Journal of Chromatography B (v.879, #13-14)

Mixed-bed chromatography is based on the use of multiple sorbents mixed together and packed in a single column. Solid-phase combinatorial libraries are a current example of mixed-bed chromatography with a large number of immobilized affinity ligands each of them attached to a different bead. They have been repeatedly reported to reduce the dynamic protein concentration range from biological extracts when used in large overloading conditions. By that way trace proteins can easily be enhanced and analyzed. When mixed libraries of ligands are used in under-saturation conditions they constitute a generic way to remove minor impurity traces still present in even highly purified biological product. Whereas ligand libraries are generally used in a neutral environment for the capturing phase, they can also be used in acidic or alkaline conditions with specific advantages due to the modulation of affinity constants. Mixed-bed cascades involving affinity libraries or ion exchangers are also approaches allowing protein fractionation. This review reports various applications of mixed-bed chromatography related to protein fractionation not only for proteomics investigations, but also for preparative purposes.
Keywords: Mixed beds; Proteomics; Peptide library; Identification; Polishing;

Designing new monoclonal antibody purification processes using mixed-mode chromatography sorbents by Magali Toueille; Audrey Uzel; Jean-François Depoisier; René Gantier (836-843).
Current platforms for purification of monoclonal antibodies, mostly relying on Protein A as a first capture step, are robust and efficient but significantly increase downstream purification costs, mainly due to Protein A resins. To decrease manufacturing costs, industry is increasingly considering the use of purification schemes without affinity Protein A resins. Mixed-mode chromatography can be used as a powerful alternative to standard purification platforms as it offers new selectivity and separation mechanisms exploiting a combination of both ionic and hydrophobic characteristics of antibodies and contaminating proteins. By using a design of experiments (DoE) approach and high throughput screening in 96-well plates, we developed four different two-steps MAb purification processes, based on the use of mixed-mode sorbents. Finally, three of the tested processes resulted in final purified Mab fractions containing less than 100 ppm of residual CHO proteins (CHOP), with overall process yields above 70%. These data show that mixed-mode chromatography sorbents, used at capture or intermediate purification steps, really expand the options of MAb purification process development with or without Protein A affinity chromatography.
Keywords: Monoclonal antibody; Purification; Mixed-mode; Host cell proteins; DoE; High throughput screening;

The protein binding characteristics of the immobilized binucleating chelate system, 1,4-bis(1,4,7-triazacyclononan-1-yl)butane (tacn2butane), complexed with Cu2+ ions have been investigated with hen egg white lysozyme, horse skeletal muscle myoglobin and horse heart cytochrome C, as well as three histidine-rich proteins, serum albumin, transferrin, and α2-macroglobulin, present in partially fractionated human serum. The effects of pH, ionic strength and elution buffers on protein binding have been examined and compared with those of the analogous immobilized mononuclear copper complex of 1,4,7-triazacyclononane (tacn). The Cu2+-tacn2butane system was generally found to exhibit higher protein binding affinities than the Cu2+-tacn system, suggesting that the presence of immobilized binuclear copper(II) species leads to enhanced coordinative interaction with surface-exposed amino acid residues of the studied proteins. However, under some buffer conditions the dependencies of protein binding and elution on pH and ionic strength with these immobilized metal ion affinity chromatographic (IMAC) systems were consistent with electrostatic, hydrophobic and π-bonding interactions playing a significant secondary role in addition to the dominant coordinative interactions. As such, the results indicated that the selectivities were not solely dependent on the histidine content of the protein. In accord with this conclusion, differences in the selectivities of the Cu2+-tacn and Cu2+-tacn2butane adsorbents for serum albumin, transferrin, and α2-macroglobulin were observed depending on the choice of elution buffer. This attribute suggests that additional selectivity features can be realised for the separation of specific proteins with this new class of adsorbent.
Keywords: Immobilized metal affinity chromatography; Protein selectivity; Macrocyclic ligands; Copper(II) complexes; 1,4,7-Triazacyclononane derivatives;

Affinity purification of a cholesterol oxidase expressed in Escherichia coli by Yu Xin; Hailin Yang; Xiaole Xia; Ling Zhang; Chen Cheng; Guocui Mou; Jiebing Shi; Yunfei Han; Wu Wang (853-858).
Keywords: Cholesterol oxidase (COD); Affinity chromatography; Brevibacterium sp.; Escherichia coli;

Oral fluid is of increasing interest as an alternative sample matrix in drugs of abuse analysis. Compared to the conventional sample matrix blood, sample volumes of oral fluid are smaller and the concentrations of some drugs can be much lower. This imposes some restrictions on the analysis method, which has to be able to detect and quantify multiple analytes from a small sample volume at low concentrations. A sensitive multi-component method for quantitative determination of 50 drug compounds from oral fluid samples collected with the StatSure SalivaSampler™ device was developed. The compounds analysed include cannabis, cocaine, amphetamines, opioids, benzodiazepines and other psychoactive medicines. Both liquid–liquid-extraction (LLE) and solid-phase-extraction (SPE) are employed in the sample pre-treatment and the samples are analysed using gas chromatography–mass spectrometry (GC–MS) with the mass selective detector (MSD) operating in either electron ionization (EI) or negative-ion chemical ionization (NICI) mode. The method was fully validated, and the validated parameters included linearity, selectivity, accuracy, precision, and extraction efficiency. Stability of the collected samples during storage at −18 °C was also studied, and even after over a year's storage all analyte concentrations were more than 60% of the original concentrations. The described method is suitable for routine analysis of oral fluid samples and it has been applied to analysis of more than 4000 oral fluid samples collected anonymously from volunteer road users in Finland during 2007–2009 as a part of the EU project DRUID (Driving under the Influence of Drugs, Alcohol and Medicines). At the moment the developed method is the most comprehensive validated analysis method for oral fluid samples.
Keywords: Gas chromatography–mass spectrometry; Drugs of abuse; Oral fluid; Multi-component analysis;

A novel method for simultaneous determination of atenolol, metoprolol and esmolol was proposed by capillary electrophoresis (CE) separation and electrochemiluminescence (ECL) detection. Poly-β-cyclodextrin (Poly-β-CD) was used as an additive in the running buffer to improve the separation of three analytes. The conditions for CE separation, ECL detection and effect of Poly-β-CD were investigated in detail. The three β-blockers with very similar structures were well separated and detected under the optimum conditions. The linear ranges of the standard solution for atenolol and esmolol were 2.5–125 μmol/L with a detection limit (S/N = 3) of 0.5 μmol/L, and for metoprolol was 0.5–25 μmol/L with a detection limit of 0.1 μmol/L. For three β-blockers from spiked aqueous and urine samples, the accuracy and precision including intraday and interday experiments were performed by calculating the recovery, the relative standard deviations of the ECL intensity and the migration time, respectively. The developed method was applied to the determination of metoprolol content in commercial pharmaceutical, and the analytical results are in good agreement with the nominal value with recoveries in the range of 98.7–105%. The proposed method was also applied to the monitoring of pharmacokinetics for metoprolol in human body.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Poly-β-cyclodextrin; β-Blocker; Urine; Pharmacokinetics;

Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC–MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair pigmentation did not have any significant effect on the degree of the deposition of drugs and their metabolites in hair.
Keywords: Drug abuse; Drug facilitated crime; Benzodiazepines; Hair analysis; LC–MS/MS;

Method validation should focus on demonstrating that an assay is fit for its intended purpose. We have applied the β-expectation tolerance interval – a statistical approach that predicts the accuracy of assay measurements in the future – to the validation of two different cell death biomarker assays, the M30 and M65 ELISAs. A meta-analysis was conducted on a total of 57 different M30 and M65 assays run over a 2 year period. All code utilised in calculations was developed using MATLAB. The optimal fit to the calibration curve for the M30 assay was shown to be a quartic curve which yielded a β-expectation tolerance interval of +20.5% and −23.6% at β  = 95% over a wide range of QC standards (88–810 U/L). However, such a fit required at least 7 points to avoid problems with over fitting. A linear fit to the M65 calibration curve normally produced a tolerance interval of less than ±20%, however, marked inter-batch variations were evident. Amelioration of batch to batch variations was accomplished by fitting M65 calibration data preferably to a 4-parameter logistic function or a cubic spline. The minimum number of QC replicates and different assays required to produce reliable accuracy profiles was determined. The β-expectation tolerance interval approach has resulted in further optimisation of the M30 and M65 ELISAs as biomarker assays that should translate into greater accuracy in results generated from clinical trials samples.
Keywords: M30 ELISA; M65 ELISA; Method validation; β-expectation tolerance interval;

Matrine-imprinted monolithic stationary phase for extraction and purification of matrine from Sophorae flavescentis Ait by Qiang Fu; Qi Fang; Bianling Feng; Sijuan Sun; Wei Du; Enijian Amut; Aiping Xiao; Chun Chang (894-900).
A matrine-imprinted monolithic stationary phase (MIP monolith) was prepared by in situ polymerization for extraction and purification of matrine from Sophorae flavescentis Ait. Matrine was used as the template molecule, methacrylic acid as the function monomer, ethylene glycol dimethacrylate as the cross-linking agent, and toluene and dodecanol as the porogenic solvents. Scanning electron microscope study revealed that a monolithic structure with mesopores and 36 μm diameter nodules was obtained. The molecular recognition process and the effect of varying chromatographic conditions on separation were examined by high-performance liquid chromatography (HPLC). Hydrogen bonding, electrostatic, hydrophobic interactions and the molecular shape matching in MIP monolith cavities were proposed to be responsible for the recognition mechanism. The use of MIP monolith as a solid-phase extraction (SPE) sorbent for extraction and purification of matrine from S. flavescentis Ait was investigated. The extraction yield was 89.2% (for 3.0 mmol l−1 matrine) with enrichment factor 29.
Keywords: Solid phase extraction; Molecularly imprinted polymer; Matrine; Monolithic stationary phase;

Determination of tobramycin in soil by HPLC with ultrasonic-assisted extraction and solid-phase extraction by Shun He; Qiyou Chen; Yan Sun; Yuncong Zhu; Laixin Luo; Jianqiang Li; Yongsong Cao (901-907).
Pharmaceuticals residues in the environment have become a growing scientific interest worldwide. In the light of the possible harmful effects of tobramycin, a rapid and sensitive analytical method for determination of tobramycin in soil was developed. The extraction and purification methods, derivatization conditions, and chromatographic conditions in the determination of tobramycin in soil have been fully investigated. Extraction was carried out by a combination of vortex mixer and ultrasonic oscillation using acetone/water as the extraction agent. The extract was concentrated to 1 mL and passed through the C18 SPE cartridge rinsed with water (3 mL), methanol (3 mL). The derivatization procedure was followed by the reaction of tobramycin with 4-Chloro-3,5-dinitrobenzotrifluoride at 60 °C for 10 min in pH 9.0 H3BO3–Na2B4O7 medium. The labeled tobramycin was determined by high performance liquid chromatography at 245 nm. Separation was accomplished within 15 min in gradient elution mode with trifluoroacetic acid in mobile phase as ion-pair reagent. The correlation coefficient for the method was 0.9999 in concentrations ranging from 0.10 to 100.0 μg/g. The limit of detection was 0.02 μg/g for tobramycin in soil at a signal-to-noise ratio of 3. The calculated recoveries of the proposed method were from 78.0 to 91.0% and RSDs were 3.38–9.79% in the application to the quantitative determination of tobramycin in all types of soil. The method will help to establish adequate monitoring of tobramycin residue in soil and make the contribution to environmental behavior evaluation.
Keywords: Tobramycin; Soil; Ultrasonic-assisted extraction; Solid-phase extraction; Pre-column derivatization;

As a part of the project for screening unequivocal biomarkers after sulfur mustard exposure, a quantitative method for the determination of β-lyase metabolites 1,1′-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) was validated. Full validation was conducted according to the FDA guidelines for method validation using pooled human urine as a sample matrix. The metabolites were extracted from urine with an optimized sample preparation procedure using ENV+ solid phase extraction cartridge with reduced volume of sample and solvents. Metabolites were detected by improved and faster liquid chromatography–heated electrospray ionization–tandem mass spectrometry (LC–HESI–MS/MS) method with two transitions of each chemical using non-buffered eluents, post-column splitter and higher flow-rate. These provided over five times faster analysis than previously published method providing 4.5 min/sample cycle time, to achieve up to 200 samples per day (24 h). Quantification was performed using deuterium labelled internal standard of SBMSE. The method was linear over the concentration range of 5–200 ng/ml (average correlation coefficients were R 2  = 0.997 and R 2  = 0.989) for both β-lyase metabolites, SBMSE and MSMTESE, respectively. The average retention times for SBMSE and MSMTESE were 1.96 ± 0.01 min and 3.24 ± 0.03 min (n  = 54). Calculated limits of detection were 4 ng/ml for both SBMSE and MSMTESE, respectively. Lower limits of quantification were 10 ng/ml and 11 ng/ml for SBMSE and MSMTESE, respectively. This validated method was successfully used in the First Confidence Building Exercise on Biomedical Samples Analysis organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Identification criteria for analysing unequivocal biomarkers of sulfur mustard with LC–MS/MS after alleged use is discussed and proposed based on the validation and exercise results.
Keywords: β-Lyase metabolites; Sulfur mustard; Biomarker; SPE; LC–HESI–MS/MS; Validation;

5-Fluoro-2′-deoxyuridine (floxuridine, 5-FdUrd) and 5-fluorouracil (5-FU) are widely used for the treatment of colorectal cancers. The mechanisms of action of 5-FdUrd and 5-FU, as well as the biochemical pathway responsible for their metabolism, are well understood. Identification of every metabolite and achieving mass balance by conventional UV absorption-based HPLC analysis are not feasible because the metabolites beyond 5-FU in the 5-FdUrd metabolic pathway are undetectable by UV light. We therefore established a mass spectrometry method, designed for fast and convenient analysis, for simultaneously measuring 5-FdUrd, 5-FU, and their metabolites. Linearity, precision and accuracy were validated in the concentration ranges studied for each compound. Hydrolysis studies of 5-FdUrd and amino acid mono ester prodrugs of 5-FdUrd in Capan-2 cell homogenates were carried out and the achievement of mass balance was established with this method (recovery of 5′-O-l-leucyl-FdUrd was 96.6–108.2% and that of 5-FdUrd was 79.4–117.4%). This simple LC–MS method achieves reliable quantitation and mass balance of 5-FdUrd, 5-FU, and their metabolites and can be effectively utilized for further kinetic studies.
Keywords: α-Fluoro-β-alanine (FBAL); α-Fluoro-β-ureidopropionate (FUPA); 5-Fluorodihydrouracil (FUH2); 5-Fluorouracil (5-FU); Floxuridine (5-FdUrd); Floxuridine prodrug; Metabolite; LC–MS; Mass balance;

A capillary electrophoresis method with contactless conductivity detection was developed for the quantification of carnitine and six acylcarnitines in plasma and urine samples. The running buffer employed consisted of 500 mmol/L acetic acid, 1.0 mmol/L hydroxypropyl-β-cyclodextrin and 0.05% Tween at a pH of 2.6. Under these conditions, the isomeric valproyl- and octanoyl-carnitines could be distinguished. The linearity was in the range from 5.0 to 200.0 μmol/L with correlation coefficients between 0.9992 and 0.9997. The limits of detection were between 1.0 and 3.2 μmol/L. Intra- and inter-day precisions as %RSD were better than 10%. The method allows for direct determination without derivatisation or extraction processes. The method was applied for the quantification of carnitine and acetylcarnitine in plasma pre- and post-exercise, and to measure valproylcarnitine in plasma and urine of patients undergoing valproate therapy.
Keywords: Capillary electrophoresis; Conductivity detection; Carnitine; Acylcarnitines; Clinical samples;

A novel method for determination of pseudolycorine in the bulb of lycoris radiata by capillary electrophoresis coupled with online electrochemiluminescence detection with ultrasonic-assisted extraction has been developed. The effects of several factors such as detection potential, concentration and pH of phosphate buffer, separation voltage, injection time, ultrasonic power and extraction time were investigated. Under optimal conditions, the linear concentration range for pseudolycorine was 0.002–2 μg/mL with a correlation coefficient of 0.9991. Relative standard deviations of migration time and peak areas were 1.4 and 3.2%, respectively. The limit of detection (S/N = 3) was 0.46 ng/mL. The proposed method can be successfully applied to the determination of pseudolycorine in the bulb of lycoris radiata.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Pseudolycorine; Ultrasonic extraction; Lycoris radiata;

Morphine is present in varying amounts as an endogenous product in human urine. Derivatization of morphine contained in urine with dansyl chloride yields a known product, which can be quantified by liquid chromatography mass spectrometry with high selectivity and sensitivity. Urine samples of 51 healthy individuals were spiked with stable-isotope labeled morphine, hydrolyzed and subjected to solid phase extraction followed by derivatization of morphine with dansyl chloride. The dansyl derivatives of naturally occurring morphine and deuterated internal standard were then detected by liquid chromatography–triple quadrupole mass spectrometry. Using the [N-CD3]-labeled internal standard and solid-phase extraction, a limit of detection of 35 fmol/ml (10 pg/ml) and a limit of quantification of 87.5 fmol/ml (25 pg/ml) was determined for morphine in human urine. This new LC–MS/MS method allowed the detection of endogenous morphine in human urine of 51 volunteers with an average value of 156.4 fmol/ml (44.7 ng/ml).
Keywords: Morphine; Urine; Dansyl chloride derivatization; Liquid chromatography mass spectrometry;

Plant hormones play crucial roles in plant growth and development. However, up to date, identification and quantification of acidic plant hormones with trace amount in complicated plant matrix is still a challenge. In current study, we developed a high sensitive assay for the determination of acidic plant hormones in rice by combining capillary electrophoresis and electrospray ionization-time of flight-mass spectrometry (CE-ESI-TOF-MS). To improve the detection sensitivity of acidic plant hormones, 3-bromoactonyltrimethylammonium bromide (BTA) was synthesized as a new mass probe, which can react efficiently with acidic plant hormones in acetonitrile containing triethylamine (TEA). The positively charged BTA-derivatives were separated by CE using amino-coated capillary, which provided a reversed electroosmotic flow (EOF) at low pH, as well as reduced the adsorption of BTA-derivatives on the inner wall of capillary. Using the CE-ESI-TOF-MS method developed in current study, 15 acidic plant hormones, including 10 gibberellins (GAs), were identified and quantified with good linearities from 1.3 to 850 ng/mL with linear coefficient R 2 values of >0.99. The limits of detection (LODs) were in the range of 0.34–4.59 ng/mL. Recoveries of compounds from spiked beverage samples ranged from 84.6 to 112.2%. And a good reproducibility was obtained by evaluating the intra and inter-day precisions with relative standard deviations (RSDs) less than 6.7 and 9.9%, respectively.
Keywords: Acidic plant hormone; Mass probe; CE-ESI-TOF-MS;

A method which involves the combination of pH-zone-refining counter-current chromatography (pH-zone-refining CCC) and conventional high-speed counter-current chromatography (HSCCC) was established for the preparative separation of alkaloids from the crude extracts of Stephania kwangsiensis. pH-zone-refining CCC was first performed with the solvent system composed of n-hexane–ethyl acetate–methanol–water (3:7:1:9, v/v), where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 2.0 g of crude extract, 370 mg of sinoacutine and 600 mg of a mixture of three other alkaloids were obtained. Then, the mixture was further separated by conventional HSCCC with the solvent system composed of n-hexane–ethyl acetate–methanol–water (7:3:6:4, v/v), yielding 42 mg of (−)-crebanine, 50 mg of (−)-stephanine and 30 mg of l-romerine from 150 mg mixture of three other alkaloids, respectively. The purities of the four compounds were all over 98% as determined by HPLC, and the chemical structures of the four compounds were confirmed by positive ESI–MS and 1H NMR data. Results of the present study successfully indicated that this method was efficient for the preparative separation of alkaloids from natural plants.
Keywords: Stephania kwangsiensis; pH-zone-refining CCC; Conventional HSCCC; Preparative chromatography; Alkaloids;

A chemical method for the determination of hyaluronan (hyaluronic acid, HA) has been developed and applied to the human blood plasma. Human blood plasma HA was converted to the ΔDi-HA by digestion with hyaluronidase SD and determined by a sensitive and selective high-performance liquid chromatography (HPLC). The HPLC includes the separation and detection of ΔDi-HA using a graphitized carbon column and fluorometric reaction with 2-cyanoacetamide in an alkaline eluent. The calibration graph for ΔDi-HA was linear over the range 0.2 ng–1 μg. It was revealed that the concentration of HA in normal human blood plasma is very low levels (about 24 ng/ml) in comparison to low-sulfated chondroitin 4-sulfate (about 13 μg/ml).
Keywords: Hyaluronan; Human blood plasma; HPLC; Graphitized carbon column; Fluorometric detection;

Susceptibility of tryptophan (Trp) in a complementarity-determining region (CDR) to oxidation is a significant issue for recombinant monoclonal antibody (mAb) therapeutics due to the clinical efficacy and stability concerns. Here we present a case study using hydrophobic interaction chromatography (HIC) to separate an oxidized Trp containing population of an IgG1. The best separation was achieved using dual Dionex ProPac HIC-10 columns, and the oxidized Trp population was isolated as a separated pre-peak. Peptide map analysis revealed that the oxidized Trp is located in a heavy chain CDR. In addition, the HIC method was capable of monitoring the oxidation status of the CDR Trp, as the oxidation rate of the CDR Trp measured by HIC directly correlated with the results of the peptide maps. The same method conditions were also capable of separating oxidized methionine (Met) and isomerization/deamidation products, which co-elute as another pre-peak at a different retention time from the oxidized Trp species. These observations indicate that the HIC procedure can be utilized to monitor the oxidative status of the CDR Trp in the IgG1.
Keywords: IgG1; Tryptophan; Complementarity-determining region; Oxidation; Hydrophobic interaction chromatography; Mass spectrometry;

A protocol for the metabolic profiling of rat liver was developed based on ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF/MS) to explore metabolic state directly. Methanol/water (4:1, v:v) was selected as the optimal extraction solvent. The established method was validated with a linearity over the 10–5000 ng/mL for internal standards (IS) and got an average correlation coefficient of 0.9986. The intra-day and inter-day RSD for most endogenous compounds were below 15%. And the absolute recovery of IS was from 84.8% to 109.1%. Liver tissues from diabetic and control rats were enrolled in the subsequent study to show the usefulness of the method. A clear classification between the control and model animals was achieved, some significant metabolites were successfully filtered. These metabolites reflected the abnormal metabolism of diabetic rats. This initial application indicated that the method is suitable and reliable for liver tissue metabolic profiling. It is expected this protocol could also be extended to metabonomic studies of other tissues.
Keywords: Metabonomics; Liver; UPLC–Q-TOF/MS; Metabolic profiling; Type 2 diabetes;

Identification and elimination of ion suppression in the quantitative analysis of sirolimus in human blood by LC/ESI-MS/MS by Nariyasu Mano; Marina Nozawa; Mayumi Sato; Masaru Mori; Hiroaki Yamaguchi; Katsuhiro Kanda; Makoto Nogami; Junichi Goto; Miki Shimada (968-974).
Ion suppression can negatively affect the performance characteristics of LC/ESI-MS/MS based methods, and we wished to identify sources of ion suppression in an assay for quantitating sirolimus in human whole blood. We first compared the peak areas of sirolimus and ascomycin added to human blood samples treated with and without extraction using octadecyl silyl (ODS)-silica gel after protein precipitation, and we found that water-soluble compounds cause the ion suppression for both drugs. Post-column infusion studies indicated that compounds retained in the sample after ODS extraction and protein precipitation caused ion suppression. MS analysis of these compounds suggested they were hydroxyl group-possessing phosphocholines, and this was confirmed using purified lysophosphatidylcholine variants. Therefore, we included a HybridSPE treatment step after the ODS extraction into the preanalytical workflow to remove phosphocholines, and this successfully eliminated the observed ion suppression for determining sirolimus concentration in human whole blood by LC/ESI-MS/MS. Sirolimus is a highly lipophilic molecule, and this study demonstrates the impact that preanalytical extraction and purification steps can have on a laboratory's ability to accurately detect and quantitate this and other lipophilic drugs.
Keywords: Electrospray ionization; Ion suppression; Mass spectrometry; Phospholipids; Sirolimus;

We developed an ionic liquid based ultrasonic assisted extraction (ILUAE) method for the extraction of the three isoflavones, namely tectoridin, iristectorin B and iristectorin A from Iris tectorum Maxim of the Iridaceae family. Three kinds of 1-alkyl-3-methylimidazolium ionic liquids with different alkyl chain and anion were investigated. The results indicated that ionic liquids (ILs) showed remarkable effects on the extraction yield of isoflavones. In addition, the ILUAE, including several ultrasonic parameters, such as the concentration, extraction time and solvent to solid ratio have been optimized. Under these optimal conditions (e.g., with 30 min extraction time and the solvent to solid ratio of 30 ml/g), this approach gained the highest extraction yields of tectoridin (37.45 mg/g), iristectorin B (2.88 mg/g) and iristectorin A (5.28 mg/g). Meanwhile, tectoridin, iristectorin B and iristectorin A in the ILUAE extract were separated and purified successfully through the high-speed counter-current chromatography (HSCCC) with a two-phase solvent system consisting of n-butanol–water (1:1, v/v). The additional advantage of this approach is that 60.21 mg tectoridin, 4.33 mg iristectorin B and 8.24 mg iristectorin A with more than 95.0% purities have been obtained from 400 mg ILUAE extract of I. tectorum within 5 h and one-step elution under the most optimized conditions (e.g., a flow rate of 2.0 ml/min, 900 rpm and the wavelengh of 280 nm). The obtained fractions were successfully analyzed by HPLC and identified by 1H-NMR and 13C-NMR.
Keywords: Ionic liquids; Ultrasonic assisted extraction; Isoflavones; High-speed counter-current chromatography; Iris tectorum Maxim;

Concentration and purification of rubella virus using monolithic chromatographic support by Dubravko Forcic; Marija Brgles; Jelena Ivancic-Jelecki; Maja Šantak; Beata Halassy; Miloš Barut; Renata Jug; Maja Markušić; Aleš Štrancar (981-986).
The production of economically acceptable viral vaccines of high quality requires simple and efficient methods for purification and concentration of viral particles. Ion-exchange chromatography (IEC) has become one of commonly used methods for large-scale downstream purification of viruses. Viruses possess different biological and/or biochemical properties and therefore IEC conditions must be established specifically for each virus. Live attenuated rubella virus vaccines have been manufactured and successfully used widely to protect people from rubella and congenital rubella syndrome for almost 40 years. The aim of this study was to search for an efficient method for concentration and purification of rubella virus using IEC. The selected operating conditions using quaternary amine monolithic supports enabled highly efficient binding, purification and concentration of rubella virus from complex biological suspension without additional procedures. Eluted viral particles maintained their infectivity and viral recovery was almost 100%. At the same time, viral preparation was successfully depleted from host cell protein and DNA. This work indicates the possibility of using monoliths to improve the rubella virus yields in productions where high virus titers during cultivation can hardly be achieved.
Keywords: Rubella virus; Virus purification; Monolithic column; Chromatography; Virus concentration;

An accurate quantitative LC/ESI–MS/MS method for sirolimus in human whole blood by Nariyasu Mano; Mayumi Sato; Marina Nozawa; Yotaro Matsumoto; Masaru Mori; Hiroaki Yamaguchi; Junichi Goto; Miki Shimada (987-992).
Sirolimus is a widely used immunosuppressant that requires therapeutic drug monitoring (TDM). We optimized a preanalytical procedure that allows for the accurate quantiation of sirolimus in whole blood by LC/ESI–MS/MS with minimal matrix effects. Sirolimus is highly lipophilic, and solvents containing greater than 50% methanol were required to maintain sirolimus recovery. The final pretreatment procedure developed consists of a zinc sulfate protein precipitation, an extraction using octadecyl silyl-silica gel for eliminating water-soluble and hydrophilic compounds, and HybridSPE cartridge treatment to eliminate phospholipids. Using this procedure prior to LC/ESI–MS/MS led to the accurate and reproducible quantitation of sirolimus in human whole blood. The linear range of detection was 0.5–50 ng/mL, a range appropriate for TDM, and the method demonstrated good repeatability and intermediate precision within this quantitative range. In order to investigate the quantitative performance of this method, we compared it to two commercially available sirolimus immunoassays and our previously reported LC/ESI–MS/MS method. The immunoassays gave consistently greater values for the sirolimus concentration, and this may be related to antibody cross-reactivity with sirolimus metabolites and/or other matrix effects. Although our procedure is too long to support real-time TDM for outpatients, it can serve as reference method to assess the performance of other analytical methods that are currently available or may be developed in the future.
Keywords: Immunoassay; LC/ESI–MS/MS; Sirolimus; Therapeutic drug monitoring;

Purification of 6×His-tagged phycobiliprotein using zinc-decorated silica-coated magnetic nanoparticles by Yingjie Chen; Peng Jiang; Shaofang Liu; Hui Zhao; Yulin Cui; Song Qin (993-997).
Zinc-decorated silica-coated magnetic nanoparticles (ZnSiMNPs) were prepared by adsorbing zinc onto colloidal silica. These nanoparticles were used for the rapid purification of 6×His-tagged recombinant phycobiliprotein. The surface changes in the magnetic nanoparticles after zinc adsorption were characterized by transmission electron microscopy. The adsorption of the 6× His-tagged phycobiliprotein onto ZnSiMNPs in 10 mM PBS at 25 °C was found to follow the Langmuir isotherm. ZnSiMNPs could be used to extract 6×His-tagged phycobiliprotein from lysate to single-band purity, as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. No spectral variation was observed in the purified phycobiliprotein. Thus, ZnSiMNPs served as a useful tool for the magnetic separation and delivery of the 6×His-tagged phycobiliprotein.
Keywords: 6×His tag; Phycobiliprotein; Silica-coated magnetic nanoparticles (SiMNPs); Zn(II); Purification; Adsorption behavior;

Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue by Yijun Li; Ryan Cleary; Mark Kellogg; Janet S. Soul; Gerard T. Berry; Frances E. Jensen (998-1002).
We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC–MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC–MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood–brain barrier.
Keywords: Bumetanide; Liquid chromatography/tandem mass spectrometry (LC–MS/MS); Stable isotope dilution;

The condition of chymotrypsin (ChTRP)–Eudragit® (Eu) insoluble complex formation was studied with the aim of applying this information to the separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate. The optimal pH of the complex precipitation was 4.60 for ChTRP–Eudragit® L100 and 5.40 for ChTRP–Eudragit® S100. The polyelectrolyte concentration necessary for the commercial enzyme precipitation was lower than 0.1% (w/v). The complex formation was inhibited by NaCl for both polyelectrolytes. The method was applied to recover the enzyme from bovine homogenate; ChTRP was precipitated by Eudragit® addition. The non-soluble complexes were separated by simple centrifugation and re-dissolved by a pH change to 8.20. The best conditions to recover ChTRP brought about a purification factor of around 4 and 90% yield.
Keywords: Polyelectrolyte precipitation; Chymotrypsin; Eudragit; Protein purification;

Determination of isometamidium residues in animal-derived foods by liquid chromatography–tandem mass spectrometry by Y.G. Li; Y. Zou; L. Zhang; C.X. Xi; G.M. Wang; X.L. Li; J.Z. Zhang; Z.G. Li (1008-1012).
In this paper, a new determination method for isometamidium residues in animal-derived foods was developed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Isometamidium residues in bovine tissues and milk were extracted with the mixed solution of acetonitrile and 0.25 mol/L of ammonium formate–methanol (v/v, 1:1), concentrated and degreased, and determined by LC–MS/MS with quantification by external standard method. The results showed that the peak area of chromatogram was linearly related to the concentration of isometamidium in the range of 1–100 μg/L, and the limits of detection (LOD) and quantification (LOQ) were 0.05 μg/kg and 5 μg/kg, respectively. The average recoveries of spiked samples were in the range of 73.8–93.9% with relative standard deviations ranged from 2.3% to 7.5%. This method is simple, accurate and suitable for the identification and quantification for isometamidium in animal-derived foods.
Keywords: HPLC–MS/MS; Isometamidium; Animal-derived food; Residual detection;

Stable isotope-dilution liquid chromatography/tandem mass spectrometry method for determination of thyroxine in saliva by Tatsuya Higashi; Takuya Ichikawa; Chikara Shimizu; So Nagai; Shinsuke Inagaki; Jun Zhe Min; Hitoshi Chiba; Shigeo Ikegawa; Toshimasa Toyo’oka (1013-1017).
A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of thyroxine (T4) in human saliva has been developed and validated. The saliva was deproteinized with methanol, purified using a Strata-X™ cartridge, and subjected to LC/ESI-MS/MS. Quantification was based on selected reaction monitoring, and [13C6]-T4 was used as the internal standard. This method allowed the reproducible (intra- and inter-assay relative standard deviations, <4.8%) and accurate (analytical recovery, 96.5–99.6%) quantification of the salivary T4 using a 400 μl sample, and the limit of quantification was 25.0 pg/ml. A preliminary study using the developed method found that there is a diagnosable difference in the salivary T4 concentration between the euthyroid subjects and the patients with Graves disease.
Keywords: Thyroxine; Saliva; Liquid chromatography/electrospray ionization-tandem mass spectrometry; Isotope dilution; Graves disease;

Rapid quantification of major reaction products formed during thermochemical pretreatment of lignocellulosic biomass using GC–MS by James F. Humpula; Shishir P.S. Chundawat; Ramin Vismeh; A. Daniel Jones; Venkatesh Balan; Bruce E. Dale (1018-1022).
Accurate quantification of reaction products formed during thermochemical pretreatment of lignocellulosic biomass would lead to a better understanding of plant cell wall deconstruction for production of cellulosic biofuels and biochemicals. However, quantification of some process byproducts, most notably acetamide, acetic acid and furfural, present several analytical challenges using conventional liquid chromatography methods. Therefore, we have developed a high-throughput gas chromatography based mass spectrometric (GC–MS) method in order to quantify relevant compounds without requiring time-consuming sample derivatization prior to analysis. Solvent extracts of untreated, ammonia fiber expansion (AFEX) treated and dilute-acid treated corn stover were analyzed by this method. Biomass samples were extracted with acetone using an automated solvent extractor, serially diluted and directly analyzed using the proposed GC–MS method. Acetone was the only solvent amongst water, methanol and acetonitrile that did not contain detectable background levels of the target compounds or facilitate a buildup of plant-derived residues in the GC injector, which decreased analytical reproducibility. Quantitative results were based on the method of standard addition and external standard calibration curves.
Keywords: Gas chromatography; Mass spectrometry; AFEX pretreatment; Acetamide; Acetic acid; Furfural; Lignocellulose; Biofuels;

Identification of flavonoids in the stems and leaves of Scutellaria baicalensis Georgi by Guozhu Liu; Nautiyal Rajesh; Xiaosong Wang; Mingshan Zhang; Qin Wu; Shengjun Li; Bo Chen; Shouzhuo Yao (1023-1028).
Scutellaria baicalensis Georgi (S. baicalensis), a perennial herb of the Labiatae family, is a well-known traditional Chinese medicine. In the present study, a comprehensive qualitative analysis of flavonoids in the stems and leaves of S. baicalensis was performed. Under the optimized experimental conditions, 21 flavonoids were clearly detected. 17 of them were successfully identified based on the on-line UV and MS n data and were sequentially confirmed by the literature search. The rest 4 flavonones, which were not on-line identified, were successfully isolated and were identified by 1D and 2D NMR. One of them, 5,6,7,3′,4′-pentahydroxy flavanone-7-O-glucuronide (2) is a new compound.
Keywords: Scutellaria baicalensis Georgi; Stems and leaves; Flavonoids; Global qualitative analysis; HPLC-UV/MS; NMR;

Direct-injection HPLC method of measuring micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column by Hiroaki Uranishi; Mitsuhiro Nakamura; Hiroki Nakamura; Yukari Ikeda; Mayuko Otsuka; Zenichiro Kato; Teruo Tsuchiya (1029-1032).
A direct-injection HPLC-based method has been developed for determining amounts of micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column. The method is easy to perform and requires only 10 μL of a filtered plasma sample. The chromatographic separations were carried out with a gradient mode. The fluorescence detection wavelengths of excitation and emission were set at 273 nm and 464 nm, respectively. Retention times for micafungin and IS were 22.4 and 23.7 min, respectively. Micafungin and FR195743 (IS) peaks were completely separated with little tailing, and no interference was observed. The calibration curve of micafungin showed good linearity in the range of 0.5–20.0 μg/mL (r 2  = 1.00). The intra-day accuracy ranged from −4.5 to 5.3%. The inter-day accuracy ranged from −9.8 to 1.5%. The precisions were less than 10%. This method is useful for the determination of micafungin in human plasma.
Keywords: Micafungin; Liquid chromatography; Direct injection; Fluorescence detection;