with methanol (MeOH) containing 27-demethoxy-sirolimus as internal standard. After evaporation of the supernatant under nitrogen, the residue was reconstituted in MeOH, further diluted with water and injected onto a column-switching unit. On-line solid-phase extraction was performed using a C8 column with an acidic aqueous mobile phase containing 5% MeOH. The analytes were transferred in the back-flush mode on a C18 column with 65% MeOH and the chromatographic separation performed with a MeOH gradient (65–90%). The detection was carried out with a single quadrupole analyzer and the sodium adducts [M + Na]+ were monitored for quantification. This sensitive method was fully validated in the range of 5–400 ng/mL. This allowed the measurement of very small CsA amounts present in cells up to 0.5 fg/PBMC in clinical samples. Trueness (95.0–113.2%), repeatability (5.1–9.9%) and intermediate precision (7.0–14.7%) were found to be satisfactory. This method represents a new potential tool for therapeutic drug monitoring of CsA and could be used in clinical conditions if the utility of intracellular measurements is confirmed in prospective clinical trials.
Keywords: Cyclosporine A; PBMCs; Intracellular; LC–MS; Column-switching; TDM;

A sensitive and homogeneous immunoassay (IA) based on capillary electrophoresis (CE) with enhanced chemiluminescence (CL) detection has been developed for the determination of hepatitis B surface antigen (HBsAg) and antibody (HBsAb) in human serum. The conditions for the CL reaction and electrophoresis were investigated in detail using horseradish peroxidase (HRP) labeled HBsAg (HBsAg*) as a marker because of its catalytic effects on the luminol–hydrogen peroxide reaction. The CL reaction was enhanced by para-iodophenol and the CL detector was designed uniquely without any dead volume or diluents effect. The present method has been used for assaying HBsAg and HBsAb in human serum using a competitive format and a non-competitive format, respectively. Under the optimal conditions, the linear ranges were from 1 to 400 pmol/L (R  = 0.9988) for HBsAg and 2 to 200 mIU/mL (R  = 0.9981) for HBsAb. The detection limits were 0.4 pmol/L and 1 mIU/mL for HBsAg and HBsAb, respectively. The relative standard deviations of peak area were 4.2% and the errors of it were from −0.03% to +0.05% for 80 pmol/L HBsAg* (n  = 7). In this study, the free HBsAg* and the bound HBsAg* (HBsAg*–HBsAb) were separated in the separation capillary within 6 min using a borate run buffer. To verify the experimental reliability, the result was comparable with that of enzyme linked immunosorbent assay (ELISA) and demonstrated the feasibility of the CE–CL immunoassay method for clinical diagnosis.
Keywords: Immunoassay; HBsAg; HBsAb; Capillary electrophoresis; Chemiluminescence;

Ostrich pancreatic phospholipase A2: Purification and biochemical characterization by Abir Ben Bacha; Youssef Gargouri; Sofiane Bezzine; Habib Mosbah; Hafedh Mejdoub (108-114).
Ostrich pancreatic phospholipase A2 (OPLA2) was purified from delipidated pancreases. Pure protein was obtained after heat treatment (70 °C), precipitation by ammonium sulphate and ethanol, respectively followed by sequential column chromatography on MonoQ Sepharose and size exclusion HPLC column. Purified OPLA2, which is not a glycosylated protein, was found to be monomeric protein with a molecular mass of 13773.93 Da. A specific activity of 840 U/mg for purified OPLA2 was measured at optimal conditions (pH 8.2 and 37 °C) in the presence of 4 mM NaTDC and 10 mM CaCl2 using PC as substrate. This enzyme was also found to be able to hydrolyze, at low surface pressure, 1,2-dilauroyl-sn-glycero-3 phosphocholine (di C12-PC) monolayers. Maximal activity was measured at 5–8 mN m−1. The sequence of the first 22 amino-acid residues at the N-terminal extremity of purified bird PLA2 was determined by automatic Edman degradation and showed a high sequence homology with known mammal pancreatic secreted phospholipases A2.
Keywords: Phospholipase A2; Phosphatidylcholine; Bile salts; Lipid monolayer;

Quantification of trans-4-hydroxy-2-nonenal enantiomers and metabolites by LC–ESI-MS/MS by Ales Honzatko; Jiri Brichac; Matthew J. Picklo (115-122).
Lipid peroxidation is a causal factor in multiple diseases including Alzheimer's disease, atherosclerosis, and alcoholic liver disease. One of the most studied products of lipid peroxidation, trans-4-hydroxy-2-nonenal (HNE), has multiple cell signaling and cytotoxic effects. In this work, we developed an LC–MS/MS method for the quantitation of HNE enantiomers, the metabolite trans-4-hydroxy-2-nonenoic acid, and HNE-glutathione adducts in a single chromatographic run. In this method, (R)-HNE and (S)-HNE are derivatized by (S)-carbidopa to form diastereomers that are separated by a reversed-phase column. This method was successfully validated and tested using respiring rat brain mitochondria that enantioselectively metabolize HNE. Metabolic profiles of HNE biotransformation, including the enantiomeric disposition of HNE, will provide useful biomarker data regarding lipid peroxidation in disease states.
Keywords: 4-Hydroxynonenal; Chiral separation; Lipid peroxidation; Aldehyde dehydrogenase; Glutathione; Mitochondria; Biomarker;

Direct determination of glucuronide and sulfate of 4-hydroxy-3-methoxymethamphetamine, the main metabolite of MDMA, in human urine by Noriaki Shima; Hiroe Kamata; Munehiro Katagi; Hitoshi Tsuchihashi; Tsutomu Sakuma; Nobuo Nemoto (123-129).
A sensitive and reliable LC-ESI–MS procedure for the simultaneous determination of MDMA and its five metabolites including 4-hydroxy-3-methoxymethamphetamine (HMMA) conjugates has been established following the synthesis of two HMMA conjugates, 4-hydroxy-3-methoxymethamphetamine-glucuronide (HMMA-Glu) and 4-hydroxy-3-methoxymethamphetamine-sulfate (HMMA-Sul). Pretreatment of urine samples with methanol and LC–MS employing a C18 semi-micro column with a gradient elution program provided the successful separations and MS determinations of these analytes within 20 min. Upon applying the method to MDMA users’ urine specimens, HMMA-Glu and HMMA-Sul have been directly determined, suggesting the superiority of sulfation to glucuronidation in the HMMA phase II metabolism.
Keywords: MDMA; 4-Hydroxy-3-methoxymethamphetamine; Sulfate; Glucuronide; LC–MS;

In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 μm ID × 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20 mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25 kV/25 °C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector (λ ex  = 488 nm and λ em  = 520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ng mL−1, and the linear range of the calibration curves was 0.5–100 ng mL−1. The intra-day and inter-day precisions were both less than 13.22%.
Keywords: Ephedrines; Amphetamines; Capillary electrophoresis; Laser-induced fluorescence; Pre-column derivatization; Fluorescein isothiocyanate; Solid-phase extraction;

A fast and sensitive approach for determination of erythromycin in rat plasma was described. The method used capillary electrophoresis coupled with end-column electrochemiluminescence (ECL) detection of Ru(bpy)3 2+. The separation column used had an inner diameter of 75 μm. The running buffer was 15 mmol/L sodium phosphate (pH = 7.5). The solution in the detection cell was 50 mmol/L sodium phosphate (pH = 8.0) and 5 mmol/L Ru(bpy)3 2+. ECL intensity varied linearly with erythromycin concentration from 1.0 ng/mL to 10 μg/mL. The detection limit (S/N = 3) was 0.35 ng/mL. The relative standard deviations, of ECL intensity and migration time for eight consecutive injections of 1.0 μg/mL erythromycin (n  = 8), were 1.3% and 1.8%, respectively. The method was successfully applied to erythromycin determination in rat plasma. The recovery ranged from 92.5 to 97.5%.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Erythromycin; Tris(2,2′-bipyridyl) ruthenium(II); Plasma;

Simultaneous determination of selected veterinary antibiotics in gilthead seabream (Sparus Aurata) by liquid chromatography–mass spectrometry by R. Romero-González; J.C. López-Martínez; E. Gómez-Milán; A. Garrido-Frenich; J.L. Martínez-Vidal (142-148).
A method was optimised and validated for simultaneous monitoring of several drugs of different classes of antibiotics such as quinolones (oxilinic acid and flumequine), tetracyclines (oxytetracycline), sulfonamides (sulfadiazine) and trimethoprim in fish muscle and skin. The method is based on solid–liquid extraction without further sample clean up followed by liquid chromatography–mass spectrometry (LC–MS) determination with electrospray ion source (ESI) in positive mode. The limits of quantification (LOQs) were lower than 20 μg/kg for all compounds and repeatability, expressed as relative standard deviations (RSD), were lower than 15%. Therefore, the LC–MS method was successfully applied for the quantitative determination of antibiotics in gilthead sea bream muscle and skin and oxytetracycline in medicated fishes.
Keywords: Multiclass; Antibiotics; Liquid chromatography; Mass spectrometry; Gilthead sea bream;

Concanavalin A (Con A) was selected as ligand and thus immobilized onto two different supports, namely the polymeric Toyopearl and the inorganic silica, with the protection of its binding sites provided during the coupling procedure. The prepared Con A affinity adsorbents were then employed to evaluate their adsorption behaviour for the enzyme glucose oxidase (GOD). The immobilization kinetics showed that the immobilization of Con A on silica supports was much faster than that on Toyopearl supports, which could highly reduce the possibility of the denaturation of Con A. The optimal adsorption conditions for binding of GOD onto the ligand were determined in terms of the pH value and the ionic strength of the adsorption medium. The adsorption isotherms for binding GOD onto two Con A affinity adsorbents fitted well with the Langmuir equation. The maximum adsorption capacity q m of Toyopearl Con A and silica Con A were 7.9 mg/ml and 4.9 mg/ml, with a dissociation constant K d of 4.8 × 10−7  M and 2.6 × 10−6  M, respectively. Due to the less diffusive resistance, silica Con A showed both higher adsorption and desorption rates for GOD when compared with Toyopearl Con A. The nonspecific adsorption of GOD was less than 8% for both end-capped Toyopearl and silica supports. The dynamic adsorption of GOD for five times repeated processes showed a high stability for both prepared adsorbents. All the results indicate a good suitability of both Con A adsorbents for affinity adsorption of GOD.
Keywords: Affinity chromatography; Affinity adsorbents; Concanavalin A; Glucose oxidase; Adsorbent optimization;

An improved HPLC assay for phosphatidylcholine hydroperoxides (PCOOH) in human plasma with synthetic PCOOH as internal standard by Shu-Ping Hui; Hitoshi Chiba; Toshihiro Sakurai; Chitose Asakawa; Hironori Nagasaka; Tsuyoshi Murai; Hajime Ide; Takao Kurosawa (158-163).
Quantitative and qualitative analyses of 1-palmitoyl-2-linoleoyl-phosphatidylcholine monohydroperoxide [PC 16:0/18:2-OOH] and 1-stearoyl-2-linoleoyl-phosphatidylcholine monohydroperoxide [PC 18:0/18:2-OOH] in human plasma were improved by chemiluminescence HPLC using synthetic 1-stearoyl-2-erucoyl-phosphatidylcholine monohydroperoxide (PC 18:0/22:1-OOH) as internal standard. The calibration curves of synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, obtained by their direct injections with the IS into the HPLC system, were linear throughout the calibration range (10–1000 pmol). Within-day and between-day coefficients of variation were below 8%, and the recoveries were between 84% and 101%. Plasma concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were 102 ± 59 nM (mean ± SD) and 36 ± 20 nM, respectively, in the 33 healthy volunteers. The present method might help understanding incompletely understood pathway of plasma phosphatidylcholine hydroperoxides.
Keywords: HPLC; Chemiluminescence; Luminol; Phosphatidylcholine hydroperoxide; PC; Human plasma;

A method for the quantification of salidroside, a major biologically active compound in Rhodiola, in rat plasma by on-line SPE LC/MS/MS in negative electrospray mode was developed and validated. A column-switching instrument and two HPLC pumping systems were employed, and salicin was used as the internal standard. A Waters Oasis HLB extraction column and an Agilent TC-C18 analytical column in a column-switching set-up with gradient elution were utilized. The MS/MS ion transitions monitored were m/z 299.0/119.0 and 285.1/122.9 for salidroside and salicin, respectively. The standard curves were linear within a range of 50–5000 ng/mL using weighted linear regression analysis (1/x). The intra- and inter-day coefficients of variance ranged from 1% to 9%. The recovery was above 90%. The freeze/thaw and long-term stability were validated. This method was subsequently applied to a pharmacokinetic study of salidroside in rats.
Keywords: Salidroside; On-line SPE; LC–MS/MS; Pharmacokinetics;

The amphetamine-derived designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) is an upcoming substance on the illicit drug market. In the current study, the identification of its metabolites in rat urine and their toxicological detection in the authors’ systematic toxicological analysis (STA) procedure were examined. DOI is extensively metabolized by O-demethylation and beside small amounts of parent compound it was found to be excreted mainly in form of metabolites. The STA procedure using full-scan GC–MS allowed proving an intake of a common drug users’ dose of DOI by detection of the two O-demethyl metabolite isomers in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOI in human urine.
Keywords: 4-Iodo-2,5-dimethoxy-amphetamine; DOI; Designer drug; Metabolism; GC–MS;

Punicalagin, the main ingredient of pomegranate (Punica granatum L.) husk, is a high molecular weight polyphenolic compound. It has shown remarkable pharmacological activities attributed in the presence of dissociable ―OH groups. To isolate punicalagin, previous methods included labor intensive and expensive solid phase extractions by column chromatography (C-18, polyamides, dellulose, Sephadex Lipophilic LH-20, Diaion HP20). High-speed countercurrent chromatography (HSCCC) was used for isolation and purification of punicalagin from pomegranate husk. Using preparative HSCCC about a 350 mg amount of the crude extract was separated, yielding 105 mg of punicalagin at a high-purity of over 92%. Eighty milligrams of gallic acid was simultaneously separated as another product, at a purity of 75%.
Keywords: Countercurrent chromatography; Punicalagin; Pomegranate husk;