Journal of Chromatography B (v.880, #C)

The purpose of this study was to develop and validate a high-performance liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for analysis of the deacetyl mycoepoxydiene in rat plasma. The analyte and internal standard (I.S.), benorilate, were extracted from rat plasma by precipitation protein and separated on a C18 column using acetonitrile–0.5% formic acid as mobile phase. Detection was performed using a turbo-spray ionization source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) at an ion voltage of +4800 V. The assay was linear over the concentration range 5–5000 ng/mL with the lowest limit of quantification (LLOQ) of 5 ng/mL. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD < 5.8%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to a preclinical pharmacokinetic study in rats after a single intravenous administration of deacetyl mycoepoxydiene 10 mg/kg.
Keywords: Deacetyl mycoepoxydiene; LC–MS/MS; Pharmacokinetics; Rat; Plasma;

The determination of budesonide and fluticasone in human sputum samples collected from COPD patients using LC–MS/MS by B.A.P. Buscher; H. Jägfeldt; H. Sandman; R. Brust-van Schaik; F. van Schaik; L.P. Brüll (6-11).
► We validated an LC–MS/MS method for budesonide and fluticasone in human sputum. ► The LLOQ was 5 nM. ► Intra- and inter-run RSD were within 6.9% (budesonide) and 8.0% (fluticasone). ► The accuracy was within ±15%. ► The method was applied to clinical sputum samples.A bioanalytical method for the quantitative determination of budesonide and fluticasone in human sputum was developed. Sputolysin® Reagent was added to the sputum samples. After incubation (37 °C; 60–70 min under shaking) and automated solid phase extraction the extracts were analysed using LC–MS/MS. Budesonide and fluticasone showed good linearity (r  > 0.99) over the range 0.1–100 nM in the first and second validation batch, and over the range 0.25–10,000 nM in the third and fourth validation batch. The lower limit of quantification (LLOQ) achieved was 5 nM for budesonide and fluticasone in 100 μL human sputum. Intra-run and inter-run RSD for four quality control levels (5–100 nM) were within 6.9% (budesonide) and 8.0% (fluticasone). The accuracy ranged from −11.4% to −1.6% (budesonide), and from −11.8% to 0.4% (fluticasone). The validated method was applied to clinical sputum samples from COPD patients.
Keywords: Sputum; HPLC; Mass spectrometry; Validation; Sample pre-treatment; Bioanalysis;

► FMOC-Cl is a suitable labeling agent for derivatization of chemicals amines or hydroxyl groups. ► The reagent was applied in labeling of valproate (VPA) an anticonvulsant with carboxylic acid moiety. ► To document reaction between FMOC-Cl and carboxylic acid moiety a LC–MS/MS method was developed. ► The method is simple, rapid, sensitive which needs using lower sample volume. ► Potential application of FMOC in assay of carboxylic acids, makes our method attractive.In High Performance Liquid Chromatographic (HPLC) determination of chemicals with acidic functions, different labeling agents are used to improve sensitivity of the assay. 9-Fluorenylmethyl chloroformate (FMOC-Cl), on the other hand, is a suitable labeling agent, which reacts with both primary and secondary amines and less readily with hydroxyl groups in alkaline conditions. However, the reagent has not been applied in labeling of chemicals with acidic function yet. In this study which is the first report on application of FMOC-Cl in derivatization and analysis of a drug with acidic function, valproic acid (VPA), one of a series of fatty carboxylic acids with anticonvulsant activity, was derivatized using the reagent and quantified in serum samples by HPLC with fluorescence detection. In addition, to document the reaction between the labeling agent and carboxylic acid moiety of the drug, we developed a liquid chromatography–tandem MS/MS (LC–MS/MS) method. Following liquid–liquid extraction, derivatization of the drug and an internal standard was achieved in alkaline medium. The elute was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The present method is more sensitive comparing with other published HPLC procedures for analysis of VPA. The assay is sensitive enough to measure drug levels obtained in human single dose studies with a limit of quantification of 0.01 μg/mL. Also the method is linear over the concentrations range of 0.01–32 μg/mL of VPA in human serum using 100 μL serum sample and 5 μL injection. The coefficient variation values of both inter and intra day analysis were less than 12% and the percentage error was less than 4%. The method performance was studied and the validated procedure applied in a randomized cross-over bioequivalence study of two different VPA preparations in 24 healthy volunteers.
Keywords: Fluorenylmethyl chloroformate; FMOC Cl; Derivatization; LC–MS/MS; Valproic acid; Bioequivalence study;

► We purified a plant-produced HPV16 subunit vaccine candidate. ► High purity and good overall process yield was achieved for the product. ► The process was scaled-up from 50 g to 1 kg biomass scale. ► Product quality was confirmed by a diverse set of analysis methods.Several studies indicated that biopharmaceuticals based on the recombinant protein E7 of human papillomavirus (HPV) can serve as therapeutic vaccines preventing the development of cancer in women infected with high-risk types of HPV such as HPV16. Here, we report effective extraction and purification of a plant-produced E7GGG-lichenase fusion protein, an HPV16 subunit vaccine candidate, from Nicotiana benthamiana plants, to a high yield. The target contains the modified HPV16 E7 protein internally fused to the surface loop of a truncated, hexa-His- and KDEL-tagged variant of bacterial lichenase, and has been previously shown to possess anti-cancer activity in an animal model . We purified the protein using a combination of immobilized metal-ion affinity chromatography and gel filtration. The achieved purity of the final product was 99% as confirmed by Coomassie or SYPRO Ruby staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical size exclusion chromatography coupled with multi-angle laser light scattering. The overall yield was 50% corresponding to 0.1 g of protein per 1 kg plant biomass. Only slight changes in these parameters were observed during the process scale-up from 50 g to 1 kg of processed leaf biomass.
Keywords: Plant-produced vaccine; Purification; Recombinant protein; Biopharmaceutical; HPV16; Transient expression;

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins by Brian D. Soriano; Lei-Ting T. Tam; Hsieng S. Lu; Violeta G. Valladares (27-33).
► A fluorescent-based assay for the quantification of thiol adducts is described. ► E. coli-derived recombinant proteins require refolding for activity. ► Cysteine and cystamine from the redox-couple can generate thiol-protein adducts. ► Released thiols are derivatized and separated by reversed-phase HPLC. ► Quantification of thiol adducts is accomplished by comparison to standard curves.Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein.
Keywords: Peptibody; Cysteine; Cysteamine; Adduct; Thiol; AccQTag; HPLC; Mass spectrometry;

► Combination of chemometrics and HPLC was used for optimization of separation of flavonoids. ► Flavonoids from different classes were separated using an isocratic mode of HPLC. ► By using the isocratic mode we could overcome the limitations of gradient elution. ► Good separation of flavonoids was obtained in reasonable analysis time.The chemometrics approach was applied for simultaneous optimization of resolution and analysis time of seven flavonoids in reverse phase liquid chromatography. Derringer's desirability function, a multi-criteria decision making method, was used for the evaluation of two different chromatographic performance goals including resolution and analysis time. The selected flavonoids belong to different classes of flavonoids including: flavonols (myricetin, morin, quercetin and kaempferol) flavones (apigenin and luteolin) and flavanones (naringenin). The effect of five experimental factors on a chromatographic response function, formed using two sigmoidal desirability functions, was investigated. The sigmoidal functions were used to transform the optimization criteria, resolution and analysis time, into the desirability values. The factors studied were percentages of methanol, phosphoric acid and THF in mobile phase as well as flow rate and temperature. A rotatable, orthogonal central composite design was used to map the chromatographic response surface and then calculated chromatographic response functions were fitted to a polynomial model. The obtained regression model was characterized by both descriptive and predictive ability (R 2  = 0.96). The model was verified, as good agreement was observed between the predicted and experimental values of the chromatographic response function in the optimal condition. The most effective factor on retention of flavonoids was percentage of methanol in mobile phase followed by temperature, flow rate, THF and H3PO4 percentages. Optimum condition for separation of flavonoids was as follows: methanol:0.4% phosphoric acid in water:THF (45.3:54.4:0.3, v/v/v) as mobile phase with flow rate of 1 mL min−1 at 30 °C. The optimum condition was applied for separation of flavonoids of Satureja sahendica Bornm. which is an endemic medicinal plant of Iran.
Keywords: Flavonoids; Separation optimization; Derringer's desirability function; Central composite design;

Determination of bioactive compounds in fermented soybean products using GC/MS and further investigation of correlation of their bioactivities by Chuan Chai; Hyun Kyoung Ju; Sang Cheol Kim; Jeong Hill Park; Johan Lim; Sung Won Kwon; Jeongmi Lee (42-49).
► Comparison between active ingredients and bioactivities of soybean and 5 soybean products. ► GC/MS analysis of soybean and 5 soybean products and validation. ► Correlation analysis between components and bioactivities (two estimators: R and R*).The active ingredients and bioactivities (anti-oxidant, anti-tyrosinase, anti-proliferative and estrogenic activities) of soybean and soybean products (Cheonggukjang, Meju, Makjang, Doenjang and soy sauce) produced by different fermentation processes were compared. There were high correlations between active ingredients and bioactivities. Free phenolic acids extracted from soybean products were identified and quantified by gas chromatography/mass spectrometry (GC/MS). Overall, the components and activities in fermented soybean products were different than those in soybeans. Total phenolic content (TPC), protein content (PC) and anti-oxidant activity increased as fermentation time increased. TPC and PC showed strong negative correlations with anti-oxidant activity. Doenjang and soy sauce, two long-term fermented products, showed lower total flavonoid content (TFC) and estrogenic activities than short-term fermented soybean products. This might be explained by the decomposition and hydrolysis of flavonoids due to the long fermentation time and high temperature. Strong anti-proliferative activity against cancer cell lines, which was highly correlated with TFC, was found in Meju and Cheonggukjang. Soybean and all fermented products except Meju exhibited effective tyrosinase inhibitory activities. Fermented products showed stronger estrogenic activity than soybeans, which was highly correlated with syringic acid.
Keywords: Soybean products; Fermentation; Active ingredients; Bioactivities; GC/MS;

► Fast and quantitative analysis method is presented for STX for verification purposes for CWC and also for food analyses and environmental monitoring. ► The developed method separates STX from other PSP toxins which are also identified by full scan MS/MS analysis. ► Food and Drug Administration's (FDA's) bioanalytical method validation recommendations were used and for identification, WADA criteria have been evaluated for verification of STX by using four diagnostic ions in MS/MS analysis. ► HILIC–MS/MS method has proven to be linear at concentration ranges from 5 to 50 ng/ml and from 25–200 ng/ml, and accurate and precise in both LLOQ and ULOQ levels. ► The presented HILIC–MS/MS method has been found to be applicable in proficiency testing of STX and quantitative analysis of STX in algal samples.Hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was validated with algal samples for verification and quantification of saxitoxin (STX), a potent neurotoxin which is listed in the Chemical Weapons Convention (CWC) in Schedule 1A. Isocratic elution, conventional bore HILIC column and high flow rate together with accurate post-column splitter provided detection of STX in 6.5 min with total analysis time of 9 min per sample. STX analogue, gonyautoxin 1 (GTX 1) was used as an internal standard. Sample preparation of freeze-dried algae included liquid extraction and centrifugal filtering with mean recovery of 99.9% at concentration level of 10 ng/ml (n  = 3). Retention times for STX and GTX 1 were 6.47 ± 0.03 min and 4.44 ± 0.01 min (n  = 45), respectively. Four diagnostic product ions were used for reliable verification of saxitoxin. Method was found to be precise and linear (R 2  = 0.9714 and R 2  = 0.9768) in concentration ranges of 5–50 ng/ml and 25–200 ng/ml, respectively. For saxitoxin, calculated LOD was 3 ng/ml and LLOQ 11 ng/ml. Validation was conducted using spiked algal matrix since this method is not only needed for verification analysis for the CWC but also for safety analysis of other environmental samples for presence of STX. Identification criteria for verification of STX with HILIC–MS/MS method are discussed.
Keywords: Saxitoxin; Quantification; HILIC; MS/MS; Validation; Identification criteria;

In vivo monitoring of cerebral agmatine by microdialysis and capillary electrophoresis by Luis Betancourt; Pedro Rada; Daniel Paredes; Luis Hernández (58-65).
► The combination of microdialysis and CZE and laser induced fluorescence detection (LIFD) for agmatine has not been attempted before. ► We used capillary zone electrophoresis and laser induced fluorescence detection (CZE–LIFD) to measure nanomolar concentrations of agmatine in submicroliter sample volumes. ► This analytical technique proved to detect 0.49 attomole of agmatine improving the sensitivity of previous analytical techniques. ► We successfully applied the technique to measure agmatine in brain dialysates from the hippocampus where agmatine acts as a putative neurotransmitter.Agmatine is a putative neurotransmitter in the brain. Current analytical techniques do not allow the detection of agmatine in extracellular fluid, making it difficult to study its physiological role. However, a new method for in vivo monitoring agmatine in the brain was developed. Capillary zone electrophoresis and laser induced fluorescence detection (CZE–LIFD) was used to measure nanomolar concentrations of agmatine in submicroliter sample volumes. This analytical technique proved to detect 0.49 attomole of agmatine improving the sensitivity of previous analytical techniques. On the other hand, the hippocampus is a brain region well known for having a population of agmatine containing neurons. Therefore, intracerebral microdialysis was performed in the hippocampus and agmatine was extracted from the extracellular environment. Detectable amounts of agmatine were found in dialysates from probes located in the hippocampus but not from the probes located in the lateral ventricle. Furthermore, extracellular agmatine was calcium and impulse dependent and depolarization of hippocampal neurons increased extracellular agmatine concentration. The methods reported here are sensitive enough to study the physiological role of brain agmatine in freely moving animals.
Keywords: Capillary electrophoresis; Neurotransmission; Chemical messengers; Bioamines; Probe recovery;

► We develop an analytical method of 15 urinary metabolites in general population. ► They are exposed to organophosphorus compounds and moth repellents. ► The metabolites from hydrolyzed urine are analyzed by GC/MS(EI) after derivatization. ► They can be measured accurately and precisely (quantification limits: 0.8–4 μg/l). ► The collected urine samples can be stored for up to 1 month at −20 °C in a freezer.An analytical method was developed for simultaneous measurement of urinary metabolites in the general population exposed to organophosphorus compounds (insecticides, flame retardants and plasticizers) and moth repellents used in Japanese households. Fifteen metabolites, dimethylphosphate, dimethylthiophosphate, diethylphosphate, diethylthiophosphate, di-n-butylphosphate, diphenylphosphate, bis(2-ethylhexyl)phosphate, 2-isopropyl-6-methyl-4-pyrimidinol, 3,5,6-trichloro-2-pyridinol, 3-methyl-4-(methylthio)phenol, 3-methyl-4-nitrophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 1-naphthol and 2-naphthol, were extracted from hydrolyzed urine by using a sorbent (hydroxylated polystyrene-divinylbenzene copolymers), and then desorbed with methylacetate and acetonitrile, concentrated, and after transformation to their tert-butyldimethylsilyl derivatives, analyzed by gas chromatography/mass spectrometry in the electron impact ionization mode. They could be determined accurately and precisely (quantification limits: 0.8–4 μg/l). The collected urine samples could be stored for up to 1 month at −20 °C in a freezer.
Keywords: Gas chromatography/mass spectrometry; Urinary metabolites; Indoor air pollution; Insecticides; Flame retardants; Plasticizers;

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system by N. Mendoza; A. Casao; I. Del Valle; E. Serrano; S. Nicolau; M.E.O.A. Asumpção; T. Muiño-Blanco; J.A. Cebrián-Pérez; R. Pérez-Pé (74-81).
► CCCD differentiates sperm subpopulations of different quality characteristics. ► These include apoptotic changes and capacitation state. ► Sperm recovered from a two-phase system are suitable for fertilization assays. ► Separated subpopulations differ in ability of binding to zona pellucida of oocytes.Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 °C or cooled down to 5 °C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/PI. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V, and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 °C than at 20 °C, which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P  < 0.01) of membrane integrity, lower PS translocation (P  < 0.05) and DNA damage (P  < 0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature.
Keywords: Sperm subpopulations; Apoptosis; Refrigeration; Two-phase partition;

A monolith purification process for virus-like particles from yeast homogenate by Claire S. Burden; Jing Jin; Aleš Podgornik; Daniel G. Bracewell (82-89).
► Separation of HBsAg virus-like particles from yeast is achieved with a monolith. ► Dynamic binding capacity for VLP with untreated feed is 3–4x that of a packed bed. ► Confocal microscopy is used to visualise the degree and position of lipid fouling. ► A lipid layer is seen on the monolith even after regeneration with 30% isopropanol. ► Reducing the lipid in the yeast homogenate causes the capacity to increase 2.5x.Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. The monolith structure provides a more open pore structure to improve accessibility for these materials and better mass transport from convective flow and reduced pressure drops. To examine the performance of this format for bioprocessing we selected the challenging capture of a VLP from clarified yeast homogenate. Using a recombinant Saccharomyces cerevisiae host it was found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity was shown to be three-fold superior for the monolith with equivalent 90% recovery of the VLP. To understand the impact of the crude feed material confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. It was seen that the lipid formed a layer on top of the column, even after regeneration of the column with isopropanol, resulting in increasing pressure drops with the number of operational cycles. Removal of the lipid pre-column significantly reduces the amount and rate of this fouling process. Using Amberlite/XAD-4 beads around 70% of the lipid was removed, with a loss of VLP around 20%. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25 mg/mL column.
Keywords: Monoliths; Fouling; Confocal; Vaccines; Virus-like particle;

► The first HPLC method for nor-NOHA assay was developed and validated. ► Sub-3-um fused core sorbent was used for the separation. ► Method is compatible with fluorimetric and MS detection. ► nor-NOHA metabolite was identified as nor-arginine using LC–MS/MS. ► Pharmacokinetic experiment with nor-NOHA was conducted on rats.For the purpose of in vivo pharmacokinetic studies, an HPLC method was developed and validated for the quantification of N-(ω)-hydroxy-nor-l-arginine, l-arginine and N-(ω)-ethyl-l-arginine (internal standard) in rat plasma. Sample processing involved a solid-phase extraction on the Waters MCX cartridges and on-line pre-column derivatization of the analytes with o-phthaldialdehyde and 3-mercaptopropionic acid. Separation of the derivatives was carried out on a core–shell Kinetex C18 column in a gradient elution mode with a mobile phase consisting of methanol and water (pH = 3.00 adjusted with formic acid). Fluorimetric detection with the excitation/emission wavelengths of 235/450 nm was used. The method was validated according to the FDA guidelines and applied to pilot pharmacokinetic experiments. An unknown metabolite was extracted from the plasma of Wistar rats after a single bolus of N-(ω)-hydroxy-nor-l-arginine (i.v. 10 mg kg−1). The metabolite was identified as nor-l-arginine using mass spectrometry. Validated method was successfully used for pilot pharmacokinetic experiment on rats.
Keywords: N-(ω)-Hydroxy-nor-l-arginine; nor-l-Arginine; Core–shell column; Pharmacokinetics; OPA-derivatization; HPLC;

Development and validation of a simple reversed-phase HPLC method for the determination of camptothecin in animal organs following administration in solid lipid nanoparticles by Susana M. Martins; Thierry Wendling; Virgínia M.F. Gonçalves; Bruno Sarmento; Domingos C. Ferreira (100-107).
► Development of a HPLC method for camptothecin (CPT) pharmacokinetic studies. ► CPT was administered in solid lipid nanoparticles to rats. ► Fluorescence detection allowed quantification limit value ≤0.5 ng/mL. ► The method was specific, accurate and precise in all biological matrices. ► CPT was stable after 24 h at RT or at 4 °C, or after three freeze/thaw cycles.A simple, sensitive and specific high-performance liquid chromatography (HPLC) assay for the quantification of camptothecin (CPT), a potent anticancer candidate, incorporated into solid lipid nanoparticles (SLN) in several rat organs (brain, heart, kidneys liver, lung, spleen) and serum was developed and validated. The sample pre-treatment involved organs homogenisation followed by CPT extraction. The samples were injected onto an analytical reversed-phase (RP) Mediterranea™ Sea18 column maintained at 30 °C. The chromatographic separation was achieved by gradient elution consisting of triethyamine buffer pH 5.5 and acetonitrile at a flow rate of 1.2 mL/min in 16 min of run time and retention time of 9.8 min (lactone). Fluorescence detection was used at the excitation and emission of 360 and 440 nm, respectively. The calibration curves in the different organs, serum and in PB3 were linear (r 2  > 0.9999) over CPT concentrations ranging from 1 to 200 ng/mL or 0.5 to 200 ng/mL (n  = 6), respectively. The method was shown to be specific, accurate (between 94.4 ± 4.5% and 108.9 ± 0.6%) and precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV < 6.3%) at three different concentrations (10, 50 and 100 ng/mL) in all matrices. Stability studies showed that CPT was stable in all matrices after 24 h of incubation at room temperature (RT), after 24 h in the autosampler or after three freeze/thaw cycles. The mean recoveries of CPT in suspension, loaded into SLN and in a physical mixture with SLN at three concentrations of 10, 50 and 200 ng/mL were higher than 86.4%. The detection limit (DL) was ≤0.2 ng/mL and the quantification limit (QL) was ≤0.5 ng/mL. The method developed is reliable, precise and accurate and can be used in the determination of CPT amount in rat organ samples after i.v. administration of CPT in suspension, in physical mixture with SLN and incorporated in SLN.
Keywords: Solid lipid nanoparticles (SLN); Camptothecin (CPT); Anticancer; RP-HPLC; Fluorescence detector; In vivo;

► This work reports the extraction of oxymatrine from Sophora flavescens Ait. extract to silica confined ionic liquids by adsorption with subsequent solid phase extraction. ► Target compound was adsorbed on the sorbent without much interference, which decreased the requirement of washing solvents. ► This method can be developed as an integrated process including extraction, concentration and separation.This study highlighted the application of a two-stepped extraction method for extraction and separation of oxymatrine from Sophora flavescens Ait. extract by utilizing silica-confined ionic liquids as sorbent. The optimized silica-confined ionic liquid was firstly mixed with plant extract to adsorb oxymatrine. Simultaneously, some interference, such as matrine, was removed. The obtained suspension was then added to a cartridge for solid phase extraction. Through these two steps, target compound was adequately separated from interferences with 93.4% recovery. In comparison with traditional solid phase extraction, this method accelerates loading and reduces the use of organic solvents during washing. Moreover, the optimization of loading volume was simplified as optimization of solid/liquid ratio.
Keywords: Silica-confined ionic liquid; Extraction; Separation; Alkaloid; Plant food;

► We developed a HPLC–UV method to detect goldenseal root alkaloids. ► Testing for goldenseal prior to urine drug testing eliminates false positives. ► We extracted and detected berberine and hydrastine alkaloids from goldenseal. ► The method can be used to analyze goldenseal alkaloids in powder, liquid and urine.A screening method was developed to extract and detect berberine and hydrastine alkaloids from goldenseal root powder and urine samples using HPLC with UV detection. The isocratic method was developed to detect alkaloids in 5 mL of urine prior to drug screening. Urine samples were spiked with the alkaloids at varying concentrations and extracted twice with 3:1 chloroform:2-propanol (CHCl3:2-propanol). The extracts were combined, concentrated using nitrogen gas and the residue was then reconstituted with a mobile phase of acetonitrile:buffer (32:68). A 17 min isocratic run time was performed with a flow rate of 2.0 mL/min, and UV detection at 230 nm using a C18 (250 mm × 4.6 mm) column at room temperature. The method showed good linearity for berberine (r 2  = 0.9990) and hydrastine (r 2  = 0.9983) over a range of 11.80 ng/mL to 17.64 μg/mL. The LOD for berberine in urine was 12.74 ng/mL and the LOD for hydrastine in urine was 54.48 ng/mL. Urine samples were spiked with goldenseal root powder and liquid extract as part of a blinded study to determine whether berberine and hydrastine alkaloids could also be extracted in vitro from goldenseal and show a presence in urine samples. Out of the 37 blinded urine samples extracted the two spiked samples were correctly identified based on the presence or absence of berberine and hydrastine. The results demonstrated that this method will enable laboratories to test for the herbal supplement in submitted urine samples prior to drug testing, avoiding false negative results.
Keywords: Forensic science; Forensic toxicology; Goldenseal root; High performance liquid chromatography; Urine; Hydrastis canadensis L.; Berberine; Hydrastine; Alkaloids;

Rapid quantification of histamine in human psoriatic plaques using microdialysis and ultra high performance liquid chromatography with fluorescence detection by Elizabeth Guihen; Wen Lyn Ho; Anna-Marie Hogan; Marie-Louise O’Connell; Martin J. Leahy; Bart Ramsay; William T. O’Connor (119-124).
► Development of a rapid and ultra-sensitive liquid chromatographic method for the separation and detection of histamine. ► This represents one of the fastest reported separations of histamine using fluorescence detection. ► Intracutaneous histamine was measured 70 min after catheter implantation, a second fraction was collected 190 min after implantation and revealed similar levels with no difference in intracutaneous histamine between the control, peri-lesional and lesional skin regions. ► When combined with a novel rapid sensitive analytical method microdialysis may be used to explore the role of mast cells in psoriasis.Psoriasis is a chronic skin disease resulting from abnormal immune function and is characterized by the presence of scaly psoriatic plaques which are areas of inflammation and excessive skin production . The psoriatic plaques contain mast cells which are increased in number in the uppermost dermis of the psoriatic lesion and which may play a role in the initiation and maintenance of the lesion. These processes are thought to be mediated via the local release of histamine along with other mediators from the mast cells; however their precise role still remains a mystery . Our study involved the development of a rapid and ultra-sensitive liquid chromatographic method for the separation and detection of histamine. To this end a state-of-the-art ultra high pressure liquid chromatography (UHPLC) system incorporating the latest technology in fluorescence detection system was employed which allowed for the rapid and reliable trace level detection of histamine in human derived microdialysate samples. This new reverse phase method utilized a sub-two-micron packed C18 stationary phase (50 mm × 4.6 mm, 1.8 μm particle size) and a polar mobile phase of ACN:H2O:acetic acid (70:30:0.05) (v/v). The column temperature was maintained at (30 ± 2 °C), the injection volume was (8 μl), with a flow rate of (1.1 ml/min). Dermal microdialysis was used to collect (20 μl) samples from healthy, peri-lesional and lesional skin regions, in the forearms of a small cohort of subjects (n  = 6), and the ultra sensitive liquid chromatographic method allowed for nanomolar quantitation of histamine in 6.7 min. To date this represents one of the fastest reported separations of histamine using fluorescence detection with very high chromatographic efficiency (258,000/m) and peak symmetry of (0.88). Prior to sample analysis being performed method linearity, precision and limit of detection (LOD) were investigated. The results showed that intracutaneous histamine measured at 70 min after catheter implantation was (3.44 ± .52 nmol) (mean ± SEM) in non-lesional (control) skin and was not dissimilar to that observed in either lesional (3.10 ± .76 nmol) or peri-lesional skin (2.24 ± .20 nmol). A second fraction collected 190 min after implantation also revealed similar levels with no difference in intracutaneous histamine observed between control (2.41 ± .56 nmol), lesional (2.69 ± .54 nmol), or peri-lesional skin (2.25 ± .50 nmol).
Keywords: Histamine; Psoriasis; Psoriatic plaques; Intracutaneous microdialysis; Mast cells; Fluorescence detection; Ultra high pressure liquid chromatography (UHPLC);

► We have developed a new procedure to measure CO in human blood. ► CO has never been quantified with internal labeled standard 13CO. ► We have validated the technique according SFSTP requirements and tested it on real postmortem blood samples. ► Our method is satisfactory to measure CO in human blood.The aim of our study was to provide an innovative headspace-gas chromatography–mass spectrometry (HS-GC-MS) method applicable for the routine determination of blood CO concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature for CO measurement is the absence of a specific CO internal standard necessary for performing quantification. Even if stable isotope of CO is commercially available in the gaseous state, it is essential to develop a safer method to limit the manipulation of gaseous CO and to precisely control the injected amount of CO for spiking and calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in a vial in situ, an internal labeled standard gas (13CO) formed by the reaction of labeled formic acid formic acid (H13COOH) with sulfuric acid. As sulfuric acid can also be employed to liberate the CO reagent from whole blood, the procedure allows for the liberation of CO simultaneously with the generation of 13CO. This method allows for precise measurement of blood CO concentrations from a small amount of blood (10 μL). Finally, this method was applied to measure the CO concentration of intoxicated human blood samples from autopsies.
Keywords: Forensic toxicology; Carbon monoxide; Headspace gas extraction;

A sensitive and rapid ultra HPLC–MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma by Cody J. Peer; Shawn D. Spencer; Dustin A.H. VanDenBerg; Michael A. Pacanowski; Richard B. Horenstein; William D. Figg (132-139).
► We developed and validated a novel uHPLC–MS/MS assay for the simultaneous quantification of clopidogrel and its derivatized active metabolite. ► Clinically relevant calibration ranges for clopidogrel (0.01–50 ng/mL) and active metabolite (0.1–150 ng/m) were used. ► Successfully applied to clinical phase I study using 3 subjects.A sensitive, selective, and rapid ultra-high performance liquid chromatography–tandem mass spectrometry (uHPLC–MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix®) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3′-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2 μm-C18 column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically relevant concentration range of 0.01–50 ng/mL for parent clopidogrel and 0.1–150 ng/mL (r 2  = 0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) <±12% and precision (%CV) of <±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range.
Keywords: Clopidogrel; Active metabolite; Ultra HPLC–MS/MS;

Enantioselective analysis of unbound tramadol, O-desmethyltramadol and N-desmethyltramadol in plasma by ultrafiltration and LC–MS/MS: Application to clinical pharmacokinetics by Natália Valadares de Moraes; Gabriela Rocha Lauretti; Marcio Nogueira Napolitano; Naiane Ribeiro Santos; Ana Leonor Pardo Campos Godoy; Vera Lucia Lanchote (140-147).
► Binding of drugs to plasma proteins can be stereoselective. ► Unbound concentrations of tramadol, M1 and M2 enantiomers were analyzed by MS/MS. ► Kinetics of tramadol and M2 was enantioselective in patients with neuropathic pain. ► Fraction unbound of tramadol enantiomers was higher than that of the (+)-M1.This study describes the enantioselective analysis of unbound and total concentrations of tramadol and its main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in human plasma. Sample preparation was preceded by an ultrafiltration step to separate the unbound drug. Both the ultrafiltrate and plasma samples were submitted to liquid/liquid extraction with methyl t-butyl ether. Separation was performed on a Chiralpak® AD column and tandem mass spectrometry consisting of an electrospray ionization source, positive ion mode and multiple reaction monitoring was used as the detection system. Linearity was observed in the following ranges: 0.2–600 and 0.5–250 ng/mL for analysis of total and unbound concentrations of the tramadol enantiomers, respectively, and 0.1–300 and 0.25–125 ng/mL for total and unbound concentrations of the M1 and M2 enantiomers, respectively. The lower limits of quantitation were 0.2 and 0.5 ng/mL for analysis of total and unbound concentration of each tramadol enantiomer, respectively, and 0.1 and 0.25 ng/mL for total and unbound concentrations of M1 and M2 enantiomers, respectively. Intra- and interassay reproducibility and inaccuracy did not exceed 15%. Clinical application of the method to patients with neuropathic pain showed plasma accumulation of (+)-tramadol and (+)-M2 after a single oral dose of racemic tramadol. Fractions unbound of tramadol, M1 or M2 were not enantioselective in the patients investigated.
Keywords: Tramadol; Metabolites; Enantiomers; Pharmacokinetics; Ultrafiltration; LC–MS/MS; Fraction unbound;

Continuous aqueous two-phase extraction of human antibodies using a packed column by P.A.J. Rosa; A.M. Azevedo; S. Sommerfeld; W. Bäcker; M.R. Aires-Barros (148-156).
► We evaluated the performance of a pilot scale packed column for the continuous extraction of IgG. ► Aqueous two-phase systems of PEG and salt enable IgG recoveries yields of 85%. ► 85% of total protein contaminants were removed. ► The mass transfer was controlled by the PEG-rich phase.The performance of a pilot scale packed differential contactor was evaluated for the continuous counter-current aqueous two-phase extraction (ATPE) of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant (CS) enriched with pure protein. Preliminary studies have been firstly performed in order to select the dispersed phase (phosphate-rich or polyethylene glycol 3350 Da (PEG)-rich phase) and the column packing material. The PEG-rich phase has been selected as the dispersed phase and the stainless steel as the preferred material for the column packing bed since it was not wetted preferentially by the selected dispersed phase. Hydrodynamic studies have been also performed, and the experimental results were successfully adjusted to the Richardson–Zaki and Mísek equations, typically used for the conventional organic-aqueous two-phase systems. An experimental set-up combining the packed column with a pump mixer-settler stage showed to have the best performance and to be advantageous when compared to the IgG batch extraction. An IgG recovery yield of 85% could be obtained with about 50% of total contaminants and more than 85% of contaminant proteins removal. Mass transfer studies have revealed that the mass transfer was controlled by the PEG-rich phase. A higher efficiency could be obtained when using an extra pump mixer-settler stage and higher flow rates.
Keywords: Continuous aqueous two-phase extraction; Packed column; Hold-up; Mass transfer; Human antibodies;

Quick identification of kuraridin, a noncytotoxic anti-MRSA (methicillin-resistant Staphylococcus aureus) agent from Sophora flavescens using high-speed counter-current chromatography by Ben Chung-Lap Chan; Hua Yu; Chun-Wai Wong; Sau-Lai Lui; Claude Jolivalt; Carine Ganem-Elbaz; Jean-Marc Paris; Barbara Morleo; Marc Litaudon; Clara Bik-San Lau; Margaret Ip; Kwok-Pui Fung; Ping-Chung Leung; Quan-Bin Han (157-162).
► High-speed counter-current chromatography provided successful fractionation with high sample recovery to drug discovery from Sophora flavescens. ► Two anti-MRSA agents, sophoraflavanone G and were identified from the active fraction. ► Sophoraflavanone G showed potent cytotoxicity against human peripheral blood mononuclear cells (PBMCs). ► Kuraridin was noncytotoxic against human PBMCs.Bacterial resistance to antibiotics has become a serious problem of public health that concerns almost all currently used antibacterial agents and that manifests in all fields of their application. To find more antibacterial agents from natural resources is all the time considered as an important strategy. Sophora flavescens is a popularly used antibacterial herb in Chinese Medicine, from which prenylated flavones were reported as the antibacterial ingredients but with a major concern of toxicity. In our screening on the antibacterial activities of various chemicals of this herb, 18 fractions were obtained from 8 g of 50% ethanol extract on a preparative high-speed counter-current chromatography (HSCCC, 1000 ml). The system of n-hexane/ethyl acetate/methanol/water (1:1:1:1) was used as the two-phase separation solvent. A chalcone named kuraridin was isolated from the best anti-MRSA fraction, together with sophoraflavanone G, a known active ingredient of S. flavescens. Their structures were elucidated by analysis of the NMR spectra. Both compounds exhibited significant anti-MRSA effects, compared to baicalein that is a well known anti-MRSA natural product. More important, kuraridin showed no toxicity on human peripheral blood mononuclear cells (PBMC) at the concentration up to 64 μg/ml while sophoraflavanone G inhibited over 50% of cellular activity at 4 μg/ml or higher concentration. These data suggested that opening of ring A of the prenylated flavones might decrease the toxicity and remain the anti-MRSA effect, from a viewpoint of structure–activity relationship.
Keywords: MRSA; Antibacteria; Sophora flavescens; Kuraridin; High-speed counter-current chromatography;

Quantification of andrographolide sodium bisulphite in urine after intravenous injection to rats by LC–MS/MS by Shuang-Qing Zhang; Xiang-Hong Chen; Min Yu; Xu Sun; Zuo-Gang Li (163-167).
► A LC–MS/MS was firstly established for measurement of andrographolide sodium bisulphite in biological matrix. ► The method was fully validated. ► The method was applied to the urinary excretion of the drug in rats.A liquid chromatography–tandem mass spectrometry method for the determination of andrographolide sodium bisulphite (ASB) in rat urine was established and validated. To our knowledge, the analytical method is the first developed assay for the determination of ASB in urine samples. Dehydroandrographolide (DAG) was used as an internal standard. ASB and DAG were separated on a C18 column and detected at negative ion mode using the mass transitions of m/z 413.2 → 287.2 and m/z 331.2 → 303.3, respectively. Good linearity was obtained over the range of 50–5000 ng/mL and the correlation coefficient was better than 0.99. The intra- and inter-day accuracy at all levels fell in the ranges of 85.8–101.4% and 87.9–97.5%, and the intra- and inter-day precision (RSD) were in the ranges of 4.3–11.2% and 8.4–13.3%, respectively. The recovery ranged from 96.1% to 98.3% and the matrix effects from 96.2% to 98.1%. Good stability was found under tested conditions. The method was successfully applied to a urinary excretion study of ASB in rats following intravenous administration of 80 mg/kg ASB.
Keywords: Andrographolide sodium bisulphite; LC–MS/MS; Excretion;

► A sensitive LC–MS/MS method with a lower limit of quantification (LLOQ) of about 2 ng mL−1 was firstly developed and validated for the simultaneous determination of three catechins (catechin, epicatechin and epicatechin gallate) in rat plasma. ► After single oral administration of 15.25 g kg−1 Cynomorium songaricum extract, C max of catechin, epicatechin and epicatechin gallate in rat plasma were respectively 86.69 ± 38.65, 32.57 ± 15.00 and 36.93 ± 12.62 ng mL−1 while T max values were respectively 0.15 ± 0.09, 0.20 ± 0.10 and 0.20 ± 0.13 h. ► The developed LC–MS/MS method was sensitive enough for pharmacokinetic study of catechins following oral administration of C. songaricum extract.A rapid and valid method was developed for simultaneous determination catechin, epicatechin and epicatechin gallate in rat plasmas using scopoletin (103 ng mL−1) as an internal standard (IS). The separation was performed on Eclipse plus C18 column (100 mm × 4.6 mm, 1.8 μm) at a flow rate of 0.3 mL min−1, and acetonitrile–0.1% formic acid was used as mobile phase. The recoveries of three analytes and IS were more than 78.9%. The lower limits of quantitation (LLOQ) in rat plasma were 2.14, 2.38 and 2.08 ng mL−1 respectively for catechin, epicatechin and epicatechin gallate. Intra-day and inter-day precisions were within 12%. The accuracies were more than 85%. After single oral administration of 15.25 g kg−1 Cynomorium songaricum extract, C max of catechin, epicatechin and epicatechin gallate in rat plasma were respectively 86.69 ± 38.65, 32.57 ± 15.00 and 36.93 ± 12.62 ng mL−1 while T max values were respectively 0.15 ± 0.09, 0.20 ± 0.10 and 0.20 ± 0.13 h. The results demonstrated that the present LC–MS/MS method was sensitive enough for pharmacokinetic study of catichins following oral administration of C. songaricum extract.
Keywords: Catechin; Epicatechin; Epicatechin gallate; LC–MS/MS; Cynomorium songaricum Rupr;