Journal of Chromatography B (v.879, #25)

Quantitative liquid chromatographic analysis of anthracyclines in biological fluids by Kristof E. Maudens; Christophe P. Stove; Willy E. Lambert (2471-2486).
Anthracyclines are amongst the most widely used drugs in oncology, being part of the treatment regimen in most patients receiving systemic chemotherapy. This review provides a comprehensive summary of the sample preparation techniques and chromatographic methods that have been developed during the last two decades for the analysis of the 4 most administered anthracyclines, doxorubicin, epirubicin, daunorubicin and idarubicin in plasma, serum, saliva or urine, within the context of clinical and pharmacokinetic studies or for assessing occupational exposure. Following deproteinization, liquid–liquid extraction, solid phase extraction or a combination of these techniques, the vast majority of methods utilizes reversed-phase C18 stationary phases for liquid chromatographic separation, followed by fluorescence detection, or, more recently, tandem mass spectrometric detection. Some pros and cons of the different techniques are addressed, in addition to potential pitfalls that may be encountered in the analysis of this class of compounds.
Keywords: Anthracyclines; Biological fluids; Sample preparation; Chromatography; Fluorescence detection; Tandem mass spectrometry; Occupational exposure;

Substrates and products of soluble epoxide hydrolase (sEH) such as 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), leukotoxin, and leukotoxin diol are potential biomarkers for assessing sEH activity in clinical trial subjects. To quantify them, we have developed and validated a semi-automated and relatively high-throughput assay in a 96-well plate format using liquid chromatography–mass spectrometry. 14,15-EET, 14,15-DHET, leukotoxin and leukotoxin diol, as well as their deuterium labeled internal standards were extracted from human plasma by liquid–liquid extraction using ethyl acetate. The four analytes were separated from other endogenous lipid isomers using liquid chromatography coupled with tandem mass spectrometry. The method was validated over a concentration range of 0.05–50 ng/mL. The validation results show that the method is precise, accurate and well-suited for analysis of clinical samples. The turn-around rate of the assay is approximately 200 samples per day.
Keywords: Soluble epoxide hydrolase (sEH); Epoxyeicosatrienoic acid (EET); Dihydroxyeicosatrienoic acid (DHET); Epoxyoctadec-12(Z)-enoic acid (leukotoxin); Dihydroxyoctadec-12(Z)-enoic acid (leukotoxin diol); LC/MS; Quantitation; Human plasma;

Analysis of urinary aristolactams by on-line solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry by Su-Yin Chiang; Wei-Chung Shih; Ho-Tang Liao; Po-Chi Shu; Ming-Tsai Wey; Hei-Feng Huang; Kuen-Yuh Wu (2494-2500).
Aristolochic acids (AAs), nephrotoxicants and known human carcinogens, are a mixture of structurally related derivatives of nitrophenanthrene carboxylic acids with the major components being aristolochic acid I and aristolochic acid II. People may ingest small amounts of AAs from its natural presence in medicinal plants and herbs of the family Aristolochiaceae, including the genera Aristolochia and Asarum, which have been used worldwide in folk medicine for centuries. In order to assess AA intake, an on-line solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry (on-line SPE-LC/MS/MS) method was developed to analyze their most abundant corresponding metabolites, aristolactams (ALs), in urine to serve as biomarkers. The limits of quantitation were 0.006 ng for aristolactam I (AL-I), and 0.024 ng for aristolactam II (AL-II) on column. Recovery varied from 98.0% to 99.5%, and matrix effects were within 75.3–75.4%. This method was applied to analyze ALs in the urine samples collected on days 1, 2, 4, and 7 from mice treated with 30 mg/kg or 50 mg/kg AAs. Their half lives were estimated to be 3.55 h and 4.00 for AL-I, and 4.04 and 4.83 h for AL-II, depending on AAs doses. These results demonstrated that the first simple on-line SPE-LC/MS/MS method was successfully developed to analyze urinary ALs with excellent sensitivity and specificity to serve as biomarkers to assess current AA intake from AAs-containing Chinese herbs.
Keywords: Aristolochic acid; Aristolactam; Online SPE; LC/MS/MS;

Capillary electrophoresis coupled with mass spectrometry for the evaluation of substance P enzymatic degradation by SaOS-2 human osteosarcoma by Antonella Cavazza; Claudio Corradini; Mario Marini; Luigi Giorgio Roda; Angela Valenti (2501-2506).
A new analytical method for the detection and the quantitative evaluation of the undecapeptide substance P by capillary electrophoresis coupled with ion trap mass spectrometry (CE–MS) by a co-axial sheath liquid interface has been developed. Conditions of analysis employed an acidic buffer and a 60 cm fused silica capillary installed by overcoming the UV window position, thus allowing to perform the analysis in a brief time. The method has been applied to the evaluation of substance P enzymatic hydrolysis during incubation with the human osteosarcoma SaOS-2 cell line. The analysis of amino acids derived from the cleavage of substance P has been also carried out simultaneously under the same electrophoretic conditions allowing the description of a kinetic of amino acid formation, parallel with substance P disappearance. The amounts of intact substance P and of free amino acids were monitored along 600 s of incubation time. A steady decrease of substance P as function of reaction time was observed. Peptide's half-life was found to be about 4.3 s, indicating an extremely fast hydrolysis in the presence of the SaOS-2 cells. Proline, phenilalanine and methionine were the predominant free amino acids recorded. Obtained results lead to hypothesize the occurrence of endopeptidases activity, followed by aminopeptidases responsible for the release of free amino acids originated after primary bond cleavage.
Keywords: Substance P; Capillary electrophoresis; Mass spectrometry; Free amino acids; Peptidases;

A new method was developed for the simultaneous determination of six phthalate esters in bottled milks using ultrasound-assisted dispersive liquid–liquid microextraction (UA-DLLME) followed by gas chromatography–flame ionization detection (GC–FID). 0.8 mL of methanol (dispersant) and 40 μL of CCl4 (extractant) were injected into 8.0 mL of milk solution and then emulsified the mixture by ultrasound for 2.0 min to form the cloudy solution. Under the optimum condition, the enrichment factors of the analytes ranged from 220 to 270 fold and the recovery ranged from 93.2% to 105.7%. Good linearity was observed for all analytes in a range of 0.8–51 ng g−1 with the correlation coefficient (r 2) ≥0.9992. The limits of detection (LODs) based on signal to noise of 3 were 0.64–0.79 ng g−1. The repeatability evaluated as intra-day and inter-day precision (relative standard deviation, RSD) were less than 4.0% (n  = 5). The presented UA-DLLME–GC–FID method was successfully applied to determine the six phthalate esters in different bottled milk products.
Keywords: Ultrasound-assisted dispersive liquid–liquid microextraction; Phthalate esters; Gas chromatography; Bottled milk products;

A rapid and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of sibiricaxanthone F in rats by Xiaojuan Yang; Guanshen Zhou; Pingping Zou; Ying Ning; Ke Zan; Pengfei Tu; Yong Jiang (2513-2518).
A simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantifying sibiricaxanthone F (SF) in rat plasma following oral and intravenous dosings. After addition of the internal standard (IS) kaempferol and the antioxidant, 20% ascorbic acid, plasma samples were precipitated with acetonitrile and separated on an Aglient Zorbax XDB-C18 column (50 mm × 4.6 mm I.D., 2.1 μm) with gradient acetonitrile and water (both containing 0.01% formic acid) as the mobile phase. The detection was performed on a Sciex API 4000 LC–MS/MS with electrospray ionization (ESI) inlet in the negative multiple reaction monitoring (MRM) mode. Good linearity was achieved over the concentration range of 0.5–500.0 ng/mL (r  > 0.996). Intra- and inter-day precisions were less than 7.60%, and accuracy ranged from 97.18% to 99.84%. The lower limit of quantification for SF was 0.5 ng/mL, and analytes were stable under various conditions (during freeze-thaw, at room temperature and under deep-freeze conditions). This validated method was successfully applied to the preliminary pharmacokinetic study of SF in rats for the first time, and the absolute bioavailability of SF was found to be only 0.22 ± 0.15%.
Keywords: Sibiricaxanthone F; Xanthone; Pharmacokinetic; Bioavailability; LC–MS/MS; Polygala;

Prohibition of some synthetic cannabimimetics (e.g., JWH-018, JWH-073 and CP 47497) in a number of countries has led to a rise in new compounds in herbal mixtures that create marijuana-like psychotropic effects when smoked. The cannabimimetic JWH-250 (1-pentyl-3-(2-methoxyphenylacetyl)indole) was identified in May 2009 by the German Federal Criminal Police as an new ingredient in herbal smoking mixtures. The absence or low presence of the native compound in urine samples collected from persons who had consumed JWH-250 necessitates a detailed identification of their metabolites, which are excreted with urine and present in blood. Using gas and liquid chromatography–mass spectrometry (GC–MS and LC–MS/MS), we identified a series of metabolites in urine samples and serum sample from humans and urine samples from rats that were products of the following reactions: (a) mono- and dihydroxylation of aromatic and aliphatic residues of the parent compound, (b) trihydroxylation and dehydration of the N-alkyl chain, (c) N-dealkylation and (d) N-dealkylation and monohydroxylation. The prevailing urinary metabolites in humans were the monohydroxylated forms, while N-dealkylated and N-dealkyl monohydroxylated forms were found in rats. The detection of the mono- and dihydroxylated metabolites of JWH-250 in urine and serum samples by GC–MS and LC–MS/MS proved to be effective in determining consumption of this drug.
Keywords: JWH-250; Herbal mixture; Metabolites; GC–MS; LC–MS/MS;

Circadian disruption can have several possible health consequences, but is not well studied. In order to measure circadian disruption, in relation to shift or night work, we developed a simple and sensitive method for the simultaneous determination of melatonin, cortisol and testosterone in human saliva. We used liquid–liquid extraction (LLE) followed by liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI–MS/MS) recorded in positive ion mode. Saliva samples were collected by spitting directly into tubes and 250 μL were used for analysis. The limits of detection were 4.1 pmol/L, 0.27 nmol/L and 10.8 pmol/L for melatonin, cortisol, and testosterone, respectively. The developed method was sensitive enough to measure circadian rhythms of all 3 hormones in a pilot study among four healthy volunteers. It can therefor be used to study the impact of night work and working in artificial light on the workers circadian rhythms. To our knowledge this is the first LC–ESI–MS/MS method for simultaneous determination of salivary melatonin, cortisol and testosterone.
Keywords: Liquid chromatography tandem mass spectrometry; Saliva; Melatonin; Cortisol; Testosterone;

In this paper, a method for the rapid and sensitive analysis of juvenile hormone III (JH III) and 20-hydroxyecdysone (20E) in queen larvae and drone pupae samples was presented. Ultrasound-assisted extraction provided a significant shortening of the leaching time for the extraction of JH III and 20E and satisfactory sensitivity as compared to the conventional shake extraction procedure. After extraction, determination was carried out by liquid chromatography–tandem mass spectrometry (LC–MS/MS) operating in electrospray ionization positive ion mode via multiple reaction monitoring (MRM) without any clean-up step prior to analysis. A linear gradient consisting of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid, and a ZORBAX SB-Aq column (100 mm × 2.1 mm, 3.5 μm) were employed to obtain the best resolution of the target analytes. The method was validated for linearity, limit of quantification, recovery, matrix effects, precision and stability. Drone pupae samples were found to contain 20E at concentrations of 18.0 ± 0.1 ng/g (mean ± SD) and JH III was detected at concentrations of 0.20 ± 0.06 ng/g (mean ± SD) in queen larvae samples. This validated method provided some practical information for the actual content of JH III and 20E in queen larvae and drone pupae samples.
Keywords: High-performance liquid chromatography–mass spectrometry; Juvenile hormone III; 20-Hydroxyecdysone; Queen larvae; Drone pupae;

A new way of solid-phase microextraction fibers preparation for selected antibiotic drug determination by HPLC–MS by Pawel Olszowy; Malgorzata Szultka; Jacek Nowaczyk; Boguslaw Buszewski (2542-2548).
The polypyrrole (PPy) and polythiophene (PTh) solid phase microextraction (SPME) coatings were obtained with the use of the electropolymerisation and linear sweep voltammetry. Such fibers were modified by an ozone treatment in a gaseous phase in the concentration of 2.1 ± 0.2 × 10−5  mol dm−3. Both kinds of fibers were applied in the microextraction of linezolid from standard solutions to compare the extraction efficiencies displayed by these sorption phases. In these investigations a better adsorption capacity was obtained for polypyrrole fibers and hence only these kinds of fibers were utilized in the measurements from human plasma. In all measurements the concentrations of the drugs were in the range from 1 to 20 μg ml−1 (standard solutions) and 1 to 15 μg ml−1 (human plasma). Before the measurements, an optimization of the desorption solution experiments was performed. The correlation coefficients (R) obtained in the standard solution and human plasma were in the range from 0.8399 to 0.9970. The relative standard deviations (RSDs) were in the range of 0.1–7.6%.
Keywords: Electropolymerisation; Ozonization; Solid phase microextraction; Linezolid; High performance liquid chromatography; Mass spectrometry;

Determination of muscarine in human urine by electrospray liquid chromatographic–mass spectrometric by Barbora Merová; Peter Ondra; Marie Staňková; Miroslav Soural; Jan Stříbrný; Lenka Hebká; Karel Lemr (2549-2553).
A liquid chromatography–mass spectrometry based method for determination of muscarine in human urine was developed and validated. The method involved a solid phase extraction of muscarine from urine using Strata X-CW column. Separation of muscarine was achieved within 16.0 min on a reversed phase Gemini C18 analytical column (150 mm × 2.0 mm i.d., 5 μm) with a mobile phase consisted of aqueous 8 mmol/L heptafluorobutyric acid and acetonitrile in a gradient mode. Mass spectrometric detection was performed at m/z 174 and m/z 216 for muscarine and acetylmuscarine (internal standard), respectively. The linearity was satisfactory with a coefficient of determination (R 2) 0.9993 at concentration range from 0.3 ng/mL to 2.0 μg/mL, LOD and LOQ for muscarine was 0.09 ng/mL and 0.3 ng/mL, respectively. The found out recoveries of muscarine were 96% or 95% for concentration 0.3 ng/mL and 0.2 μg/mL or 2.0 μg/mL, respectively. The precision in the intra-assay-study varied from 0.48% to 1.39% and in the inter-assay-study from 2.39% to 5.49%. The accuracy ranged from −3.3% to −6%. The validation results demonstrated that the method fulfilled satisfactory requirements for precision and accuracy across the calibration curve. The applicability of the method has been demonstrated by analyzing clinical urine samples. The method offers the fast objective identification of intoxication by muscarine and can become a routine screening alternative to more difficult microscopic examination of spores in the gastric content in clinical practice.
Keywords: Muscarine; Determination; Human urine; Liquid chromatography; Mass spectrometry;

Development of a novel LC–MS/MS method for the determination of letosteine in human plasma and its application on pharmacokinetic studies by Xinxia Liu; Heng Zheng; Wei Tang; Zhenyu Qian; Yinsong Zhu; Jing Wang; Shiying Yuan; Xin Wen; Cuiling Cao; Hui Chen (2554-2560).
Letosteine has been found to be effective in treating patients with chronic bronchopneumopathies in clinical practice. To provide robust support for its pharmacokinetic and clinical studies, a rapid and sensitive method based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was developed and validated for the analysis of letosteine in plasma samples. After protein precipitation, the plasma samples were separated on a reversed-phase C18 column in less than 1.5 min. The LC–MS/MS system was performed in the positive ion multiple-reaction-monitoring (MRM) mode to produce intensive product ions of m/z 280.1 → 160.0 for letosteine and m/z 248.1 → 121.1 for the internal standard, tinidazole. The method was found to have excellent linearity (r  ≥ 0.9974), precision (RSD ≤ 5.83%), extraction recovery (71.8–73.0%) and stability (RE, −8.45% to 9.03%) over a concentration range of 0.1140–152.0 μg L−1. Compared to the previous published radioactive method, LC–MS/MS method showed many advantages including shorter analysis time, simpler preparation procedure, increased sensitivity as well as lower safety risks. In addition, this method was successfully applied to study the pharmacokinetics of letosteine following a single and multiple dose oral administration in Chinese healthy volunteers.
Keywords: Liquid chromatography–tandem mass spectrometry; Letosteine; Human plasma; Pharmacokinetics;

Determination of vandetanib in human plasma and cerebrospinal fluid by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) by Feng Bai; Jennifer Johnson; Fan Wang; Lei Yang; Alberto Broniscer; Clinton F. Stewart (2561-2566).
A sensitive and precise LC-ESI-MS/MS method for the determination of vandetanib (ZD6474) in human plasma and cerebrospinal fluid (CSF) using [13C,d3]-ZD6474 as an internal standard (ISTD) was developed and validated. Sample preparation consisted of a simple liquid–liquid extraction with tert-butyl methyl ether containing 0.1% or 0.5% ammonium hydroxide. ZD6474 and ISTD were separated on a Kinetex C18 column (2.6 μm, 50 mm × 2.1 mm) at ambient temperature with an isocratic mobile phase (acetonitrile/10 mM ammonium formate = 50/50, v/v, at pH 5.0) delivered at 0.11 mL/min. The retention time of both compounds was at 1.60 min in a runtime of three min. Detection was achieved by an API-3200 LC–MS/MS system, monitoring m/z 475.1/112.1 and m/z 479.1/116.2 for vandetanib and ISTD, respectively. The method was linear in the range of 0.25–50 ng/mL (R 2  ≥ 0.990) for the CSF curve and from 1.0 to 3000 ng/mL (R 2  ≥ 0.992) for the plasma curve. The mean recovery for vandetanib was 80%. Within-day and between-day precisions were ≤8.8% and ≤5.9% for CSF and plasma, respectively. Within-day and between-day accuracies ranged from 95.0 to 98.5% for CSF, and from 104.0 to 108.5% for plasma. Analysis of plasma from six different sources showed no matrix effect for vandetanib (MF = 0.98, %CV ≤ 4.97, n  = 6). This method was successfully applied to the analysis of pharmacokinetic samples from children with brain tumors treated with oral vandetanib.
Keywords: Vandetanib (ZD6474); Human plasma and cerebrospinal fluid (CSF); Liquid–liquid extraction (LLE); Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS); Lower limit of quantitation (LLOQ);

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution(aqueous). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution(aqueous) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C18 (250 × 4.6 mm, 5 μm) analytical column using methanol–0.05% trifluoroacetic acid solution(aqueous) (05/95, v/v), containing 0.1 mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min−1 at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity (r 2  > 0.999 in the range of 0.2–10,000 ng mL−1 for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250–400 fg and limit of quantification: 0.8–1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.
Keywords: Column liquid-chromatography; Vitamin C; Aminothiols; Methionine; Electrochemical detection; Optimization; Validation;

Development and validation of an EI-GC/MS method for the determination of sertraline and its major metabolite desmethyl-sertraline in blood by Alaa Khraiwesh; Ioannis Papoutsis; Panagiota Nikolaou; Constantinos Pistos; Chara Spiliopoulou; Sotirios Athanaselis (2576-2582).
A sensitive and specific GC/MS method for the determination of sertraline and its main metabolite desmethyl-sertraline in whole blood has been developed, optimized and validated. Sample preparation included solid-phase extraction of both analytes and their derivatization with heptafluorobutyric anhydride (HFBA). Protriptyline was used as internal standard for the determination of both analytes. Limits of detection and quantification for both sertraline and desmethyl-sertraline were 0.30 and 1.00 μg/L, respectively. The calibration curves were linear within the dynamic range of each analyte (1.00–500.0 μg/L) with a correlation coefficient (R 2) exceeding 0.991. Extraction efficiency ranged from 90.1(±5.8)% to 95.4(±3.0)% for sertraline, and from 84.9(±8.2)% to 107.7(±4.4)% for desmethyl-sertraline. The precision for sertraline and desmethyl-sertraline was between 3.6–5.5% and 4.7–7.2%, respectively, while the accuracy was in the range of −6.67% to 2.20% and −6.33% to 2.88% for sertraline and desmethyl-sertraline, respectively. The method was applied to real blood samples obtained from patients that follow sertraline treatment and also in cases of forensic interest. The developed method can be used in routine every day analysis by clinical and forensic laboratories, for pharmacokinetic studies, for therapeutic sertraline monitoring or for the investigation of forensic cases where sertraline is involved.
Keywords: Sertraline; Blood; GC/MS; Solid-phase extraction; HFBA-derivatization;

The effects of hitchhiker antigens co-eluting with affinity-purified research antibodies by Lilly Mechetner; Radhika Sood; Van Nguyen; Pete Gagnon; Missag H. Parseghian (2583-2594).
The popularity of Protein G for the purification of antibodies has given rise to an entire industry that supplies scientists with research grade immunoreagents; however, many times the supplied product is contaminated with antigens bound to the antibody's complementarity-determining regions (CDRs). These “hitchhikers” are a category of host cell proteins that are elusive to detect due to their interaction with the antibody in the final product and yet their impact on an experiment or an entire field of study can be far reaching. In an earlier work, the role of hitchhikers on a human anti-histone antibody destined for clinical usage was explored and a stringent purification scheme developed. Here we use a murine monoclonal, which reflects the type of commercial antibody usually purchased for research. We evaluate three purification schemes: a traditional approach using a one-step, low pH elution buffer (pH 2.5); a gentler approach using a pH gradient elution scheme (pH 7 down to pH 2.5); and finally, a more stringent purification patterned on our earlier published method that uses a quaternary amine guard column and a high salt wash during antibody immobilization on the Protein G. We stress that the stringent purification incorporates the pH gradient scheme and is gentler than the low-pH approach. The resulting product from all three purifications is directly compared for binding potency, histone content (using an ELISA based assay) and residual DNA (using quantitative PCR). The results demonstrate that the first two methods are inadequate for hitchhiker removal. The traditional one-step, low pH approach produces a single elution peak containing histone contaminated antibody with picogram quantities of residual DNA, however, the trailing end of the same peak is loaded with antibody complexed to nanogram amounts of DNA, in some cases, over 100 ng. The pH gradient approach provided antibodies accompanied by only picograms of residual DNA and, on average, 1 out of every 10–20 CDRs occupied by a histone antigen. The more stringent approach, using the salt wash prior to elution with the pH gradient, has an average of 1 out of every 75 CDRs contaminated with a histone while the majority of the residual DNA is captured by the quaternary amine column placed in front of the Protein G. The consequences of these contaminants is illustrated by showing how they manifest themselves in unusual antibody potency values ranging from 558% for antibody bound to histone hitchhikers down to 15% for antibody contaminated with DNA hitchhikers. Those samples purified by the recommended stringent approach show potency values between 90 and 101%. Most importantly, we repeatedly demonstrate in a simulated chromatin immunoprecipitation (ChIP) assay the ability to precipitate clean plasmid DNA with histone contaminated antibody that had been purified using the traditional one-step, low pH elution approach. Expectedly, those antibodies stringently purified and showing 100% binding potency were unable to precipitate DNA in the absence of histone hitchhikers.
Keywords: Hitchhiker antigens; Antibody purification; Commercial-scale purification; Affinity chromatography; Host cell proteins; Chromatin immunoprecipitation;

Self-assembly molecularly imprinted polymers of 17β-estradiol on the surface of magnetic nanoparticles for selective separation and detection of estrogenic hormones in feeds by Shu Wang; Yun Li; Meijuan Ding; Xiaoli Wu; Jinhui Xu; Ruoyu Wang; Tingting Wen; Wenyu Huang; Ping Zhou; Kunfang Ma; Xuemin Zhou; Shuhu Du (2595-2600).
This paper reports a surface molecular self-assembly strategy for molecular imprinting on magnetic nanoparticles for selective separation and detection of estrogens in feeds. First, γ-methacryloxypropyltrimethoxysilane (MEMO) was successfully assembled at the surface magnetic nanoparticles through simple free radical polymerization, and subsequently, the copolymerization was further assembled between methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) in the presence of templates 17β-estradiol (E2). The synthesized magnetic molecularly imprinted polymers for E2 (E2-MMIPs) showed quick separation, large adsorption capacity, high selectivity and fast binding kinetics for E2. Meanwhile, a dispersive solid-phase extraction (DSPE) based on E2-MMIPs has been established for efficient separation and fast enrichment of estrogens from the feeds. The assay exhibited a linear range of 0.1–4 μM for E2 and estriol (E3) with the correlation coefficient above 0.9996 and 0.9994, respectively. Recoveries of E2 from three kinds of feeds spiked at different concentration levels ranged from 92.7% to 97.0% with RSD < 4.7%, and recoveries of E3 ranged from 71.9% to 83.1% with RSD < 4.9%, respectively. The method is simple and sensitive, and can be used as an alternative tool to effectively separate and enrich the trace of estrogens in agricultural products by HPLC–UV.
Keywords: Self-assembly molecularly imprinted polymers; Magnetic nanoparticles; Dispersive solid-phase extraction; 17β-Estradiol; Feeds;

The aim of this study was to develop and validate an analytical method to simultaneously determine European Union-regulated β-lactams (penicillins and cephalosporins) and quinolones in cow milk. The procedure involves a new solid phase extraction (SPE) to clean-up and pre-concentrate the three series of antibiotics before analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). LC–MS/MS and UPLC–MS/MS techniques were also compared. The method was validated according to the Directive 2002/657/EC and subsequently applied to 56 samples of raw cow milk supplied by the Laboratori Interprofessional Lleter de Catalunya (ALLIC) (Laboratori Interprofessional Lleter de Catalunya, Control Laboratory Interprofessional of Milk of Catalunya).
Keywords: Antibiotics; Milk; LC–MS/MS; UPLC–MS/MS; Matrix effect; Validation;

The purpose of this study was develop and validate a sensitive and specific enantioselective liquid–chromatography/tandem mass spectrometry (LC–MS/MS) method, for the simultaneous quantification of eslicarbazepine acetate (ESL), eslicarbazepine (S-Lic), oxcarbazepine (OXC) and R-licarbazepine (R-Lic) in human plasma. Analytes were extracted from human plasma using solid phase extraction and the chromatographic separation was achieved using a mobile phase of 80% n-hexane and 20% ethanol/isopropyl alcohol (66.7/33.3, v/v). A Daicel CHIRALCEL® OD-H column (5 μm, 50 mm × 4.6 mm) was used with a flow rate of 0.8 mL/min, and a run time of 8 min. ESL, S-Lic, R-Lic, OXC and the internal standard, 10,11-dihydrocarbamazepine, were quantified by positive ion electrospray ionization mass spectrometry. The method was fully validated, demonstrating acceptable accuracy, precision, linearity, and specificity in accordance with FDA regulations for the validation of bioanalytical methods. Linearity was proven over the range of 50.0–1000.0 ng/mL for ESL and OXC and over the range of 50.0–25,000.0 ng/mL for S-Lic and R-Lic. The intra- and inter-day coefficient of variation in plasma was less than 9.7% for ESL, 6.0% for OXC, 7.7% for S-Lic and less than 12.6% for R-Lic. The accuracy was between 98.7% and 107.2% for all the compounds quantified. The lower limit of quantification (LLOQ) was 50.0 ng/mL for ESL, S-Lic, OXC and R-Lic in human plasma. The short-term stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability and stock solution stability all met acceptance criteria. The human plasma samples, collected from 8 volunteers, showed that this method can be used for therapeutic monitoring of ESL and its metabolites in humans treated with ESL.
Keywords: Eslicarbazepine acetate; Eslicarbazepine; R-licarbazepine; Oxcarbazepine; Antiepileptic drugs; LC–MS/MS;

The use of SAX-HPLC–CD as a heparin screening strategy by A.J. Chmielewski; F.E. Stanley; A.M. Stalcup (2619-2623).
Heparin, a heterogeneous polysaccharide, has been widely used as an anticoagulant for decades. Recently, however, international events involving the sudden onset of allergic-type reactions following heparin administration led to numerous fatalities, and demanded the use of multiple laborious, time consuming techniques to identify an economically motivated adulterant. Using these methods cooperatively, the semi-synthetic molecule known as oversulfated chondroitin sulfate (OSCS), was found to be present at significant concentrations. Since the discovery of this adulterant, several analytical methods have been put forth or updated to advance the process of screening pharmaceutical heparins; of these, strong anion exchange high performance liquid chromatography (SAX-HPLC) methods have now become routine. In this preliminary work, we report the use of circular dichroism (CD) detection in conjunction with existing SAX-HPLC methods to quantitate various sulfated polysaccharides. The proposed strategy exploits the selectivity associated with CD detection of heparin and heparin-like polysaccharides, while taking advantage of the method's insensitivity to the use of mobile phase additives and programmed gradients. The limit of detection of heparin by CD was found to be ∼0.22 mg/mL, whereas traditional UV/Vis detection yielded a detection limit of ∼1.09 mg/mL. The success of CD detection varied for other polymers, however no significant modifications were made to the separations method to capitalize on the advantages of CD detection.
Keywords: Circular dichroism; Dermatan sulfate; Glycosaminoglycan; Heparin; Oversulfated chondroitin sulfate; SAX-HPLC;

A column switching high performance liquid chromatographic method with estimable sensitivity and accuracy was developed for the determination of cetirizine and ambroxol in human plasma using nebivolol as the internal standard. Plasma samples were prepared by liquid–liquid extraction in methylene chloride and a mixture of diethylether (80:20, v/v). The extracted samples were injected into a multifunctional clean-up column SupelcosilTM LCABZ (50 mm × 4.6 mm, 5 μm particle size) using mobile phase 1 comprising acetonitrile–phosphate buffer (pH 3.5; 20 mM) (20:80, v/v). The eluate of cetirizine and ambroxol were separated to an analytical Kromasil C8 micro bore column (50 mm × 0.3 mm, 5 μm particle size) via a column switching device. A Kromasil C18 analytical column (250 mm × 2.1 mm, 5 μm particle size) was used as a separation column. Mobile phase 2 consisting acetonitrile–triethylamine (0.5%) in phosphate buffer (pH 3.5; 20 mM) (55:45, v/v) was used for the compound elution. The eluents were detected at 230 nm with photodiode array detector. An aliquot of 150 μl of plasma sample was introduced into the pretreatment column via the auto sampler using mobile phase 1 at a flow rate of 0.5 ml/min, column switching valve being positioned at A. The pretreatment column retained cetirizine, ambroxol and nebivolol (IS) in the column leaving the residual proteins of plasma eluted in void volume and drained out. The switching valve was shifted to position B at 7.5 min. Cetirizine, ambroxol and IS were eluted from the pretreatment column between 7. 5 and 11.5 min and introduced to the concentration column. Finally, cetirizine, ambroxol and IS were introduced to the separation column by switching valve using mobile phase 2 at a flow rate of 0.4 ml/min. During the analysis the pretreatment column was washed for the next analysis and resume to the position A. The total run time was 25 min for a sample. The procedure was repeated for urine analysis also. The method was linear from 2 to 450 ng/ml and 7–300 ng/ml for cetirizine and ambroxol respectively in plasma and 1–500 ng/ml and 5–400 ng/ml, respectively for cetirizine and ambroxol in urine. Intra-day and inter-day precision of cetirizine and ambroxol was below 15% in terms of coefficient of variation and accuracy of cetirizine and ambroxol was ranged from 94 to 101.6% and 91.1 to 100.2%, respectively. The method demonstrated high sensitivity and selectivity and therefore, applied to evaluate pharmacokinetics of cetirizine and ambroxol in healthy human volunteer after a single oral administration. Urine samples obtained from healthy human volunteers and clinical subjects with renal impairment have also been analyzed by the method to compare the elimination pattern. The method was precise and accurate for the estimation of cetirizine and ambroxol both in blood and in urine.
Keywords: Cetirizine HPLC; Cetirizine ambroxol HPLC; Column switch HPLC; Cetirizine ambroxol switching elution; Column switching PK;

Dalbavancin is a novel second-generation lipoglycopeptide antibiotic with activity against broad range of Gram-positive pathogens. In order to determine the pharmacokinetics (PK) of dalbavancin in pediatric patients, a new High Performance Liquid Chromatography–Tandem Mass Spectrometry (HPLC–MS/MS) bioanalytical method has been developed for quantification of dalbavancin in plasma and in urine. The plasma method was validated for dalbavancin in the linear range from 0.5 μg/mL to 500 μg/mL using 50 μL of K2 EDTA plasma. For dalbavancin spiked in urine, non-specific binding (NSB) of the drug to polypropylene (PP) urine collection containers was observed. The loss amounted to about 10% per transfer. After successfully establishing the collection/sampling procedure for urine by addition of Triton X-100 to the collection vessels (with a purpose of preventing NSB), the method was validated for dalbavancin in the range from 0.05 μg/mL to 50 μg/mL, using 100 μL of urine. These methods were used to quantify dalbavancin in plasma and urine of hospitalized children in a pediatric dalbavancin PK study. Eighteen percent of the total number of plasma study samples was reassayed for incurred samples reproducibility (ISR) and all the reassayed dalbavancin concentrations were within the ±20% limits. For urine, all the collected samples were reassayed for ISR and the original dalbavancin concentration was confirmed within the ±20% limits for 17 (94%) samples; the one remaining urine sample had its reassayed concentration confirmed within ±25% of the original result.
Keywords: Dalbavancin HPLC–MS/MS in plasma; Non-specific binding; Pediatric pharmacokinetic sampling; Urine sample collection; Validation; ISR;

A rapid LC–MS/MS method for confirmatory testing of five major categories of drugs of abuse (amphetamine-type substances, opiates, cocaine, cannabis metabolites and benzodiazepines) in urine has been developed. All drugs of abuse mandated by the Australian/New Zealand Standard AS/NZS 4308:2008 are quantified in a single chromatographic run. Urine samples are diluted with a mixture of isotope labelled internal standards. An on-line trap-and-flush approach, followed by LC–ESI-MS/MS has been successfully used to process samples in a functioning drugs of abuse laboratory. Following injection of diluted urine samples, compounds retained on the trap cartridge are flushed onto a reverse-phase C18 HPLC column (5-μm particle size) with embedded hydrophylic functionality. A total chromatographic run-time of 15 min is required for adequate resolution. Automated quantitation software algorithms have been developed in-house using XML scripting to partially automate the identification of positive samples, taking into account ion ratio (IR) and retention times (Rt). The sensitivity of the assay was found to be adequate for the quantitation of drugs in urine at and below the confirmation cut-off concentrations prescribed by AS/NZS 4308:2008.
Keywords: LC–MS/MS; Drugs of abuse; Online extraction;

The residues of sulfonamides (SAs) in the foods of animal origin are of the major concern because they are harmful to the consumer's health and could induce pathogens to develop resistance. Rapid and efficient determination methods are urgently in need. A quantitative high performance liquid chromatography method (HPLC) and a confirmative liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous determination of 18 sulfonamides such as sulfamidinum, sulfanilamide, sulfisomidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfisoxazole, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadoxine, sulfaclozine, sulfadimethoxine, sulfaquinoxaline in the muscles, livers and kidneys of swine, bovine and chicken were developed and validated. The sample preparation procedures included a pressurized liquid extraction (PLE) with acetonitrile conducted at elevated temperature (70 °C) and pressure (1400 psi). After clean-up with hydrophilic–lipophilic balance cartridge, the extraction solution was concentrated and analyzed by HPLC and LC–MS/MS analysis. 18 SAs were separated by the HPLC with a Zorbax SB-Aq-C18 column and the mobile phase of methanol/acetonitrile/1% acetic acid with a gradient system. The wavelength of UV for the HPLC detection was set at 285 nm. The LC–MS/MS analysis was achieved with a Hypersil Golden column and the mobile phase of acetonitrile and 0.1% formic acid aqueous solution with two gradient systems. The Limits of detection (LOD) and the limits of quantitation (LOQ) were 3 μg/kg and 10 μg/kg, respectively, for both of the HPLC and LC–MS/MS. Linearity was obtained with an average coefficient of determination (R) higher than 0.9980 over a dynamic range from the LOQ value up to 5000 μg/kg. The recoveries of the methods range from 71.1% to 118.3% with the relative standard derivation less than 13%. The peaks of interest with no interferences were observed throughout the chromatographic run. The sample pretreatment provided efficient extraction and cleanup that enables a sensitive and rugged determination of 18 SAs, the obtained results revealed that PLE, in comparison with other sample preparation methods applied, has significantly higher efficacy for SAs isolation from animal tissues.
Keywords: Sulfonamides; Pressurized liquid extraction; High performance liquid chromatography; Liquid chromatography–tandem mass spectrometry; Edible tissues; Food animals;

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma by Hongliang Jiang; YuRong Wang; Manjunath S. Shet; Yang Zhang; Duane Zenke; Douglas M. Fast (2663-2668).
A rapid, specific, and reliable LC–MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-d-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤6.5% relative standard deviation (RSD) and −8.3 to −2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples.
Keywords: Naloxone; 6β-Naloxol; Naloxone-3β-d-glucuronide; LC–MS/MS; Quantification;

Determining the pharmacokinetics of psilocin in rat plasma using ultra-performance liquid chromatography coupled with a photodiode array detector after orally administering an extract of Gymnopilus spectabilis by Jianbo Chen; Meijia Li; Xitao Yan; Enqi Wu; Hongmei Zhu; Kwan Jun Lee; Van Men Chu; Lifeng Zhan; Wonjae Lee; Jong Seong Kang (2669-2672).
This study established ultra-performance liquid chromatography coupled with a photodiode array detector for determining psilocin and its pharmacokinetics in rat plasma after orally administering an extract of Gymnopilus spectabilis. The extract was separated on an ODS C18 column (2.3 μm, 100 mm × 2.1 mm I.D.) by gradient elution with (A) water containing 50 mM AcONH4 and (B) acetonitrile. The wavelength was set at 265 nm and the injection volume was 10 μL. Under these conditions, the calibration curve was linear over the concentration range 0.2–20 μg/mL with a correlation coefficient of r 2  = 0.9992. The inter- and intraday precision levels were less than 7% and the accuracies (%) were within the range 92.0–102.5%. The method was sufficiently valid to be applied to a pharmacokinetics study of psilocin in rat plasma. The pharmacokinetic parameters of psilocin in rat plasma after the oral administration of a G. spectabilis extract were as follows: C max, 0.43 ± 0.12 μg/mL; T max, 90 ± 2.1 min; AUC0→t , 1238.3 ± 96.4 (μg/mL) min; and T 1/2, 117.3 ± 40.3 min.
Keywords: Pharmacokinetic; Psilocin; Gymnopilus spectabilis; UPLC-PDA;

A validated method for the determination of Combretastatin A1 phosphate (CA1P, OXi4503), a bisphosphate prodrug of the vascular disrupting agent Combretastatin A1 in human plasma has been developed using fluorescence detection after post-column photolysis. The separation used the ion-pairing agent tetrabutylammonium hydrogen sulphate, and this agent was also required to give consistently high recovery from plasma. Initially, the range was shown to be linear (r 2  > 0.995) from the LOQ of 0.025 μM to 5 μM, but as the trial progressed to much higher doses, using a lower injection volume, the assay was subsequently subject to limited revalidation to cover the range from 0.05 to 50 μM. Intra-assay precision and accuracy ranged from 2.2 to 11.8% and 1.8 to 13% respectively, and for inter-assay from 4.4 to 14.9% and 1.7 to 6.5%. Mean recovery of OXi4503 from plasma was 80.2%.
Keywords: OXi4503; CA1P; Plasma; Ion-pairing; HPLC; Fluorescence;

Corrigendum to “Dried blood spot assay for estimation of metronidazole concentrations in rats and its application in single animal drug pharmacokinetic study” [J. Chromatogr. B 879 (2011) 1713] by Prashant Laxman Kole; Rita Majithia; Thakur Raghu Raj Singh; Martin J. Garland; Katarzyna Migalska; Helen O. McCarthy; Ryan F. Donnelly; James McElnay (2677).