Journal of Chromatography B (v.939, #C)

Formation and characteristics of aqueous two-phase systems formed by a cationic surfactant and a series of ionic liquids by Xi-Lian Wei; Xiu-Hong Wang; A-Li Ping; Pan-Pan Du; De-Zhi Sun; Qing-Fu Zhang; Jie Liu (1-9).
Aqueous two-phase systems (ATPS) were obtained in the aqueous mixtures of a cationic surfactant and a series of ionic liquids (ILs). The effects of IL structure, temperature and additives on the phase separation were systematically investigated. The microstructures of some ATPS were observed by freeze-fracture replication technique. Lyotropic liquid crystal was found in the bottom phase besides micelles under different conditions. Remarkably, both IL structure and additives profoundly affected the formation and properties of the ATPSs. The phase separation can be attributed to the existence of different aggregates and the cation-π interactions of the cationic surfactant with the ILs, which has a significant role in the formation of ATPS. The extraction capacity of the studied ATPS was also evaluated through their application in the extraction of two biosubstances. The results indicate that the ILs with BF4 as anion show much better extraction efficiencies than the corresponding ILs with Br as anion do under the same conditions. l-Tryptophan was mainly distributed into the NPTAB-rich phase, while methylene blue and capsochrome were mainly in the IL-rich phase.
Keywords: Aqueous two-phase systems; Cationic surfactant; Ionic liquids; Additives; Partitioning;

Performance evaluation of affinity ligands for depletion of abundant plasma proteins by Majlinda Kullolli; Jonathan Warren; Maria Arampatzidou; Sharon J. Pitteri (10-16).
Human plasma is a commonly used diagnostic fluid in clinical chemistry. In-depth plasma proteomic analysis is performed to search for disease biomarkers, however the large dynamic range of protein abundance in plasma presents a substantial analytical challenge. Removal of abundant plasma proteins using antibody capture approaches is a common and attractive means to reduce sample complexity and to aid the analysis of lower abundance proteins of interest. A novel class of heavy chain camelid-derived affinity ligands produced in Saccharomyces cerevisiae, has recently been developed as an alternative to antibody-based depletion methods. Here, we evaluate the performance characteristics of these ligands for removal of high abundance plasma proteins. Affinity ligands were tested for the removal of 14 abundant human plasma proteins. The performance characteristics were evaluated by gel-electrophoresis and LC–MS/MS of the bound and flow-through fractions. The capacity of a 5.6 mL column was found to be 125 μL of plasma. Replicate analysis demonstrated high column reproducibility and linearity, efficient removal of abundant proteins, and enrichment of lower abundance proteins observed after depletion. The novel class of affinity ligands provides an attractive alternative to traditional antibody-based immunodepletion methods.
Keywords: Affinity ligands; Protein depletion; Immunoaffinity/immunodepletion alternative; Ligand affinity; Plasma proteomics;

HPLC method development for determination of doxycycline in human seminal fluid by Slavica M. Sunarić; Marko S. Denić; Zoran Ž. Bojanić; Vladmila V. Bojanić (17-22).
The present paper reports the development and validation of an analytical method for doxycycline quantification in human seminal fluid by HPLC with UV detection. The separation of doxycycline was achieved at 40 °C on a reversed-phase C18 column using isocratic elution. The mobile phase consisted of acetonitrile (A) and water buffered at pH 2.5 with a concentrated orthophosphoric acid (B) in the volume ratio of 20:80 (v/v), respectively. The detection was performed at 350 nm. As an internal standard (IS), tetracycline was used. The proposed method involves the extraction of doxycycline from seminal fluid based on acidic precipitation of the proteins using perchloric acid. The method showed good intra- and inter-day precisions (RSD < 7.0%), good accuracy (recovery for doxycycline > 80%), and high correlation coefficient (r  = 0.998) for standards subjected to the entire procedure. The detection and quantification limits were 0.087 μg/ml and 0.264 μg/ml. The developed method was used to analyze doxycycline in the seminal fluids obtained from male subjects who were treated with doxycycline-hyclate. The mean doxycycline concentrations of 0.89 ± 0.07 μg/ml and 0.45 ± 0.26 μg/ml were detected in seminal fluid after 6 h and 12 h, respectively. This is the first study reporting extraction and HPLC determination of doxycycline in this complex sample and can be very useful in support of clinical and pharmacokinetic studies on this antibiotic.
Keywords: Doxycycline; HPLC determination; Human seminal fluid;

A new method using ultra-fast liquid chromatography and tandem mass spectrometry (UFLC–MS/MS) was developed for the simultaneous determination of buprenorphine and the metabolites norbuprenorphine, buprenorphine-3β-glucuronide, and norbuprenorphine-3β-glucuronide in plasma and urine. Sample handling, sample preparation and solid-phase extraction procedures were optimized for maximum analyte recovery. All four analytes of interest were quantified by positive ion electrospray ionization tandem mass spectrometry after solid-phase microextraction. The lower limits of quantification in plasma were 1 pg/mL for buprenorphine and buprenorphine glucuronide, and 10 pg/mL for norbuprenorphine and norbuprenorphine glucuronide. The lower limits of quantitation in urine were 10 pg/mL for buprenorphine, norbuprenorphine and their glucuronides. Overall extraction recoveries ranged from 68–100% in both matrices. Interassay precision and accuracy was within 10% for all four analytes in plasma and within 15% in urine. The method was applicable to pharmacokinetic studies of low-dose buprenorphine.
Keywords: Buprenorphine; Norbuprenorphine; Glucuronide; Mass spectrometry;

Increased circulating concentrations of homocysteine (HCY) and asymmetric dimethylarginine (ADMA) are associated with vascular disease and vascular risk factors. HCY has been shown to inhibit the activity of endothelial dimethylaminohydrolase (DDAH), causing the accumulation of ADMA and the inhibition of nitric oxide synthesis. The concentrations of HCY and ADMA in biological fluids are used in the clinical diagnosis of cardiovascular diseases and this necessitates the development of a rapid and sensitive method for simultaneous determination of HCY and ADMA. A rapid, simple and sensitive method for simultaneous determination of HCY and ADMA by liquid chromatography–tandem mass spectrometry (LC–MS/MS) coupled with electro spray ionization (ESI) in human urine was reported here. The methodology designed here was used to estimate these molecules in urine samples collected from patients reported to Cardiology Department of our hospital. Chromatographic separation was performed on Atlantis HILIC silica (100 mm × 2.1 mm, 5 μm, Waters). Positive multiple reactions monitoring (MRM) mode was chosen for quantification of each analyte and cystamine dihydrochloride (CYA) was used as the internal standard (IS) for the assay. The intra-assay precision and accuracy were in the range of 2.4–4.8 and −1.8% to 3.1%, respectively. The inter-assay precision and accuracy were in the range of 3.0–4.2% and −1.2% to 3.2%, respectively. The recoveries were between 94.9% and 101.4%. Our approach is simple, rapid and could be extended to routine urine assay.
Keywords: Liquid chromatography mass spectrometry (LC–MS); Electro spray ionization (ESI); Homocysteine (HCY); Asymmetric dimethylarginine (ADMA); Urine;

Development and validation of an LC–ESI/MS/MS method with precolumn derivatization for the determination of betulin in rat plasma by Zhiwei Hu; Na Guo; Ziming Wang; Yong Liu; Yu Wang; Weimin Ding; Dehui Zhang; Yang Wang; Xiufeng Yan (38-44).
Neutral pentacyclic triterpenes with only one or two hydroxyl groups, such as betulin, are not easily ionized by electrospray ionization (ESI). However, because betulin is reactive and neutral, derivatization may improve ionization efficiency. In the present study, the potency of different derivatization reagents was evaluated and p-toluenesulfonyl isocyanate (PTSI) was proven to be the optimal. The derivative generated by the reaction of betulin with PTSI was ionizable and fragmentable in the negative mode by liquid chromatography–electrospray ionization/tandem mass spectrometry (LC–ESI/MS/MS). Based on this chemical derivatization, an LC–ESI/MS/MS method was developed and validated for the quantification of betulin in rat plasma. The sample was extracted with ethyl acetate, derivatized with PTSI, separated on an ACQ UPLC BEH phenyl column, and analyzed in negative multiple reaction monitoring (MRM) mode. The calibration curve was linear over the betulin concentration range 2.5–200 ng/mL. The lower limit of quantification was 2.5 ng/mL. The inter- and intra-day accuracy and precision were within ±15%. Betulin recoveries were 86.7% or higher at three quality control levels (5, 50, and 160 ng/mL). This validated method was subsequently applied to a pharmacokinetic study of betulin in rat plasma after oral administration.
Keywords: Betulin; Precolumn derivatization; LC–ESI/MS/MS; p-Toluenesulfonyl isocyanate; Pharmacokinetics;

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of sudan I, sudan II, sudan III, sudan IV, and their metabolites such as 4-aminoazobenzene and ortho-aminoazotoluole in 12 animal derived foods (including the muscle and liver of swine, muscle, liver and skin of chicken and duck, muscle and skin of fish, as well as the eggs of hen and duck). Sample preparation procedure included ultrasound-assisted extraction with acetonitrile, defatting with n-hexane and final clean-up with solid phase extraction (SPE) on Aluminum B cartridges. The detection and quantification of the 6 sudan dyes and their metabolites were performed by a reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS). The CCαs and the CCβs of various samples varied from 0.03 μg/kg to 0.12 μg/kg, 0.09 μg/kg to 0.19 μg/kg, respectively. The recoveries of spiked sample from 0.2 μg/kg to 0.8 μg/kg ranged from 61.9% to 87.4% with the relative standard deviations of less than 19.1%. Performances of the whole analytical procedure meet the criteria established by the European Commission for mass spectrometric detection.
Keywords: Sudan dyes; Metabolites; LC–MS/MS; Residues; Animal derived food;

Metabolite profiling and identification of triptolide in rats by Jia Liu; Liang Li; Xin Zhou; Xiaoyan Chen; Haihua Huang; Shunbo Zhao; Xiuli Li; Dafang Zhong (51-58).
The purpose of the current study was to investigate the metabolite profile of [3H]triptolide in rats. The separation and characterisation techniques used to identify the major metabolites were high-performance liquid chromatography-online radiodetector, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and nuclear magnetic resonance. In all, 33 major metabolites were detected. The major components found in the rat plasma included the parent drug and its monohydroxy- and dihydroxy-metabolites. Reference standards for the monohydroxy-metabolites were obtained either by the incubation of the parent drug with rat liver microsomes or by microbial transformation with Cunninghamella blakesleana. The metabolites’ structures were identified as 17-hydroxytriptolide, 16-hydroxytriptolide, tripdiolide, and 15-hydroxytriptolide. The major metabolites found in male rat urine included the monohydroxy-, dihydroxy-, and trihydroxy-metabolites. The major metabolites in female rat urine were the monohydroxy- and dihydroxy-metabolites, as well as sulphates of the monohydroxy-metabolites. A glutathione adduct, multiple hydroxy-metabolites, and a number of unidentified metabolites were detected in the bile and faeces of male rats. Sulphates of monohydroxy-metabolites were detected in the bile and faeces of female rats.
Keywords: Triptolide; Metabolite profiling; High-performance liquid chromatography-online radiodetector; Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry;

A sensitive and rapid method based on ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS) was developed and validated for the simultaneous determination of blonanserin, its major active metabolite (N-deethyl form) and other three metabolites (N-oxide form, Ethylenediamine form and Carboxylate form) in human plasma. Plasma samples were pre-purified by solid-phase extraction (SPE) and analyzed using a gradient chromatographic separation over an Acquity UPLC CSH C18 column. The mobile phase consisted of acetonitrile–water containing 5 mM ammonium formate and 0.1% formic acid at a flow rate of 0.5 mL/min. Positive electrospray ionization was employed as the ionization source in the multiple reaction monitoring (MRM) mode. The analysis time was about 3.5 min. The method was fully validated over the concentration range of 0.01–1 ng/mL for all analytes. The lower limit of quantification (LLOQ) was 0.01 ng/mL. Inter- and intra-batch precision was less than 15% and the accuracy was within 85–115%. The mean extraction recoveries of all analytes at two concentration levels were consistent. Selectivity, matrix effect and stability were also validated. The method was applied to the pharmacokinetic study of blonanserin in Chinese healthy subjects.
Keywords: Determination; Blonanserin; Metabolite; UPLC–MS/MS; Human plasma;

A novel and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to determine the exposure of 3-n-butylphthalide (NBP) enantiomers in human plasma. The NBP enantiomers were extracted from human plasma using methyl tert-butyl ether. The baseline separation of R-(+)-NBP and S-(−)-NBP was achieved within 11.0 min using a teicoplanin-based Astec Chirobiotic T column (250 mm × 4.6 mm i.d., 5 μm) under isocratic conditions at a flow rate of 0.6 mL/min. The selection of the chiral stationary phase and the effect of the mobile phase composition on the resolution of the enantiomers were discussed. The selectivity, linearity, precision, accuracy, matrix effect, recovery, and stability were evaluated under optimized conditions. The LC–MS/MS method using 200 μL of human plasma was linear over the concentration range of 5.00–400 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for both enantiomers was 5.00 ng/mL. The intra- and inter-assay precision values of the replicated quality control samples were within 8.0% for each enantiomer. The mean accuracy values for the quality control samples were within ±6.1% of the nominal values for R-(+)-NBP and S-(−)-NBP. No chiral inversion was observed during sample storage, preparation, and analysis. The method proved suitable for enantioselective pharmacokinetic studies of NBP after an oral administration of a therapeutic dose of racemic NBP.
Keywords: 3-n-Butylphthalide (NBP); Teicoplanin-based chiral column; LC–MS/MS; Enantioselective pharmacokinetics;

A highly selective, sensitive, and rapid high performance liquid chromatographic (HPLC) method has been developed and validated to quantify dasatinib, a tyrosine kinase inhibitor, in rabbit plasma. Montelukast was used as internal standard (IS). Dasatinib and IS were extracted by deproteinization technique, followed by injection of aliquot of supernatant into chromatographic system. Chromatographic separation was achieved on a reversed phase C18 column with a mobile phase of 0.02 M potassium dihydrogen phosphate:methanol (10:90, v/v) pumped at flow rate of 2.0 mL/min. The analytes were detected at 340 and 374 nm for excitation and emission, respectively. The assay exhibited a linear range of 50.0–3000 ng/mL, with a lower detection limit of 15.0 ng/mL. The method was statistically validated for linearity, accuracy, precision, selectivity and stability following FDA guidelines. The intra- and inter-assay coefficients of variation did not exceed 13.5% from the nominal concentration. The accuracy of dasatinib was within ±15% of the theoretical value. The assay has been applied successfully in a pharmacokinetic study.
Keywords: Dasatinib; HPLC; Method of validation; Rabbit plasma; Pharmacokinetic study;

Thiopurine efficacy is partly reflected by the genetic polymorphism of the thiopurine methyltransferase (TPMT) enzyme, which is responsible for variation in the metabolism, toxicity and therapeutic efficacy of the thiopurines azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Determination of TPMT activity before administration of thiopurines is thus crucial for individualized dosing in order to prevent toxicity in TPMT deficient individuals. These individuals must be treated with markedly lower (eg, 5–10% of the standard) doses of the prescribed medications. This paper describes a comparison of three different methods for the quantification of TPMT activity in red blood cells (RBC) and cultured human cell lines. We succeeded to perform the measurement of TPMT activity in a minimum amount of 1 × 106 cultured cells with an HPLC-UV system modified and optimized in our laboratory. The TPMT activity was linearly correlated with the cell concentration of the cultured cell line in a range of 1–10 × 106 cells. A significant correlation of determination of TPMT activity in RBC between radiometric detection by HPLC, classic radiochemical detection and UV detection by HPLC, was observed, correlation coefficient (r) were 0.72 and 0.73, respectively. The within-day and day-to-day coefficients of variation of the HPLC-UV-based method were 8% and 16%, respectively. The evaluation of the methods was demonstrated by studying the TPMT activity in RBC isolated from 198 patients, as well as in MOLT4 leukemic cell line and its sub-cell lines with acquired resistance to 6-MP and 6-TG.
Keywords: 6-Mercaptopurine; Thiopurine methyltransferase; High performance liquid chromatography;

A rapid, sensitive, and selective ultra fast liquid chromatography–tandem mass spectrometry method was developed for quantitative determination of arenobufagin in rat plasma. Sample pretreatment involved a one-step protein precipitation with methanol using 0.1 mL rat plasma. The separation was carried out on a Shim-pack XR-ODS II (75 mm × 2.0 mm, i.d. 2.1 μm) column with gradient elution at a flow rate of 0.30 mL min−1. The mobile phase was acetonitrile and 0.1% formic acid in water. A post-column switching valve was applied to reduce the matrix effect. The detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode after electrospray ionization. Linear calibration curves for arenobufagin were obtained over the concentration range 1.056–1056 ng mL−1, with a lower limit of quantification of 1.056 ng mL−1. The intra-day and inter-day precision values were lower than 15% and the accuracy ranged from 5.4% to 9.8% at all quality control levels. The method was successfully applied to the determination and pharmacokinetic study of arenobufagin in rat plasma following intraperitoneal administration.
Keywords: Arenobufagin; UFLC–ESI-MS/MS; Pharmacokinetics; Rat plasma; Intraperitoneal administration;

Glycine is an important amino acid neurotransmitter in the central nervous system (CNS) and a useful biomarker to indicate biological activity of drugs such as glycine reuptake inhibitors (GRI) in the brain. Here, we report how a liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the fast and reliable analysis of glycine in brain microdialysates and cerebrospinal fluid (CSF) samples has been established. Additionally, we compare this method with the conventional approach of high performance liquid chromatography (HPLC) coupled to fluorescence detection (FD). The present LC–MS/MS method did not require any derivatisation step. Fifteen microliters of sample were injected for analysis. Glycine was detected by a triple quadrupole mass spectrometer in the positive electrospray ionisation (ESI) mode. The total running time was 5 min. The limit of quantitation (LOQ) was determined as 100 nM, while linearity was given in the range from 100 nM to 100 μM. In order to demonstrate the feasibility of the LC–MS/MS method, we measured glycine levels in striatal in vivo microdialysates and CSF of rats after administration of the commercially available glycine transporter 1 (GlyT1) inhibitor LY 2365109 (10 mg/kg, p.o.). LY 2365109 produced 2-fold and 3-fold elevated glycine concentrations from 1.52 μM to 3.6 μM in striatal microdialysates and from 10.38 μM to 36 μM in CSF, respectively. In conclusion, we established a fast and reliable LC–MS/MS method, which can be used for the quantification of glycine in brain microdialysis and CSF samples in biomarker studies.
Keywords: LC–MS/MS; Glycine; In vivo microdialysis; CSF; Biomarker; GlyT1;

This study describes the development and validation of a simple, accurate and sensitive ultra high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) method for the simultaneous quantification of 10 mycotoxins and 29 pesticides in ginseng. The method featured a fast and straightforward one-step extraction procedure using acetonitrile/water/formic acid (99:33:1, v/v/v) without further cleanup. Rapid LC separation in 8 min was successfully achieved on a Phenomenex Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm) with a flow rate of 0.30 mL/min using a mobile phase of water containing 0.1% formic acid and methanol. Simultaneous acquisition was performed in the positive and negative ion modes. For some analytes, enhanced responses were acquired in negative ion mode (e.g., Zearalenone, α-Zearalenol and β-Zearalenol); however, the majority of analytes were monitored in positive ion mode with multiple reaction monitoring (MRM). Two MS/MS transitions for each analyte were acquired to ensure reliable identification and accurate quantification. The method was validated in house through linearity, selectivity, precision, and recovery studies. Analytical data were satisfactory with typical recoveries of 70–120% and relative standard deviations (RSDs) below 20%. The limits of detection (LODs) ranged from 0.01 to 0.25 ng/mL, which are below the maximum residue levels (MRLs) established by European legislation for mycotoxins or pesticides in foods and foodstuffs. Forty-three ginseng samples (ginseng (n  = 30), American ginseng (n  = 6), red ginseng (n  = 7)) collected from Chinese markets were analyzed and the most frequently detected pesticide was chlorpyrifos with an incidence of 97% and ranged from 37.63 to 158.60 μg/kg. Ion ratios, retention times and experimental Q/q ratios were also compared with those of the corresponding reference standard in order to avoid false-positive results.
Keywords: UHPLC–ESI-MS/MS; Ginseng; Mycotoxins; Pesticides; One-step extraction; Trace analysis;