Journal of Chromatography B (v.923-924, #C)

► A sensitive method for analysis of bioactives of ginseng is proposed. ► Method combined equilibrium dialysis and UPLC-MS/MS. ► The study of the interactions between ginseng and liposome is firstly reported. ► The method offers a theoretical direction of potential bioactivities in ginseng.To study the interactions between components of Panax Ginseng and liposome biomembrane, we applied the equilibrium dialysis system combined with ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach to analyze and identify the bioactive components of ginseng. Moreover, the effect of pH value has also been investigated on their interactions between the ginsenosides of ginseng extract and biomembrane. The result shows that seven kinds of ginsenosides have obvious interactions with biomembrane in comparison with the standards in terms of tandem mass spectrometry (MS/MS) data along with retention time, including four panaxadiol ginsenosides (Rb1, Rb2, Rc, Rd) and three panaxatriol ginsenosides (Re, Rf, Rg2). The value of binding degree decreased with the increase of molecular weight. The sugar moieties which are attached to C-20 were the main factor affecting the binding degree of panaxadiol ginsenosides. The interactions between panaxadiol ginsenosides and biomembrane correlate to the type and number of sugar moieties in ginsenosides. The sugar moieties which are at C-6 and C-20 have been shown to influence the value of binding degree for panaxatriol ginsenosides. In addition, the pH value has been shown to have an impact on the interactions. Overall, ginsenoside Rd has a better absorption character among the seven ginsenosides. In the study, we have screened the potential bioactive components of ginseng in vitro using the equilibrium dialysis-UPLC-MS/MS method, and then predicted the potential bioactivities of ginseng, which contribute to the investigation of the efficacy of ginseng.
Keywords: Equilibrium dialysis; UPLC-MS/MS; Liposome biomembrane; Ginsenosides; Active components;

► Fully automated on-line SPE–HPLC utilized for PK evaluation of WM-5. ► Response surface methodology used for optimization of on-line SPE parameters. ► On-line SPE removed the time-consuming, tedious and costly manual process. ► The method is simple, fast, specific and sensitive.Response surface methodology (RSM) was utilized for rapid and systematic optimization of on-line solid-phase extraction (SPE) parameters to maximize the response and separation of WM-5. The optimization was performed with Box–Behnken designs. Four major parameters were investigated for their contributions to the response and separation of WM-5, with a total of 29 experiments being performed for each instrument, respectively. Quantitative determination of WM-5 in mouse plasma was performed to evaluate the statistical significance of the parameters on chromatographic response. A fully automated on-line SPE and high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for the determination of WM-5 in mouse plasma. Calibration curve with good linearity (r  = 0.9989) was obtained in the range of 20–4000 ng/mL in mouse plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) of the assay were 6 ng/mL and 20 ng/mL, respectively. The overall intra-day and the inter-day variations were less than 1.90%. The recovery of the method was in the range of 93.74–96.33% with RSD less than 3.06%. The optimized method demonstrated good performance in terms of specificity, LLOQ, linearity, recovery, precision and accuracy, and was successfully applied to quantify WM-5 in mouse plasma to support the pharmacokinetic study.
Keywords: WM-5; On-line solid-phase extraction; HPLC-DAD; Pharmacokinetic study; Response surface methodology;

► Deglycosylation systematically improved proteomic analyses containing glycoproteins. ► AspN and trypsin provided complementary analysis with deglycosylation. ► Improvement in identification correlated with the density of glycosylation sites. ► Cis3p, important for cell wall integrity, was upregulated in Δalg3 yeast.Post-translational modification of proteins with glycosylation is of key importance in many biological systems in eukaryotes, influencing fundamental biological processes and regulating protein function. Changes in glycosylation are therefore of interest in understanding these processes and are also useful as clinical biomarkers of disease. The presence of glycosylation can also inhibit protease digestion and lower the quality and confidence of protein identification by mass spectrometry. While deglycosylation can improve the efficiency of subsequent protease digest and increase protein coverage, this step is often excluded from proteomic workflows. Here, we performed a systematic analysis that showed that deglycosylation with peptide-N-glycosidase F (PNGase F) prior to protease digestion with AspN or trypsin improved the quality of identification of the yeast cell wall proteome. The improvement in the confidence of identification of glycoproteins following PNGase F deglycosylation correlated with a higher density of glycosylation sites. Optimal identification across the proteome was achieved with PNGase F deglycosylation and complementary proteolysis with either AspN or trypsin. We used this combination of deglycosylation and complementary protease digest to identify changes in the yeast cell wall proteome caused by lack of the Alg3p protein, a key component of the biosynthetic pathway of protein N-glycosylation. The cell wall of yeast lacking Alg3p showed specifically increased levels of Cis3p, a protein important for cell wall integrity. Our results showed that deglycosylation prior to protease digestion improved the quality of proteomic analyses even if protein glycosylation is not of direct relevance to the study at hand.
Keywords: ALG3; N-glycosylation; Saccharomyces cerevisiae; PNGase F; Deglycosylation; Proteomics;

Determination of methylphenidate in plasma and saliva by liquid chromatography/tandem mass spectrometry by A. Seçilir; L. Schrier; Y.A. Bijleveld; J.H. Toersche; S. Jorjani; J. Burggraaf; J. van Gerven; R.A.A. Mathôt (22-28).
► A LC–MS/MS method was validated for the determination of methylphenidate (MPH) in plasma and saliva. ► The method allows fast, accurate and precise quantification of MPH in both plasma and saliva. ► The use of deuterated MPH as an internal standard is essential, due to matrix effects of plasma. ► MPH is degraded in plasma and saliva; clinical samples should be stored at −20 °C directly after sampling.Methylphenidate (MPH) is a phenethylamine derivative used in the treatment of attention-deficit hyperactivity disorder (ADHD). In adults, clinical monitoring of MPH therapy is usually performed by measuring plasma MPH concentrations. In children blood sampling is however undesirable. Saliva may be an alternative matrix for monitoring MPH concentrations with the advantage that it can be obtained non-invasively. Therefore, we developed an analytical method for the quantification of MPH in both plasma and saliva. We present the validation of a liquid chromatography–tandem mass spectrometric method using a hydrophilic interaction liquid chromatography column (HILIC). In 100 μL sample, proteins were precipitated with 750 μL acetonitrile/methanol 84/16 (v/v) containing d9-methylphenidate as the internal standard. Standard curves were prepared over the MPH concentration range of 0.5–100.0 μg/L. The total analysis time was 45 s. Accuracy and within- and between-run imprecision were in the range of 98–108% and less than 7.0%, respectively. Matrix effects were greater for plasma than saliva with 46% and 8% ionization suppression. The matrix effects were adequately compensated by the use of deuterated MPH as internal standard. MPH significantly degraded in plasma and saliva at room temperature and 5 °C. Samples were stable at −20 °C for at least 4 weeks. The method was successfully applied for the determination of MPH concentrations in plasma and saliva samples from an adult healthy volunteer. Using protein precipitation and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry, this method allows fast, accurate and precise quantification of MPH in both plasma and saliva.
Keywords: Methylphenidate; LC–MS/MS; Saliva; Plasma; HILIC;

Evaluation of the effect of TM208 on the activity of five cytochrome P450 enzymes using on-line solid-phase extraction HPLC–DAD: A cocktail approach by Wensi Lin; Jianmei Zhang; Xiaomei Ling; Ning Yu; Jing Li; Haisong Yang; Runtao Li; Jingrong Cui (29-36).
► On-line solid-phase extraction HPLC–DAD method was applied to determine five specific probe substrates in vivo. ► The method was applied to evaluate the effects of TM208 on rat five CYP450 isoforms. ► TM208 had the potential to induce the metabolic activities of CYP2E1 and CYP3A4 in rats. ► TM208 might not significantly affect CYP1A2, CYP2D6 and CYP2C19-mediated metabolism in rats.A rapid, simple, and sensitive on-line solid-phase extraction HPLC–DAD method for simultaneous evaluation of the activity of five CYP450 isoforms (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vivo has been developed and validated. The five specific probe substrates include caffeine (1A2), metoprolol (2D6), dapsone (3A4), omeprazole (2C19) and chlorzoxazone (2E1). Automated pre-purification of plasma and enrichment of analytes were performed using a C18 on-line solid-phase extraction cartridge. After being eluted from the cartridge, the analytes and the internal standard antipyrine were separated on a C18 RP analytical column and analyzed by DAD. The method was validated to quantify the concentration ranges of 0.05–50.0 μg/ml for dapsone and omeprazole, 0.1–50.0 μg/ml for caffeine and 0.2–50.0 μg/ml for metoprolol and chlorzoxazone. The linearity (R 2) for all analytes tested was exceeded 0.99. The intra-day precision ranged from 0.29 to 13% and the inter-day precision ranged from 5.0 to 15%, respectively. The intra-day and inter-day accuracy were between 86.7% and 113.6%. The extraction recoveries were in the range 82.8–109.9% for all the analytes and internal standard antipyrine. This method was successfully applied to evaluate the effects of TM208 on rat five CYP450 isoforms.
Keywords: Probe drug cocktail; Online solid-phase extraction; HPLC; TM208; Rat plasma;

► A method for determination of PCBs in marine products was developed. ► 1,1,1,2-Tetrafluoroethane (R134a) is used as subcritical fluid. ► Several factors were employed to optimize the conditions of the extraction. ► High extraction efficiency could be obtained at low pressure and temperature. ► The proposed method was successfully applied to the real samples.This paper describes a new procedure for extracting polychlorinated biphenyls (PCBs) from marine samples using subcritical 1,1,1,2-tetrafluoroethane (R134a). The extraction procedure was optimized at temperatures varying from 20 to 70 °C and pressures ranging from 3 to 15 MPa. The volume of the co-solvent was then optimized using 1,1,1,2-tetrafluoroethane (R134a) as the subcritical phase. PCBs were characterized by GC–MS using the optimized conditions of 3 MPa, 30 °C, and a co-solvent volume of 6 mL. The average yields of PCBs from subcritical fluid extraction of spiked oyster samples were measured and found to be greater than 90%, with relative standard deviations (RSD) of less than 10%. Detection limits of this method were in the range of 0.045–0.108 ng/g of dry mass. The method was compared to Soxhlet extraction and then applied for monitoring PCBs in oysters from Qingdao, Shandong, China.
Keywords: Subcritical R134a extraction; Polychlorinated biphenyls; Marine samples; GC–MS;

Determination of Raddeanin A in rat plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study by Xin Luan; Ying-Yun Guan; Chao Wang; Mei Zhao; Qin Lu; Ya-Bin Tang; Ya-Rong Liu; De-Hong Yu; Xiao-Lin Wang; Hong Qi; Chao Fang; Hong-Zhuan Chen (43-47).
► A sensitive HPLC–MS/MS method for in vivo quantitative assay of Raddeanin A was developed. ► The method was characteristic of short running time and simple preparation process. ► The pharmacokinetics of Raddeanin A in rat after intravenous and intraperitoneal injection was evaluated.A simple, rapid and sensitive LC–MS/MS analysis method was developed and validated for the determination of Raddeanin A (RA) in rat plasma. Protein precipitation with three volumes of methanol as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Thermo Syncronis C18 column with the mobile phase of methanol–water (containing 5 mM ammonium formate, pH 2.2) (85:15, v/v). RA and glycyrrhetinic acid (internal standard) were monitored under negative electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of RA and IS were 2.1 min and 3.5 min, respectively. The limit of detection was 5 ng/mL and the linear range was 50–50,000 ng/mL. The intra-day and inter-day precision was 1.87–2.94% and 3.25–5.36%, and the intra-day and inter-day accuracy ranged from 5.9% to 10.5% and 5.6% to 11.1%, respectively. The absolute recovery was above 90.3%. The method has been successfully translated to the pharmacokinetic study of RA in rats after intravenous and intraperitoneal administration (0.75 mg/kg).
Keywords: Raddeanin A; HPLC–MS/MS; Pharmacokinetics; Rat plasma;

► UF-LC–MS method for screening XOD inhibitors was developed. ► Method is suited for rapid screening and characterization of XOD inhibitors from TCM. ► UF-LC–MS is a rapid, sensitivity and high throughput tool to screen XOD inhibitors. ► Eleven XOD inhibitors were screened from the Radix Salvia Miltiorrhizae extract. ► The relationship between the structure and activity of inhibitors was estimated.Xanthine oxidase (XOD) inhibitors play an important role in the treatment of gout and many other diseases related to the superoxide anion metabolism. In this study, an ultrafiltration-liquid chromatography–mass spectrometry (UF-LC–MS) method was developed for the screening and identification of potential XOD inhibitors from Radix Salviae Miltiorrhizae extract. Eleven lipophilic diterpenoid quinines were identified as XOD inhibitors from the extract. The relationship between the structure and activity of the detected compounds was estimated on the basis of the UF-LC–MS data. The results demonstrate that the 1,2-naphthoquinone group is necessary for the XOD inhibitory activity of the compounds, and that furan and hydroxyl on the alicyclic ring could enhance the activity of the compounds at different levels. These results may explain and support the medical use of the extract of Radix S. Miltiorrhizae for the prevention and treatment of hyperuricemia and gout.
Keywords: Radix Salviae Miltiorrhizae; Gout; Xanthine oxidase inhibitors; Screening; Ultrafiltration LC–MSS;

Analysis of iridoid glucosides from Paederia scandens using HPLC–ESI-MS/MS by Zhi-Jun Wu; Jian-Hua Wang; Dong-Mei Fang; Guo-Lin Zhang (54-64).
► A total of 24 iridoid glucosides (14 new ones) were characterized using HPLC–ESI-MS. ► The difference mainly focuses on the side chain of iridoid moiety for simple ones. ► The connection order of iridoid, sugar and phenolic acid moieties can be confirmed. ► The acylation of 6-OH of sugar by phenolic acid can be determinated. ► Connection order between asperuloside and paederoside moieties can be confirmed.Iridoid glycosides are an important class of natural products and have many biological activities. Iridoid glucosides in an extract of the plant species Paederia scandens were investigated using reversed-phase high performance liquid chromatography and electrospray quadrupole time-of-flight-type tandem mass spectrometry. The elemental composition of most of the compounds was determined by accurate mass and relative isotopic abundance (RIA) measurements. In positive ion mode, the fragmentation of [M+NH4]+ precursor ions was carried out using low energy collision-induced electrospray ionization tandem spectrometry. The neutral losses of NH3, H2O, Glc, and the side chain of the iridoid moiety were the main fragmentation patterns observed. For simple iridoid glycosides, the main differences were related to the side chains. Fragmentation of the [M−H]precursor ions was achieved for the compounds possibly having phenolic acid group. The connection order of the iridoid, sugar, and phenolic acid moieties, and the linkage of the 6-OH group of the sugar to the phenolic acid were unambiguously confirmed using a combination of MS/MS spectra in both positive and negative ion modes, and our previous work. For some trace dimeric iridoid glucosides, the connection order between the asperuloside and paederoside moieties was determined by the characteristic product ions; this was supported by D-labeling experiments. A total of 24 iridoid glucosides, including 14 new species, were identified or tentatively characterized based on exact mass, RIA values, tandem mass spectra, and D-labeling experiments.
Keywords: Iridoid glycosides; Electrospray; Natural product; Paederia scandens; Fragmentation; HPLC–ESI-MS/MS;

A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots by N. Mohamed Shah; A.F. Hawwa; J.S. Millership; P.S. Collier; J.C. McElnay (65-73).
► We report a simple method for the analysis of four antiepileptic drugs in DBS samples. ► The method was applied to DBS samples collected from children with epilepsy. ► Such technique has potential in assessing adherence to AEDs using home sampling.An increasing number of publications on the dried blood spot (DBS) sampling approach for the quantification of drugs and metabolites have been spurred on by the inherent advantages of this sampling technique. In the present research, a selective and sensitive high-performance liquid chromatography method for the concurrent determination of multiple antiepileptic drugs (AEDs) [levetiracetam (LVT), lamotrigine (LTG), phenobarbital (PHB)], carbamazepine (CBZ) and its active metabolite carbamazepine-10,11 epoxide (CBZE)] in a single DBS has been developed and validated. Whole blood was spotted onto Guthrie cards and dried. Using a standard punch (6 mm diameter), a circular disc was punched from the card and extracted with methanol: acetonitrile (3:1, v/v) containing hexobarbital (Internal Standard) and sonicated prior to evaporation. The extract was then dissolved in water and vortex mixed before undergoing solid phase extraction using HLB cartridges. Chromatographic separation of the AEDs was achieved using Waters XBridge™ C18 column with a gradient system. The developed method was linear over the concentration ranges studied with r  ≥ 0.995 for all compounds. The lower limits of quantification (LLOQs) were 2, 1, 2, 0.5 and 1 μg/mL for LVT, LTG, PHB, CBZE and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for within and between day were <20% at the LLOQs and <15% at all other concentrations tested. This method was successfully applied to the analysis of the AEDs in DBS samples taken from children with epilepsy for the assessment of their adherence to prescribed treatments.
Keywords: Antiepileptic drugs; Dried blood spot; HPLC; Children;

► Evaluation of LC/UV/MS conditions for quantitative analysis of modified nucleosides. ► Minor (low abundance) RNA modifications reproducibly analyzed. ► Biological variability in modified nucleosides < 10% RSD. ► tRNA modifications easier to quantify than rRNA modifications. ► Column temperature can be adjusted to improve separation window.Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less.
Keywords: tRNA; rRNA; HPLC; Posttranscriptional modification; Metabolites;

► Polar endogenous compounds in plasma identified to interfere in HILIC–ESI-MS. ► Both enhancement and suppression of the analyte signal was observed. ► Choosing columns with different selectivity alters the regions of suppression/enhancement.Matrix effects on electrospray ionization were investigated for plasma samples analysed by hydrophilic interaction chromatography (HILIC) in gradient elution mode, and HILIC columns of different chemistries were tested for separation of plasma components and model analytes. By combining mass spectral data with post-column infusion traces, the following components of protein-precipitated plasma were identified and found to have significant effect on ionization: urea, creatinine, phosphocholine, lysophosphocholine, sphingomyelin, sodium ion, chloride ion, choline and proline betaine. The observed effect on ionization was both matrix-component and analyte dependent. The separation of identified plasma components and model analytes on eight columns was compared, using pair-wise linear correlation analysis and principal component analysis (PCA). Large changes in selectivity could be obtained by change of column, while smaller changes were seen when the mobile phase buffer was changed from ammonium formate pH 3.0 to ammonium acetate pH 4.5. While results from PCA and linear correlation analysis were largely in accord, linear correlation analysis was judged to be more straight-forward in terms of conduction and interpretation.
Keywords: Electrospray ionization; Principal component analysis; Linear correlation analysis; Hydrophilic interaction chromatography; Gradient elution; Matrix effects;

► First report on n-octanol/water partition of treosulfan and its epoxy-transformers. ► Results of log  P calculated by ALOGPs correlated best with the experimental values. ► Negative correlation between log  P of the analytes and their retention in RP-HPLC. ► Treosulfan and its epoxides do not constitute a ‘congeneric’ series of compounds. ► Hydrophilic nature of treosulfan corresponds to its pharmacokinetic parameters.For the last decade an alkylating agent treosulfan (TREO) has been successfully applied in clinical trials in conditioning prior to hematopoietic stem cell transplantation. Pharmacological activity of the pro-drug depends on its epoxy-transformers, monoepoxide (S,S-EBDM) and diepoxide (S,S-DEB), which are formed in a non-enzymatic consecutive reaction accompanied by a release of methanesulfonic acid. In the present study partition coefficient n-octanol/water (P OW) of TREO as well as its biologically active epoxy-transformers was determined empirically (applying a classical shake-flask method) and in silico for the first time. In vitro the partition was investigated at 37 °C in the system composed of the pre-saturated n-octanol and 0.05 M acetate buffer pH 4.4 adjusted with sodium and potassium chloride to ionic strength of 0.16 M. Concentration of the analytes was quantified by reversed-phase high performance liquid chromatography (RP-HPLC) method in which retention time increased from S,S-DEB to TREO. It was shown that neither association nor dissociation of the tested compounds in the applied phases occurred. Calculated log  P OW (TREO: −1.58 ± 0.04, S,S-EBDM: −1.18 ± 0.02, S,S-DEB: −0.40 ± 0.03) indicate the hydrophilic character of the all three entities, corresponding to its pharmacokinetic parameters described in the literature. Experimentally determined log  P OW of the compounds were best comparable to the values predicted by algorithm ALOGPs. Interestingly, the P OW values determined in vitro as well as in silico were inversely correlated with the retention times observed in the endcapped RP-HPLC column. It might be explained by the fact that a cleavage of methansulfonic acid from a small molecule of TREO generates significant changes in the molecular structure. Consequently, despite the common chemical origin, TREO, S,S-EBDM and S,S-DEB do not constitute a ‘congeneric’ series of compounds. We concluded that this might occur in other low-weight species, therefore measurement of their P OW by RP-HPLC had to be applied with a special care.
Keywords: Epoxides; In silico; Lipophilicity; Shake-flask method; HPLC; Retention;

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of specific IgY by Soujanya Ratna Edupuganti; Om Prakash Edupuganti; Richard O’Kennedy; Eric Defrancq; Stéphanie Boullanger (98-101).
► Immobilization of T-2 toxin on amine-activated sepharose beads. ► Utilization of T-2 toxin-immobilized beads for increasing specificity of IgY with reduction in cross reactivity. ► Development of SPR-based inhibition assay and its validation.An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.
Keywords: Affinity purification; T-2 toxin immobilized amine-activated beads; Enzyme-linked immunoassay (ELISA); SPR-based inhibition assay;

Native chromatographic sample preparation of serum, plasma and cerebrospinal fluid does not comprise a risk for proteolytic biomarker loss by Jelena Pesek; Thomas Krüger; Nadine Krieg; Michael Schiel; Johannes Norgauer; Julian Großkreutz; Heidrun Rhode (102-109).
► We recently developed a native fractionation method for serum, plasma and CSF. ► Proteolysis was tested during usual and prolonged sample handling and preparation. ► No evidence of increased peptide fractions was found after variable incubation time. ► Virtually no proteolytic activity was observed after moderate trypsin addition. ► There is practically no risk of biomarker loss by intrinsic proteinases with our method.We recently developed a native multidimensional chromatographic method for serum and plasma fractionation for proteomic biomarker search. This method has several advantages:parallelization and automation, high reproducibility and proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, optional enzymatic and immunological analytics additional to mass spectrometry, retaining metabolites, and information on complex formation, modification, and fragmentation of constituents. Nevertheless, native conditions have the probable risk of proteome alteration and biomarker loss by intrinsic proteinases.Hence, we tried to quantify here intrinsic proteolytic activity in native samples and fractions from serum, plasma and cerebrospinal fluid, as well as the effectiveness of intrinsic anti-proteinases during sample handling and preparation under our fractionation conditions. Therefore, we used several quantitative measures: (1) total proportion of intrinsic protein and peptide fractions, (2) azocasein hydrolysis and (3) mass spectrometric protein coverage and peptide numbers. To 1: In all non-fractionated specimens, neither decrease of protein concentration or molecular weight nor increase of peptide concentration was found after variable clotting or pre-incubation time. To 2: No azocasein hydrolysis was seen in these samples when prepared within a few hours at room temperature. Trypsin, when added in concentrations not higher than 0.85 μg/mL (0.04 μM), even was completely inhibited. Moreover, in native 1-D fractions no proteinase activity could be observed. To 3: Mass spectrometry confirmed that neither protein coverage nor peptide numbers differ significantly in 1-D or 2-D fractions after variable incubation time. These results suggest that intrinsic, native proteinase inhibitors potentially protect the proteomes considered, enabling “top-down” proteomic approaches under native conditions with serum, plasma and cerebrospinal fluid.
Keywords: Native sample fractionation; Serum; Plasma; Cerebrospinal fluid; Proteinase activity; Peptide quantification;

Quantitative determination of nebivolol from human plasma using liquid chromatography–tandem mass spectrometry by Jatin Nandania; S.J. Rajput; Pritesh Contractor; Pragnesh Vasava; Bhavik Solanki; Mohsin Vohra (110-119).
► Liquid–liquid extraction method was used to extract nebivolol from human plasma. ► Liquid chromatography with tandem mass spectrometry was used to analyze and quantify nebivolol. ► Method was validated in terms of accuracy, precision, selectivity, absolute recovery and stability. ► This method is free from ion suppression, ion enhancement and any type of abnormal ionization. ► Developed bioanalytical method is a highly sensitive as lower limit of quantification was proved 30 pg/mL.In the present work, a rapid, sensitive, specific, precise and accurate liquid chromatography–tandem mass spectrometry method for determination of nebivolol in human plasma was developed and validated with a large calibration curve range (50–5000 pg/mL) which can be used for routine drug analysis and bioequivalence studies. Liquid–liquid extraction method was used to extract the analyte from the human plasma. The separation was achieved using Waters symmetry, C18, 4.6 × 150 mm, 5 μm column with formic acid in water, 0.01%, v/v: Acetonitrile (40:60) as a mobile phase. A flow rate of 0.8 mL/min, no splitting and run time 2.00 min was used for the chromatographic analysis of nebivolol. Sensitivity of this method was found to be 30 pg/mL. The analyte was analyzed by mass spectrometry in the multiple reaction monitoring mode. A Turbo-Ion spray source was interfaced between the HPLC and triple quadrupole mass spectrometer (MDS Sciex API 4000). The precursor-product ion m/z 406.00–151.00 for nebivolol and m/z 410.20–151.00 for nebivolol-D4 were used for quantification of an analyte and its IS. The method was validated in terms of accuracy, precision, selectivity, absolute recovery, freeze-thaw stability, bench-top stability, dry extract stability, short and long term stock solution stability, wet extract stability and re-injection reproducibility. The within- and between-batch accuracy was found to lie within the range of 87.00–100.40% and within- and between-batch precision was obtained within the range 0.33–8.67%. The mean recovery of all three concentration levels for drug was obtained 67.67% where as the mean recovery of IS was 68.74%. The %RSD value at higher concentration and lower concentration in all stability experiments was within 15%. This method is free from ion suppression, ion enhancement and any type of abnormal ionization.
Keywords: Nebivolol; Liquid–liquid extraction; Pharmacokinetic profile; LC/MSMS; Bioavailability; Bioequivalence;

Determination of 3β-hydroxy-Δ5-bile acids and related compounds in biological fluids of patients with cholestasis by liquid chromatography–tandem mass spectrometry by Tsuyoshi Murai; Kana Oda; Terutake Toyo; Hiroshi Nittono; Hajime Takei; Akina Muto; Akihiko Kimura; Takao Kurosawa (120-127).
► We developed an LC–MS/MS method for determination of 3β-hydroxy-Δ5-bile acids. ► We analyzed urine and serum from patients with 3β-dehydrogenase deficiency. ► These bile acids were the major bile acids in urine and serum from those patients. ► This method is suitable for determination of these bile acids in clinical analysis.A method for the determination of conjugated and unconjugated 3β-hydroxy-Δ5-bile acids and related bile acids in human urine and serum has been developed using high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry. Calibration curves for the bile acids were linear over the range of 10–2000 pmol/mL, and the detection limit was less than 4 pmol/mL for all bile acids using selected reaction monitoring analysis. The bile acids in urine and serum samples from two patients with severe cholestatic liver disease were measured by this analytical method. Glycine-conjugated 3β-hydroxy-Δ5-bile acid 3-sulfates were determined to be the major bile acids in the urine and serum from patients with a 3β-hydroxy-Δ5-C27-steriod dehydrogenase/isomerase deficiency or dysfunction.
Keywords: 3β-Hydroxy-Δ5-bile acids; 3β-Hydroxy-Δ5-C27-steriod dehydrogenase/isomerase deficiency; High-performance liquid chromatography–electrospray ionization-tandem mass spectrometry;

Detection of main urinary metabolites of β2-agonists clenbuterol, salbutamol and terbutaline by liquid chromatography high resolution mass spectrometry by Juan C. Domínguez-Romero; Juan F. García-Reyes; Rubén Martínez-Romero; Esther Martínez-Lara; María L. Del Moral-Leal; Antonio Molina-Díaz (128-135).
► Urinary metabolism study of clenbuterol, salbutanol and terbutaline in rat. ► Methodology based on solid-phase extraction followed by LC-Electrospray-TOFMS. ► Use of diagnostic fragments ions and accurate mass shifts from biotransformations. ► Up to eleven metabolites detected, four of them non previously reported in urine. ► Three clenbuterol metabolites were detected over a longer period than parent drug.Clenbuterol, terbutaline and salbutamol are B2-agonists drugs included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this article, the excretion of urinary metabolites of clenbuterol, terbutaline and salbutamol have been studied using liquid chromatography electrospray time-of-flight mass spectrometry (LC-TOFMS), after a single therapeutic dose administration in rats. Urine collected was processed with solid-phase extraction prior to LC-TOFMS analyses using electrospray in the positive ion mode and pseudo MS/MS experiments from in-source collision induced dissociation (CID) fragmentation (without precursor ion isolation). The strategy applied for the identification of metabolites was based on the search of typical biotransformations with their corresponding accurate mass shift and the use of common diagnostic fragment ions from the parent drugs. The approach was satisfactory applied, achieving the identification of 11 metabolites (5 from clenbuterol, 4 from salbutamol and 3 from terbutaline), 4 of them not previously reported in urine. Novel metabolites identified in rat urine included N-oxide-salbutamol, hydroxy-salbutamol, methoxy-salbutamol glucuronide and terbutaline N-oxide, which are all reported here for the first time.
Keywords: B2-agonists; Clenbuterol; Terbutaline; Salbutamol; Urine; Liquid chromatography; Mass spectrometry; Metabolites;

► A new MI-MSPD-HPLC method is developed for rapid screening of clenbuterol in chicken. ► Dummy molecularly imprinted microspheres are synthesized by suspension polymerization. ► The extraction selectivity is significantly improved. ► The effect of template leaking on quantitative analysis is avoided.A simple and selective molecularly imprinted matrix solid-phase dispersion (MI-MSPD) method coupled with high performance liquid chromatography (HPLC) ultraviolet detection was developed for rapid screening of clenbuterol hydrochloride (CH) in chicken samples. The new molecularly imprinted microspheres (MIM) were synthesized by using butylamine and chloroaniline as dummy template with aqueous suspension polymerization and revealed good affinity to CH in aqueous solution. The application of the obtained MIM as sorbent of matrix solid-phase dispersion (MSPD) improved the selectivity of extraction procedure and avoided the effect of template leakage on quantitative analysis. Under the optimized conditions, good linearity of CH was obtained in a range of 0.059–18.30 μg mL−1 with the correlation coefficient (R) of 0.9996. The recoveries of CH at three spiked levels were ranged from 92.0 to 99.1% with the relative standard deviation less than 4.0% (n  = 3). The presented MI-MSPD-HPLC method combined the superiority of MIM and MSPD, and therefore could be potentially applied for the determination of CH in complicated biological samples.
Keywords: Molecularly imprinted microspheres; Matrix solid-phase dispersion; Selective extraction; Clenbuterol hydrochloride; Chicken samples;