Journal of Chromatography B (v.878, #21)

A method for the analysis of six thyroid hormones in thyroid gland by liquid chromatography–tandem mass spectrometry by Tatsuya Kunisue; Jeffrey W. Fisher; Babatope Fatuyi; Kurunthachalam Kannan (1725-1730).
Perchlorate can competitively inhibit iodide uptake by the thyroid gland (TG) via the sodium/iodide symporter, consequently reducing the production of thyroid hormones (THs). Until recently, the effects of perchlorate on TH homeostasis are being examined through measurement of serum levels of TH, by immunoassay (IA)-based methods. IA methods are fast, but for TH analysis, they are compromised by the lack of adequate specificity. Therefore, selective and sensitive methods for the analysis of THs in TG are needed, for assessment of the effects of perchlorate on TH homeostasis. In this study, we developed a method for the analysis of six THs: l-thyroxine (T4), 3,3′,5-triiodo-l-thyronine (T3), 3,3′,5′-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2), 3,3′-diiodo-l-thyronine (3,3′-T2), and 3-iodo-l-thyronine (3-T1) in TG, using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). TGs used in this study were from rats that had been placed on either iodide-deficient diet or iodide-sufficient diet, and that had either been provided with perchlorate in drinking water (10 mg/kg/day) or control water. TGs were extracted by pronase digestion and then analyzed by LC–MS/MS. The instrumental calibration range for each TH ranged from 1 to 200 ng/ml and showed a high linearity (r  > 0.99). The method quantification limits (LOQs) were determined to be 0.25 ng/mg TG for 3-T1; 0.33 ng/mg TG for 3,3′- and 3,5-T2; and 0.52 ng/mg TG for rT3, T3, and T4. Rats were placed on an iodide-deficient or -sufficient diet for 2.5 months, and for the last 2 weeks of that period were provided either perchlorate (10 mg/kg/day) in drinking water or control water. Iodide deficiency and perchlorate administration both reduced TG stores of rT3, T3, and T4. In iodide-deficient rats, perchlorate exacerbated the reduction in levels of THs in TG. With the advances in analytical methodology, the use of LC–MS/MS for measurement of hormone levels in TG will allow more comprehensive evaluations of the hypothalamic-pituitary–thyroid axis.
Keywords: Thyroid hormones; Thyroid gland; Perchlorate; Iodide deficiency; LC–MS/MS;

The surface imprinting of basic protein lysozyme (Lys) was carried out by designing a new route. The copolymerization of N-vinylpyrrolidone (NVP) and 2-hydroxyethyl methacrylate (HEMA) was first conducted in an inverse suspension polymerization system, and the crosslinked copolymeric microspheres HEMA/NVP were prepared. Subsequently, the esterification reaction of methacryloyl (MAO) chloride with the hydroxyl groups on the surfaces of HEMA/NVP microspheres was performed, and the modified microspheres MAO–HEMA/NVP, on which a mass of polymerisable double bonds were introduced, were obtained. In the presence of lysozyme, by initiating of K2S2O8–NaHSO3, the monomer methacrylic acid (MAA) in the solution was crosslink-polymerized on the surfaces of MAO–HEMA/NVP microspheres, resulting in the surface imprinting of lysozyme. After removing the template molecules, the lysozyme molecule-surface-imprinted material MIP-HEMA/NVP was obtained. Because there were strong interactions between lysozyme and monomer MAA, electrostatic interaction and hydrogen bonding, the lysozyme molecule-surface imprinting was successfully realized. The MIP-HEMA/NVP microspheres have very high binding affinity for lysozyme, and the binding capacity gets up to 216 mg/g. It is more important that MIP-HEMA/NVP microspheres have specific recognition selectivity for lysozyme, and the selectivity coefficient for lysozyme with respect to bovine hemoglobin (BHb), which was used as a contrast protein in the experiments, actually reaches 31.07. In the respect of protein imprinting, the imprinting material with such high performance is unwonted.
Keywords: Lysozyme; Surface imprinting; Methacrylic acid; 2-Hydroxyethyl methacrylate; Recognition selectivity;

A liquid chromatography method for quantifying caffeine dissolution from pharmaceutical formulations into colloidal, fat-rich media by Hywel D. Williams; David A. Barrett; Robert Ward; Ian J. Hardy; Colin D. Melia (1739-1745).
A simple and rapid high-performance liquid-chromatography method is presented that permits quantification of caffeine in colloidal fat emulsions proposed as new ‘biorelevant’ dissolution media (Intralipid™ and various milks). Using a mobile phase of 0.1 M sodium acetate (pH 4.0) and acetonitrile (89.5:10.5, v/v) at 1 ml min−1, the drug and internal standard (7-β-hydroxyethyltheophylline) were eluted within 8 min. Caffeine extraction was undertaken by protein precipitation in ice-cold 12% (w/v) trichloroacetic acid and centrifugation at 10,000 rpm for 15 min. This simple extraction method generated caffeine recovery values (corrected for % fat content) of 75.4 ± 1.4–100.6 ± 5.5%. The limit of detection was within the range 0.25–0.4 μg ml−1 and linearity was demonstrated in each medium up to 125 μg ml−1. Precision was <11.5% RSD and intra- and inter-day accuracy was 93.4–109.3%. The validated method was applied to in vitro USP dissolution tests in milk which compared the kinetics of caffeine release from (i) extended release matrices containing hydroxypropyl methylcellulose (HPMC) and (ii) an immediate release commercial analgesic tablet. Good reproducibility was obtained in both extended and immediate release dissolution tests. The method provides high-throughput quantification of this common drug in fat emulsions used as biorelevant dissolution media.
Keywords: Caffeine; HPLC; Fat emulsions; Milk; Intralipid; HPMC matrices;

A new method has been developed for determination and confirmation of amitraz and its main metabolite, 2,4-dimethylaniline, in food animal tissues using gas chromatography-electron capture detector (GC-ECD) and gas chromatography–mass spectrometry detector (GC–MS). This method is based on a new extraction procedure using accelerated solvent extraction (ASE). It consists of an n-hexane/methanol extraction step, a cleaning-up step by BakerBond octadecyl C18 silica bonded cartridge, hydrolysis and derivatization to 2,4-dimethyl-7-F-butyramide for GC-ECD analysis. For confirmation using GC–MS, hydrolysis and derivatization were not needed. Parameters for extraction pressure, temperature and cycle of ASE, clean-up, derivatization and analysis procedure have been optimized. Spike recoveries from 50 to 300 μg/kg levels were found to be between 72.4 and 101.3% with relative standard deviation less than 11.5% in GC-ECD, from 5 to 20 μg/kg levels were found to be between 77.4 and 107.1% with relative standard deviation less than 11.6% in GC–MS. The LOD and LOQ are 5 and 10 μg/kg, respectively, for these two analytes using GC-ECD. For GC–MS, LOD and LOQ were 2 and 5 μg/kg, respectively. The rapid and reliable method can be used for characterization and quantification of residues of amitraz and its main metabolite, 2,4-dimethylaniline, in liver and kidney samples of swine, sheep and bovine.
Keywords: Amitraz; 2,4-Dimethylaniline; Accelerated solvent extraction; Gas chromatography; Liver and kidney;

Cumulative exposure assessment for trace-level polycyclic aromatic hydrocarbons (PAHs) using human blood and plasma analysis by J.D. Pleil; M.A. Stiegel; J.R. Sobus; S. Tabucchi; A.J. Ghio; M.C. Madden (1753-1760).
Humans experience chronic cumulative trace-level exposure to mixtures of volatile, semi-volatile, and non-volatile polycyclic aromatic hydrocarbons (PAHs) present in the environment as by-products of combustion processes. Certain PAHs are known or suspected human carcinogens and so we have developed methodology for measuring their circulating (blood borne) concentrations as a tool to assess internal dose and health risk. We use liquid/liquid extraction and gas chromatography–mass spectrometry and present analytical parameters including dynamic range (0–250 ng/ml), linearity (>0.99 for all compounds), and instrument sensitivity (range 2–22 pg/ml) for a series of 22 PAHs representing 2–6-rings. The method is shown to be sufficiently sensitive for estimating PAHs baseline levels (typical median range from 1 to 1000 pg/ml) in groups of normal control subjects using 1-ml aliquots of human plasma but we note that some individuals have very low background concentrations for 5- and 6-ring compounds that fall below robust quantitation levels.
Keywords: Human blood; Systems biology; Polycyclic aromatic hydrocarbons; Exposure assessment; Internal dose; GC–MS;

One-pot microwave derivatization of target compounds relevant to metabolomics with comprehensive two-dimensional gas chromatography by Konstantinos A. Kouremenos; James J. Harynuk; William L. Winniford; Paul D. Morrison; Philip J. Marriott (1761-1770).
Metabolomics has been defined as the quantitative measurement of all low molecular weight metabolites (sugars, amino acids, organic acids, fatty acids and others) in an organism's cells at a specified time under specific environmental/biological conditions. Currently, there is considerable interest in developing a single method of derivatization and separation that satisfies the needs for metabolite analysis while recognizing the many chemical classes that constitute the metabolome. Chemical derivatization considerably increases the sensitivity and specificity of gas chromatography–mass spectrometry for compounds that are polar and have derivatizable groups. Microwave-assisted derivatization (MAD) of a set of standards spanning a wide range of metabolites of interest demonstrates the potential of MAD for metabolic profiling. A final protocol of 150 W power for 90 s was selected as the derivatization condition, based upon the study of each chemical class. A study of the generation of partially derivatized components established the conditions where this could potentially be a problem; the use of greater volumes of reagent ensured this would not arise. All compounds analyzed by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry in a standard mixture showed good area ratio reproducibility against a naphthalene internal standard (RSD < 10% in all but one case). Concentrations tested ranged from 1 μg/mL to 1000 μg/mL, and the calibration curves for the standard mixtures were satisfactory with regression coefficients generally better than 0.998. The application to gas chromatography–quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry for a typical reference standard of relevance to metabolomics is demonstrated.
Keywords: Metabolic profiling; Microwave-assisted derivatization; Gas chromatography; Quadrupole mass spectrometry; Comprehensive two-dimensional gas chromatography; Time-of-flight mass spectrometry;

Efficient isolation of egg white components is desired due to its potential uses. Existing methods mainly targeted on one specific protein; an attempt has been made in the study to co-extract all the valuable egg white components in a continuous process. Ovomucin was first isolated by our newly developed two-step method; the resultant supernatant obtained after ovomucin isolation was used as the starting material for ion-exchange chromatography. Anion-exchange chromatography of 100 mM supernatant yielded a flow-through fraction and three other fractions representing ovotransferrin, ovalbumin and flavoproteins. The flow-through fraction was further separated into ovoinhibitor, lysozyme, ovotransferrin and an unidentified fraction which represents 4% of total egg white proteins. Chromatographic separation of 500 mM supernatant resulted in fractions representing lysozyme, ovotransferrin and ovalbumin. This co-extraction protocol represents a global recovery of 71.0% proteins.
Keywords: Egg white proteins; Co-extraction; Ion-exchange chromatography;

Matrix metalloproteinase 9 (MMP-9) plays an important role in cancer invasion and metastasis and has been an attractive target for therapeutic intervention of cancer metastasis. However, considering the high cost and intricacy associated with the expression, isolation and purification of the recombinant enzyme for the screening of drug candidates, alternative methods that explore the recycling of enzymes become desirable. In this study, a new immobilized enzyme reactor (IMER) containing human recombinant MMP-9 enzyme was developed and characterized for the on-line screening of MMP-9 inhibitors. The MMP-9 IMER containing active unit of the enzyme (U  = 0.08 μmol/min) on the disk was inserted into a HPLC system connected to a UV–vis detector for on-line chromatographic screening. The resulting conjugated enzyme was shown to be an active and stable catalyst for the hydrolysis of MMP-9 chromogenic thiopeptide substrate Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5. The kinetics profile of the enzyme in IMER and free solution were determined and compared. Selected reversible MMP inhibitors, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycyl hydroxamic acid (NNGH), doxycycline and minocycline were further characterized using the MMP-9 IMER and free enzyme solution assays. Our system demonstrated applicability as a rapid and cost-effective screening tool for inhibitors of the MMP-9 enzyme.
Keywords: Matrix metalloproteinase; MMP-9; Immobilized enzyme reactor; High throughput screening;

Aqueous two-phase systems (ATPS) have been widely adopted for the combined purpose of solid liquid separation, and recovery and purification of bioproducts such as proteins, viruses and organelles from biological feedstocks and fermentation broth. However, in spite of potential advantages over other techniques applied to concentrated biological feedstocks, ATPS have been applied at process scale only by a few industries and research establishments. ATPS are sensitive to loading with modest to extreme quantities of biological feedstock due to the contribution of that material to phase formation in combination with the conventional phase-forming chemicals. This causes problem associated with the definition and manipulation of loaded working systems, which may be addresses as in the present study with the aid of distribution analysis of radiolabel led analytes (DARA) in representative process samples. The present study focussed on establishing a generic description for characterising ATPS loaded with biological feedstocks and the redefinition of the biological feedstock loaded system composition in terms of phase forming chemical equivalents. This evaluation will be useful to achieve ATPS process implementation where phase recycle/reuse is adopted without compromise to process operations and consistent protein recovery performance.
Keywords: Aqueous two-phase systems; Biological feedstock; Phase chemical composition; DARA; Protein;

Purification and kinetic properties of rabbit liver paraoxonase 1 by Tülin Bayrak; Ahmet Bayrak; Ediz Demirpençe; Kamer Kılınç (1791-1795).
Paraoxonase 1 (PON1) is synthesized in the liver and secreted into the blood, where it is associated exclusively with HDL. In this study, rabbit liver PON1 enzyme was purified to homogeneity using a new purification approach, and the kinetic properties of the enzyme were investigated using phenyl acetate and homocysteine thiolactone as substrates. Rabbit liver PON1 was purified through the preparation of liver microsomal fraction, Sephacryl S300 HR gel filtration chromatography, DEAE Trisacryl M ion-exchange chromatography and hydroxyapatite chromatography steps. Using this method, rabbit liver PON1 was purified 576 times with a specific activity of 2726 U/mg protein. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the obtained enzyme as a single protein band close to 40 kDa. The Km of the this enzyme was found as 0.55 ± 0.024 mM for phenyl acetate and 17.31 ± 1.2 mM for homocysteine thiolactone. In this study, a new approach was used to purify PON1 enzyme from rabbit liver and for the first time in the literature, its kinetics was studied with homocysteine thiolactone as substrate.
Keywords: Paraoxonase 1; Liver; Purification; Homocysteine thiolactone;

Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects by Marek Sanak; Anna Gielicz; Krzysztof Nagraba; Marek Kaszuba; Jagoda Kumik; Andrew Szczeklik (1796-1800).
Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry.To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals.Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor.Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E2 was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D2 products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B4 has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes–eoxins were detectable and metabolized to eoxin E4. Two main vascular prostanoids: prostacyclin and thromboxane B2 were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF isoprostane was increased in smokers.Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment.
Keywords: Exhaled breath condensate; Lipidomics; Eicosanoids; Tobacco smoking; Mass spectrometry;

Analysis and stability study of temozolomide using capillary electrophoresis by Melinda Andrasi; Rose Bustos; Attila Gaspar; Frank A. Gomez; Almos Klekner (1801-1808).
The applicability of micellar electrokinetic capillary chromatography (MEKC) for the analysis of temozolomide (TMZ) and its degradants, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and 5-amino-imidazole-4-carboxamide (AIC) has been studied. Using short-end injection, the analysis of TMZ and its degradants could be performed within 1.2 min. The obtained precision of migration times was better than 1.6 RSD%, and the limit of quantitation (LOQ) was 0.31–0.93 μg/mL. The therapeutic concentration of TMZ in blood samples can be determined after direct sample injection and conventional on-capillary UV detection. The proposed MEKC method was applied to study the stability of TMZ in water and serum at different pH values. It was established that the half-life of the TMZ in vitro serum at room temperature was 33 min, close to the half-life (28 min) obtained in water at pH 7.9.
Keywords: Micellar electrokinetic capillary chromatography; Temozolomide; Solution stability;

A convenient procedure for determination of seven betaine analogs and dimethylsulfoniopropionate (DMSP) in extracts of coral tissues using LC–MS stable isotope dilution is described. Extraction procedures were optimized for selective extraction of polar metabolites from coral tissues. The LC–MS protocol employed a pentafluorophenylpropyl (PFPP) column for HPLC separation, with chromatographic resolution of isobaric and isomeric zwitterionic metabolites optimized by adjusting the acidity of the mobile phase. A ternary gradient was used to exploit the unusual retention characteristics of cationic metabolites on the PFPP column, with incorporation of ammonium acetate in a later gradient stage promoting elution of more hydrophobic betaines which are retained at high organic content in the absence of ammonium acetate. We demonstrate that the new LC–MS based method provides accurate measurements from nanomolar to high micromolar concentrations, and can be applied for profiling of betaine metabolites and DMSP in corals or other aquatic organisms.
Keywords: Coral; Betaines; Dimethylsulfoniopropionate; LC–MS; Pentafluorophenylpropyl column; Metabolite profiling; Stable isotope dilution;

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry by Wei Zeng; Yang Xu; Marvin Constanzer; Eric J. Woolf (1817-1823).
A simple offline LC–MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm × 50 mm, 3 μm) using a mobile phase of ACN/H2O (80/20, v/v) containing 10 mM NH4Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408 → 235 for sitagliptin and m/z 412 → 239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 μL of plasma is processed. The linear calibration range is 1–1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n  = 6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.
Keywords: LC–MS/MS; Sitagliptin; DPP-IV inhibitor; Protein precipitation;

Haloacetic acids (HAAs) are water disinfection byproducts (DBPs) formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromine. HAAs are second to trihalomethanes as the most commonly detected DBPs in surface drinking water and swimming pools. After oral exposure (drinking, showering, bathing and swimming), HAAs are rapidly absorbed from the gastrointestinal tract and excreted in urine. Typical methods used to determine these compounds in urine (mainly from rodents) only deal with one or two HAAs and their sensitivity is inadequate to determine HAA levels in human urine, even those manual sample preparation protocols which are complex, costly, and neither handy nor amenable to automation. In the present communication, we report on a sensitive and straightforward method to determine the nine HAAs in human urine using static headspace (HS) coupled with GC–MS. Important parameters controlling derivatisation and HS extraction were optimised to obtain the highest sensitivity: 120 μl of dimethylsulphate and 100 μl of tetrabutylammonium hydrogen sulphate (derivatisation regents) were selected, along with an excess of Na2SO4 (6 g per 12 ml of urine), an oven temperature of 70 °C and an equilibration time of 20 min. The method developed renders an efficient tool for the precise and sensitive determination of the nine HAAs in human urine (RSDs ranging from 6 to 11%, whereas LODs ranged from 0.01 to 0.1 μg/l). The method was applied in the determination of HAAs in urine from swimmers in an indoor swimming pool, as well as in that of non-swimmers. HAAs were not detected in the urine samples from non-swimmers and those of volunteers before their swims; therefore, the concentrations found after exposure were directly related to the swimming activity. The amounts of MCAA, DCAA and TCAA excreted from all swimmers are related to the highest levels in the swimming pool water.
Keywords: Static headspace technique; Haloacetic acids; Swimming pools; Urine sample;

The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5–1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG–protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.
Keywords: Trypsinogen; Alpha-chymotrypsinogen; Isothermal titration calorimetry; Aqueous two-phase systems; Protein average surface hydrophobicity;

A sensitive and accurate ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) method was developed and validated for the simultaneous determination of vitexin-4″-O-glucoside (VGL), vitexin-2″-O-rhamnoside (VRH), rutin (RUT) and vitexin (VIT) in rat plasma after intravenous administration of hawthorn leaves flavonoids (HLF). Following protein precipitation by methanol, the analytes were separated on an ACQUITY UPLC BEH C18 column packed with 1.7 μm particles by gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.20 mL/min. The analytes and diphenhydramine (internal standard, IS) were detected in the multiple reaction monitoring (MRM) mode by means of an electrospray ionization (ESI) interface (m/z 292.96 for vitexin-4″-O-glucoside, m/z 293.10 for vitexin-2″-O-rhamnoside, m/z 299.92 for rutin, m/z 310.94 for vitexin and m/z 166.96 for IS). The calibration curve was linear over the range 10–40,000 ng/mL for vitexin-4″-O-glucoside, 10–50,000 ng/mL for vitexin-2″-O-rhamnoside, 8–1000 ng/mL for rutin and 16–2000 ng/mL for vitexin. The intra- and inter-run precisions (relative standard deviation, RSD) of these analytes were all within 15% and the accuracy (the relative error, RE) ranged from −10% to 10%. The stability experiment indicated that the four analytes in rat plasma samples and plasma extracts under anticipated conditions were stable. The developed method was applied for the first time to pharmacokinetic studies of the four bioactive compounds of hawthorn leaves flavonoids following a single intravenous administration of 20 mg/kg in rats.
Keywords: Vitexin-4″-O-glucoside; Vitexin-2″-O-rhamnoside; Rutin; Vitexin; Hawthorn leaves flavonoids; UPLC–ESI-MS/MS;

A sensitive and reliable liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) has been developed and validated for simultaneous determination of active components, i.e., xanthone glycosides (neomangiferin and mangiferin), timosaponins (timosaponin E1, timosaponin B-II and timosaponin B) and alkaloids (palmatine and berberine) in rat plasma after oral administration of Zi-Shen Pill extract. Plasma samples were pretreated by protein precipitation with acetonitrile containing the internal standards ginsenoside Re (for xanthone glycosides and timosaponins) and tetrahydroberberine (for alkaloids). LC separation was achieved on a Zorbax SB-C18 column (150 mm × 2.1 mm I.D., 3.5 μm) with gradient elution using a mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.25 mL/min. The detection was carried out by a triple–quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between negative (for xanthone glycosides and timosaponins) and positive (for alkaloids) ionization mode. Linear calibration curves were obtained over the concentration range of 5–1000 ng/mL for mangiferin, 0.5–100 ng/mL for neomangiferin, timosaponin E1, timosaponin B-II and timosaponin B, and 0.05–10 ng/mL for palmatine and berberine. The mean recovery of all the analytes ranged from 64.7 to 93.8%. The intra- and inter-day precision (% R.S.D.) was within 11.7% and accuracy (% bias) ranged from −9.0 to 10.9%. This fully validated method was successfully applied to pharmacokinetic study of the above seven compounds in rats.
Keywords: Xanthone glycoside; Timosaponin; Alkaloid; Zi-Shen Pill; Pharmacokinetics; LC–MS/MS;

Purification of bacteriophage M13 by anion exchange chromatography by Razieh Monjezi; Beng Ti Tey; Chin Chin Sieo; Wen Siang Tan (1855-1859).
M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious.
Keywords: Filamentous bacteriophage; Virus purification; Anion exchange chromatography; Fast protein liquid chromatography; Cesium chloride centrifugation;

Simultaneous diagnostic method for phenylketonuria and galactosemia from dried blood spots using high-performance liquid chromatography-pulsed amperometric detection by Nam-Hee Kim; Ji-Seon Jeong; Ha-Jeong Kwon; Yong-Moon Lee; Hye-Ran Yoon; Kyoung Ryul Lee; Seon-Pyo Hong (1860-1864).
We developed a simultaneous diagnostic method for phenylketonuria (PKU) and galactosemia through simultaneous determination of phenylalanine (Phe) and galactose (Gal) by high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD). The intra- and inter-day precisions were <5.8%, with satisfactory mean recoveries (98.2–105%). For all PKU-positive samples, Phe levels were above the cut-off value (>30.0 mg/L), but Gal levels were nearly zero. For 77% of galactosemia-positive samples, Phe levels were above the cut-off value, but Gal levels were above the cut-off value (>80.0 mg/L) for all samples. Our HPLC-PAD method can reduce the false-positive rate of misdiagnosis for PKU and galactosemia.
Keywords: Newborn screening; Simultaneous diagnosis; Phenylketonuria; Galactosemia; HPLC; Dried blood spot;

A simple reversed phase high-performance liquid chromatography method for polysorbate 80 quantitation in monoclonal antibody drug products by Michael Adamo; Lawrence W. Dick; Difei Qiu; An-Horng Lee; John Devincentis; Kuang-Chuan Cheng (1865-1870).
In this paper, we discuss an improved high-performance liquid chromatography (HPLC) method for the quantitation of polysorbate 80 (polyoxyethylenesorbitan monooleate), a commonly used stabilizing excipient in therapeutic drug solutions. This method is performed by quantitation of oleic acid, a hydrolysis product of polysorbate 80. Using base hydrolysis, polysorbate 80 releases the oleic acid at a 1:1 molar ratio. The oleic acid can then be separated from other polysorbate 80 hydrolysis products and matrices using reversed phase HPLC. The oleic acid is monitored without derivatization using the absorbance at 195 nm. The method was validated and also shown to be accurate for the quantitation of polysorbate 80 in a high protein concentration monoclonal antibody drug product. For the measured polysorbate 80 concentrations, the repeatability was less than 6.2% relative standard deviation of the mean (% RSD) with the day-to-day intermediate precision being less than 8.2% RSD. The accuracy of the oleic acid quantitation averaged 94–109% in different IgG1 and IgG4 drug solutions with variable polysorbate 80 concentrations. In this study, polyoxyethylene, a by-product of the polysorbate 80 hydrolysis was also identified. This peak was not identified by previous methods and also increased proportionally to the polysorbate 80 concentration. We have developed and qualified a method which uses common equipment found in most laboratories and is usable over a range of monoclonal antibody subclasses and protein concentrations.
Keywords: Polysorbate 80; Tween-80; RP-HPLC; Oleic acid; Surfactant;

Quantitation of fatty acid ethyl esters in human meconium by an improved liquid chromatography/tandem mass spectrometry by Ho-Seok Kwak; Young-Sun Kang; Ki-Ok Han; Jong-Taek Moon; Young-Chul Chung; June-Seek Choi; Jung-Yeol Han; Moon-Young Kim; E. Yadira Velázquez-Armenta; Alejandro A. Nava-Ocampo (1871-1874).
This paper reports the development and validation of an improved assay for quantitation of fatty acid ethyl esters (FAEEs) in human meconium using liquid chromatography/tandem mass spectrometry (LC–MS/MS). FAAEs (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate) and the internal standard (I.S.), ethyl heptadecanoate, were separated by reverse phase HPLC and quantified by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ionization mode. The absolute recovery of FAEEs varied from 55 ± 10% for 0.33 nmol/g (100 ng/g) of ethyl linoleate up to 86 ± 8% for 1.55 nmol/g (500 ng/g) of ethyl miristate. The LODs and LOQs varied from 0.01 to 0.08 nmol/g and from 0.02 to 0.27 nmol/g, respectively. The assay has been successfully applied to examine the FAEE levels in 81 meconium samples from babies born to mothers reporting alcohol consumption, to varying degrees, during pregnancy.
Keywords: Mass spectrum analysis; Newborns; Transplacental exposure;

A high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method employing electrospray ionization (ESI) has been developed for simultaneous determination of lancemaside A (3-O-β-d-glucuronopyranosyl-3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester) and its metabolites in mouse plasma. When lancemaside A (60 mg/kg) was orally administered to mice, echinocystic acid was detected in the blood. T max and C max of the echinocystic acid were 6.5 ± 1.9 h and 56.7 ± 29.1 ppb. Orally administered lancemaside A was metabolized to lancemaside X (3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester) by intestinal microflora in mice, which was metabolized to echinocystic acid by intestinal microflora and/or intestinal tissues. Human intestinal microflora also metabolized lancemaside A to echinocystic acid via lancemaside X. These results suggest that the metabolism by intestinal microflora may play an important role in pharmacological effects of orally administered lancemaside A.
Keywords: Codonopsis lanceolata; Lancemaside A; Echinocystic acid; Metabolism; LC–MS/MS; Pharmacokinetic study;

In this paper, five isoquinoline alkaloids were successfully separated from a crude extract of Stephania yunnanensis using pH-zone-refining counter-current chromatography in single-step. With a two-phase solvent system composed of methyl-tert-butyl ether (MtBE)–acetonitrile–water (2:2:3, v/v) where triethylamine (10 mM) was added to the upper organic phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.4 g crude extract, 68.7 mg isocorydine, 78.2 mg corydine, 583.4 mg tetrahydropalmatine, 36.3 mg N-methylasimilobine, and 47.3 mg anonaine were separated with purities over 90%. Their structures were identified by 1H NMR, 13C NMR, ESI-MS data.
Keywords: pH-zone-refining counter-current chromatography; Stephania yunnanensis; Alkaloids; Preparative chromatography;

The steroidal liver X receptor agonist, 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane (ATI-829) is a potential therapeutic agent for the treatment of atherosclerosis. A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the quantification of ATI-829 in mouse plasma was developed and validated. Proteins in a 25 μL aliquot of mouse plasma were precipitated, and ATI-829 was extracted from the precipitate by the addition of 125 μL methanol. The overall extraction efficiency was greater than 99%. LC–MS–MS with negative ion electrospray and selected reaction monitoring was used for the quantitative analysis of ATI-829. The lower limit of quantitation of ATI-829 corresponded to 5.0 ng/mL (9.7 nM) plasma. Interference from matrix was negligible. The calibration curve was linear over the range 5–2000 ng/mL. The intra-day precision and inter-day precision of the analyses were <4.5% and <6%, respectively, and the accuracy ranged from 92% to 103%. ATI-829 in plasma was stable for at least 6 h at room temperature, 1 week at 4 °C, and 3 weeks at −20 °C. The validated method was then utilized for pharmacokinetic studies of ATI-829 administered to mice.
Keywords: Atherosclerosis; Cholanic acid; Pharmacokinetics; Quantitative analysis; Method validation; LC–MS–MS;

Validation of an isocratic HPLC method to detect 2-fluoro-β-alanine for the analysis of dihydropyrimidine dehydrogenase activity by Kinta M. Serve; Jennifer L. Darnell; Jody K. Takemoto; Neal M. Davies; Margaret E. Black (1889-1892).
The efficacy of the chemotherapeutic drug 5′-fluorouracil is reduced by catabolism to 2′-fluoro-β-alanine (FBAL), a three-step reaction in which dihydropyrimidine dehydrogenase (DPD) catalyzes the rate-limiting step. To study in vitro DPD activity, we developed and validated an isocratic, reverse-phase HPLC method to detect and quantify FBAL without using multiple columns or radiolabeled substrates. Pre-column derivatization of FBAL was performed using o-phthalaldehyde in the presence of two sulfur donors, ethanthiol or β-mercaptoethanol, and the resulting products assayed. Calibration curves were linear over a range of 10–200 μg/ml and the method was successfully applied to the examination of DPD activity in cultured cells.
Keywords: Reverse-phase liquid chromatography; Isocratic; Dihydropyrimidine dehydrogenase; Fluoro-β-alanine; Cytosine deaminase;

An improved on-line solid phase extraction coupled HPLC–MS/MS system for quantification of Sifuvirtide in human plasma by Qing-Qing Wang; Shen-Si Xiang; Yan-Bo Jia; Lun Ou; Fang Chen; Hai-Feng Song; Qing Liang; Dan Ju (1893-1898).
An improved liquid chromatographic method with on-line solid phase extraction (SPE) and tandem mass spectrometric detection was optimised for quantification of the anti-HIV peptide Sifuvirtide in human plasma. The SPE sorbents, loading buffer composition and other aspects of the on-line SPE column were investigated in detail for efficiently extracting the interesting peptides and simultaneously discarding the large amount of proteins. The gradient elution program was optimised on the analysis column to decrease the matrix effect and obtain excellent selectivity. The multiple charge ion at m/z 946.4 of Sifuvirtide was quantified by a linear ion trap mass spectrometer, operating in the positive mode, and selective reaction monitoring (SRM) acquisition. Method validation results demonstrated that the linear calibration curve covered a range of 6.1–6250 ng/mL, and the correlation coefficients (r 2) were above 0.992. The lower limit of detection (LLOD) with a signal-to-noise (S/N) ratio higher than 10 was 6.1 ng/mL. The accuracy ranged from −7.6 to 10.6%, and the intra- and inter-batch precisions were less than 8.7% and 5.5%, respectively. Finally, more than nine hundred of samples from a clinical trial was completely analyzed using this on-line SPE coupled HPLC–MS/MS system in one single week, due to the rapid run-time of individual sample (6.5 min).
Keywords: Sifuvirtide; On-line solid phase extraction; HPLC–MS/MS; Pharmacokinetics;

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n  = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.
Keywords: Capillary electrophoresis; Aminomethylbenzoic acid; Cefminox sodium; Etamsylate; Human urine;