Journal of Chromatography B (v.879, #30)

Quantitative analysis of peptides in biological matrices remains a challenging task. This is due to the low dosage and the complexity of both the matrix and the analytical characteristics of peptides. SS-20 is a tetrapeptide compound developed for the treatment of Parkinson's disease. To investigate the pharmacokinetics of SS-20, a sensitive and rapid liquid chromatography coupled with mass spectrometry method was developed and validated. An aliquot of 50 μL plasma sample was extracted via solid phase extraction. The extracts were separated using a hydrophilic interaction liquid chromatography column, and were then detected with a triple quadrupole mass spectrometer using electrospray ionization in positive-ion mode and selected reaction monitoring. The use of a deuterium-labeled internal standard provided acceptable accuracy, precision, and matrix effect. The lower limit of quantification was 0.30 ng/mL. The linear range of the method was from 0.30 to 1000 ng/mL. The intraday and interday precisions were lower than 10.2% in terms of relative standard deviation, and the accuracy was within ±2.1% in terms of relative error. The validated LC–MS/MS method was successfully applied to a pharmacokinetic study of SS-20 following an intravenous or subcutaneous injection administration of 1.0 mg/kg to Sprague-Dawley rats.
Keywords: Hydrophilic interaction liquid chromatography; Liquid chromatography tandem mass spectrometry; Peptide; Pharmacokinetics;

Determination of volatile organic compounds as biomarkers of lung cancer by SPME–GC–TOF/MS and chemometrics by Joanna Rudnicka; Tomasz Kowalkowski; Tomasz Ligor; Bogusław Buszewski (3360-3366).
A method for qualitative and quantitative the determination of concentrations volatile organic compounds (VOCs) in human breath samples using solid phase microextraction (SPME) and gas chromatography–time of flight–mass spectrometry (GC–TOF/MS) has been carried out. They are employed for the preconcentration, separation and analysis of biological samples. The technique to rapid determination compounds present in human air, at the level of parts per billion (ppb) is applied. This method was optimized and evaluated. It showed linear correlations ranging from 0.83 to 234.05 ppb, limit of detection in the range of 0.31 to 0.75 ppb and precision, expressed as the RSD, was less then 10.00%. The unique combination of statistical methods allowed reduce the number of compounds to significant ones only and indicate the potential way to find the biomarkers of the lung cancer. Presented an analytical and statistical methods for detection composition of exhaled air could be applied as a potential non-intrusive tool for screening of lung cancer.
Keywords: Lung cancer; Volatile organic compounds; Solid phase microextraction; Gas chromatography–time of flight–mass spectrometry; Statistics;

Oral fluid (OF) is an alternative matrix for monitoring drugs of abuse in workplace, clinical toxicology, criminal justice, and driving under the influence of drugs (DUID). OF is suitable for detection of drugs that have been taken recently. It is unproblematic to observe the collection and hence avoid the possibility of the samples being tampered. OF often contains compounds in low concentrations, and small volumes are often collected. It is therefore necessary to have a sensitive, multi component method for drug detection. In this study an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS) method has been developed. The samples were prepared by liquid–liquid extraction (LLE) with ethyl acetate/heptane (4:1) and the separation was achieved by an Acquity HSS T3-column (2.1 mm × 100 mm, 1.8 μm particles). Mass detection was performed by positive ion mode electrospray MS–MS. 32 drugs of abuse were determined with a cycle time of 9 min. Stability of drugs in oral fluid before analysis is an important factor that must be evaluated for each sampling device. The collection devices Intercept® and StatSure Saliva Sampler™ were tested using pools of real samples containing various drugs. The testing showed that 6-MAM (6-acetylmorphine), cocaine and zopiclone were the least stable compounds. In the testing for short term stability, StatSure Saliva Sampler™ showed better results. The testing of 1 year of storage at −20 °C showed that most of the compounds were stable for both sampling devices, except for 6-MAM, cocaine and zopiclone. Samples of OF should be analysed as soon as possible after collection, and they should be kept frozen if immediate analysis is not possible.
Keywords: Drugs of abuse; Oral fluid; Stability; Ultra-performance liquid chromatography–tandem mass spectrometry; Multi-component analysis;

Quantification of intracellular and extracellular prostanoids stimulated by A23187 by liquid chromatography/electrospray ionization tandem mass spectrometry by Ayako Furugen; Hiroaki Yamaguchi; Nobuaki Tanaka; Hajime Ito; Kazuaki Miyamori; Asuka Fujikawa; Natsuko Takahashi; Jiro Ogura; Masaki Kobayashi; Takehiro Yamada; Nariyasu Mano; Ken Iseki (3378-3385).
Prostanoids are bioactive substances that contribute to various biological and pathological processes. To evaluate both extracellular and intracellular levels of prostanoids at the same time, we developed methods for quantification of extracellular and intracellular levels of prostanoids, including prostaglandin E2 (PGE2), PGD2, PGF, 6-keto PGF, and TXB2, in cultured cells using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and we validated the LC/MS/MS methods. A solid-phase extraction cartridge was used for extraction of prostanoids. The prostanoids were separated by a C18 column with an isocratic flow of acetonitrile/water/acetic acid (40:60:0.1, v/v/v). Calibration curves of extracellular measurement for the prostanoids were linear in the range from 0.1 to 100 ng/mL (r 2  > 0.999), and those of intracellular measurement were linear in the range from 0.05 to 50 ng (r 2  > 0.999). Validation assessment showed that both methods of extracellular and intracellular measurements were highly reliable with good accuracy and precision. We also applied the methods to human airway epithelial Calu-3 cells and human lung adenocarcinoma epithelial A549 cells.
Keywords: Prostanoid; LC/MS/MS; Intracellular measurement;

► Multiple reaction monitoring (MRM) quantifies TJ0711 HCl. ► A MRM-IDA-EPI method supports metabolite identification of TJ0711 HCl. ► Demethylation and hydroxylation are the principle metabolism pathways for TJ0711 HCl.In this paper, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous analysis of metabolic stability and metabolite profiling of 1-[4-(2-methoxyethyl) phenoxy]-3-[[2-(2-methoxyphenoxy) ethyl]amino]-2-propanol hydrochloride (TJ0711 HCl), a new vasodilatory β-blocker. Multiple reaction monitoring (MRM) was used as a survey scan to quantify the parent compound and to trigger the acquisition of enhanced product ions (EPI) for the identification of formed metabolites. In addition, comparison between MRM-only and MRM-information dependent acquisition-EPI (MRM-IDA-EPI) methods was conducted to determine analytical variables, including linearity, limit of detection (LOD), lower limit of quantification (LLOQ), as well as intra-day and inter-day accuracy and precision. Results demonstrated that MRM-IDA-EPI quantitative analysis was not affected by the addition of EPI scans to obtain qualitative information during the same chromatographic run, compared to MRM-only method. Thereafter, metabolic stability and metabolite identification of TJ0711 HCl were investigated using human liver microsomes (HLM) by the MRM-IDA-EPI method. The in vitro metabolic stability parameters were calculated and t 1/2, microsomal intrinsic clearance (CLint), as well as hepatic CL, were 13.0 min, 106.5 μL/min/mg microsomal protein, and 1082.2 mL/min, respectively. The major formed metabolites were also simultaneously monitored and the metabolite profiling data demonstrated that this MRM-IDA-EPI method was capable of targeting a large number of metabolites, in which demethylation and hydroxylation were the principle metabolism pathways during the in vitro incubation with HLM.
Keywords: LC–MS/MS; Metabolic stability; Metabolite profiling; TJ0711 hydrochloride;

Quantitative subproteomic analysis of age-related changes in mouse liver peroxisomes by iTRAQ LC–MS/MS by Hanna Amelina; Marcus O.D. Sjödin; Jonas Bergquist; Susana Cristobal (3393-3400).
Aging is a complex multifactorial phenomenon, which is believed to result from the accumulation of cellular damage to biological macromolecules. Peroxisomes recently emerged as another important source of reactive oxygen species (ROS) production in addition to mitochondria. However, the role of these organelles in the process of aging is still not clear. The aim of this study was to characterize the changes in protein expression profiles of young (10 weeks old) versus old (18 months old) mouse liver peroxisome-enriched fractions. We have applied shotgun proteomic approach based on liquid chromatography and tandem mass spectrometry (LC–MS/MS) combined with iTRAQ (isobaric tags for relative and absolute quantitation) labeling that allows comparative quantitative multiplex analysis. Our analysis led to identification and quantification of 150 proteins, 8 out of which were differentially expressed between two age groups at a statistically significant level (p  < 0.05), with folds ranging from 1.2 to 4.1. These proteins involved in peroxisomal β-oxidation, detoxification of xenobiotics and production of ROS. Noteworthy, differences in liver proteome have been observed between as well as within different age groups. In conclusion, our subproteomic quantitative study suggests that mouse liver proteome is sufficiently maintained until certain age.
Keywords: Liquid chromatography; Tandem mass spectrometry; Peroxisomes; Aging; Liver; iTRAQ;

Bioanalysis in clinical development of tasquinimod using liquid chromatography/tandem mass spectrometry by G.P. Hansson; M. Olin; C. Svanström; L.D. Svensson; C.J. Sennbro (3401-3406).
Tasquinimod (ABR-215050) is an oral drug in clinical development for treatment of patients with castrate resistant prostate cancer. This paper describes a method for the determination of tasquinimod in human plasma. The method is based on liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) using stable isotope labeled tasquinimod as internal standard (IS). The plasma samples were processed by protein precipitation using acidic acetonitrile containing the IS. The precipitated samples were centrifuged and the supernatant was injected directly into the LC–MS/MS system. Chromatographic separation was performed on a reversed phase column using fast gradient elution, with a total run cycle time of 4 min. The method was validated with respect to accuracy, precision, dynamic range, lower limit of quantification, selectivity and robustness. Furthermore, the stability of tasquinimod in spiked plasma, in processed extracts and in incurred samples was thoroughly studied. The method was validated in the range of 1.0–2400 nmol/L, defining the lower and upper limits of quantification. The repeatability, reproducibility and overall bias were 1.5–7.1%, 3.5–7.4%, and 1.3–4.7%, respectively, in the range of 1–2000 nmol/L. Excellent selectivity was demonstrated in the validation, as well as in study samples from both healthy volunteers and cancer patients. Robustness was demonstrated by the calculated carry-over as low as 0.06%, and by an incurred sample reproducibility (ISR) experiment where 97% of the reanalyzed samples fulfilled the acceptance criteria of 20% deviation from initial analysis result. Also, tasquinimod was found to be stable in all investigated matrices, including in incurred samples. In an incurred sample stability (ISS) investigation, tasquinimod was demonstrated to be stable for 24 months, and 97% of the reanalyzed samples were within 20% from the initial analysis result. In conclusion, the method was demonstrated to be accurate, precise, robust and reliable for the determination of tasquinimod. The method was successfully used in several clinical studies for the support of pharmacokinetic and pharmacodynamic evaluations.
Keywords: Tasquinimod; Prostate cancer; Bioanalysis; Incurred sample stability; Stable isotope; Internal standard; LC–MS;

► Positive/negative ion-switching ESI and segmental monitoring were used in the method. ► Two internal standards were used for the analytes in the different segments. ► Sodium acetate was used to stabilize and promote the ionization effect of the [M+Na]+. ► The MRM collision energy was optimized to eliminate the interference from plasma. ► Pharmacokinetics of the analytes in rats was reported for the first time.A new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method operated in the positive/negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma. After addition of internal standards diazepam (for asperosaponin VI) and glycyrrhetic acid (for hederagenin), the plasma sample was deproteinized with acetonitrile, and separated on a reversed phase C18 column with a mobile phase of methanol (solvent A)–0.05% glacial acetic acid containing 10 mM ammonium acetate and 30 μM sodium acetate (solvent B) using gradient elution. The detection of target compounds was done in multiple reaction monitoring (MRM) mode using a tandem mass spectrometry equipped with positive/negative ion-switching ESI source. At the first segment, the MRM detection was operated in the positive ESI mode using the transitions of m/z 951.5 ([M+Na]+) → 347.1 for asperosaponin VI and m/z 285.1 ([M+H]+) → 193.1 for diazepam for 4 min, then switched to the negative ESI mode using the transitions of m/z 471.3 ([M−H]) → 471.3 for hederagenin and m/z 469.4 ([M−H]) → 425.4 for glycyrrhetic acid, respectively. The sodiated molecular ion [M+Na]+ at m/z 951.5 was selected as the precursor ion for asperosaponin VI, since it provided better sensitivity compared to the deprotonated and protonated molecular ions. Sodium acetate was added to the mobile phase to make sure that abundant amount of the sodiated molecular ion of asperosaponin VI could be produced, and more stable and intensive mass response of the product ion could be obtained. For the detection of hederagenin, since all of the mass responses of the fragment ions were very weak, the deprotonated molecular ion [M−H] m/z 471.3 was employed as both the precursor ion and the product ion. But the collision energy was still used for the MRM, in order to eliminate the influences induced by the interference substances from the rat plasma. The validated method was successfully applied to study the pharmacokinetics of asperosaponin VI and its active metabolite hederagenin in rat plasma after oral administration of asperosaponin VI at a dose of 90 mg/kg.
Keywords: Asperosaponin VI; Hederagenin; Liquid chromatography–tandem mass spectrometry; Ion-switching; Pharmacokinetics;

A robust and sensitive method using liquid chromatography–tandem mass spectrometry was developed and validated for the simultaneous determination of a novel topoisomerase 1 inhibitor CH0793076 (3076), the prodrug CH4556300 (TP300), and the active metabolite CH0793011 (3011) in human plasma. All plasma analyzed with this method was acidified with 1 M HCl and 46% citric acid solution in a ratio of 100:10:1 (v:v:v) to avoid the pH-based degradation of TP300 and to shift the equilibria of 3076 and 3011 between the lactone and carboxylate forms towards the lactone forms. After the plasma proteins were precipitated with methanol:acetonitrile:HCl 1 M (50:50:1, v:v:v) containing stable isotopic internal standards, the analytes were trapped on an Xterra MS C18 column (10 × 2.1 mm i.d., 5 μm) and separated on a Gemini C18 column (50 × 2.0 mm i.d., 5 μm) using column-switching liquid chromatography. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to quantify the analytes with transitions m/z 587.2 > 441.2 for TP300, 459.1 > 415.2 for 3076, and 475.1 > 361.1 for 3011. The inter- and intra-day precisions were below 12%, and the accuracy was between −16% and 16% at the lower limit of quantitation (LLOQ) and between −11% and 14% at the other quality controls. The LLOQs of TP300, 3076, and 3011 were 0.8, 0.04, and 0.04 ng/mL, respectively. The validated method was successfully applied to clinical sample analysis and incurred sample reanalysis was also conducted.
Keywords: LC–MS/MS; Human plasma; Topoisomerase 1 inhibitor; Column switching; Pharmacokinetics;

► A modified QuEChERS method was used to extract pesticide residues from peanut oil. ► Multi-walled carbon nanotubes and alumina were used as adsorbent for clean-up step. ► The method has stronger purifying ability and higher sensitivity.The organophosphorus pesticides including phorate, diazinon, tolclofos-methyl, fenitrothin, malathion, fenthion, isocarbophos, quinalphos and phenamiphos, in peanut oils were determined by liquid–liquid extraction coupled with dispersive solid phase extraction and gas chromatography–mass spectrometry (GC–MS). The mixture of multi-walled carbon nanotubes and alumina was used as adsorbent in dispersive solid phase extraction. The effects of some experimental conditions, such as types of multi-walled carbon nanotubes, amount of adsorbents and extraction time were examined. The limits of detection for the analytes were between 0.7 and 1.6 μg kg−1. The obtained recoveries of the analytes in the samples were between 85.9 and 114.3% and relative standard deviations were lower than 8.48%.
Keywords: Multi-walled carbon nanotubes; Organophosphorus pesticides; Peanut oil; Dispersive solid phase extraction; Gas chromatography–mass spectrometry;

► A preconcentration method of the celastrol was first developed with UA IL-DLLME. ► Sonication contributed to dispersion of IL into the sample solution. ► 110-fold enrichment factor was obtained and the limit of detection was 1.6 μg/L (S/N).Ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction (UA IL-DLLME) coupled with high-performance liquid chromatography (HPLC) has been developed for the determination of celastrol in human urine samples. In the microextraction procedure, ionic liquid (IL) was used as extraction solvent and dispersed into the aqueous sample solution as fine droplets by means of dispersive solvent and ultrasonication which promoted the analyte to migrate into IL phase more easily. Several important parameters affecting the extraction efficiency were studied and optimized, including the type and volume of extraction solvent and dispersive solvent, sample pH, ultrasonication time, cooling time, centrifugation time and salting-out effect. Under the optimized conditions, 110-fold enrichment factor was obtained and the limit of detection (LOD) was 1.6 μg/L at a signal-to-noise ratio of 3. The calibration curve was linear over the range of 10–1000 μg/L for celastrol in human urine sample, with a correlation coefficient of 0.9980. Intra- and inter-assay precision were 0.43% and 2.78%, respectively. The proposed method was successfully applied to the real human urine samples and good spiked recoveries in the range of 93.2–109.3% were obtained.
Keywords: Ultrasound-assisted IL-DLLME; HPLC; Celastrol; Urine sample;

Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV by Fazli Nasir; Zafar Iqbal; Abad Khan; Lateef Ahmad; Yasar Shah; Amir Zada Khan; Jamshaid Ali Khan; Salimullah Khan (3434-3443).
► We developed a simple validated HPLC-UV method for the simultaneous analysis of timolol maleate (TM), rosuvastatin (RST), and diclofenac sodium (DS). ► The developed method was successfully applied in pharmaceuticals and physiological fluids. ► The suggested method will be used for the pharmacokinetics studies of these drugs in animal models.A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C18 column (250 mm × 4.6 mm, 5 μm), applying 0.2% triethylamine (TEA) and acetonitrile (ACN) (40:60, v/v), in isocratic mode as mobile phase, pH 2.75 adjusted with 85% phosphoric acid at a flow rate of 1 ml/min. The column oven temperature was kept at 45 °C and the peak response was monitored at 284 nm after injecting a 50 μl sample into HPLC system. The direct liquid–liquid extraction procedure was applied to human plasma and bovine aqueous humor samples using mobile phase as an extraction solvent after deproteination with methanol. The different HPLC experimental parameters were optimized and the method was validated according to standard guidelines. The recoveries of the suggested method in human plasma were 98.72, 96.04, and 95.14%, for TM, RST, and DS, while in aqueous humor were 94.99, and 98.23%, for TM, and DS, respectively. The LOD values were found to be 0.800, 0.500, and 0.250 ng/ml, for TM, RST, and DS, respectively, while their respective LOQ values were 2.00, 1.50, and 1.00 ng/ml. The co-efficient of variation (CV) were in the range of 0.1492–1.1729% and 1.0516–4.0104%, for intra-day and inter-day studies, respectively. The method was found accurate in human plasma and bovine aqueous humor and will be applied for the quantification of these compounds in plasma, and aqueous humor samples using animal models and in pharmaceuticals.
Keywords: Timolol; Rosuvastatin; Diclofenac sodium; Plasma; Aqueous humor; Recovery;

Preparation of high purity biphenyl cyclooctene lignans from Schisandra extract by ion exchange resin catalytic transformation combined with macroporous resin separation by Chun-hui Ma; Ting-ting Liu; Lei Yang; Yuan-gang Zu; Feng-jian Yang; Chun-jian Zhao; Lin Zhang; Zhong-hua Zhang (3444-3451).
► Analysis of four kinds of biphenyl cyclooctene lignans by LC–ESI-MS simultaneously. ► Hydrolysis ester-bound lignans by ion exchange resin instead of inorganic catalyst. ► The adsorption isotherm analysis by Langmuir and Freundlich model simultaneously.In this study, ester-bond biphenyl cyclooctene lignans were efficiently hydrolytically degraded into free biphenyl cyclooctene lignans by ion exchange resin transformation and simultaneous removal of impurities by macroporous resin. The OH-type strongly basic anion exchange resin 201 × 7 was the best one, and the dynamic hydrolysis efficiency was 146.7 ± 5.0%. HPD5000 macroporous resin, which offered higher adsorption and desorption capacities and faster adsorption than other resins. The purity of free biphenyl cyclooctene lignans in the product increased from 5.14 ± 0.24% to 79.67 ± 0.0.67%. After dynamic catalytic transformation by 201 × 7 resin combined with purification of HPD5000 resin, the yield and the purity of free biphenyl cyclooctene lignans in the product were 132.1 ± 4.7% and 80.91 ± 3.53%, respectively.
Keywords: Schisandra chinensis; Biphenyl cyclooctene lignans; Catalytic hydrolysis; Ion exchange resin; Adsorption and desorption; Macroporous resin;

Determination of HS270, a new histone deacetylase inhibitor, in rat plasma by LC–MS/MS—Application to a preclinical pharmacokinetic study by Guo-Ping Zhong; Jiang-Ying Chen; Hui-Chang Bi; Xiao-Ling Qin; Chang-Liang Dai; Jing Liu; Xiao Chen; Gui-Xiong Zeng; Zhi-Ying Huang; Min Huang (3452-3458).
► HS270, a new histone deacetylase inhibitor, is a promising anticancer drug. ► We developed a LC–MS/MS method to determine HS270 in rat blood. ► The method was validated with high sensitivity, specificity and efficiency. ► The method was also with acceptable precision and accuracy. ► The method was successfully applied in a preclinical pharmacokinetic study.A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated to determine HS270, a new histone deacetylase (HDAC) inhibitor, in rat plasma using SAHA as the internal standard (IS). After a single step liquid–liquid extraction with acetoacetate, analytes were subjected to LC–MS/MS analysis using positive electro-spray ionization (ESI+) under selected reaction monitoring mode (SRM). The chromatographic separation was achieved on a Hypurity C18 column (50 mm × 2.1 mm, i.d., 5 μm). The MS/MS detection was conducted by monitoring the fragmentation of m/z 392.3 → 100.1 for HS270, m/z 265.1 → 232.1 for IS. The method had a chromatographic running time of 2.5 min and linear calibration curves over the concentrations of 0.5–1000 ng/mL. The recovery of the method was 70.8–82.5% and the lower limit of quantification (LLOQ) was 0.5 ng/mL. The intra- and inter-batch precisions were less than 15% for all quality control samples at concentrations of 1.0, 100.0, and 750.0 ng/mL. The validated LC–MS/MS method has successfully applied to a HS270 pharmacokinetic study after oral doses of 25, 50, 100, 200 mg/kg, and i.v. dose of 5 mg/kg to rats.
Keywords: Histone deacetylase inhibitor; HS270; SAHA; Liquid chromatography/tandem mass spectrometry (LC–MS/MS); Pharmacokinetics;

Simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat plasma using HPLC with on-line solid-phase extraction by Dewei Shang; Xipei Wang; Xiutai Zhao; Fengming Huang; Ganzhong Tian; Wei Lu; Tianyan Zhou (3459-3464).
► Simultaneous determination of two chemical compounds with different polarity and solubility (NTDP and HCTZ) in SHR plasma using HPLC with on-line solid-phase extraction. ► Many anti-hypertension drugs were lipophilic, and the polarity and solubility was different from HCTZ. So this method may be used for the simultaneous quantification in combination therapy with HCTZ. ► The method is simple, sensitive and reliable.A HPLC method with on-line solid phase extraction (SPE) and DAD detection was developed for the simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat (SHR) plasma. Plasma samples (100 μL) were injected directly onto a CAPCELL MF C8 SPE column. High-abundance proteins and most matrixes in plasma were removed by on-line SPE technology, while nitrendipine and hydrochlorothiazide trapped on the SPE column were effectively separated on a C18 analytical column. The column temperature was maintained at 20 °C. The optimal detection wavelength was 237 nm for NTDP and 271 nm for HCTZ. The total analytical run time was 34 min. The proposed method was linear over the range 5–500 ng mL−1 for nitrendipine and 10–1000 ng mL−1 for hydrochlorothiazide. The lower limit of detection (LLOD) was 0.5 and 0.6 ng mL−1 for nitrendipine and hydrochlorothiazide, respectively. The sensitivity and precision of the method were within acceptable limits during validation period. The method was successfully used to investigate the pharmacokinetic characteristics of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rats.
Keywords: Nitrendipine; Hydrochlorothiazide; On-line solid-phase extraction; Spontaneously hypertensive rat; Pharmacokinetics;

Simultaneous quantification of nicotine and metabolites in rat brain by liquid chromatography–tandem mass spectrometry by Paula L. Vieira-Brock; Eleanor I. Miller; Shannon M. Nielsen; Annette E. Fleckenstein; Diana G. Wilkins (3465-3474).
► We validated a LC–MS/MS method for quantification of nicotine, eight metabolites and two alkaloids in brain. ► We evaluated and reported imprecision, accuracy, matrix effect, extraction recovery and process efficiency. ► We utilized this method to measure concentration of nicotine and metabolites in rat brain. ► This method supports research of nicotine and its pharmacokinetics in the brain.A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of nicotine (NIC), cotinine (COT), nornicotine (NNIC), norcotinine (NCOT), nicotine-N-β-d-glucuronide (NIC GLUC), cotinine-N-β-d-glucuronide (COT GLUC), nicotine-1′-oxide (NNO), cotinine-N-oxide (CNO), trans-3′-hydroxycotinine (3-HC), anabasine (AB) and anatabine (AT) was modified and validated for quantification of these selected analytes in rat brain tissue. This analytical method provides support for preclinical NIC pharmacokinetic and toxicological studies after controlled dosing protocols. After brain homogenization and solid-phase extraction, target analytes and corresponding deuterated internal standards were chromatographically separated on a Discovery® HS F5 HPLC column with gradient elution and analyzed by LC–MS/MS in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. Method linearity was assessed and calibration curves were determined over the following ranges: 0.1–7.5 ng/mg for NIC, COT GLUC and AB; and 0.025–7.5 ng/mg for COT, NNIC, NCOT, NIC GLUC, NNO, CNO, 3-HC and AT (R 2  ≥ 0.99 for all analytes). Extraction recoveries ranged from 64% to 115%, LC–MS/MS matrix effects were ≤21%, and overall process efficiency ranged from 57% to 93% at low and high quality control concentrations. Intra- and inter-assay imprecisions and accuracy for all analytes were ≤12.9% and ≥86%, respectively. The method was successfully applied to quantification of NIC and metabolites in the brain of post-natal day 90 rats that were sacrificed 2-h after a single 0.8 mg/kg s.c. administration of (−)NIC. In these tissues, striatal concentrations were 204.8 ± 49.4, 138.2 ± 14.2 and 36.1 ± 6.1 pg/mg of NIC, COT and NNIC, respectively. Concentrations of NIC, COT and NNIC in the remaining whole brain (RWhB) were 183.3 ± 68.0, 130.0 ± 14.1 and 46.7 ± 10.3 pg/mg, respectively. Quantification of these same analytes in plasma was also performed by a previously validated method. NIC, COT, NNIC, NCOT, NNO and CNO were detected in plasma with concentrations comparable to those reported in previous studies. However, and in contrast to brain tissues, COT concentrations in plasma were significantly higher than were those of NIC (194.6 ± 18.6 ng/mL versus 52.7 ± 12.9 ng/mL). Taken together, these results demonstrate that a sensitive and selective method has been developed for the determination of NIC biomarkers in rat brain.
Keywords: Liquid chromatography–mass spectrometry; Nicotine; Metabolites; Brain; Rat;

Simultaneous determination of ipratropium and salbutamol in rat plasma by LC–MS/MS and its application to a pharmacokinetic study by Jingwen Wu; Cungang Ding; Qinghua Ge; Zhou Li; Zhen Zhou; Xiaojin Zhi (3475-3483).
► A novel LC–MS/MS method for the simultaneous determination of ipratropium and salbutamol in rat plasma was described. ► The LLOQs for IPR and SAL were 8 pg/mL and 50 pg/mL, respectively. ► SPE was applied to extract analytes with only 200 μL plasma. ► The drug concentration could be detected until 6 h after administration by this method.A novel, sensitive and specific LC–MS/MS method with silica-based solid-phase extraction was developed for simultaneous determination of ipratropium (IPR) and salbutamol (SAL) in rat plasma. Chromatographic separation was achieved on a Shiseido Capcell Pak CR column (SCX:C18  = 1:4, 150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of methanol/water (85:15, v/v) containing 20 mmol/L ammonium formate and 0.1% formic acid at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. This method was validated in terms of specificity, linearity, accuracy (within ±115.4%), intra- and inter-day precision (<11.4%) over the concentration range of 8–1612 pg/mL for IPR and 50–10,000 pg/mL for SAL. In addition, stability and matrix effects of IPR and SAL in plasma were evaluated. This method has been successfully applied to the pharmacokinetic study of compound ipratropium bromide aerosol mainly containing ipratropium bromide (IB) and salbutamol sulphate (SS) after inhalation in rats.
Keywords: Ipratropium; Salbutamol; LC–MS/MS; Simultaneous determination; Pharmacokinetics;

A sensitive and very fast analytical method has been developed for the simultaneous quantification of sixteen sulfonylurea herbicides in surface water. An ultra-high-pressure liquid chromatography coupled with tandem mass spectrometry method with solid phase extraction for sample cleanup has been developed for screening sixteen sulfonylurea herbicides (oxasulfuron, thifensulfuron-methyl, cinosulfuron, metsulfuron methyl, sulfometuron methyl, triasulfuron, rimsulfuron, ethametsulfuron methyl, sulfosulfuron, tribenuron methyl, bensulfuron methyl, iodosulfuron methyl, pyrazosulfuron ethyl, prosulfuron, chlorimuron ethyl, ethoxysulfuron) in water samples simultaneously within 12 min. Water samples were acidified, and the target herbicides were extracted by passing through ProElut C18 extraction cartridges. After drying by nitrogen flow, the cartridges were eluted with elution solvents, and the eluate was then evaporated to dryness, redissolved and analyzed. The mobile phase composed of 0.02% formic acid and acetonitrile using gradient elution. A triple quadrupole mass spectrometer equipped with an electrospray ionization source operated in the positive ion with selective reaction monitoring mode. Each of the analytes in all the samples was monitored using protonated molecule and its two characteristic fragment ions for confirmation. The limits of detection for all analytes were below 1.0 ng/mL, except for sulfosulfuron and prosulfuron, and limits of quantitation were between 1 and 8 ng/mL for this method. Three water types were used for the validation of the method.
Keywords: Sulfonylurea herbicides; Surface water; Ultra-high-pressure liquid chromatography; Tandem mass spectrometry; Solid phase extraction;

Development and validation of ultra high performance liquid chromatography–mass spectrometry method for LBH589 in mouse plasma and tissues by A. Estella-Hermoso de Mendoza; I. Imbuluzqueta; M.A. Campanero; D. Gonzalez; A. Vilas-Zornoza; X. Agirre; H. Lana; G. Abizanda; F. Prosper; M.J. Blanco-Prieto (3490-3496).
► The method is specific, accurate and reproducible for biological analysis of LBH589. ► Liquid–liquid extraction allowed acceptable recovery and matrix effect values. ► The method is valuable for the determination of the pharmacokinetic behavior of LBH589 in mice.An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed and validated for the quantitation of LBH589, a novel histone deacetylase inhibitor (HDACi), in mouse plasma and tissues (liver, spleen, kidney and lung). Tobramycin was employed as the internal standard. Separation was performed on an Acquity UPLC™ BEH column, with a mobile phase consisting of 10% water (with 0.1% of trifluoroacetic acid) and 90% methanol (with 0.1% trifluoroacetic acid). LBH589 and tobramycin were determined using an electrospray ionization (ESI) interface. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of LBH589 at m/z 349.42 → 157.95 and of tobramycin at 468.2 → 163. Calibration curves for the UHPLC method (0.0025–1 μg/mL for plasma and tissue homogenates, equivalent to 0.0357–14.2857 μg/g for tissue samples) showed a linear range of detector responses (r  > 0.998). Intra-batch and inter-batch precision expressed as coefficient of variation (CV) ranged from 0.92 to 8.40%. Accuracy expressed as bias, ranged from −2.41 to 2.62%. The lower limit of quantitation (LLOQ) was 0.0025 μg/mL for both plasma and tissue homogenate samples, equivalent to 0.0357 μg/g tissue. This method was successfully applied to quantify LBH589 in plasma and tissue samples obtained after the intraperitoneal administration of a single dose of 20 mg/kg of LBH589 in BALB/c mice.
Keywords: LBH589; Panobinostat; UHPLC-MS/MS; Tissue biodistribution; Bioanalysis;

Development of a liquid chromatography–tandem mass spectrometry assay of six antimicrobials in plasma for pharmacokinetic studies in premature infants by Michael Cohen-Wolkowiez; Nicole R. White; Arlene Bridges; Daniel K. Benjamin; Angela D.M. Kashuba (3497-3506).
► The majority of premature infants receive multiple antimicrobials simultaneously. ► PK sampling in these infants with limited blood volume is challenging. ► Our method successfully quantified 6 antimicrobials simultaneously in human plasma. ► Our method has many applications to pediatric research using micro-volume samples.This method provides a simple extraction procedure, as well as a validated, sensitive, and specific liquid chromatography–tandem mass spectrometry assay for the simultaneous quantification of ampicillin, piperacillin, tazobactam, meropenem, acyclovir, and metronidazole in human plasma. The method was validated over concentration ranges specific for each compound, with a lower limit of quantification of 50–300 ng/mL and a sample volume of 50 μL. The method is accurate and precise, with within- and between-day accuracy ranging from 85 to 110% and 92 to 110%, respectively, and within- and between-day precision of 89–111% and 91–109%, respectively. Simplicity, low plasma volume, and high throughput make this method suitable for clinical pharmacokinetic studies in premature infants.
Keywords: Prematurity; Neonates; Antibiotics; Antivirals; HPLC; Mass spectrometry;

► Lysine chromatography selectively recognizes different nucleic acids. ► Plasmid DNA was efficiently purified with pharmaceutical-grade by the lysine support. ► Impurities quantification and transfection studies confirmed the plasmid purity.Gene therapy and DNA vaccination cover a variety of applications using viral and non-viral vectors as vehicles of choice for treatment of genetic or acquired diseases. Recently, most therapeutic applications have been performed with non-viral biological agents preparations highly enriched in supercoiled plasmid molecules and it has been concluded that this isoform is more efficient at gene transfection than open circular isoform. This work describes for the first time a new strategy that uses lysine-chromatography to efficiently eliminate Escherichia coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate to purify and obtain pharmaceutical-grade supercoiled plasmid DNA. The quality control tests indicated that the levels of impurities in the final plasmid product were below the generally accepted specifications. Furthermore, the delivery of the purified product to eukaryotic cells, the cell uptake and transfection efficiency were also analyzed. The results showed that the transfection efficiency reached with the application of the supercoiled plasmid conformation, purified with lysine-agarose, was higher than the values achieved for other plasmid topologies. Therefore, this study presents a new enabling technology to obtain the completely purified non-viral vector, able to act with good efficiency as gene therapy delivery vehicle in several diseases like cancer.
Keywords: Lysine-affinity chromatography; Non-viral vectors; Supercoiled plasmid DNA; Transfection efficiency;

A novel liquid chromatography–atmospheric-pressure chemical ionization–mass spectrometry (LC–APCI/MS) method was developed and validated for the simultaneous determination of four sesquiterpene pyridine alkaloids (wilfortrine, wilfordine, wilforgine and wilforine) in human plasma. The chromatographic separation was performed on a Shim-pack XR-ODS column using an ammonium acetate buffer solution–acetonitrile in a gradient program. The detection was achieved by an ion trap mass spectrometry in the positive selected ion monitoring (SIM) mode. The method utilized acetonitrile as protein precipitation solvent and followed by solid-phase extraction (SPE). Calibration curves were linear for the four alkaloids over the range of 0.5–100.0 μg/L with the limits of quantification of 0.5 μg/L, while the method exhibited the recovery of 86.5–98.6%, intra- and inter-day RSDs of less than 8.2% and 12.8%, respectively. Methodology was validated in line with the EU requirements (Commission Decision 2002/657/EC). Results of incurred samples demonstrated excellent reproducibility. To our knowledge, this is the first analytical method for simultaneous determination of the four sesquiterpene pyridine alkaloids in plasma. The method was applicable to clinical pharmaceutical research of alkaloids in rheumatoid arthritis volunteer patients after oral administrations.
Keywords: Tripterygium wilfordii Hook. f.; Pyridine alkaloids; High performance liquid chromatography coupled with mass spectrometry; Human plasma;

Synthesis of imprinted beads by aqueous suspension polymerisation for chiral recognition of antihistamines by Rachel Walsh; Qendresa Osmani; Helen Hughes; Patrick Duggan; Peter McLoughlin (3523-3530).
A novel non-stabilised aqueous suspension polymerisation methodology for the preparation of spherical molecularly imprinted polymers is described with chlorpheniramine (CP), d-chlorpheniramine (d-CP), brompheniramine (BP) and d-brompheniramine (d-BP) as the templates, respectively. Using this rapid and simple technique, controlled polymer beads in the low micron range with narrow size distributions were generated by photo-polymerisation. The use of agitation speed as a method of controlling bead size distribution was demonstrated. Enantioselective properties of the imprinted beads were examined and the polymers prepared using d-chlorpheniramine and d-brompheniramine were capable of discriminating between the enantiomers of the template. Cross-selectivity studies were performed by batch rebinding with the influence of template size and functional group orientation of analytes on the recognition properties of the imprinted polymers investigated. Physical characteristics of all polymers were studied by nitrogen sorption porosimetry, particle size analysis and scanning electron microscopy (SEM) in order to gain an insight into the role of such properties on retention behaviour.
Keywords: Molecularly imprinted polymers; Chiral separation; Aqueous suspension polymerisation; Chlorpheniramine; Brompheniramine; Microspheres;

Selective sample cleanup by immunoaffinity chromatography for determination of fenvalerate in vegetables by Yanru Wang; Qi Zhang; Peiwu Li; Wen Zhang; Ying Li; Xiaoxia Ding (3531-3537).
► An immunoaffinity chromatography was developed for extraction of fenvalerate from vegetables. ► The extraction conditions were carefully optimized. ► High recoveries of fenvalerate were obtained by using IAC combined with GC detection. ► The calculated LOD of the whole developed method towards different vegetables were all acceptable.This paper describes the establishment of an immunoaffinity chromatography (IAC) for selective extraction of fenvalerate from vegetable samples. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against fenvalerate to CNBr-activated Sepharose 4B and packed into a cartridge. The extraction conditions were carefully optimized, including loading, washing and eluting solutions. Under the optimal conditions, the IAC column was able to capture fenvalerate with the maximum capacity of 4000 ng. An average recovery of 94.5% and a RSD of 8.8% were obtained with six IAC columns prepared on six different days. Three vegetable samples spiked with fenvalerate at four different concentrations were extracted with IAC column and determined by gas chromatography with electron capture detection (GC–ECD). Chromatograms of final extracts were clean and fenvalerate could be easily detected without the interferences. The extraction recoveries and RSD were 74.7–96.5% and 2.5–5.2%, respectively, and the calculated limit of detection of the whole method was 0.008–0.012 ng g−1.
Keywords: Immunoaffinity chromatography; Fenvalerate; Vegetables; Gas chromatography (GC);

► 8-OHdG in plasma and urine were quantified in humans. ► A one-step membrane extraction method for the plasma sample preparation. ► Simple, rapid on-line SPE LC–MS/MS for the detection, identification, and quantification.A quantitative analytical method using automated on-line solid phase extraction (SPE) and liquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS) for the determination of 8-OHdG (8-hydroxy-2′-deoxyguanosine) in human plasma was developed and validated. A one-step membrane extraction method for the plasma sample preparation and a C18 SPE column with simple extraction and purification were used for the on-line extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. 15N5-8-OHdG (15N5-8-hydroxy-2′-deoxyguanosine) was used as an internal standard for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve as one strategy to reduce the matrix effect and simultaneously improve detection sensitivity. Identification and quantification were based on the following transitions: m/z 284 → 168 for 8-OHdG and m/z 289 → 173 for 15N5-8-OHdG. Satisfactory recovery was obtained, and the recovery ranged from 95.1 to 106.1% at trace levels in human plasma and urine, with a CV lower than 5.4%. Values for intraday and interday precision were between 2.3 and 6.8% for plasma and between 2.7 and 4.5% for urine, respectively. Values for the method accuracy of intraday and interday assays ranged from 93.0 and 100.5% for plasma and 110.2 and 119.4% for urine, respectively. The limits of detection (LOD) and LOQ were 0.008 ng/mL and 0.02 ng/mL, respectively.The applicability of this newly developed method was demonstrated by analysis of human plasma samples for an evaluation of the future risk of oxidative stress status in human exposure to nanoparticles and other diseases.
Keywords: On-line SPE; 8-OHdG; LC–MS/MS;

► We applied the metabolomics approach to assess the mechanisms of melamine-induced nephrolithiasis in young children. ► Using the nephrolithiasis patients with no history of melamine exposure to filter out biomarkers unique to the melamine-induced nephrolithiasis group. ► Examining hypoxanthine and proline significantly distinguishing the controls/melamine exposure groups. ► The predictive model showed the good performance to identify the markers.Milk products contaminated with melamine caused renal disease in young children in mainland China in 2008. The present study was designed to identify potential markers and assess the underlying metabolomic mechanisms of melamine-induced nephrolithiasis in young children. Urine samples were collected from healthy children (n  = 74) and from children diagnosed with nephrolithiasis (n  = 73) with either a positive (n  = 40) or a negative (n  = 33) history of melamine exposure. Ultra high-performance liquid chromatography coupled to time of flight mass spectrometry (U-HPLC–MS/MS) was applied to profile the abundances of metabolites. Partial least squares-discriminant analysis (PLS-DA) was used to discriminate between the samples. Seven compounds were found to highly discriminate between healthy controls and nephrolithiasis patients with a history of melamine exposure. The critical markers such as proline and 5C-aglycone were the predominant markers in the control group and detected only rarely in nephrolithiasis patients with a history of melamine exposure. In contrast, hypoxanthine at was the most significant compound that distinguished nephrolithiasis patients with a history of melamine exposure. It was increased to 116.12 ± 23.34 μg/L (mean ± S.D.) in the melamine-induced nephrolithiasis group, whereas the non-melamine group was at the level of 67.47 ± 9.33 μg/L (p  < 0.001). The biomarkers for melamine-induced nephrolithiasis identified by this study may have clinical application in determining the aetiology of renal disease in young children.
Keywords: Melamine exposure; Biomarker; Nephrolithiasis; U-HPLC–Q-TOF/MS;

► Simultaneous analysis of fluoroquinolones and xanthine derivatives in serum. ► Selective molecularly imprinted matrix solid-phase dispersion coupled with liquid chromatography method. ► New molecularly imprinted polymer synthesized using ofloxacin and theophylline as mixed template. ► Good selectivity, recoveries and purification efficiency were observed for human serum samples.A new molecularly imprinted polymer was synthesized using ofloxacin and theophylline as template and methacryclic acid as function monomer and it was employed as a special dispersant of matrix solid-phase dispersion for selective extraction of fluoroquinolones (ofloxacin, ciprofloxacin and enrofloxacin) and xanthine (caffeine and theophylline) from human serum samples. To eliminate the influences of template leaking on quantitative analysis, acetonitrile–trifluoracetic acid (99:1, v/v) was used as the template removing solution. By using water and acetonitrile–trifluoracetic acid (99.5:0.5, v/v) as the washing and elution solvent, respectively, satisfactory recoveries and clean enough chromatogram could obtained. Good linearity of all the analytes was observed in a range of 0.35–150 μg g−1 with the correlation coefficient (r 2) ≥ 0.9991. The recoveries of spiked human serum samples were in a range of 89.5–104.0% for fluoroquinolones and xanthine derivatives with RSD less than of 5.0%.
Keywords: Matrix solid-phase dispersion; Molecularly imprinted polymer; Human serum; Fluoroquinolones; Xanthine derivatives;

Reversed phase LC/MS/MS method for targeted quantification of glycerophospholipid molecular species in plasma by Olaf Uhl; Claudia Glaser; Hans Demmelmair; Berthold Koletzko (3556-3564).
► RP-LC–MS/MS in negative ion mode enables detection of GP molecular species. ► Major classes of human plasma are covered (LPC, LPE, PC, PE). ► Investigation of dietary effects and large observational studies. ► Studies in infants due to small sample volume.The relationship between lipid status and metabolism, infant development and health has widely been studied, but the importance of individual glycerophospholipid species for biological functions in infants has hardly been considered. We developed a method for quantitative analyses of plasma glycerophospholipids from small sample volume. Proteins were precipitated with methanol, which eliminated further sample preparation. The supernatant was analysed by reversed-phase HPLC using a gradient of water, methanol and isopropanol as mobile phase. Electrospray ionisation in negative mode in combination with tandem mass spectrometry enabled detection of specific fatty acids as fragments of glycerophospholipid species. With this combination of chromatography and mass spectrometry, PC, lyso-PC, PE and lyso-PE species and their relevant isobaric compounds were quantified. Method validation showed a linear working range between 0.05 μmol/L and 10 μmol/L in diluted plasma samples. The intra-assay coefficients of variation (n  = 6) ranged from 1.1% to 13.9%. Results were comparable with data of the human metabolome database and gas chromatographic fatty acid analyses. All quantitatively important PE and PC species are covered. The method can be applied for investigating dietary effects on plasma GP composition from small plasma volumes.
Keywords: Glycerophospholipids; RP-HPLC; MS/MS; Plasma; Fatty acids; Quantification;

A novel “target constituent knock-out” strategy coupled with TLC, UPLC–ELSD and microcalorimetry for preliminary screening of antibacterial constituents in Calculus bovis by Wei-Jun Kong; Jia-Bo Wang; Qing-Ce Zang; Cheng Jin; Zhe-Wei Wang; Xiao-Yan Xing; Yu-Yue Wu; Yan-Ling Zhao; Mei-Hua Yang; Xiao-He Xiao (3565-3573).
► The single constituents in Calculus bovis were separated and knocked out by TLC. ► These knocked-out constituents were identified by UPLC–ELSD. ► The antibacterial activities of the knocked-out constituents and C. bovis were evaluated by microcalorimetry. ► The proposed “target constituent knock-out” strategy is useful and novel for screening active constituents in C. bovis.A novel “target constituent knock-out” strategy was proposed and applied for preliminary screening of antibacterial constituents in Calculus bovis (C. bovis). This strategy was accomplished through the following steps: (1) the single constituents (A–F) in C. bovis samples were knocked out on the Silica Gel thin-layer plates by thin-layer chromatography (TLC); (2) these knocked-out constituents were identified by ultra performance liquid chromatography–evaporative light scattering detection (UPLC–ELSD); (3) the antibacterial activities of these knocked-out constituents and C. bovis samples on Staphylococcus aureus (S. aureus) were evaluated by microcalorimetry combined with principal component analysis (PCA); (4) the activities of these knocked-out constituents and the total extract of C. bovis, also the interaction properties between these single constituents and the total extract were elucidated. The results showed that the sum of inhibitory ratio (I) of constituents A–F (202.0%) was 5-fold of the I of C. bovis sample (38.01%), showing that these knocked-out constituents had strong antagonistic effects on each other in C. bovis sample and the antagonistic extent was 81.18%. And we found that the key antibacterial composition of C. bovis was not a single component, also not the high content component (cholic acid, CA), but constituent F, which was the combinatorial composition of deoxycholic acid (DCA) and hyodeoxycholic acid (HDCA). Constituent F revealed over 33-fold high activity of the sum of DCA and HDCA activity in solo-use, showing strong synergistic effect between DCA and HDCA. In addition, constituents A–E had significant antagonistic effects on constituent F. Our study indicates that this proposed “target constituent knock-out” strategy is a useful approach for screening active constituents and elucidating the multi-component interactions in C. bovis, further providing some reference for understanding the pharmacodynamic actions, controlling the quality of Chinese materia medicas (CMMs) and discovering new drugs.
Keywords: Target constituent knock-out; C. bovis; TLC; UPLC–ELSD; Microcalorimetry; Multi-component interactions;

► Nicotine, metabolites and varenicline were simultaneously quantified by UPLC–MS/MS. ► Hydrophilic interaction liquid chromatography was used to increase sensitivity. ► The procedure was sensitive and specific, and matrix effects were insignificant. ► Validation led to good results concerning accuracy, precision, trueness, stability. ► The assay was successfully applied to human plasma samples from a clinical study.A sensitive and specific ultra performance liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3′-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3′-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5 mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1 × 100 mm, 1.7 μm). A gradient program was used, with a 10 mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4 mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2–500 ng/mL for nicotine, 1–1000 ng/mL for cotinine, 2–1000 ng/mL for trans-3′-hydroxycotinine and 1–500 ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2–113.6%), repeatability (1.9–12.3%) and intermediate precision (4.4–15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.
Keywords: UPLC–MS/MS; HILIC; Nicotine; Nicotine metabolites; Varenicline;

► We developed a simple and sensitive method for the simultaneous detection of imatinib mesylate (IM) and its active metabolite, N-desmethyl imatinib (M1), in human serum samples. ► The method was successfully applied to calculate the pharmacokinetic parameters of chronic myeloid leukemia patients receiving imatinib. ► The method is suitable to be routinely applied for determination of IM and M1 in serum.We developed a simple and sensitive method for the simultaneous detection of imatinib mesylate (IM) and its active metabolite, N-desmethyl imatinib (M1), in human serum samples. Separation was successfully achieved using an Agilent® ZORBAX Eclipse plus C18 reversed phase column (50 mm × 2.1 mm, i.d.; 1.8 μm) under isocratic mobile phase conditions consisting of acetonitrile: 0.02 M potassium dihydrogen phosphate with 0.2% triethylamine at pH 3 (25:75, v/v) and ultra-violet detection was achieved at 235 nm. Extraction of the target compounds was completed using 100% cold acetonitrile. Good linearities (r 2  > 0.99) for both IM and M1 were achieved for the concentration ranges of 50–1800 ng/mL and 50–360 ng/mL, respectively. The detection limits were 20 ng/mL and 10 ng/mL for M1 and IM, respectively. The intra- and inter-day precisions were less than 1% with percent recoveries of more than 90%. The method was successfully applied to calculate the pharmacokinetic parameters of chronic myeloid leukemia patients receiving imatinib. The method is suitable to be routinely applied for determination of IM and M1 in serum.
Keywords: Imatinib mesylate; N-Desmethyl imatinib; Protein precipitation; UHPLC;

Method development for the measurement of quinone levels in urine by Dianne Lim; Akihiro Ikeda; Kennedy K.-T. Vu; Kent T. Yamaguchi; Tim R. Tyner; Alam S. Hasson (3592-3598).
► A method was developed to measure 1–4 ring quinones in urine samples. ► Detection limits for ten quinones are 1–2 nmol dm−3. ► The method was tested with samples from Sprague-Dawley rats and humans. ► Urinary quinone levels in the animal model are correlated with exposure. ► Eight quinones were detected in human samples, at levels up to 3 μmol dm−3.A method was developed for the quantification of 1–4 ring quinones in urine samples using liquid–liquid extraction followed by analysis with gas chromatography–mass spectrometry. Detection limits for the ten quinones analyzed are in the range 1–2 nmol dm−3. The potential use of this approach to monitor urinary quinone levels was then evaluated in urine samples from both Sprague-Dawley rats and human subjects. Rats were exposed to 9,10-phenanthraquinone (PQ) by both injection and ingestion (mixed with solid food and dissolved in drinking water). Urinary levels of PQ were found to increase by up to a factor of ten compared to control samples, and the levels were found to depend on both the dose and duration of exposure. Samples were also collected and analyzed periodically from human subjects over the course of six months. Eight quinones were detected in the samples, with levels varying from below the detection limit up to 3 μmol dm−3.
Keywords: Quinones; Polyaromatic hydrocarbons; Urine analysis;

LC–MS determination of oxidized and reduced glutathione in human dermis: A microdialysis study by Sophie Robin; Nathalie Leveque; Carol Courderot-Masuyer; Philippe Humbert (3599-3606).
► We have developed a LC–ESI-MS to determine reduced and oxidized glutathione. ► LOQ was improved by at least a factor of 20 for reduced and oxidized glutathione. ► We quantify reduced and oxidized glutathione in human dermal microdialysates.A simple, highly selective, sensitive and reproducible liquid chromatography-electrospray ionization mass spectrometry method has been developed for the direct and simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in microdialysis samples from human dermis. Chromatographic separation was carried out on an MODULO CART QS KROMASIL 5C18 (250 mm × 2 mm × 5 μm) analytical column at a flow rate of 0.25 ml/min. An isocratic mode was used and consisted of acidified water and acetonitrile (50/50, v/v). To improve the sensitivity, silver nitrate was added as post-column reagent. A trap mass spectrum was used equipped with an ESI interface. The limits of detection and quantification were respectively 0.12 and 0.4 ng/ml for GSH and 0.2 and 0.5 ng/ml for GSSG. Intra-day and inter-day accuracy and precision were determined and the variability was less than 6.2% (R.S.D.).
Keywords: Oxidized glutathione; Reduced glutathione; Liquid chromatography; Mass spectrometry; Silver adducts;

LC–MS/MS determination of helicid in human plasma and its application in pharmacokinetic studies by Haitang Xie; Yuanwei Jia; Zhirong Tan; Wei Zhang; Rao Chen; Hua Sun; Jie Shen; Honghao Zhou (3607-3611).
► An LC–MS/MS assay for the measurement of helicid in human plasma has been established. The method is specific, sensitive, and accurate over a concentration range of 0.2–20 μg/L. ► This method demonstrated a relatively short analysis time and the good precision, selectivity, recovery and stability. The lowest standards in the calibration curves of plasma, whose signal-to-noise ratio (S/N) were larger than 10, were 0.2 μg/L, which is the lowest detection level reported so far. ► The developed method was fully validated and successfully applied to the human pharmacokinetics studies following a single oral dose of 100 mg helicid.Helicid is a traditional Chinese medicine used to treat headache and insomnia with definite effects. To facilitate pharmacokinetic studies of helicid in man, a sensitive and specific LC–MS/MS method for the quantitative detection of helicid in human plasma was developed and validated. The method involved the addition of bergeninum as the internal standard (IS), protein precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor → product ion transitions were monitored at m/z 282.8 → 120.9 for helicid and m/z 326.9 → 192.2 for the IS, respectively. The lower limit of quantification (LLOQ) was 0.2 μg/L. The calibration curves for helicid was linear over a concentration range of 0.2–20 μg/L. The intra- and inter-batch analyses of QC samples at 0.4, 2, 20 μg/L indicated good precision (%R.S.D. between 2.69 and 5.47%) and accuracy (between 96.15 and 105.05%). The helicid was stable in human plasma stored at room temperature for at least 24 h, 4 °C for at least 24 h, −20 °C for at least 1 month, and for routine three freeze–thaw cycles. This accurate and specific assay provides a useful method for evaluating the pharmacokinetic profile of helicid in humans.
Keywords: Helicid; LC–MS/MS; Pharmacokinetics; Quantitation;

► We measured process-related impurities (yeastolates, triton X-100 and methotrexate) in rHuPH20 in-process and manufacturing materials. ► We developed and validated an RP-HPLC method for simultaneous determination of yeastolates, triton X-100 and methotrexate. ► We examined the method with respect to selectivity, linearity, LOD, LOQ, precision and accuracy.Yeastolates, triton X-100 (TX-100) and methotrexate (MTX) are common process-related impurities (PRI) in cell-based bioproduction of many active biopharmaceuticals. In this study, a reverse phase high performance liquid chromatography (RP-HPLC) method coupled with ultraviolet (UV) detection was developed for simultaneous determination and quantitation of these impurities. The chromatographic separation was achieved using a Jupiter C4 column and analyses of yeastolates, TX-100 and MTX were monitored at 257, 280 and 302 nm, respectively. The method was further validated with respect to selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. The limits of quantitation for yeastolates, TX-100 and MTX were determined to be 27 ppm, 10 ppm and 41 ppb, respectively. Finally, the suitability of the method for analyses of recombinant human hyaluronidase (rHuPH20) in-process (viral inactivation, QFF, PS, APB and CHT filtered, final viral filtrate) and final manufacturing materials was demonstrated, and trace levels of yeastolates, TX-100 and MTX were reliably measured except for three matrices early in the purification process in which TX-100 was not accurately determined due to interfering effects.
Keywords: Method validation; Liquid chromatography; Yeastolates; Methotrexate; Triton X-100; Process-related impurities (PRI); Hyaluronidase; rHuPH20;

Direct, simultaneous measurement of liposome-encapsulated and released drugs in plasma by on-line SPE–SPE–HPLC by Eiichi Yamamoto; Kenji Hyodo; Naozumi Ohnishi; Takuya Suzuki; Hiroshi Ishihara; Hiroshi Kikuchi; Naoki Asakawa (3620-3625).
► Liposomal and released drug in plasma were separated by restricted-access media. ► During the extraction, drug release and/or leakage from liposomes was not observed. ► Liposomal and released drug were simultaneously measured by column-switching HPLC.A method for the simultaneous measurement of liposome-encapsulated and released drugs in mouse plasma by on-line solid phase extraction (SPE)–SPE–HPLC with direct plasma injection was developed using a doxorubicin (DXR)-containing liposome formulation as the model drug. During SPE, the released DXR was extracted on the 1st restricted-access media (RAM) SPE column, whereas the liposomes were eluted. The eluted liposomes were collapsed on-line, and the released DXR was delivered to the 2nd RAM SPE column for extraction. The retained DXR on the SPE columns was analyzed via HPLC–fluorescent detector by switching the valves. The method was validated and applied to the pharmacokinetic study of DXR in mice after intravenous injection of DXR-containing liposomes.
Keywords: Liposomes; Plasma; Doxorubicin; Restricted-access media; Solid-phase extraction;

► We describe the application of DBS sampling and extraction to screen posaconazole in human whole blood. ► We demonstrate the advantages of our assay for potential use in therapeutic drug monitoring of sick patients. ► We examine the posaconazole DBS method's ruggedness and robustness by implementing guidelines commonly used in FDA regulated bioanalytical environments. ► Blood spot homogeneity was maintained and yielded accurate, precise and reproducible quantitation results.A rugged and robust liquid chromatographic tandem mass spectrometric (LC–MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2 h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex™ C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC–MS/MS system equipped with a TurboIonSpray™ source operated in the positive ion mode. The assay was linear over the concentration range of 5–5000 ng/mL. The inter-run accuracy and precision of the assay were −1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.
Keywords: Dried blood spots (DBSs); Posaconazole; LC–MS/MS; UPLC™;

A simple, accurate and precise high-performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin (GEM) in rat plasma using furosemide as internal standard (I.S.). Plasma samples were pretreated by direct deproteinization and all samples and standard solutions were chromatographed at 45 °C using triethylamine solution (0.5%, v/v, pH 3.0 ± 0.1), methanol and acetonitrile (63:30:7, v/v/v) as the mobile phase. Chromatographic resolution was achieved using a RP-C18 column (Atlantis, Waters, 150 mm × 4.6 mm, 5 μm) at a flow rate of 1.0 mL min−1 and an injection volume of 30 μL. The analytes were measured by fluorescence detection with excitation and emission wavelengths of 344 nm and 399 nm, respectively. The retention times for GEM and I.S. were approximately 7.5 and 12.6 min, respectively. The lower limit of quantitation (LLOQ) was 20 ng mL−1 and the calibration curves were linear over a concentration range of 20–5000 ng mL−1. The intra- and inter-day precisions, expressed by relative standard deviation (R.S.D.) were lower than 6.24% and 4.49%, respectively. The accuracy ranged from 91.3% to 112% and from 98.8% to 106% for the lower and upper limit of quantitation of the calibration curve, respectively. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. No interferences from endogenous substances were found. The recovery of GEM and I.S. from plasma was greater than 90%. Drug stability in plasma was shown at room temperature for 4 h, after three freeze–thaw cycles for 24 h, in freezer at −80 °C for 60 days, and in the autosampler after processing for 12 h. The utility of the assay was confirmed by the successful analysis of plasma samples from GEM pharmacokinetics studies in the rats after intravenous administration.
Keywords: LC; Gemifloxacin; Fluorescence detection; Rat plasma; Pharmacokinetics;

► Ammonium sulfate precipitation was used for depleting highly abundant proteins from plasma. ► 40% ammonium sulfate saturation allowed depleting 39% albumin and 82% α-1-antitrypsin. ► Ammonium sulfate precipitation (ASP) showed high reproducibility in 2D-PAGE analysis. ► ASP is a reliable depletion method that may help in plasma proteomics.Ammonium sulfate precipitation (ASP) was explored as a method for depleting some highly abundant proteins from blood plasma, in order to reduce the dynamic range of protein concentration and to improve the detection of low abundance proteins by 2D-PAGE. 40% ammonium sulfate saturation was chosen since it allowed depleting 39% albumin and 82% α-1-antitrypsin. ASP-depletion showed high reproducibility in 2D-PAGE analysis (4.2% variation in relative abundance of albumin), similar to that offered by commercial affinity-depletion columns. Besides, it allowed detecting 59 spots per gel, very close to the number of spots detected in immuno-affinity-depleted plasma. Thus, ASP at 40% saturation is a reliable depletion method that may help in proteomic analysis of blood plasma. Finally, ASP-depletion seems to be complementary to hydrophobic interaction chromatography (HIC)-depletion, and therefore an ASP-step followed by a HIC-step could probably deplete the most highly abundant plasma proteins, thus improving the detection of low abundance proteins by 2D-PAGE.
Keywords: Ammonium sulfate precipitation; Two-dimensional gel electrophoresis; Plasma proteomics; Protein depletion;

The development of an optimized sample preparation for trace level detection of 17α-ethinylestradiol and estrone in whole fish tissue by Ahmed M. Al-Ansari; Ammar Saleem; Linda E. Kimpe; Vance L. Trudeau; Jules M. Blais (3649-3652).
► An optimized method is presented to analyze steroidal estrogens in fish by GC/MS. ► The method detection limit (ng/g) was 0.68 for E1 and 0.67 for EE2. ► A derivitization with pentafluorobenzoyl chloride was employed. ► This method permits further study in the environmental fate of steroidal estrogens.The purpose of this study was to develop an optimized method for the extraction and determination of 17α-ethinylestradiol (EE2) and estrone (E1) in whole fish tissues at ng/g levels. The optimized procedure for sample preparation includes extraction of tissue by accelerated solvent extraction (ASE-200), lipid removal by gel permeation chromatography (GPC), and a cleanup step by acetonitrile precipitation followed by a hexane wash. Analysis was performed by gas chromatography/mass spectrometry (GC/MS) in negative chemical ionization (NCI) mode after samples were derivatized with pentafluorobenzoyl chloride (PFBCl). The method was developed using high lipid content wild fish that were exposed to the tested analytes. The whole procedure recoveries ranged from 74.5 to 93.7% with relative standard deviation (RSD) of 2.3–6.2% for EE2 and 64.8 to 91.6% with RSD of 9.46–0.18% for E1. The method detection limits were 0.67 ng/g for EE2 and 0.68 ng/g for E1 dry weight. The method was applied to determine EE2 levels in male goldfish (Carrasius auratus) after a 72 h dietary exposure. All samples contained EE2 averaging 1.7 ng/g (±0.29 standard deviation, n  = 5). This is the first optimized protocol for EE2 extraction from whole fish tissue at environmentally relevant concentrations. Due to high sensitivity and recovery, the developed method will improve our knowledge about the environmental fate and uptake of synthetic steroidal estrogens in fish populations.
Keywords: 17α-Ethinylestradiol; Whole tissue extraction; Sample preparation; Goldfish;

► 1-N-butyl-3-methylimidazolium chloride as cell wall dissolution medium. ► Microwave accelerates sample dissolution. ► Microwave radiation-accelerated ionic liquid pretreatment can be used as a potential alternative method for the pretreatment of medicinal plants.A microwave radiation-accelerated ionic liquid pretreatment (MRAILP) was developed to enhance extraction of patchouli alcohol from Pogostemon cablin. 1-N-butyl-3-methylimidazolium chloride ([C4mim]Cl) was selected as microwave absorbing and cellulose dissolution medium and microwave was applied to accelerate sample dissolution. The conditions of MRAILP including particle size, solvent, microwave pretreatment time and power and the ratio of ionic liquid (IL) to sample were optimized. Under the optimized conditions, the extraction yield of patchouli alcohol by the MRAILP was 1.94%, which has increased by 166% compared with microwave-assisted extraction. The recovery was in the range of 95.71–103.7% with relative standard deviation lower than 3.0%. It was a novel alternative extraction method for the fast extraction and determination of patchouli alcohol from Pogostemon cablin.
Keywords: Microwave radiation-accelerated ionic liquid pretreatment; Ionic liquid; Pogostemon cablin; Patchouli alcohol; Gas chromatography;

► A method for determination of butenafine hydrochloride in human plasma was developed. ► The method was successfully applied to study the pharmacokinetics of butenafine hydrochloride in healthy Chinese volunteers. ► Results of the pharmacokinetic study following its topically administration are provided.An HPLC/MS/MS method for determination of butenafine hydrochloride in human plasma with testosterone propionate as the internal standard (IS) was developed and validated. Plasma samples were extracted with an n-hexane/diethyl ether (1:2, v/v) mixture and separated using a C18 column by a gradient elution with the mobile phase containing acetonitrile and 5 mM ammonium acetate buffer. Quantification was performed using multiple reaction monitoring (MRM) mode with transition of m/z 318.4 → 141.0 for butenafine hydrochloride and m/z 345.5 → 97.0 for testosterone propionate (IS). This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The lower limit of quantification (LLOQ) of this method was 0.0182 ng/ml and the calibration curve was linear over the 0.0182–1.82 ng/ml. The intra- and inter-run coefficient of variance was less than 11.53% and 10.07%, respectively. The samples were stable under all the tested conditions. The method was successfully applied to study the pharmacokinetics of butenafine hydrochloride in healthy Chinese volunteers following its topical administration.
Keywords: Butenafine hydrochloride; LC–MS/MS; Pharmacokinetics;

Simple and rapid liquid chromatography method for determination of moxifloxacin in saliva by A. K. Hemanth Kumar; V. Sudha; R. Srinivasan; Geetha Ramachandran (3663-3667).
► Developed and validated a sensitive, specific, rapid and accurate method utilising a single step extraction for quantitative determination of moxifloxacin in saliva. ► Good intra- and inter-day precision, with chromatogram yielding well resolved peaks for moxifloxacin. ► Good correlation between plasma and saliva concentrations of moxifloxacin (r 2  = 0.847; p  < 0.001). ► Saliva to plasma ratio was 0.54.A high performance liquid chromatographic method for determination of moxifloxacin in human saliva was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C18 column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.25 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from saliva was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.
Keywords: Moxifloxacin; Saliva; HPLC;

Determination of sucrose in equine serum using liquid chromatography–mass spectrometry (LC/MS) by E. D’Arcy-Moskwa; L. Weston; G.N. Noble; S.L. Raidal (3668-3671).
Mucosal integrity may be objectively assessed by determination of the absorption of exogenous substances such as sucrose. Gas chromatography–mass spectrometry (GC/MS) and liquid chromatography–mass spectrometry (LC/MS) have been reported for the accurate quantification of low concentrations of sucrose in serum. LC/MS offered the advantage of high sensitivity and mass selectivity without the need for extensive sample derivatization required for GC/MS methods. However, the high polarity and non-volatile nature of the sucrose molecule renders LC/MS techniques challenging. Previously published reports lacked sufficient detail to permit replication of methodology. Problems encountered with existing protocols included poor peak resolution and weak fragmentation of the parent molecule. This communication describes a LC/MS protocol developed to provide improved resolution and product detection.
Keywords: Liquid chromatography–mass spectrometry; Peak resolution; Sodiated sucrose;