Journal of Chromatography B (v.969, #C)

Application of a liquid chromatography–tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of sweroside in rats by Ning Sheng; Lin Yuan; Xuran Zhi; Cheng Cui; Zhiyong Zhang; Peipei Jia; Xiaoxu Zhang; Lantong Zhang; Xinguo Wang (1-11).
A sensitive, reliable and accurate high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) was developed and validated for the quantification of sweroside in rat plasma, tissue and excretion. A single-step protein precipitation by methanol was used to prepare samples. Sweroside and swertiamarin (internal standard, IS) were separated by using a C18 column and a mobile phase consisted of methanol and water containing 0.1% formic acid running at a flow rate of 0.8 ml/min for 6 min. Detection and quantification were performed using a mass spectrometer by the multiple-reaction monitoring (MRM) in positive electrospray ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were [M+H]+359.1 → 197.2 for sweroside and [M+Na]+397.4 → 165.3 for swertiamarin (IS), respectively. The inter-day precision (RSD %) was less than 11.20% and intra-day precision (RSD %) was less than 10.90%, while the inter-day accuracy (RE %) was ranged from −9.69 to 9.17% and intra-day accuracy (RE %) was ranged from −10.56 to 13.47%. The mean elimination half-life (t 1/2) of sweroside for 5, 10 and 15 mg/kg dose were 78.8, 67.6 and 77.2 min, respectively. And sweroside follows linear plasma pharmacokinetics across the investigated dosage range in rats (5–15 mg/kg). The absolute bioavailability (F %) of sweroside was 11.90% on average. The results of tissue distribution showed the higher sweroside concentrations were found in kidney, liver, spleen and lung, and the small amount of drug was distributed into the brain tissue. The high distribution in liver confirms the reports that sweroside has hepatoprotective activity and promoted liver regeneration, and there was no long-term accumulation of sweroside in rat tissues. Total recoveries of sweroside within 48 h were 0.67% in bile, 1.55% in urine and 0.46% in feces, which might be resulted from liver first-pass effect. The above results suggested that sweroside was mainly excreted as the metabolites.
Keywords: Pharmacokinetics; Tissue distribution; Excretion study; Sweroside; LC–ESI-MS/MS;

Determination and pharmacokinetic studies of arecoline in dog plasma by liquid chromatography–tandem mass spectrometry by Bing Li; Xu-Zheng Zhou; Jian-Yong Li; Ya-Jun Yang; Jian-Rong Niu; Xiao-Juan Wei; Xi-Wang Liu; Jin-Shan Li; Ji-Yu Zhang (12-18).
A rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of arecoline concentration in dog plasma. Plasma sample was prepared by protein precipitation using n-hexane (containing 1% isoamyl alcohol) with β-pinene as an internal standard. Chromatographic separation was achieved on an Agilent C18 column (4.6 × 75 mm, 3.5 μm) using methanol: 5 mM ammonium acetate as the mobile phase with isocratic elution. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve for arecoline was linear over a concentration range of 2–500 ng/mL. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations. In summary, the LC–MS/MS method described herein was fully validated and successfully applied to the pharmacokinetic study of arecoline hydrobromide tablets in dogs after oral administration.
Keywords: Arecoline hydrobromide; Arecoline hydrobromide tablets; LC–MS/MS; Pharmacokinetics; Dog plasma;

Propylthiouracil quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry: Application in a bioequivalence study by Gustavo D. Mendes; Samara Bittencourt; Celso Francisco Pimentel Vespasiano; Tainah Babadópulos; Thiago Gagliano-Jucá; André Moreira Martins Arruda; Elisa Perissutti; Francesco Frecentese; Gilberto De Nucci (19-28).
A rapid, sensitive and specific method for quantifying propylthiouracil in human plasma using methylthiouracil as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid–liquid extraction using an organic solvent (ethyl acetate). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC–MS/MS) in negative mode (ES−). Chromatography was performed using a Phenomenex Gemini C18 5 μm analytical column (4.6 mm × 150 mm i.d.) and a mobile phase consisting of methanol/water/acetonitrile (40/40/20, v/v/v) + 0.1% of formic acid. For propylthiouracil and I.S., the optimized parameters of the declustering potential, collision energy and collision exit potential were −60 (V), −26 (eV) and −5 (V), respectively. The method had a chromatographic run time of 2.5 min and a linear calibration curve over the range 20–5000 ng/mL. The limit of quantification was 20 ng/mL. The stability tests indicated no significant degradation. This HPLC–MS/MS procedure was used to assess the bioequivalence of two propylthiouracil 100 mg tablet formulations in healthy volunteers of both sexes in fasted and fed state. The geometric mean and 90% confidence interval CI of Test/Reference percent ratios were, without and with food, respectively: 109.28% (103.63–115.25%) and 115.60% (109.03–122.58%) for C max, 103.31% (100.74–105.96%) and 103.40% (101.03–105.84) for AUClast. Conclusion: This method offers advantages over those previously reported, in terms of both a simple liquid–liquid extraction without clean-up procedures, as well as a faster run time (2.5 min). The LOQ of 20 ng/mL is well suited for pharmacokinetic studies. The assay performance results indicate that the method is precise and accurate enough for the routine determination of the propylthiouracil in human plasma. The test formulation with and without food was bioequivalent to reference formulation. Food administration increased the T max and decreased the bioavailability (C max and AUC).
Keywords: Bioavailability; Pharmacokinetics; Healthy volunteer;

Seabird guano is one of the main sources of nutrient fertilizers in remote coastal island areas, but guano-derived contaminants such as arsenic may cause serious threats to local ecosystems and public health issues. In this study, a new method was developed to analyze arsenic speciation in guano and ornithogenic sediments. Good extraction efficiencies of As(III) (arsenite), DMA (dimethylarsinate), MMA (monomethylarsonate) and As(V) (arsenate) were obtained by using 1.0 mol L−1 orthophosphoric acid and 0.1 mol L−1 ascorbic acid, followed by microwave-assisted extraction and high-performance liquid chromatography coupled to hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS) detection. Under the optimized conditions, the extraction efficiencies of four arsenic species were over 80%. The relative standard deviations (RSDs) were 9.60, 6.15, 6.34 and 2.93% (n  = 7), and the detection limits (μg L−1) were 0.82, 2.38, 1.45 and 2.31 for As(III), DMA, MMA and As(V), respectively. This method was successfully used to determine arsenic speciation in the guano samples collected from the Xisha Islands of the South China Sea, and the results indicated that As(III) and As(V) were the dominant arsenic species in modern and ancient guano, respectively.
Keywords: Arsenic speciation; Guano; Transformation; Enrichment; Microwave-assisted extraction; Atomic fluorescence spectrometry;

This study proposes a new analytical methodology for the determination of trace levels of testosterone (T) and epitestosterone (E) in urine matrices using bar adsorptive microextraction combined with liquid desorption followed by high-performance liquid chromatography with diode array detection (BAμE-LD/HPLC-DAD). The comparison of different sorbent coatings (five activated carbons, one styrene-divinylbenzene, two modified pyrrolidone, one ciano and one n-vinylpyrrolidone polymers) through BAμE showed that the latter phase presented much higher selectivity and capacity offering multiple mechanisms of interaction. Assays using this phase were performed on 25 mL of water samples spiked at the 8.0 μg/L level, yielded average recoveries of 92.1 and 93.4% for T and E, respectively, under optimized experimental conditions; BAμE (n-vinylpyrrolidone): 16 h (1000 rpm), pH 5.5; LD: acetonitrile, 30 min under sonication treatment. From the developed analytical methodology, suitable detection limits were achieved (0.4 μg/L) and good linear dynamic ranges (1.4–16.0 μg/L) with remarkable determination coefficients (r 2  > 0.9978). By using the standard addition methodology, the application of the present analytical approach on urine samples revealed good sensitivity. The proposed method, which operated under the floating sampling technology, proved to be a suitable sorption-based static microextraction alternative for screening T, E and the T/E ratio in urine samples for doping control purposes. The methodology showed to be easy to implement, demonstrating good reproducibility, sensitivity and robustness, allowing the possibility to choose the most selective sorbent coating according to the compounds of interest.
Keywords: Testosterone; Epitestosterone; Urine matrices; Floating sampling technology; BAμE.;

Metabolic profiling of Gynostemma pentaphyllum extract in rat serum, urine and faeces after oral administration by Dao-Jin Chen; Hua-Gang Hu; Shao-Fang Xing; Ya-Jun Gao; Si-Fan Xu; Xiang-Lan Piao (42-52).
Folk drug Gynostemma pentaphyllum (Thunb.) Makino contains many biologically active phytochemicals which have been demonstrated to be effective against chronic diseases. As in vivo anti-tumor experiments of G. pentaphyllum extract (GP) show much stronger antitumor activities than in vitro, it is important and necessary to understand the metabolic study of GP. A sensitive and specific U-HPLC–MS method was utilized for the first time to rapidly identify gypenosides and its possible metabolites in rat serum, urine, and faeces after oral administration. Solid phase extraction was utilized in the sample preparation. Negative Electrospray ionisation (ESI) mass spectrometry was used to discern gypenosides and its possible metabolites in rat samples. As a result, after oral administration, a total of seven metabolites of G. pentaphyllum extract were assigned, two from the rat serum and seven both from the rat urine and faeces. As metabolites of G. pentaphyllum extract, all of them have never been reported before.
Keywords: Gynostemma pentaphyllum extract (GP); Metabolic profiling; U-HPLC–MS; Oral administration;

A new selective and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the quantification of natamycin (NAT) in rabbit corneas with amphotericin B as the internal standard (IS). The cornea samples were processed by a simple and protective methanol soaking extraction technology. The NAT could be extracted completely from rabbit cornea after 24 h of soaking with methanol under a mild condition. Chromatographic separation was performed on a C18 column (2.1 mm × 50 mm, 3.5 μm) using mobile phase with ammonium acetate buffer (pH 4.5; 4.0 mM):acetonitrile (40:60, v/v) at a flow rate of 0.25 ml/min. Quantification was performed using the transitions 666.2 → 503.2 m/z for NAT and 924.5 → 906.6 m/z for IS by positive ion electrospray ionization in multiple reaction monitoring mode. The assay was validated over a concentration range of 8.64 ng/ml to 843 ng/ml with lower limit of detection of 4.32 ng/ml. The method was validated with respect to linearity, accuracy, precision, recovery, stability and extracting efficiency. The extraction recovery of NAT from cornea samples was approximately 100% with the new methanol soaking extraction procedure. The method has been successfully applied to the ocular pharmacokinetic studies of NAT eye drops in the cornea of Japanese white rabbit.

Use of a carboxylesterase inhibitor of phenylmethanesulfonyl fluoride to stabilize epothilone D in rat plasma for a validated UHPLC–MS/MS assay by Long Yuan; Yunlin Fu; Duxi Zhang; Yuan-Qing Xia; Qianping Peng; Anne-Françoise Aubry; Mark E. Arnold (60-68).
A sensitive, accurate and rugged UHPLC–MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed. Carboxylesterases were identified as the likely esterases responsible for the hydrolysis of EpoD in plasma ex vivo, and the cause of the species different stability. Phenylmethanesulfonyl fluoride, a carboxylesterase inhibitor, was used to stabilize EpoD in rat blood during sample collection, processing, and storage. A systematic method screening and optimization strategy was used to improve the assay sensitivity and minimize potential bioanalytical risks. The stabilized plasma samples were extracted by liquid–liquid extraction. Chromatographic separation was achieved on an Acquity UPLC BEH Phenyl column with a gradient elution. EpoD and its stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 0.100 to 100 ng/mL was fitted to a 1/x 2 weighted linear regression model. The intra-assay precision was within ±3.6% CV and inter-assay precision was within ±4.2% CV. The assay accuracy was within ±8.3% of the nominal values. Assay recovery of EpoD was high (∼90%) and matrix effect was minimal (1.02–1.05). EpoD was stable in stabilized rat plasma for at least 30 h at room temperature, 180 days at −20 °C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in toxicology studies.
Keywords: Epothilone D; BMS-241027; UHPLC–MS/MS; Esterase inhibitor; Species different stability;

We report a rapid solid-phase extraction method for glyphosate (Glyp), glufosinate (Gluf), and bialaphos (Bial) using a zirconia-coated silica cartridge, which interacts specifically with phosphorous-containing amino acid herbicides (PAAHs). We extracted PAAHs from serum and urine samples. The PAAHs were derivatized with trimethyl orthoacetate-acetic acid and analyzed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS). The intra-day and inter-day accuracy was within ±13% RE, the intra-day and inter-day precision was less than 12% RSD, and the total recovery was more than 60% for Glyp and more than 80% for Gluf and Bial. The linearity ranges of the calibration curves of the serum samples were 0.2–10,000 μg/mL for Glyp, 0.1–1000 μg/L for Gluf, and 0.5–1000 μg/L for Bial; and those of the urine samples were 0.4–20,000 μg/L for Glyp, 0.2–2000 μg/L for Gluf, and 0.1–2000 μg/L for Bial. This range covers almost all the reported poisoning cases involving these compounds, from very mild to fatal cases. The present paper offers a universal cleanup method for PAAHs in serum and urine samples for clinical and forensic analysis.
Keywords: Glyphosate; Zirconia-coated cartridge; Phosphorous containing amino acid herbicides; Liquid chromatography–tandem mass spectrometry;

The activity of 11β-hydroxysteroid dehydrogenases (11β-HSD) is traditionally assessed using the ratio of cortisol to cortisone in urine or saliva. However, these biomarkers only reflect the local activity of 11β-HSD, and are easily affected by circadian variation of cortisol secretion. The shortcomings might be overcome by hair analysis. The present study aimed to develop an enhanced assay for simultaneous measurements of cortisol and cortisone in both hair and urine samples. The samples were collected from 29 patients under methadone maintenance treatment. The cortisol and cortisone were extracted either by solid phase extraction from a 20-mg milled hair sample after a 14-h incubation in 1 ml of methanol, or by twice liquid–liquid extraction from a 20-fold diluted urine sample. The analyses were performed using high performance liquid chromatography tandem mass spectrometry with electrospray ionization in negative mode. Limits of detection and quantification were 0.5 and 1.25 pg/mg for hair steroids and 0.2 and 0.5 ng/ml for urinary steroids, respectively. The recoveries were more than 97%. The intra- and inter-day coefficients of variation were less than 10%. The ratios of cortisol to cortisone in hair and urine were both less than one, but did not correlate with each other. A possible reason for the lack of correlation was that the ratios in hair and urine might mostly reflect the activity of 11β-HSD type 2 in the eccrine sweat gland and in the kidney, respectively. Additionally, a significant correlation was observed between results obtained using external standard quantification and internal standard quantification.
Keywords: 11β-Hydroxysteroid dehydrogenases; Ratio of cortisol to cortisone; Hair; Urine; Electrospray;

A rapid and sensitive analytical method was developed for simultaneous determination of eight neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) and three specific metabolites of acetamiprid (N-desmethylacetamiprid, 5-(N-acetyl-N-methylaminomethyl)-2-chloropyridine and 5-(N-acetylaminomethyl)-2-chloropyridine) in human serum and urine. A diatomaceous earth-assisted extraction using Extrelut NT3 column with chloroform/2-propanol (3:1, v/v) as eluent was selected for the single step cleanup procedure for all the target compounds. Qualitative and quantitative analyses were conducted by liquid chromatography–tandem mass spectrometry with multiple reaction monitoring mode. The limits of detection and the limits of quantification of eleven compounds were in the ranges of 0.1–0.2 ng/mL and 0.5–10 ng/mL for serum, 0.1–1 ng/mL and 1–10 ng/mL for urine, respectively. The extraction recoveries were between 80.9% and 101.8% for serum samples, 91.9% and 106% for urine samples. The intra-day RSDs and the inter-day RSDs were less than 11.5% and 13.2% for serum, less than 8.3% and 8.8% for urine. The proposed procedure will be suitable for forensic investigations of human poisoning cases with neonicotinoid insecticides. This is the first report of simultaneous determination of eight neonicotinoids in serum and urine samples.
Keywords: Neonicotinoids; Diatomaceous earth column; Isotope dilution; Liquid chromatography–tandem mass spectrometry;

A simple, rapid and sensitive reversed-phase ultra-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous quantitative determination of oxiracetam and its degraded substance 4-hydroxy-2-oxo-1-pyrrolidine acetic acid (HOPAA) in rat plasma. After plasma samples were deproteinized with acetonitrile, the post-treatment samples were analyzed on a C18 column interfaced with a triple quadrupole tandem mass spectrometer in the positive electrospray ionization mode at a flow rate of 0.35 ml/min. Piracetam was used as internal standard (IS). Multiple selected reaction monitoring was performed using the transitions m/z 159.00 → 141.94, 159.95 → 113.98 and 142.98 → 125.97 to quantify oxiracetam, HOPAA and IS, respectively. This method was validated over the concentration range of 25–2250 μg/ml for oxiracetam (r 2  ≥ 0.99) and 0.5–250 μg/ml (r 2  ≥ 0.99) for HOPAA. Intra-run and inter-run precision values of oxiracetam and HOPAA were less than 15% and accuracy was within 85–115% at all quality control levels. The average extraction recovery was 93.9% for oxiracetam and 95.6% for HOPAA, respectively. In conclusion, a simple, sensitive and specific HPLC-MS/MS method was successfully applied to the pharmacokinetic study in rats after an intravenous administration at a high dose of 2 g/kg.
Keywords: Simultaneous determination; Oxiracetam; 4-Hydroxy-2-oxo-1-pyrrolidine acetic acid; HPLC-MS/MS; Rat plasma; Pharmacokinetics.;

Curcumin is a well-known multitherapeutic agent widely employed in neurodegenerative disorders and cancer. A selective, fast, and sensitive method employing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of curcumin in mouse plasma and brain tissue, by using salbutamol as an internal standard. Triple quadrupole mass detection with multiple reaction monitoring (MRM) mode was used to monitor the ion transitions, m/z of 369 > 285 for curcumin, and m/z of 240 > 148 for salbutamol. The method was validated for recovery, accuracy, precision, linearity, and applicability. The lower limits of quantification (LLOQ) in both matrices were 2.5 ng/mL. The inter-day and intra-day precisions and accuracy values were within the Food and Drug Administration (FDA) acceptance criteria, for both matrixes. The method was successfully applied in pharmacokinetics and brain distribution studies of curcumin after intravenous administration of free curcumin and curcumin-loaded solid lipid nanoparticles to mice. Furthermore, the application of this method along with serial blood sampling in mice has led to significant reduction in animal use and dosage and drastic improvement in speed, throughput, and quality of pharmacokinetic parameters.
Keywords: Curcumin; Serial blood sampling; Mouse plasma; Mouse brain tissue; LC-MS/MS;

An effective assessment of valproate sodium-induced hepatotoxicity with UPLC–MS and 1HNMR-based metabonomics approach by Taoguang Huo; Xi Chen; Xiumei Lu; Lianyue Qu; Yang Liu; Shuang Cai (109-116).
Valproate sodium is one of the most prescribed antiepileptic drugs. However, valproate sodium has various side effects, especially its toxicity on liver. Current markers for toxicity reflect mostly the late stages of tissue damage; thus, more efficient methods for toxicity evaluation are desired. To evaluate the toxicity of valproate sodium on liver, we performed both UPLC–MS and 1HNMR-based metabonomics analysis of serum samples from 34 epileptic patients (age: 42.0 ± 18.6, 18 male/16 female) after valproate sodium treatment. Compared to conventional markers, the serum metabolic profiles provided clear distinction of the valproate sodium induced normal liver function and abnormal liver function in epileptic patients. Through multivariate statistical analysis, we identified marker metabolites associated with the hepatotoxicity induced by valproate sodium, such as glucose, lactate, acetoacetate, VLDL/LDL, lysophosphatidylcholines, phosphatidylcholines, choline, creatine, amino acids, N-acetyl glycoprotein, pyruvate and uric acid. This metabonomics approach may provide effective way to evaluate the valproate sodium-induced toxicity in a manner that can complement current measures. This approach is expected to find broader application in other drug-induced toxicity assessment.
Keywords: Metabonomics; Valproate sodium; Hepatotoxicity; UPLC–MS; 1HNMR;

Development and validation of a HILIC–MS/MS method for quantification of decitabine in human plasma by using lithium adduct detection by Wenyi Hua; Thomas Ierardi; Michael Lesslie; Brian T. Hoffman; Daniel Mulvana (117-122).
A highly sensitive, selective, and rugged quantification method was developed and validated for decitabine (5-aza-2′-deoxycytidine) in human plasma treated with 100 μg/mL of tetrahydrouridine (THU). Chromatographic separation was accomplished using hydrophilic interaction liquid chromatography (HILIC) and detection used electrospray ionization (ESI) tandem mass spectrometry (MS/MS) by monitoring lithiated adducts of the analytes as precursor ions. The method involves simple acetonitrile precipitation steps (in an ice bath) followed by injection of the supernatant onto a Thermo Betasil Silica-100, 100 × 3.0 mm, 5 μm LC column. Protonated ([M+H]+), sodiated ([M+Na]+), and lithiated ([M+Li]+) adducts as precursor ions for MS/MS detection were evaluated for best sensitivity and assay performance. During initial method development abundant sodium [M+Na]+ and potassium [M+K]+ adducts were observed while the protonated species [M+H]+ was present at a relative abundance of less than 5% in Q1. The alkali adducts were not be able to be minimized by the usual approach of increasing acid content in mobile phases. Significant analyte/internal standard (IS) co-suppression and inter-lot response differences were observed when using the sodium adduct as the precursor ion for quantification. By adding 2 mM lithium acetate in aqueous mobile phase component, the lithium adduct effectively replaced other cationic species and was successfully used as the precursor ion for selected reaction monitoring (SRM) detection. The method demonstrated the separation of anomers and from other endogenous interferences using a 3-min gradient elution. Decitabine stock, working solution stabilities were investigated during method development. Three different peaks, including one from anomerization, were observed in the SRM transition of the analyte when it was in neutral aqueous solution. The assay was validated over a concentration range of 0.5–500 ng/mL (or 0.44–440 pg injected on column) in 50 μL of human plasma. The accuracy and precision were within 8.6% relative error and 6.3% coefficient of variation, respectively. Decitabine was stable in THU treated human plasma for at least 68 days and after 5 freeze–thaw cycles when stored at −70 °C. Stability of decitabine in THU treated human whole blood, matrix factor and recovery were also evaluated during method validation. The method was successfully used for clinical sample analysis.
Keywords: Lithiated adducts; Alkali adducts; HILIC; Tandem mass spectrometry (MS/MS); Anomerizaton; Decitabine;

A novel ultrasound-assisted dispersive liquid–liquid microextraction based on solidification of floating organic droplet method (UA-DLLME-SFO) combined with gas chromatography (GC) was developed for the determination of eight pyrethroid pesticides in tea for the first time. After ultrasound and centrifugation, 1-dodecanol and ethanol was used as the extraction and dispersive solvent, respectively. A series of parameters, including extraction solvent and volume, dispersive solvent and volume, extraction time, pH, and ultrasonic time influencing the microextraction efficiency were systematically investigated. Under the optimal conditions, the enrichment factors (EFs) were from 292 to 883 for the eight analytes. The linear ranges for the analytes were from 5 to 100 μg/kg. The method recoveries ranged from 92.1% to 99.6%, with the corresponding RSDs less than 6.0%. The developed method was considered to be simple, fast, and precise to satisfy the requirements of the residual analysis of pyrethroid pesticides.
Keywords: Dispersive liquid–liquid microextraction; Solidification of floating organic droplet; Pyrethroid pesticides; Gas chromatography; Tea;

New graphene fiber coating for volatile organic compounds analysis by GuoJuan Zhang; XiaoXi Guo; ShuLing Wang; XueLan Wang; YanPing Zhou; Hui Xu (128-131).
In the work, a novel graphene-based solid phase microextraction-gas chromatography/mass spectrometry method was developed for the analysis of trace amount of volatile organic compounds in human exhaled breath vapor. The graphene fiber coating was prepared by a one-step hydrothermal reduction reaction. The fiber with porous and wrinkled structure exhibited excellent extraction efficiency toward eight studied volatile organic compounds (two n-alkanes, five n-aldehydes and one aromatic compound). Meanwhile, remarkable thermal and mechanical stability, long lifespan and low cost were also obtained for the fiber. Under the optimal conditions, the developed method provided low limits of detection (1.0–4.5 ng L−1), satisfactory reproducibility (3.8–13.8%) and acceptable recoveries (93–122%). The method was applied successfully to the analysis of breath samples of lung cancer patients and healthy individuals. The unique advantage of this approach includes simple setup, non-invasive analysis, cost-efficient and sufficient sensitivity. The proposed method supply us a new possibility to monitor volatile organic compounds in human exhaled breath samples.
Keywords: Solid-phase microextraction; Graphene fiber; Gas chromatography/mass spectrometry; Volatile organic compounds;

An efficient and reliable enantioseparation method by high-performance liquid chromatography (HPLC) for the chiral herbicide diclofop-methyl (DM) and its primary metabolite diclofop (DC) was developed and validated, and the biological process of the enantiomers in loach liver microsomes (LLM) in vitro was investigated. The enantiomers of DM and DC were first separated on an immobilized-type stationary phase of Chiralpak IC. The best resolutions were achieved under the chromatographic condition of n-hexane/IPA/TFA 96:4:0.1(v/v/v) at 20 °C with each R s  > 2 and the two pairs of enantiomers could be eluted in about 10 min. The extraction recoveries of the analytes from LLM were 79.6–108.9% with RSD ≤ 11.5%. The enzyme kinetics of DM enantiomers were different, with the K m value 320.7 for (S)-DM and 392.1 for (R)-DM. The metabolism experiment in vitro showed DM underwent a rapid phase-I metabolism with or without NADPH, indicating the esterases in liver played a dominant role. No interconversion between the two enantiomers was observed by single-enantiomer incubation. The preferential degradation of (S)-DM was confirmed with the half-time (t 1/2) of (S)-DM 5.27 min and 21.2 min for (R)-DM. DC enantiomer was generated by its corresponding ester form and could not be further degraded during the incubation. It was the first study on biotransformation of DM and DC enantiomers in LLM in vitro. The results may help to evaluate the ecological risks of chiral pesticides.
Keywords: Diclofop-methyl; Diclofop; Metabolism; Enantioselective; Chiralpak IC; Loach liver microsome;

Parabens are a family of most widely used antimicrobial preservatives in food ingredients, cosmetic consumer products and pharmaceutical preparations. But several recent studies have cautioned that exposure to parabens may have more harmful consequences on animal and human health than what we realized previously, which made the analysis of parabens necessary. In this paper, we reviewed main sample preparation methods and chromatographic analysis methods proposed in formerly published works dealing with the analysis of parabens in different matrices. The sample preparation methods included ultrasonic assisted extraction, supercritical fluid extraction, pressurized liquid extraction, solid phase extraction, solid phase microextraction, liquid phase microextraction, dispersive liquid–liquid microextraction, stir bar sorptive extraction and matrix solid phase dispersion. The chromatographic analysis methods involved liquid chromatography, gas chromatography, and capillary electrophoresis.
Keywords: Parabens; Sample preparation; Chromatographic analysis; Analytical methods;

A systematic set of optimization experiments was conducted to design an efficient extraction and analysis protocol for screening six different sub-classes of phenolic compounds in the seed coat of various lentil (Lens culinaris Medik.) genotypes. Different compounds from anthocyanidins, flavan-3-ols, proanthocyanidins, flavanones, flavones, and flavonols sub-classes were first optimized for use as standards for liquid chromatography mass spectrometry (LC–MS) with UV detection. The effect of maceration duration, reconstitution solvent, and extraction solvent were investigated using lentil genotype CDC Maxim. Chromatographic conditions were optimized by examining column separation efficiencies, organic composition, and solvent gradient. The results showed that a 1 h maceration step was sufficient and that non-acidified solvents were more appropriate; a 70:30 acetone: water (v/v) solvent was ultimately selected. Using a Kinetex PFP column, the organic concentration, gradient, and flow rate were optimized to maximize the resolution of phenolic compounds in a short 30-min analysis time. The optimized method was applied to three lentil genotypes with different phenolic compound profiles to provide information of value to breeding programs.
Keywords: Polyphenols; Lens culinaris; Extraction; Core–shell column; HPLC condition optimization;

Simultaneous determination of most prescribed antibiotics in multiple urban wastewater by SPE-LC–MS/MS by Julia Rossmann; Sara Schubert; Robert Gurke; Reinhard Oertel; Wilhelm Kirch (162-170).
A rapid analytical method was developed for the application of a long-term monitoring (>one year) of the most prescribed and often in hospitals used antibiotics in diverse wastewaters of an urban sewage treatment plant (STP). Additionally to the selected multi-class antibiotics amoxicillin, penicillin V and piperacillin (penicillins), cefotaxime and cefuroxime (cephalosporins), azithromycin, clarithromycin and roxithromycin (macrolids), ciprofloxacin and levofloxacin–ofloxacin (fluoroquinolones), clindamycin (lincosamide), doxycycline (tetracycline), sulfamethoxazole (sulfonamide) and trimethoprim (dihydrofolate reductase inhibitor), the bioactive metabolite clindamycin-sulfoxide, the reserve antibiotic vancomycin (glycopeptide) and as tracer of the STP the anticonvulsant carbamazepine and the antifungal fluconazole were involved. The analytical method combines a low-sample-volume solid phase extraction (SPE), followed by a chromatographic separation using a reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) technique, respectively, coupled to a triple quadrupole mass spectrometer. Detection was performed with multiple reaction monitoring (MRM) measured with positive electrospray ionization (ESI+). The extraction efficiency of different SPE cartridges and optimized pH-values of the preparation procedure were tested. Finally, the extraction of antibiotics was realized with the Oasis HLB cartridge and a pH adjustment at 3.5. An external calibration curve in diluted blank urine was used for quality control of the sample set of daily composite samples of the STP for the duration of one year monitoring. The squared coefficient of determination (r 2) in the concentration range (20–20,000 ng/L or 100–100,000 ng/L) of the calibration curves for the method was higher than 0.99 for all determined substances. The limit of quantification (LoQ) ranged between 0.8 ng/L (azithromycin) and 245.1 ng/L (vancomycin). Furthermore, a standard addition was used for quantification in wastewater samples. The process efficiencies ranged from 20% (doxycycline) to 134% (cefuroxime) in influent samples and from 31% (doxycycline) to 171% (cefuroxime) in effluent samples of the STP. All selected substances have been found in wastewater samples. Cefuroxime, doxycycline, levofloxacin, piperacillin, sulfamethoxazole and carbamazepine showed highest concentrations up to 6.2 μg/L.
Keywords: Antibiotics; Wastewater; HILIC; RP; SPE-HPLC–MS/MS; Prescription;

Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction by Chen Chen; Graziella El Khoury; Christopher R. Lowe (171-180).
One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7 mg GOx/ml resin and an apparent dissociation constant (K d ) of 1.45 × 10−6  M at pH 7.4. The adsorbent can also bind 8.1 mg AGP/ml resin and displays an apparent affinity constant K d  = 1.44 × 10−5  M. The ligand has a sugar specificity in the following sequence: sorbitol > fructose > mannitol > ribose > arabinose > xylose > galactose > mannose > glucose > fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and Concanavalin A and analysis of peak elution fraction with SDS-PAGE and gel densitometry showed that ligand A13C24I8 is capable of purifying GOx to 92% (w/w) with respect to the protein specific activity of 69% under current chromatographic conditions. The solid phase Ugi scaffold provides a strategy for the development of cost-effective pseudo-biospecific ligands for glycoprotein purification.
Keywords: Affinity chromatography; Boronic acid; Glycoprotein purification; Ugi reaction;

A robust two-dimensional liquid chromatography (2D-LC) method for determining the free and total concentrations of vancomycin in plasma was developed and validated. The 2D-LC system, which exhibited a strong capacity for inhibiting interference, comprised a unique RP1–IEX–RP2 column system and an “Assistant Flow” configuration. Ultrafiltration technology was employed to separate free vancomycin from the protein-bound fraction in human plasma. The influence of ultrafiltration conditions on the free vancomycin concentration was evaluated. The calibration curve was linear over the 0.195–49.92 μg/ml range for the free and total vancomycin concentrations. The within- and between-run precision ranges were 1.5–3.9% and 2.0–4.7% for the total concentration, 1.4–3.3% and 2.4–4.0% for the free concentration, respectively. Ultrafiltration was susceptible to variations in the experimental conditions, including the centrifugation time, the centrifugal force, and the nominal molecular weight limit of the ultrafiltration membrane. A total of 101 serum samples from 84 elderly patients were analyzed by this method. The free vancomycin concentration was 5.88 ± 3.75 μg/ml (range: 0.240–16.79 μg/ml), the total concentration was 12.36 ± 5.36 μg/ml (range: 2.16–27.14 μg/ml), and the unbound fraction was 45.6 ± 18.8% (range: 11.1–96.9%). There was a poor correlation between the free and total vancomycin concentrations (R 2  = 0.596, p  < 0.05). This method appears to be sensitive, precise, selective, and suitable for use in protein-binding studies of vancomycin.
Keywords: Two-dimensional liquid chromatography; Free and total concentrations; Vancomycin; Protein binding; Elderly patients;

Isolation of two new prenylated flavonoids from Sinopodophyllum emodi fruit by silica gel column and high-speed counter-current chromatography by Yanjun Sun; Yinshi Sun; Hui Chen; Zhiyou Hao; Junmin Wang; Yanbin Guan; Yanli Zhang; Weisheng Feng; Xiaoke Zheng (190-198).
Two new prenylated flavonoids, sinoflavonoids A–B, were isolated from the dried fruits of Sinopodophyllum emodi by silica gel column chromatography (SGCC) and high-speed counter-current chromatography (HSCCC). The 95% ethanol extract was partitioned with petroleum ether, dichloromethane, ethyl acetate, and n-butanol in water, respectively. The ethyl acetate fraction was pre-separated by SGCC with a petroleum ether–acetone gradient. The eluates containing target compounds were further separated by HSCCC with n-hexane–ethyl acetate–methanol–water (4:6:4:4, v/v). Finally, 17.3 mg of sinoflavonoid A and 25.9 mg of sinoflavonoid B were obtained from 100 mg of the pretreated concentrate. The purities of sinoflavonoid A and sinoflavonoid B were 98.47% and 99.38%, respectively, as determined by HPLC. Their structures were elucidated on the basis of spectroscopic evidences (HR-ESI-MS, 1H-NMR, 13C-NMR, HSQC, HMBC). The separation procedures proved to be efficient, especially for trace prenylated flavonoids.
Keywords: Sinopodophyllum emodi; New prenylated flavonoid; Silica gel column chromatography; High-speed counter-current chromatography;

Supercritical fluid extraction as a preparation method for mass spectrometry of dried blood spots by Atsuki Matsubara; Yoshihiro Izumi; Shin Nishiumi; Makoto Suzuki; Takeshi Azuma; Eiichiro Fukusaki; Takeshi Bamba; Masaru Yoshida (199-204).
The potential of supercritical fluid extraction (SFE) as a preparation method for mass spectrometry of dried blood spots (DBS) was examined. SFE is generally used for the extraction of hydrophobic compounds, but hydrophilic metabolites such as amino acids, amines, and nucleic-acid-related metabolites could be extracted by adding a low level of methanol as a modifier. Under the optimized conditions, over 200 metabolites were detected from a dried serum spot, of which over 160 metabolites could be analyzed stably (RSD <20%). These results show that SFE is an effective extraction method of metabolites with a wide range of polarity in DBS.
Keywords: Supercritical fluid extraction (SFE); Dried blood spot (DBS); Mass spectrometry (MS); Metabolite; Metabolome analysis;

The silica-supported ionic liquid (S-SIL) was prepared by impregnation and used as the dispersion adsorbent of matrix solid phase dispersion (MSPD) for the simultaneous extraction of eight phenolic acids and flavonoids, including caffeic acid, ferulic acid, morin, luteolin, quercetin, apigenin, chrysin, and kaempferide in raw propolis. High performance liquid chromatography with a Zorbax SB-C18 column (150 mm × 4.6 mm, 3.5 μm) was used for separation of the analytes. The mobile phase consisted of 0.2% phosphoric acid aqueous solution and acetonitrile and the flow rate of the mobile phase was 0.5 mL/min. The experimental conditions for silica-supported ionic liquid-based matrix solid phase dispersion (S-SIL-based MSPD) were optimized. S-SIL containing 10% [C6MIM]Cl was used as dispersant, 20 mL of n-hexane as washing solvent and 15 mL of methanol as elution solvent. The ratio of S-SIL to sample was selected to be 4:1. The standard curves showed good linear relationship (r  > 0.9995). The limits of detection and quantification were in the range of 5.8–22.2 ng mL−1 and 19.2–74.0 ng mL−1, respectively. The relative standard deviations (RSDs) of intra-day and inter-day determination were lower than 8.80% and 11.19%, respectively. The recoveries were between 65.51% and 92.32% with RSDs lower than 8.95%. Compared with ultrasound-assisted extraction (UAE) and soxhlet extraction, the present method consumed less sample, organic solvent, and extraction time, although the extraction yields obtained by S-SIL-based MSPD are slightly lower than those obtained by UAE.
Keywords: Silica-supported ionic liquid-based matrix solid phase dispersion (S-SIL-based MSPD); Phenolic acids and flavonoids; Raw propolis; High-performance liquid chromatography (HPLC);

A simple and rapid ultra-high performance liquid chromatography–mass spectrometry/mass spectrometry (UPLC–MS/MS) method has been developed for measuring intracellular concentrations of the anticancer agent 7-ethyl-10-hydroxycamptothecin (SN-38) in tumour cells using camptothecin (CPT) as internal standard. SN-38 extraction was carried out using acidified acetonitrile. SN-38 and CPT were separated on a PFP column using gradient elution with acidified water and acetonitrile. SN-38 and CPT were quantified using a triple quadrupole mass spectrometry system. Least square regression calibration lines were obtained with average correlation coefficients of R 2  = 0.9993 ± 0.0016. The lower limit of detection (LOD) and lower limit of quantification (LOQ) for SN-38 were 0.1 and 0.3 ng/ml, respectively. CPT recovery was 98.5 ± 13% and SN-38 recoveries at low quality control (LQC, 5 ng/ml) and high quality control (HQC, 500 ng/ml) were 89 ± 6% and 95 ± 8%, respectively. The intra- and inter-day imprecision for LQC was 5.8 and 8.5%, and for HQC was 6.3 and 4.4%, respectively. The method was compared to a validated high performance liquid chromatography-fluorescent method. In addition, the method has been successfully applied to determine the intracellular accumulation of SN-38 investigating the transport through ABCB1 (P-gp) and ABCG2 (BCRP) efflux pumps in colorectal cancer cell lines.
Keywords: SN-38; Ultra-high performance LC–MS/MS; HPLC-fluorescence; Method validation; Method comparison;

A simple and rapid high performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of pioglitazone and its active metabolites hydroxypioglitazone and ketopioglitazone in human plasma. Samples were processed by protein precipitation with acetonitrile and selective phospholipid depletion in a 96-well plate format. The method used deuterated internal standards for each analyte. Chromatographic separation was achieved with gradient elution on a Hypersil GOLD C18 column. The mass spectrometer was operated in electrospray positive ion mode with detection by selected reaction monitoring using the transitions m/z 357.1 > 134.0 for pioglitazone, m/z 373.1 > 150.0 for hydroxypioglitazone, and m/z 371.0 > 148.0 for ketopioglitazone. A linear standard curve was established for the range of 10–1800 ng/mL for all three analytes. Intra-run and inter-run precision and accuracy (relative error) were less than 15%, and the mean extraction recoveries of all analytes were more than 87.8%. The validated method is sensitive and selective and was successfully applied to analyze clinical samples obtained from patients with nonalcoholic fatty liver disease taking pioglitazone.
Keywords: Pioglitazone; Metabolites; Liquid chromatography; Mass spectrometry; LC–MS/MS; Human plasma;

Determination of tea catechins in human plasma might provide a means of better evaluation of their benefits. The main difficulty in their analysis is the low catechins concentrations in plasma and their susceptible to oxidation during sample pretreatment. In the current work, a sweeping-micellar electrokinetic chromatography (sweeping-MEKC) by long alkyl chain ionic liquid was investigated for the simultaneous determination of seven principal naturally-occurring tea catechins in human plasma under acidic conditions after the intake of green tea beverage. The effects of type and concentration of three 1-tetradecyl-3-methylimidazolium ionic liquids, namely bromide, acetate and hydrogen sulfate salts were studied. The seven catechins were successfully separated within 5 min by micellar running buffer of 5 mmol L−1 1-tetradecyl-3-methylimidazolium bromide and 15 mmol L−1 phosphate buffer at pH 4.5 under optimal parameters of 50 mbar injection for 150 s, 10 kV, 25 °C and 200 nm. To prevent the possibility of IL adsorption, an appropriate rinsing protocol was established. The method has analytical ranges from 0.5, 1, 0.5, 1, 2, 1 and 1 to 500 ng mL−1 for GC, C, EC, EGCG, GCG, ECG and EGC, respectively (r ranged from 0.995 to 0.999). The intraday precision and accuracy were 0.1–0.9% RSD (n  = 10) and 97–106% recovery, respectively. Limits of detections of analytes were ranged from 0.2 to 1.2 ng mL−1. The current sweeping-MEKC achieved sensitivity enhancement factor (SEF) up to 183-fold of analytes concentrations compared to other hitherto published CE reports that is suitable to find out the trace amounts of catechins in plasma.
Keywords: Micellar electrokinetic chromatography; Sweeping; Ionic liquids; Catechins; Plasma;

Development and validation of a method for determination of plasma 25-hydroxyvitamin D3 3-sulfate using liquid chromatography/tandem mass spectrometry by Tatsuya Higashi; Ayaka Goto; Misato Morohashi; Shoujiro Ogawa; Kenji Komatsu; Takahiro Sugiura; Tetsuya Fukuoka; Kuniko Mitamura (230-234).
The quantification of plasma 25-hydroxyvitamin D3 3-sulfate [25(OH)D3S] is expected to be helpful in the assessment of the vitamin D status, especially for infants. In this study, a simple and sensitive method for the quantification of 25(OH)D3S in plasma using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was deproteinized with acetonitrile, purified using an Oasis® HLB cartridge, and subjected to LC/ESI-MS/MS operating in the negative-ion mode. Quantification was based on the selected reaction monitoring, and deuterated 25(OH)D3S was used as the internal standard. This method enabled the reproducible (intra- and inter-assay relative standard deviations, 7.9% or lower) and accurate (analytical recovery, 95.8–105.3%) quantification of the plasma 25(OH)D3S using a 20-μL sample, and the limit of quantification was 2.5 ng/mL. The developed method was applied to the determination of plasma 25(OH)D3S in infants; the result revealed that preterm infants have lower plasma 25(OH)D3S concentrations.
Keywords: 25-Hydroxyvitamin D3 3-sulfate; LC/ESI-MS/MS; Plasma; Infant; Vitamin D status;

A quantitative LC-MS/MS method for determination of SP-141, a novel pyrido[b]indole anticancer agent, and its application to a mouse PK study by Subhasree Nag; Jiang-Jiang Qin; Shivaputra Patil; Hemantkumar Deokar; John K. Buolamwini; Wei Wang; Ruiwen Zhang (235-240).
In the present study, a specific and sensitive liquid chromatography-triple quadrupole mass spectrometry method was developed and validated for the determination of SP-141, a novel pyrido[b]indole anticancer agent. After a liquid–liquid extraction with n-hexane–dichloromethane–2-propanol (20:10:1, v/v/v) mixture, the analyte was separated on a Kinetex C18 column (50 × 2.1 mm, 2.6 μm) with mobile phases comprising of water (0.1% formic acid, v/v) and acetonitrile (0.1% formic acid, v/v) at a flow rate of 0.4 mL/min. The test compound (SP-141) and the internal standard (SP-157) were analyzed in the multiple reaction-monitoring mode using the mass transitions m/z 325.1 → 282.0. The method was linear in the concentration range of 0.648–162 ng/mL with coefficients of determination (R 2) of 0.999 in mouse plasma. The lower limit of quantification was 0.648 ng/mL. The intra- and inter-day assay precisions (coefficient of variation, %CV) were less than 4.2% and accuracies (relative error, %RE) ranged from −6.1% to 2.1%. The extraction recoveries were between 97.1 and 103.1% and the relative matrix effect was minimal. In addition, SP-141 was found to be stable in the plasma after three freeze-thaw cycles, at 37 °C and 4 °C for 24 h, and at −80 °C for 4 weeks. It was also stable in the stock solution at room temperature for 24 h and after preparation in the autosampler for 36 h. The validated method was successfully applied to an initial pharmacokinetic study of SP-141 in CD-1 mice following intraperitoneal and intravenous administrations.
Keywords: SP-141; Pyrido[b]indole; LC-MS/MS; Mouse plasma; Pharmacokinetics;

Use of mep HyperCel for polishing of human serum albumin by Karl B. McCann; Yvonne Vucica; John Wu; Joseph Bertolini (241-248).
The manufacture of human serum albumin by chromatographic procedures involves gel filtration chromatography as a final polishing step. Despite this step being essential to remove high molecular weight impurity proteins and thus ensure a stable and safe final product, it is relatively inefficient. This paper explores the use of hydrophobic charge induction chromatographic media, MEP HyperCel as an alternative to Sephacryl S200HR gel filtration for the polishing of human serum albumin derived by ion exchange chromatographic purification of Cohn Supernatant I. The use of MEP HyperCel results in a product with a higher purity than achieved with gel filtration and in a less time consuming manner and with potential resource savings. MEP HyperCel appears to have great potential for incorporation into downstream processes in the plasma fractionation industry as an efficient means of achieving polishing of intermediates or capture of proteins of interest.
Keywords: MEP HyperCel; Human serum albumin; Hydrophobic charge induction chromatography; Plasma;

Plasma pharmacokinetics and tissue distribution study of roemerine in rats by liquid chromatography with tandem mass spectrometry (LCMS/MS) by Yue-Qiong Liu; Gong-Hao He; Hong-Liang Li; Jiang-Chang He; En-Fu Feng; Lan Bai; Cheng-Ying Wang; Gui-Li Xu (249-255).
In the present study, a new LC–MS/MS method for the determination of roemerine in rat plasma and tissue samples was developed and successfully used to study the pharmacokinetics and tissue distribution of roemerine after oral and intravenous (i.v.) administration in rats. The plasma and tissue samples were processed by liquid–liquid extraction with n-hexane. Isocorydine was used as the internal standard (IS) for sample processing and analysis. The MS/MS detection was carried out by monitoring the transitions of m/z 280→249 and m/z 342→279 for roemerine and the IS, respectively. The calibration curve displayed excellent linearity over the concentration range of 10–2000 ng/mL (n  = 8, r 2  ≥ 0.995), and the lower limit of quantification (LLOQ) was determined to be 10 ng/mL. This method was rapid, accurate, highly sensitive, and fully validated. The pharmacokinetic study showed that roemerine was rapidly absorbed and eliminated with a t max of 0.22 ± 0.08 h, t 1/2 of 1.59 ± 0.46 h, CL of 4.44 ± 0.42 L/h/kg, and V d of 10.16 ± 2.95 μg/L following oral administration. Additionally, roemerine showed an excellent oral bioavailability of 84% and a wide tissue distribution with brain penetration. Highest concentrations of roemerine were found in the liver and lung, followed by kidney, spleen, heart, and brain, in that order.
Keywords: Roemerine; Pharmacokinetics; Tissue distribution; LC–MS/MS;

Evaluation of a multimodal resin for selective capture of CHO-derived monoclonal antibodies directly from harvested cell culture fluid by Kimberly A. Kaleas; Margaret Tripodi; Scott Revelli; Vikas Sharma; Shelly A. Pizarro (256-263).
This proof-of-concept study examines the applicability of using multimodal chromatography to selectively capture recombinantly produced monoclonal antibodies (mAb) directly from harvested mammalian cell culture fluid (HCCF) with minimal optimization. Capto MMC is a multimodal resin that contains a ligand with the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. Twelve mAb HCCF feedstocks were examined for dynamic binding capacity (DBC) and then two representative feedstocks were selected to develop a systematic approach for elution buffer development. A range of dynamic binding capacities was observed for 10 feedstocks (24–53 g/L) and two feedstocks had poor binding properties (<10 g/L) despite load conditioning towards a more favorable pH. Analysis of the DBC versus molecular properties showed that the mAb-ligand binding interaction was predominantly charge based. Four separate elution strategies were identified to selectively recover the mAb and could be applied with minimal optimization to other mAb feedstocks. Downstream processing of the Capto MMC pools showed that it is feasible to produce material with comparable purity to a process with affinity capture after two chromatography steps.
Keywords: Capto MMC; Multimodal chromatography; Mammalian cell culture fluid; Monoclonal antibody; Selective capture;

Panax notoginseng saponins (PNS) constitute the main active components of a traditional Chinese medicine, Panax notoginseng (Burk.) F.H. Chen (Sanqi). To investigate brain distribution of Panax Notoginsenoside R1, Ginsenosides Rg1, Rb1, Re, and Rd, and the integrated PNS in rats, their contents in cortex, striatum, hypothalamus, medulla oblongata, hippocampus and olfactory bulb were simultaneously measured by UPLC-MS/MS. Sample preparation was carried out by the protein precipitation technique with an internal Digoxin standard. The method described here was highly efficient, with short run time, excellent specificity and sensitivity, and successfully applied for pharmacokinetics studies. NGR1, GRg1, GRb1, GRe and GRd from PNS have been detected in all six brain regions studied and quantified accurately. These findings provide more insight for further understanding of the main ways from the nasal cavity to brain as well as the migration of nasally applied drugs into the CNS parenchyma.
Keywords: Panax Notoginseng Saponins; Brain distribution; Pharmacokinetics; Integrated pharmacokinetics; Intranasal administration;

Simultaneous detection of 93 conventional and emerging drugs of abuse and their metabolites in urine by UHPLC-MS/MS by Magdalene H.Y. Tang; C.K. Ching; Caroline Y.W. Lee; Ying-Hoo Lam; Tony W.L. Mak (272-284).
Novel psychoactive substances (NPS) are becoming increasingly popular worldwide in recent years, some of which have been reported to cause considerable harm and even fatalities. Currently, simultaneous screening for a comprehensive panel of conventional and novel drugs of abuse is not widely available in most clinical laboratories. The aim of this study was to establish a chromatography/mass spectrometry-based analytical system for the simultaneous detection of conventional drugs of abuse and NPS in urine. Sample preparation entails enzyme digestion and solid phase extraction; analytes were then detected by liquid-chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring. Forty-seven conventional drugs (28 parent drugs, 19 metabolites) and 46 NPS analytes (44 parent drugs, two metabolites) are covered by the established method, which has been validated according to international guidelines. The method was then applied to 964 urine samples collected from drug abusers and the results revealed the presence of two NPS - TFMPP and methcathinone – as well as conventional drugs of abuse. To conclude, an LC-MS/MS method has been established that allows the simultaneous detection of over 90 conventional as well as novel psychoactive substances and metabolites in urine samples. The method was successfully applied to authentic specimens revealing the presence of conventional as well as novel drugs of abuse in the local population.
Keywords: Novel psychoactive substance; Drugs of abuse; LCMS; Urine analysis; Drug screening;

Poliumoside is one of the major phenylethanoid glycosides (PhGs) isolated from Callicarpae Caulis et Folium (CCF) which is a traditional Chinese medicine used for hemostasis in clinic. In this study, high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC/Q-TOF-MS) was applied to investigate the metabolites of poliumoside in rat feces after oral administration. A total of 66 metabolites were confirmed or tentatively identified. Poliumoside could be partly transformed into its positional isomer isopoliumoside in vivo, and poliumoside was easily hydrolyzed and metabolized into degradation products. The parent compound and its degradation products could further undergo extensive phase I and phase II metabolism. The results indicated that hydrolysis, hydroxylation, acetylation, sulfation, hydration, reduction, dehydrogenation and dimethylation were the major metabolic pathways of poliumoside. The major metabolic soft spots of poliumoside and the fragmentation patterns of the metabolites were also proposed. This study provided valuable information regarding the metabolites of poliumoside in rats.
Keywords: Poliumoside; Phenylethanoid glycosides; Rat feces; Metabolite identification; HPLC/Q-TOF-MS;