Journal of Chromatography B (v.1031, #C)

Effects of comprehensive function of factors on retention behavior of microparticles in gravitational field-flow fractionation by Shuang Guo; Bai-Ling Qiu; Chen-Qi Zhu; Ya-Ya Gao Yang; Di Wu; Qi-Hui Liang; Nan-Yin Han (1-7).
Gravitational field-flow fractionation (GrFFF) is a useful technique for separation and characterization for micrometer-sized particles. Elution behavior of micrometer-sized particles in GrFFF was researched in this study. Particles in GrFFF channel are subject to hydrodynamic lift forces (HLF), fluid inertial forces and gravity, which drive them to different velocities by carrier flow, resulting in a size-based separation. Effects of ionic strength, flow rate and viscosity as well as methanol were investigated using polystyrene latex beads as model particles. This study is devoted to experimental verification of the effect of every factor and their comprehensive function. All experiments were performed to show isolated influence of every variable factor. The orthogonal design test was used to evaluate various factors comprehensively. Results suggested that retention ratio of particles increases with increasing flow rate or the viscosity of carrier liquid by adjusting external forces acting on particles. In addition, retention ratio increases as ionic strength decreases because of decreased electrostatic repulsion between particles and channel accumulation wall. As far as methanol, there is no general trend due to the change of both density and viscosity. On the basis of orthogonal design test it was found that viscosity of carrier liquid plays a significant role in determining resolution of micrometer-sized particles in GrFFF.
Keywords: Gravitational field-flow fractionation (GrFFF); Micrometer-sized particles; Ionic strength; Viscosity; Orthogonal design test;

Ultra performance liquid chromatography tandem mass spectrometry assay for determination of kukoamine B in human blood and urine by Qian Zhao; Lili Li; Zhenlei Wang; Ji Jiang; Kai Dong; Shuai Chen; Pei Hu (8-14).
In this paper, we report a sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method which is capable of quantifying kukoamine B (KB) levels in human blood and urine. Following solid phase extraction and direct dilution process, the analyte and its internal standard (D5-KB) run on an Acquity UPLC® HSS T3 column (2.1 × 50 mm i.d., 1.8 µm) by using a gradient elution method (run time was 1.5 min). The mass spectrometric analysis was performed by using an API-5500 mass spectrometer coupled with an electro-spray ionization source. The MRM transitions of m/z 531.3+  ? 222.1+ and 536.3+  ? 222.1+ were used to quantify KB and D5-KB respectively. This assay method has been fully validated in terms of selectivity, linearity, lower limit of quantification, precision, accuracy, stability, recovery and matrix effect. The concentration range of this method is 10.0–2000.0 ng mL-1 in blood and 0.5–500.0 ng mL-1 in urine. Linearity (R2) of calibration curves were 0.9964 ± 0.0022 and 0.9935 ± 0.0053 for blood and urine, respectively (regression equation: y = ax + b). The precision (RSD%) of quality control samples is less than 10.3% for blood and less than 10.5% for urine. The accuracy (RE%) is within -4.0–11.3% and -11.7–12.5% for blood and urine respectively. KB was stable after 4 h in ice-water bath, 1 freeze/thaw cycles and 180 days at -80 °C for blood samples; and was stable after 3 h at room temperature, 3 freeze/thaw cycles and 180 days at -80 °C for urine samples. Recoveries of KB were 4.7 ± 0.9% in blood and 96.5 ± 1.3% in urine, respectively. Additionally, the applicability of this method has been proved by analyzing clinical samples from pharmacokinetic study of KB in human.
Keywords: Kukoamine B; UPLC/MS/MS; Quantitative analysis; Pharmacokinetics;

A new, rapid, sensitive, precise and validated high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) method was developed for the simultaneous determination of eight neonicotinoid insecticides with their two primary metabolites in cucumbers and soil based on QuEChERS as a pretreatment procedure.In QuEChERS procedure, cucumber samples were extracted with acetonitrile and cleaned using (C18 sorbent material), while soil samples were extracted with a mixture of acetonitrile:dichloromethane (8.3:16.7 v:v). The LC–MS/MS conditions were optimized to provide good selectivity and specificity of the developed method where neonicotinoids were separated using gradient elution of water and acetonitrile both containing 0.1% formic acid with Gemini C18 column where the last compound was eluted at 9.5 min. Average recoveries of the eight neonicotinoids and their metabolites ranged between 81.6% and 95.7% in fortified cucumber samples with relative standard deviations (RSDs) lower than 13.18% and between 80.3% and 104% in fortified soil samples with relative standard deviations (RSDs) lower than 8.44%. The limits of detection (LODs) and quantification (LOQs) for the ten compounds were in the ranges of (0.08–6.06 ng/g) and (0.26–20 ng/g), respectively. The method was applied successfully to determine residues and rate of disappearance of the eight neonicotinoids from cucumber and soil and their half-lives where a safety pre-harvest interval of 5 days for acetampirid, 12 days for imidacloprid, 15 days for nitenpyram, 12 days for thiamethoxam, 5 days for flonicamid, 8 days for clothianidin, 2 days for Dinotefuran, and 1 day for thiacloprid were suggested.
Keywords: MS/MS; QuEChERS; Neonicotinoids; Cucumber; Soil; Metabolites;

A dual-task method for the simultaneous separation and purification of (−)-epigallocatechin gallate (EGCG) and caffeine (CAF) from crude extract of green tea was established by size exclusion effect onto hydroquinone modified porous adsorbents. The results showed that hydroquinone modified porous adsorbents P4 provided the best separation power due to it has more porous structure and phenolic hydroxyl group. The adsorption–desorption behaviors of EGCG and CAF onto P4 adsorbents were investigated. Adsorption kinetics of EGCG and CAF results showed that the adsorption followed the pseudo-second-order kinetic model. The results also indicated that the equilibrium adsorption data best fit the Langmuir model. Meanwhile, EGCG and CAF were separated successfully onto P4 adsorbents packed columns in a gradient eluent process, and P4 adsorbents exhibited the size exclusion effect for small molecules CAF. Based on the phenolic hydroxyl group and size exclusion effect of P4 adsorbents, the high purity EGCG and CAF were obtained with 40% (v/v) ethanol eluent successively. The process fulfilled the task of simultaneous separation and purification of EGCG and CAF, and proved to be a promising basis for preparations of difficult to obtain active components that have similar polarity and different sizes of molecules and derived from the same natural products.
Keywords: Hydroquinone modified adsorbents; Porous structure; Size exclusion effect; (−)-Epigallocatechin gallate; Caffeine;

Bile acids (BAs) play important roles in physiological functions, including the homeostasis of cholesterol and lipids and as ligands for G protein-coupled receptors (GPCRs). With the increasing importance of BAs, analytical methods for their quantification and screening have been developed. However, due to the diverse range and variety of BAs with different activation potency, a simple, effective, and sensitive method is required to screen BAs for accurate quantification and identification. This paper presents an application of ultrahigh-performance liquid chromatography-orbitrap mass spectrometry (UHPLC-LTQ–Orbitrap MS) for profiling BAs in bile. Using this method, along with the accurate quantification of 19 targeted BAs, 22 unknown BAs were detected and characterized by their fragmentation patterns. The method is beneficial for screening most of the BAs (quantitatively and qualitatively) in rat bile with simple preparation in a single run. The sample dilution ranges of each BA were optimized depending on the concentration of BAs in the bile to obtain good peak separation and accurate data. The method validation was performed successfully using charcoal-treated bile and the intra and inter-day coefficients of variation were less than 20% for all BAs while the recovery were above 88.5% except for the lithocholic acid. The method was applied to profile the age-dependent changes in the contents of rat BAs. Through statistical analysis, we found that as the rats aged, unconjugated BAs and glycine-conjugated BAs decreased or were unaffected, while taurine-conjugated BAs were increased in general. Among the unknown BAs, 5 of the taurine-conjugated BAs increased, while a glycine-conjugated BA decreased, in agreement with the trends of the targeted BAs.
Keywords: Aging; Bile acids (BAs); Bile; UPLC–Orbitrap-MS;

An LC/MS/MS method for the simultaneous determination of individual sphingolipid species in B cells by Si Mi; Yuan-Yuan Zhao; Rebecca F. Dielschneider; Spencer B. Gibson; Jonathan M. Curtis (50-60).
Comprehensive profiling of sphingolipids is of great importance for clinical and pharmaceutical studies. An LC/MS/MS method was established for the simultaneous separation and quantification of individual sphingolipid species including ceramides, dihydroceramides, glucosylceramides, sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate. All target individual sphingolipid species were separated and quantified in a single chromatographic run of <20 min. Method validation results indicated that calibration curves were linear in the range of 2.5–10,000 nM for ceramides and glucosylceramides, 10–10,000 nM for dihydroceramides, 5–10,000 nM for sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate, respectively. The limits of detection ranged from 0.5 nM to 5 nM. Accuracies of 92.5–113% with precisions of 0.3–8.0% RSD were obtained for all of the standards over a wide range of concentrations. The application of this method was demonstrated using B cells collected from Chronic Lymphocytic Leukemia patients (n = 5) and healthy donors (n = 4). 17 sphingolipid species were successfully characterized and quantified in the lipid extract. This is a rapid method that could be readily adapted to lipidomic investigations of sphingolipids in other bio-fluids and tissues.
Keywords: Sphingolipids; LC/MS/MS; Quantification; Validation; B cells; Chronic Lymphocytic Leukemia;

Simple and fast determination of perfluorinated compounds in Taihu Lake by SPE-UHPLC–MS/MS by Pengfei Zhu; Xia Ling; Wenwei Liu; Lingcan Kong; Yuyang Yao (61-67).
A simple and fast analytical method for determination of eleven Polyfluorinated Compounds (PFCs) in source water was developed in the present work. The water sample was prepared without filtered through microfiltration membrane and 500 mL of source water was enriched by the solid phase extraction (SPE). The targent compounds were analyzed by ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). The optimized analytical method was validated in terms of recovery, precision and method detection limits (MDLs). The recovery values after correction with the corresponding labeled standard were between 97.3 and 113.0% for samples spiked at 5 ng/L, 10 ng/L and 20 ng/L. All PFCs showed good linearity and the linear correlation coefficient was over 0.99. The precisions were 1.0–9.0% (n = 6). As the result of the enrichment, the MDL values ranged from 0.03 to 1.9 ng/L and were enough for analysis of the trace levels of PFCs in the Taihu Lake. The method was further validated in determining the source water and the results showed that PFHxS, PFHxA, PFOA and PFOS were the primary PFCs in Taihu Lake which might be different from the other researches. The method can be used for determination of PFCs in water with a stable recovery, good reproducibility, low detection limit, less solvent consumption, time saving and labor saving. To our knowledge, this is the first method that describes the effect of the filter membrane on the determination of PFCs in water which might acquire more accurate concentration of PFCs in Taihu Lake.
Keywords: Perfluorinated compounds (PFCs); Taihu Lake; UHPLC–MS/MS; Filter membrane;

The metabolic and pharmacokinetic studies for HM-3 in rats based on LC-Q-TOF/MS and LC–MS/MS combing a convenient biological sample processing method by Guangji Wang; Tai Rao; Yuhao Shao; Jingcheng Xiao; Dian Kang; Boyu Shen; Huimin Chen; Xinuo Li; Zhangpei Zhu; Xiaoxi Yin; Yan Liang (68-75).
In this contribution, the metabolic and pharmacokinetic characteristics for the therapeutic peptide HM-3 were investigated using LC-Q-TOF/MS and LC–MS/MS systems combing a fast biological sample processing method. According to the accurate MS1 and MS2 data generated by LC-Q-TOF/MS, a total of 6 metabolites in rats were detected and tentatively identified as the degradation products which formed by successive loss of amino acid from HM-3. The structures of the 2 main metabolites (M1 and M2) were confirmed by comparing the chromatographic and mass spectrographic characteristics with the corresponding synthetic standards. Then, an absolute quantitative analysis method based on LC–MS/MS system was built and fully validated with respect to linearity, sensitivity, accuracy, precision, matrix effect, stability, etc. The results indicated that HM-3, M1 and M2 were linear in peak area ratios over the concentration range of 0.5–200.0 ng/mL with a correlation coefficient > 0.99. The intra-day and inter-day precisions (RSD%) were less than 15%, and the accuracy was below 10% in terms of RE%. The validated method was then successfully applied to the studies of preclinical pharmacokinetics for HM-3.
Keywords: HM-3; Pharmacokinetics; Metabolism; LC-Q-TOF/MS; LC–MS/MS;

Development and validation of an UPLC-PDA method for the determination of corilagin in rat plasma and its application to pharmacokinetic study by Bingjing Zheng; Dahui Chen; Xiangxiang Yang; Longo Phemba Igo; Zhengxue Li; Xiaoxia Ye; Zheng Xiang (76-79).
Corilagin, which was isolated from several medical herbs, has been reported to exert many pharmacological activities. A simple and rapid liquid ultra-performance liquid chromatography (UPLC) coupled to photodiode array (PDA) method has been developed to quantify corilagin in rat plasma. In this study, plasma samples were prepared by ethyl acetate extraction. Separation was performed on a HSS T3 (100 mm × 2.1 mm, 1.8 μm) column by using a mobile phase of acetonitrile and water with 0.1% trifluoroacetic acid (v/v). Corilagin and internal standard epicatechin were detected at a wavelength of 266 nm. The calibration curve was linear (r > 0.998) over a concentration range of 0.2 μg/mL to 20 μg/mL with a lower quantification limit of 0.2 μg/mL. Both intra and inter-day precision values were within 5.7% and extraction recovery were greater than 81.0%. Stability tests showed that corilagin and IS remained stable during the analytical procedure. The validated UPLC-PDA method was then used to analyze the pharmacokinetics of corilagin administered to rats intravenously (10 mg/kg) or orally (50 mg/kg). Oral bioavailability of corilagin was calculated to be 10.7%, indicating that this component is not suitable for oral administration. The results provide basis for further preclinical studies on corilagin.
Keywords: Corilagin; Pharmacokinetics; UPLC;

Liquid chromatography–tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma by Johannes J.M. Rood; Mark T.J. van Bussel; Jan H.M. Schellens; Jos H. Beijnen; Rolf W. Sparidans (80-85).
A method for the quantitative analysis by ultra-performance liquid chromatography–tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000 ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8 μm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5–102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid–liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes.
Keywords: Osimertinib; Non-small cell lung carcinoma; LC–MS/MS; Human plasma; Salting-out assisted liquid–liquid extraction;

Affinity chromatography is a widespread technique for the enrichment and isolation of biologics, which relies on the selective and reversible interaction between affinity ligands and target molecules. Small synthetic affinity ligands are valuable alternatives due to their robustness, low cost and fast ligand development. This work reports, for the first time, the use of a sequential Petasis-Ugi multicomponent reaction to generate rationally designed solid-phase combinatorial libraries of small synthetic ligands, which can be screened for the selection of new affinity adsorbents towards biological targets. As a proof of concept, the Petasis-Ugi reaction was here employed in the discovery of affinity ligands suitable for phosphopeptide enrichment. A combinatorial library of 84 ligands was designed, synthesized on a chromatographic solid support and screened in situ for the specific binding of phosphopeptides binding human BRCA1C-terminal domains. The success of the reaction on the chromatographic matrix was confirmed by both inductively coupled plasma atomic emission spectroscopy and fluorescence microscopy. Three lead ligands were identified due to their superior performance in terms of binding capacity and selectivity towards the phosphorylated moiety on peptides, which showed the feasibility of the Petasis-Ugi reaction for affinity ligand development.
Keywords: Phosphopeptide enrichment; Affinity chromatography; Petasis; Ugi;

Higher doses of cefazolin are required in obese patients for preoperative antibiotic prophylaxis, owing to its low lipophilicity. An ultra high performance liquid chromatography-tandem mass spectrometry method was developed to quantify cefazolin in serum and adipose tissue from 6 obese patients undergoing cesarean delivery, and using stable-isotope labeled cefazolin as an internal standard. The method has a 2 μg/g lower limit of quantitation. The concentration in adipose tissue was 3.4 ± 1.6 μg/mL, which is less than half of the reported minimum inhibitory concentration of 8 μg/mL for cefazolin. Serum cefazolin concentrations were more than 30-fold higher than in adipose tissue.
Keywords: Cefazolin; Mass spectrometry; Adipose tissue; Cesarean delivery; Stable-isotope internal standard;

A rapid and sensitive method for the determination of 171 pesticides in cowpea was developed using multi-walled carbon nanotubes (MWCNTs) as reversed-dispersive solid-phase (r-DSPE) extraction materials. The clean-up performance of MWCNTs was proved to be obviously superior to PSA and GCB. This method was validated on cowpea spiked at 0.01 and 0.1 mg kg−1 with five replicates. The mean recoveries for 169 pesticides ranged from 74% to 129% with relative standard deviations (RSDs) (n = 5) lower than 16.4%, except diflufenican and quizalofop-ethyl. Good linearity for all pesticides was obtained with the calibration curve coefficients (R2) larger than 0.9970. The limit of detection (LODs) and limit of quantification (LOQs) for the 171 pesticides ranged from 0.001 to 0.003 mg kg−1 and from 0.002 to 0.009 mg kg−1, respectively. The method was demonstrated to be reliable and sensitive for the routine monitoring of the 171 pesticides in cowpea samples.
Keywords: Cowpea; MWCNTs; r-DSPE; Pesticide residues;

Determination of free fatty acids in olive oils by UPHLC–MS by Saikh M. Wabaidur; Ahmad AlAmmari; Ahmad Aqel; Saad A. AL-Tamrah; Zeid Abdullah Alothman; A.Y. Badjah Hadj Ahmed (109-115).
A simple, fast, highly efficient and direct method using ultra-performance liquid chromatography coupled to mass spectrometry has been established for the simultaneous separation, identification and quantitation of a few saturated and unsaturated fatty acids in olive oils from various countries. No sample pretreatment techniques were employed such as extraction or derivatization for the analysis of target acids from oil samples, as the oil samples were just diluted, filtered and then directly injected to the instrument. The chromatographic separations of all target fatty acids were achieved on a Hypersil Gold C18 column of particle size 1.9 μm, 50 × 2.1 mm I.D, while the gradient elution using a binary mobile phase mixture of acetonitrile and water at a flow rate of 1.5 ml/min was adopted for achieving optimum separations. The identification and quantitation of target compounds was accomplished using selected ion reaction monitoring mode. The recoveries of the fatty acids were obtained higher than 89% with good validation parameters; linearity (r2  > 0.992), detection limit between 0.09 and 0.24 μg/ml, run to run and day to day precisions with percent relative standard deviation lower than 2.4% at both low (1 μg/ml) and medium (10 μg/ml) concentration levels. The total content of fatty acids in each individual oils was found in the range of 472.63–7751.20 μg/ml of olive oil, while oleic acid was found to be the major fatty acid among all analyzed oils with the amount 3785.94 μg/ml (maximum) in Syrian olive oil. The obtained validation parameters confirm that the proposed analytical method is rapid, sensitive, reproducible and simple and it could be applied for the successful evaluation of fatty acids in various oils and other matrices. All the fatty acids were efficiently eluted in a time of less than 8 min with well resolved peaks by employing the proposed method.
Keywords: Ultra performance liquid chromatography; Mass spectrometry; Fatty acids; Olive oils;

While several methods for the bioanalysis of nerve agents or their metabolites have been developed for the verification of nerve agent exposure, these methods are generally limited in the amount of time after an exposure that markers of exposure can be detected (due to rapid metabolism from biological matrices). In this study, a method for the analysis of nerve agent hydrolysis products from nail clippings was developed to allow evaluation of nails as a long-term repository of these markers. Pinacolyl methylphosphonic acid (PMPA) and isopropyl methylphosphonic acid (IMPA) were extracted from nail samples with N,N-dimethylformamide and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Limits of detection for PMPA and IMPA were 0.3 μg/kg and 7.5 μg/kg and linear ranges were 0.75–300 μg/kg and 30–1500 μg/kg, respectively. Precision was within 10% and 8% for PMPA and IMPA, respectively, and accuracy was 100 ± 12% for both analytes. The approach presented here is complementary to current methods for nerve agent exposure verification, and should allow for long-term determination of nerve agent poisoning.
Keywords: Soman; Pinacolyl methylphosphonic acid; Sarin; Isopropyl methylphosphonic acid; Liquid-chromatography mass-spectrometry; Long-term chemical warfare agent biomarker; Nerve agent exposure;

Pharmacokinetics and bioavailability assessment of Miltefosine in rats using high performance liquid chromatography tandem mass spectrometry by Guru R. Valicherla; Priyanka Tripathi; Sandeep K. Singh; Anees A. Syed; Mohammed Riyazuddin; Athar Husain; Deep Javia; Kishan S. Italiya; Prabhat R. Mishra; Jiaur R. Gayen (123-130).
Display OmittedMiltefosine (MFS) is the first effective oral drug for treatment of visceral, mucosal and cutaneous leishmaniasis. In this study, liquid chromatography coupled mass spectrometry (LC–MS/MS) method of MFS was validated in rat plasma and its practical utilization to pharmacokinetic studies in rats for the first time. A rapid, selective and sensitive LC–MS/MS method for MFS in rat plasma was linear over the calibration range of 1–500 ng/mL. MFS and Phenacetin (internal standard) were separated on Phenomenex Luna 3 μ HILIC 200A (150 × 4.6 mm) column under isocratic condition using methanol: 0.1% formic acid in triple distilled water, 90:10 (v/v) mobile phase at a flow rate of 0.8 mL/min. The total chromatographic run time was 4.0 min. The intra- and inter-day assay accuracy was observed between 99.45-102.88% and 99.92-101.58%, respectively. The intra- and inter-day assay precision was observed between 2.68-5.54% and 2.35-5.94%, respectively. The validated assay was practically applied to determine the plasma concentrations after oral and intravenous administration of MFS to rats. After oral administration, MFS showed Cmax (3200.00 ± 95.39 ng/mL) was observed at 12.00 h (tmax) and t1/2 was 102.36 ± 16.65 h. The absolute bioavailability of MFS was 60.33 ± 2.32%.
Keywords: Miltefosine; Leishmaniasis; LC–MS/MS; Pharmacokinetics; Bioavailability;

High-throughput analysis of 19 endogenous androgenic steroids by ultra-performance convergence chromatography tandem mass spectrometry by Jonathan L. Quanson; Marietjie A. Stander; Elzette Pretorius; Carl Jenkinson; Angela E. Taylor; Karl-Heinz Storbeck (131-138).
11-Oxygenated steroids such as 11-ketotestosterone and 11-ketodihydrotestosterone have recently been shown to play a putative role in the development and progression of castration resistant prostate cancer. In this study we report on the development of a high throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) method for the analysis of thirteen 11-oxygenated and six canonical C19 steroids isolated from a cell culture matrix. Using an Acquity UPC2 BEH 2-EP column we found that UPC2 resulted in superior selectivity, increased chromatographic efficiency and a scattered elution order when compared to conventional reverse phase ultra-performance liquid chromatography (UPLC). Furthermore, there was a significant improvement in sensitivity (5–50 times). The lower limits of quantification ranged between 0.01–10 ng mL−1, while the upper limit of quantification was 100 ng mL−1 for all steroids. Accuracy, precision, intra-day variation, recovery, matrix effects and process efficiency were all evaluated and found to be within acceptable limits. Taken together we show that the increased power of UPC2-MS/MS allows the analyst to complete in vitro assays at biologically relevant concentrations for the first time and in so doing determine the routes of steroid metabolism which is vital for studies of androgen responsive cancers, such as prostate cancer, and could highlight new mechanisms of disease progression and new targets for cancer therapy.
Keywords: Adrenal androgens; Gas chromatography; Steroids; Supercritical fluid chromatography; Ultra performance convergence chromatography; Ultra performance liquid chromatography;

Pharmacokinetics, tissue distribution, and plasma protein binding study of chicoric acid by HPLC–MS/MS by Yutang Wang; Guo Xie; Qian Liu; Xiang Duan; Zhigang Liu; Xuebo Liu (139-145).
Chicoric acid is a major active constituent of Echinacea purpurea and has a variety of biological functions. In this study, a liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) approach was developed and validated for the determination of chicoric acid in rat plasma and various tissues using ferulic acid as an internal standard (IS). This method was successfully applied to pharmacokinetics, tissue distribution, and plasma protein binding (PPB) study of chicoric acid in Sprague-Dawley (SD) rats dosed with 50 mg/kg by gastric gavage. The pharmacokinetic parameters were determined and showed a half-life (t1/2) of 4.53 ± 1.44 h, an apparent volume of mean residual time (MRT) of 18.58 ± 4.43 h, and an area under the curve (AUC) of 26.14 mg h L−1. The tissue distribution of chicoric acid in rats after gavage administration showed a decreasing tendency in different tissues (liver > lung > kidney > heart > spleen > brain). The PPB rates in rat plasma, human plasma, and bovine serum albumin were 98.3, 96.9, and 96.6%, respectively. These results provide insight for the further pharmacological investigation of chicoric acid.
Keywords: Chicoric acid; HPLC–MS/MS; Pharmacokinetics; Tissue distribution; Plasma protein binding;

Separation and analysis of mono-glucosylated lipids in brain and skin by hydrophilic interaction chromatography based on carbohydrate and lipid moiety by Kazuki Nakajima; Hisako Akiyama; Kaori Tanaka; Ayako Kohyama-Koganeya; Peter Greimel; Yoshio Hirabayashi (146-153).
Mono-glycosylated sphingolipids and glycerophospholipids play important roles in diverse biological processes and are linked to a variety of pathologies, such as Parkinson disease. The precise identification of the carbohydrate head group of these lipids is complicated by their isobaric nature and by substantial differences in concentration in different biological samples. To overcome these obstacles, we developed a zwitterionic (ZIC)-hydrophilic interaction chromatography (HILIC) electrospray ionization tandem mass spectrometry method. ZIC-HILIC preferentially retains inositol, followed by glucose- and galactose-featuring lipids. Comparison with unmodified silica gel HILIC stationary phase revealed different retention specificity. To evaluate the precision of ZIC-HILIC, we quantified glucosyl- (GlcCer) and galactosylceramides (GalCer) in seven different regions of the mouse brain and discovered that GlcCer and GalCer concentrations are inversely related. The highest GalCer (lowest GlcCer) content was found in the medulla oblongata and hippocampus, whereas the highest GlcCer (lowest GalCer) content was found in other regions. With a neutral loss scan, ZIC-HILIC resolved glucosylceramide species featuring non-hydroxylated fatty acid, hydroxylated fatty acid, and trihydroxy sphingoid bases in mouse epidermis samples. This demonstrates that our ZIC-HILIC-based approach is a valuable tool for characterizing the structural diversity of mono-glucosylated lipids in biological material and for quantifying these important lipids.
Keywords: Hydrophilic interaction chromatography; Zwitterionic phase; Mass spectrometry; Glucosylceramide; Phopsphatidylglucoside; Glucosylated lipid; Lipid isomer;

Pre-clinical investigation of plasma pharmacokinetics and biodistribution of a novel antithrombotic agent S002-333 in mice using LC–MS/MS by Manisha Bhateria; Rachumallu Ramakrishna; Santosh Kumar Puttrevu; Sahithi Yerrabelli; Anil K. Saxena; Rabi Sankar Bhatta (154-162).
S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide] is a novel and potent antithrombotic agent developed by CSIR-CDRI, India. The present study was aimed to develop a sensitive LC–MS/MS method for the quantification of S002-333 in mice plasma and tissues. The extraction of S002-333 from relatively small amount of mouse biomatrices (50 μL) was accomplished using protein precipitation followed by liquid-liquid extraction and the separation of analytes was achieved on C18 reversed phase column using acetonitrile and triple distilled water (75:25, v/v) as mobile phase at a flow rate of 0.6 mL/min. The instrument was operated in the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive scan mode. For all the biomatrices, linear relationship was attained over the concentration range of 0.39–200 ng/mL with correlation coefficients ≥0.992. The lower limit of quantification for mouse plasma and tissue homogenates was 0.39 ng/mL. The bioanalytical method was reproducible and reliable for all the matrices with inter-day and intra-day variability in precision being less than 15% and accuracy within ±15%. The assay was successfully applied to pharmacokinetics and tissue distribution of S002-333 in mice. The pharmacokinetic study revealed adequate gastrointestinal absorption of S002-333 into the systemic circulation of mice with absolute oral bioavailability of 45.8%. Tissue distribution data showed rapid and wide distribution of S002-333 in the following order: small intestine > liver > kidney ≈ lungs > heart > spleen > brain. The present findings may provide meaningful basis for further clinical development of this new chemical entity.
Keywords: S002-333; Antithrombotic; LC–MS/MS; Pharmacokinetics; Bioavailability; Tissue distribution;

Identification and discrimination of three common Aloe species by high performance liquid chromatography–tandem mass spectrometry coupled with multivariate analysis by Yan Zhao; Ya Nan Sun; Min Jung Lee; Young Ho Kim; Wonjae Lee; Kyeong Ho Kim; Kyung Tae Kim; Jong Seong Kang (163-171).
Aloe arborescens, Aloe barbadensis and Aloe ferox are the most widely cultivated and used among 500 aloe species due to their potent bioactivity. However, the difference of aloe species is neglected and labeled only one name Aloe in the market without specifying aloe species discrimination in general. Furthermore, differences in bioactivity and side effects from different aloe species have not been well investigated. This study develops an effective method for simultaneous qualitative and quantitative determination of three common aloe species using high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) and high performance liquid chromatography-diode array detection (HPLC-DAD). The extraction conditions were optimized by response surface methodology (RSM) based on methanol concentration, extraction time and solvent-to-material ratio. A partial least squares-discriminant analysis (PLS-DA) was used to identify the three aloe species. The developed HPLC–MS/MS method coupled with multivariate analysis can be applied to discriminate three aloe species successfully.
Keywords: Aloe; Response surface methodology; LC-ESI–MS/MS; Multivariate analysis;

Pharmacokinetic studies of novel berberine derivatives with ultra-performance liquid chromatography–tandem mass spectrometry by Wenchao Wang; Qin Shen; Hui Liang; Changlong Hua; Yuhui Liu; Fengzhi Li; Qingyong Li (172-180).
An ultra-performance liquid chromatography with tandem mass spectrometric detection method was developed for the detection of berberine and its derivatives (A4, B4) in rat plasma and other organs. This validated method was successfully applied to our pharmacokinetic study of BBR derivatives in rats. At the same dose of administration, the Cmax of B4 was about eight times higher than BBR, and its half-life was approximately two times longer than BBR, according to the bigger areas under plasma concentration curves. Inversely, the pharmacokinetic parameter levels of A4 were all inferior to BBR, suggesting a tight structure-activity relationship of these compounds. Small dose of parenteral administration was used for the study of absolute oral bioavailability of A4, B4, and BBR, and the results calculated were 0.12%, 3.4% and 0.7%, respectively. The accumulations of B4 among all organs were intestine > liver > heart > kidney > lung > spleen > plasma, proving a deeply targeting property of B4, which met our experimental assumption. Together, the experimental results proved that compared with BBR and A4, the derivative B4 had higher absolute oral bioavailability and the ability of deeply targeting so that can be likely used in some organ-targeted diseases.
Keywords: Berberine (BBR); Berberine derivatives; UPLC–MS/MS; Pharmacokinetics; Tissue distribution; Targeting;

The measurement of estrogens at sub-picogram levels is essential for research on breast cancer and postmenopausal plasma. Heretofore, these concentration levels have rarely been achieved. However, it is possible through derivatization but still represent problems for monitoring catechol estrogens and 16α-hydroxyestrone (16α-OH-E1). Estrogens possess poor ionization efficiency in MS/MS, which results in insufficient sensitivity for analyzing samples at trace concentrations. The method presented here was used to extract ten estrogen metabolites (EMs) with a derivatization step involving a new adduct. The electrospray ionization (ESI) MS/MS sensitivity for the EMs was enhanced by derivatization with 3-bromomethyl-propyphenazone (BMP). The lower limits of quantification (LLOQ) of the EMs were 12–100 femtogram on-column, equivalent to 0.3–3.6 pg/mL plasma, and the limits of detection (LOD) were 0.1–0.8 pg/mL plasma. The percentage coefficient of variation (CV%) at the LLOQ was <20 for all investigated EMs. Ionization suppression was minimized by reacting the excess reagent, BMP, with methanol. The method was successfully applied for the determination of ten EMs in the plasma of fifty healthy postmenopausal and fifty ductal breast cancer women aged 47–65 years old. 16α-OH-E1 and three catechol estrogen metabolites, 4-OH-E1, 2-OH-E2 and 4-OH-E2, were successfully measured in the plasma of healthy and breast cancer women. The methyl-propyphenazone-EM derivatives exhibited better sensitivity in ESI-MS (7.5-fold) compared to the commonly used dansylation procedure.
Keywords: Estrogen metabolites; Catechol estrogens; Breast cancer; 3-Bromomethyl-propyphenazone; Derivatization; Liquid chromatography–mass spectrometry;

Gas chromatography (GC) coupled with triple quadrupole tandem mass spectrometry (MS/MS) operated in electron ionization mode (EI) has been shown to have advantages in the trace analysis of chemical compounds. Employing the instrument, a method has been built to simultaneously determine eugenol, isoeugenol‚ and methyleugenol, which have been widely used as fish anesthetic, in the fish fillet. Procedure for the sample preparation was achieved by using hexane extraction followed by phenyl solid phase extraction (SPE) cleanup, which was free of such steps as rotary evaporation and nitrogen blowing by taking the volatility of eugenol and its isomers into consideration. The method was validated by conducting recovery studies on fortified fish fillet samples at four concentrations. The linearity in the range of 5–500 μg·L−1 was forced through the origin giving a coefficient of determination (r 2) greater than 0.9982. Limits of detection (LODs) for eugenol, isoeugenol‚ and methyleugenol were 0.4, 1.2‚ and 0.2 μg·kg−1, respectively. The limits of quantification (LOQs) were 1.2, 4‚ and 0.7 μg·kg−1 for eugenol, isoeugenol‚ and methyleugenol, respectively. The recoveries for eugenol and its isomers ranged from 76.4 to 99.9% with relative standard deviations (RSD) in a range from 2.18 to 15.5%. This method is quick, simple and suitable for determining the residues of eugenol, isoeugenol and methyleugenol simultaneously in batch samples of fish fillet.
Keywords: Eugenol; Isoeugenol; Methyleugenol; Fish; GC–MS/MS;

A novel thiol-affinity micropipette tip method using zinc(II)–cyclen-attached agarose beads for enrichment of cysteine-containing molecules by Hiroshi Kusamoto; Akio Shiba; Norinao Koretake; Haruto Fujioka; Yuhzo Hieda; Emiko Kinoshita-Kikuta; Eiji Kinoshita; Tohru Koike (195-201).
Cysteine-containing biomolecules are attractive targets in the study of thiol biology. Here we introduce a novel method for the selective enrichment of thiol-containing molecules using a thiol-capture zinc(II) complex of 1,4,7,10-tetraazacyclododecane (Zn2+–cyclen). Recognition of N-acetylcysteine amide by Zn2+–cyclen has been studied by potentiometric pH titration, revealing formation of a 1:1 thiolate-bound Zn2+–cyclen complex with a large thiolate-affinity constant of 106.2  M−1 at 25 °C and I  = 0.10 M (NaCl). The Zn2+-bound thiolate anion is unexpectedly stable in aqueous solution at pH 7.8 under atmospheric conditions for a few days. These findings have contributed to the development of a convenient method for separation of thiol compounds by using a micropipette tip. A 200 μL micropipette tip containing 10 μL of hydrophilic cross-linked agarose beads attached to Zn2+–cyclen moieties was prepared. All steps for thiol-affinity separation (binding, washing, and eluting) are conducted using aqueous buffers at room temperature. The entire separation protocol requires less than 15 min per sample. We demonstrate practical example separations of cysteine-containing molecules. This micropipette tip method would be used preferentially as an alternative to existing tools for reliable enrichment of thiol-containing molecules.
Keywords: Thiol-affinity chromatography; Thiolate recognition; Zinc(II) complex; Macrocyclic tetraamine;

A high-throughput method for simultaneous qualitative and quantitative analysis of 21 mycotoxins in Radix Paeoniae Alba (RPA) was developed by coupling the modified QuEChERS method with ultra-high performance liquid chromatography quadrupole linear ion trap mass spectrometry (UHPLC-QqLIT-MS). The 21 mycotoxins were extracted and cleaned up using QuEChERS-based procedure, then further separated on a C18 column and detected by a hybrid triple quadrupole linear ion trap mass spectrometer equipped with electrospray ionization source in the multiple reaction monitoring-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode. Under this technique, 13 mycotoxins were detected using acetonitrile and water containing 0.1% formic acid as the mobile phase in positive mode while the other 8 mycotoxins were detected using acetonitrile and water containing 0.1% ammonia as the mobile phase in negative mode. The calibration curves of all analytes showed good linearity (r 2  > 0.995) within test ranges. The limits of detection and quantification ranged from 0.031 to 5.4 µg/kg and 0.20 to 22 µg/kg, respectively. Additionally, recoveries were all above 75.3% with relative standard deviations within 15%. The method proposed herein with significant advantages including simple pretreatment, rapid determination as well as high sensitivity, accuracy and throughput would be a preferred candidate for the determination and quantification of multi-class mycotoxin contaminants in real samples.
Keywords: Radix Paeoniae Alba; Mycotoxin; QuEChERS; Ultra-high performance liquid chromatography quadrupole linear ion trap mass spectrometry; Multiple reaction monitoring-information dependent acquisition-enhanced product ion;

A highly sensitive HPLC–MS/MS assay method was established to quantify 20(S)-protopanaxadiol (PPD) in human plasma with dexamethasone as an internal standard. The electrospray ion mass spectrometry (ESI–MS) was operated under the multiple reactions monitoring mode (MRM) using positive ion mode. PPD was extracted from 500 μL plasma samples by liquid–liquid extraction then separated by a C18 analytical column with gradient elution. The concentration of PPD could be determined by this HPLC–MS/MS method over the range of 0.05–20 ng/mL with the lower limit of quantification (LLOQ) of 0.05 ng/mL. The method was successfully applied to phase IIa clinical trial of Yuxintine (PPD capsule) in which plasma samples of 87 subjects were analyzed following 6 weeks of oral administration of placebo or PPD capsules in 5 different doses. In this study, the measured concentration was linearly related to the oral dosage with R = 0.9901. The minimum and maximum values of measured concentration were 0.06 and 11.60 ng/mL, respectively. In addition, plasma concentrations of PPD in depression patients were reported for the first time in our study.
Keywords: 20(S)-Protopanaxadiol; HPLC–MS/MS; Phase IIa clinical trial; Anti-depressant agent; Qualification;

Coenzyme Q10 (CoQ10) solid dispersion was prepared to improve its oral bioavailability due to the poor solubility of CoQ10. To evaluate the pharmacokinetic behaviors of CoQ10 solid dispersion, a simple, rapid, sensitive and environment friendly method for the determination of CoQ10 in rat plasma was developed. In this study, samples were prepared by one-step protein precipitation with acetone and then the supercritical fluid chromatography-electrospray ionization tandem mass spectrometry (SFC-ESI–MS/MS) method was used. The separation was achieved by an ACQUITY UPC2™ BEH 2-EP column (100 mm × 3 mm, 1.7 μm) maintained at 35 °C, using carbon dioxide (≥99.99%) and methanol (85:15, v/v) as the mobile phase at a flow rate of 1.0 ml/min. To improve the response and sensitivity, the compensation solvent of methanol with 2 mM ammonium acetate at a flow rate of 0.2 ml/min was used and the total analysis time was only 1.5 min for each sample. The detection was carried out on a tandem mass spectrometer with electrospray ionization (ESI) source and the mass transition ion pair was m/z 881.0 → 197.0 and 285.1 → 193.0 for CoQ10 and diazepam, internal standard (IS), respectively. Calibration curve was linear over the concentration range of 2.00–500.00 ng/ml (r2  ≥ 0.998) with a lower limit of quantification of 2.00 ng/ml. The intra- and inter-day accuracy and precision were below 15% for all quality control samples. The proposed method was rapid, accurate and reproducible, which was suitable to compare the pharmacokinetic behaviors in rats after a single oral dose of 100 mg/kg CoQ10 solid dispersion or tablets.
Keywords: Coenzyme Q10; Post-column compensation; SFC-ESI–MS/MS; Pharmacokinetic study; Rat plasma;