Journal of Chromatography B (v.905, #C)

► Validated method for analysis of 35 endogenous steroids in equine serum. ► Androgens, estrogens, and progestins are monitored in one method. ► Turbulent flow chromatography allows for minimal sample processing. ► Allows for analysis of multiple steroids in one run from 300 μL serum.A method for the detection and quantitation of 35 endogenous steroids in equine serum was developed and validated. Androgens, estrogens, progestins and their metabolites potentially present in serum were simultaneously monitored in one method using on-line sample extraction by turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system and detected on a triple-stage quadrupole mass spectrometer by electrospray ionization. Analytes were detected and quantitated by single-reaction monitoring or selected-ion monitoring. Limits of detection (range 0.025–10 ng mL−1) and quantitation (range 0.125–25 ng mL−1) along with recovery and matrix effects were determined for each analyte. Inter- and intra-day accuracy and precision was assessed for with the majority of analytes having %CV less than 20% and accuracy within 20% of the expected concentrations. Eight of the 35 analytes were unable to meet these guidelines across all of the quality control concentrations monitored for each analyte. This method was used to determine the endogenous steroid profiles of Thoroughbred horses and has been modified for use in non-human primates and cell culture.
Keywords: Mass spectrometry; Liquid chromatography; Steroids; Horse; Androgens;

► LC/MS/MS method for analyzing plasma hydroxocobalamin and cyanocobalamin. ► Use of hydroxocobalamin in porcine animal models with induced cyanide poisoning. ► Plasma hydroxocobalamin and cyanocobalamin levels after hydroxocobalamin treatment.Cyanide poisoning occurs in individuals after fire smoke inhalation and after oral ingestion of cyanide. Hydroxocobalamin (HOCbl), a hydroxylated form of vitamin B12, is often used as an antidote to treat cyanide toxicity. It has a high affinity for cyanide and rapidly removes cyanide from tissue by forming cyanocobalamin (CNCbl). Little information is available on the pharmacokinetics of HOCbl and CNCbl largely because of the lack of analytical methods for analyzing HOCbl and CNCbl. In this study, we developed a new liquid chromatographic mass spectrometric (LC/MS/MS) method for the quantitative analysis of plasma HOCbl and CNCbl in the porcine (Sus scrofa) model. The method uses on-column extraction, reversed phase gradient chromatography, and multiple reaction monitoring (MRM) for quantitation. MRM transitions monitored were 664.7 → 147.3 and 664.7 → 359.2 for HOCbl and 678.8 → 147.3, 678.8 → 359.1 678.8 → 457.1 for CNCbl. The limit of detection (LOD) and the lower limit of quantitation (LLOQ) were 1.0 and 1.0 μmole/L, respectively, for plasma HOCbl and 0.1 and 0.5 μmole/L for plasma CNCbl. The within-day and between-day CVs were 4.3 and 6.4% for plasma HOCbl at 500.0 μmole/L and 5.5 and 5.7% for CNCbl at 100.0 μmole/L (n  = 6). The plasma HOCbl and CNCbl calibrations curves were linear from 100.0 to 2000.0 and 50.0 to 500.0 μmole/L, respectively. Based on 6 separate calibration curves the average linear regression coefficient (R 2) for both HOCbl and CNCbl was 0.992. The LC/M/MS method was found to be accurate and precise and has been validated by determining the plasma HOCbl and CNCbl concentrations in 11 pigs that were treated with HOCbl for cyanide poisoning.
Keywords: Hydroxocobalamin; Cyanocobalamin; LC/MS/MS analysis; Cyanide poisoning antidote; Pharmacokinetics; Porcine models;

Breath biomarkers of liver cirrhosis by Jesica Dadamio; Sandra Van den Velde; Wim Laleman; Paul Van Hee; Wim Coucke; Frederik Nevens; Marc Quirynen (17-22).
► We used TD/GC/MS to analyze the breath composition of patients with cirrhosis. ► We compared their breath profiles with healthy volunteers. ► Linear discriminant analysis identified 24 models. ► Sensitivity and specificity varied between 82% and 88%, and 96% and 100%, respectively. ► Patients with cirrhosis have a specific pattern of breath biomarkers.The diagnosis of asymptomatic cirrhosis in patients with liver disease is of importance to start screening for complications in due time. Liver biopsy is neither sensitive nor practical enough to be used as a frequent follow-up test in patients with chronic liver disease. The volatile organic compounds present in exhaled breath offer the possibility of exploring internal physiologic and pathologic process in a non invasive way. This study examined whether a specific pattern of biomarkers can be found in breath samples of patients with cirrhosis. To this aim samples of alveolar breath from patients with cirrhosis and healthy volunteers were analyzed using gas chromatography–mass spectrometry. When linear discriminant analysis was used to search for a model(s)/pattern of compounds characteristic for liver cirrhosis, 24 models of 8 independent compounds could distinguish between the groups. The sensitivity and specificity (between 82% and 88%, and 96% and 100%, respectively) of the models suggest that a specific pattern of breath biomarkers can be found in patients with cirrhosis, which may allow detecting this complication of chronic liver disease in an early stage.
Keywords: Breath biomarkers; Alveolar breath; Liver disease; VOCs; Liver cirrhosis;

► Fractionation of bioactive peptides by centrifugal partition chromatography (CPC). ► Efficiency of the ion-exchange CPC mode in the field of downstream processes. ► Optimization of the exchanger/analyte ratio. ► Quantification of the target dipeptide by RP-HPLC–MS/MS.This article reports a promising use of the mixed ion-exchange centrifugal partition chromatography (MIXCPC) technique in the field of downstream processes. A complex alfalfa white protein concentrate hydrolysate (AWPC hydrolysate) showing anti-hypertensive properties was successfully fractionated by MIXCPC to yield a l-valyl-l-tryptophan (VW) enriched fraction in one run. This dipeptide shows an interesting anti-angiotensin converting enzyme (anti-ACE) activity. An analytical method based on RP-LC/MS-MS was developed to quantify the target VW peptide in both the starting material and the enriched fractions. The best results for the MIXCPC fractionation were obtained by the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger with an exchanger (DEHPA)/peptides ratio of 15, and of two displacers: calcium chloride and hydrochloric acid. The complexity of the starting material involved the selectivity optimization by splitting the stationary phase into two sections that differed by their triethylamine concentration. From 1 g of AWPC hydrolysate containing 0.26% of VW, 30.7 mg of a VW enriched fraction were recovered with a purity of 10.9%, corresponding to a purification factor of 41 and a recovery of 97%.
Keywords: Bioactive peptide; Centrifugal partition chromatography; Ion exchange; HPLC–MS; Protein hydrolysate fractionation; Antihypertensive peptide;

High sensitivity measurement of amino acid isotope enrichment using liquid chromatography–mass spectrometry by Hans M.H. van Eijk; Karolina A.P. Wijnands; Babs A.F.M. Bessems; Steven W. Olde Damink; Cornelis H.C. Dejong; Martijn Poeze (31-36).
► Amino acid (AA) isotope enrichment down to 0.005% at 5 μM concentrations. ► Special focus on low arg and cit due to LPS treatment. ► Use OPA derivatization of AA combined with RP-HPLC and MS analysis.Measurement of the incorporation or conversion of infused stable isotope enriched metabolites in vivo such as amino acids plays a key role in metabolic research. Specific routes are frequently probed in knockout mouse models limiting the available amount of sample. Although less precise as compared to combustion-isotope ratio mass spectrometry (C-IRMS), gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) techniques are therefore often the method of choice to measure isotopic enrichment of target metabolites. However, under conditions of metabolic depletion, the precision of these systems becomes limiting. In this paper, studies were performed to enhance the sensitivity and precision of isotope enrichment measurements using LC–MS. Ion-statistics and resolution were identified as critical factors for this application when using a linear trap mass spectrometer. The combination with an automated pre-column derivatization and a carefully selected solvent mix allowed us to measure isotopic enrichments down to 0.005% at plasma concentrations as low as 5 μmol/l, an improvement by a factor of 100 compared to alternative methods. The resulting method now allowed measurement of the in vivo conversion of the amino acid arginine into citrulline as a marker for the production of nitric oxide in an in vivo murine endotoxemia model with depleted plasma levels of arginine and citrulline.
Keywords: HPLC; Mass spectrometry; Amino acids; Isotope;

Realgar (As4S4) is a traditional medicine used in China and Europe for thousands of years. As an arsenical, the toxicity from realgar has raised public concern. Several organic acids in urine are found to be potential biomarkers of realgar exposure, including taurine, citric, glutamic, lactic, pyruvic, succinic and uric acid. In this study, using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), a rapid and sensitive method was developed to separate and quantify these compounds in urine. A ZIC®-HILIC column was used for the separation at an isocratic condition of acetonitrile and 10 mM ammonium acetate in water. Analytes were detected in multiple-reaction monitoring with negative ionization mode, using ibuprofen as internal standard. Good line arities (R 2  > 0.996) were obtained for all analytes with the limits of detection from 0.2 to 0.7 μg/mL. The intra-day and inter-day accuracy ranged from 89.1 to 104.4% and the relative standard deviation (RSD) did not exceed 15.0%. The recovery was more than 80%with RSD less than 14.0%. The validated method was applied to analyze the urine samples of control and reaglar treated rats, and significant changes of these organic acids were observed.
Keywords: Organic acids; Realgar; Biomarkers; HILIC–MS/MS; Rat urine;

Post acquisition data processing techniques for lipid analysis by quadrupole time-of-flight mass spectrometry by Tong Xie; Yan Liang; Jiye A; Haiping Hao; Linsheng Liu; Xiao Zheng; Chen Dai; Yuanyuan Zhou; Tianye Guan; Yanna Liu; Lin Xie; Guangji Wang (43-53).
► Lipidomic analysis of rats’ plasma was studied. ► 145 lipids were detected by data processing techniques. ► Thirteen differential features could be highlighted upon using the described methodology.This study describes the effectiveness of post-acquisition data processing techniques in detecting the lipid species rapidly from the massive data generated by high resolution mass spectrometry. The filtering approaches by product ions or neutral losses enabled glycerophospholipids and sterol conjugates to be identified based on the investigation of their fragmentation patterns, and the filtration by mass defect facilitated the detection of fatty acyl residues and bile acids by limiting the range of mass defect values. After application of these filtering techniques to mass spectra, the background noise was significantly filtered out and characteristic peaks of lipid species were efficiently sorted out. Totally 145 individual lipids were identified and structurally elucidated. Validation results of the LCMS-Q-TOF-based quantitative performance for all the peaks showed that the accuracy, expressed as relative errors (RE%), was lower than ±15%, and values (RSD%) of the inter-batch and intra-batch precision were lower than 15% in the assay. The developed method was integrated to the evaluation of plasma lipid profile from high fat diet versus energy restricted diet fed rats. A unique discrimination of the groups was successfully achieved through a principal component analysis (PCA).
Keywords: Lipid detection; LCMS-Q-TOF; Product ion filtration; Neutral loss filtration; Mass defect filtration;

► Polyphyllin D and Paris H are two new potential antitumor active components in Paris polyphylla, one of the Traditional Chinese Medicines (TCMs). ► A rapid, sensitive and accurate LC–ESI-MS/MS method for simultaneous determination of Polyphyllin D and Paris H in rat plasma was firstly established and fully validated. ► The lower limits of quantification (LLOQs) of Polyphyllin D and Paris H were both 1.0 ng/mL using 100 μL rat plasma. ► The developed and validated method has been successfully applied in the simultaneous quantification and pharmacokinetic studies of Polyphyllin D and Paris H in rats.Polyphyllin D and Paris H are two potential antitumor active components in Paris polyphylla, one of the Traditional Chinese Medicines (TCMs). The present study details the development and validation of a rapid, sensitive and accurate LC–ESI-MS/MS method for the separation and simultaneous determination of Polyphyllin D and Paris H in rat plasma using Ginsenoside Rh2 as the internal standard (IS). A simple protein precipitation method was used for the preparation of plasma sample. Chromatographic separation was successfully achieved on an Agilent Zorbax C18 column using a step gradient program with the mobile phase of 10 mmol/L aqueous ammonium acetate and acetonitrile. Both analytes were detected by negative mode electrospray ionization mass spectrometry. Selected reaction monitoring (SRM) was applied for all monitored analytes. This method demonstrated good linearity and did not show any endogenous interference. The lower limits of quantification (LLOQs) of Polyphyllin D and Paris H were both 1.0 ng/mL using 100 μL rat plasma. The average recoveries of Polyphyllin D and Paris H from rat plasma were both above 80%. The inter-day precisions (%RSD) of both analytes determined in five days were all below 15%. The developed and validated method has been successfully applied in the simultaneous quantification and pharmacokinetic studies of Polyphyllin D and Paris H in rats.
Keywords: Paris polyphylla; Polyphyllin D; Paris H; Pharmacokinetics; LC–ESI-MS/MS;

► The analysis of glycine in biological matrix by HILIC–MS/MS is firstly reported. ► The method avoids the usage of derivatization or/and ion-pairing reagents. ► The method features the direct injection of samples, with no sample pretreatment. ► ECSCs are firstly discovered to release glycine by the validated method. ► The discovery helps to improve our understanding of the novel effects of glycine.Because glycine plays a prominent role in living creatures, an accurate and precise quantitative analysis method for the compound is needed. Herein, a new approach to analyze glycine by hydrophilic interaction chromatography (HILIC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was developed. This method avoids the use of derivatization and/or ion-pairing reagents. N-methyl-d-aspartate (NMDA) is used as the internal standard (IS). The mobile phase for the isocratic elution consisted of 10 mM ammonium formate in acetonitrile–water (70:30, v/v, adjusted to pH 2.8 with formic acid), and a flow rate of 250 μL/min was used. Two microliters of sample was injected for analysis. The signal was monitored in the positive multiple reaction monitoring (MRM) mode. The total run time was 5 min. The dynamic range was 40–2000 ng/mL for glycine in the biological matrix. The LLOQ (lower limit of quantification) of this method was 40 ng/mL (80 pg on column). The validated method was applied to determine the dynamic release of glycine from P19 embryonal carcinoma stem cells (ECSCs). Glycine spontaneously released from the ECSCs into the intercellular space gradually increased from 331.02 ± 60.36 ng/mL at 2 min in the beginning to 963.52 ± 283.80 ng/mL at 60 min and 948.27 ± 235.09 ng/mL at 120 min, finally reaching a plateau, indicating that ECSCs consecutively release glycine until achieving equilibration between the release and the reuptake of the compound; on the contrary, the negative control NIH/3T3 embryonic fibroblast cells did not release glycine. This finding will help to improve our understanding of the novel effects of neurotransmitters, including glycine, on non-neural systems.
Keywords: Glycine; HILIC; LC–MS/MS; Embryonal carcinoma stem cells;

Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC–ESI-Q-TOF-MS analysis by Ming-Luan Chen; Xiao-Meng Fu; Jia-Qi Liu; Tian-Tian Ye; Sheng-Yu Hou; Yun-Qing Huang; Bi-Feng Yuan; Yan Wu; Yu-Qi Feng (67-74).
► Using polymer monolithic column, 15 targeted hormones are well separated. ► High sensitivity of pg/mL is obtained, combined with online trapping process. ► Tandem SPE followed by LLE is developed for purification of hormones from matrix. ► Developed method is applied in the analysis of acidic phytohormones in 5 mg rice.In current study, we developed a highly sensitive method for the quantitative profiling of acidic phytohormones. Tandem solid-phase extraction (SPE) and liquid–liquid extraction (LLE) was employed to efficiently purify acidic phytohormones, which were further derived by 3-bromoactonyltrimethylammonium bromide (BTA) to increase the ionization efficiency in electrospray ionization-mass spectrometry detection. Additionally, fifteen BTA-derived acidic phytohormones, including ten gibberellins (GAs), were well separated with a salt gradient on poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolithic column. By employing online trapping system, the signal intensities of the analytes were significantly improved. The limits of detection (LODs, Signal/Noise = 3) of targeted phytohormones ranged from 1.05 to 122.4 pg/mL, which allowed the highly sensitive determination of low abundant acidic phytohormones with tiny amount plant sample. Good reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 10.9 and 11.9%, respectively. Recoveries of the target analytes from spiked rice leave samples ranged from 88.3 to 104.3%. By employing the method developed here, we were able to simultaneously determine 11 endogenous acidic phytohormones from only 5 mg of rice leave sample, which dramatically decreased the required sample amount (three orders of magnitude lower) for the profiling of low abundant acidic phytohormones compared to previous reports. Taken together, the method provided a good solution for the highly sensitive and quantitative profiling of endogenous acidic phytohormones.
Keywords: Acidic phytohormones; Derivatization; Nano-LC–ESI-Q-TOF-MS; Poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolith;

Validation of determination of plasma metabolites derived from thyme bioactive compounds by improved liquid chromatography coupled to tandem mass spectrometry by Laura Rubió; Aida Serra; Alba Macià; Xenia Borràs; Maria-Paz Romero; Maria-José Motilva (75-84).
► A rapid and sensitive method for analysis of plasma thyme metabolites is proposed. ► Method combined micro elution solid-phase extraction and UPLC–MS/MS. ► LODs ranged from 1 to 65 μg/L plasma, except thymol sulfate metabolite (240 μg/L). ► Main thyme metabolites were hydroxyphenylpropionic acid sulfate and thymol sulfate.In the present study, a selective and sensitive method, based on microelution solid-phase extraction (μSPE) plate and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) was validated and applied to determine the plasma metabolites of the bioactive compounds of thyme. For validation process, standards of the more representative components of the phenolic and monoterpene fractions of thyme were spiked in plasma samples and then the quality parameters of the method were studied. Extraction recoveries (%R) of the studied compounds were higher than 75%, and the matrix effect (%ME) was lower than 18%. The LODs ranged from 1 to 65 μg/L, except for the thymol sulfate metabolite, which was 240 μg/L. This method was then applied for the analysis of rat plasma obtained at different times, from 0 to 6 h, after an acute intake of thyme extract (5 g/kg body weight). Different thyme metabolites were identified and were mainly derived from rosmarinic acid (coumaric acid sulfate, caffeic acid sulfate, ferulic acid sulfate, hydroxyphenylpropionic acid sulfate, dihydroxyphenylpropionic acid sulfate and hydroxybenzoic acid) and thymol (thymol sulfate and thymol glucuronide). The most abundant thyme metabolites generated were hydroxyphenylpropionic acid sulfate and thymol sulfate, their respective concentrations in plasma being 446 and 8464 μM 1 h after the intake of the thyme extract.
Keywords: UPLC–MS/MS; Monoterpenes; Phenolic compounds; Plasma metabolites; Thyme;

► Cannabinoids dabsyl derivatization allows LC/MS-MS determination in microvolume sample. ► Cannabinoids dabsylation requires only a protein precipitation step as pre-treatment. ► Sensitivity is similar as in GC–MS, but using only 50 μL without extraction step. ► The method lies in rapid, specific, sensitive cannabinoids measurement in DRUID cases.Due to the non-polar nature and the absence of an ionizable group on the cannabinoids, the ionization efficiency in electrospray is low and leads to poor limits of detection (LOD). The reaction of chloride dabsyl with the phenolic OH group of cannabinoids results in a product containing a tertiary amine, which is easily protonated in positive electrospray mode and can significantly improve the cannabinoids LOD. A rapid, selective and sensitive LC/MS-MS method was developed for quantitative determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH–THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC–COOH), cannabinol (CBN) and cannabidiol (CBD), in micro volume blood samples following dabsyl derivatization to enhance signal intensity. The method comprised protein precipitation followed by derivatization with dabsyl chloride and subsequent analysis using liquid chromatography–tandem mass spectrometry (LC/MS-MS). Chromatographic separation was achieved using a 150 mm × 2.1 mm C18 analytical column maintained at 65 °C and eluted with a gradient of water and acetonitrile, both containing 0.2% formic acid. The run time was 8 min. The assay was successfully validated using the approach based on the accuracy profile. Lower limits of quantification (LOQ) were 0.25 ng/mL for THC and THC–COOH, 0.30 ng/mL for 11-OH–THC, 0.40 ng/mL for CBN and 0.80 ng/mL for CBD. A comparative study of cannabinoids in blood and plasma, as determined by the developed LC/MS-MS method or the in-house GC/MS-MS technique, demonstrated an excellent correlation between the two methods. Dabsylation was also tested on-line with a spiral of peek tubing placed in the LC/MS-MS column heater at 65 °C before the analytical column. The results obtained with on-line dabsyl derivatization were similar to those observed off-line.
Keywords: Cannabinoids; Dabsyl derivatization; LC/MS-MS; Micro volume;

Protein pre-fractionation with a mixed-bed ion exchange column in 3D LC–MS/MS proteome analysis by Luofu Zhang; Ling Yao; Yan Zhang; Ting Xue; Guangchen Dai; Keying Chen; Xiaofang Hu; Lisa X. Xu (96-104).
► A mixed-bed IEX chromatography for protein pre-fractionation using volatile solvents was used prior to classic MudPIT. ► Mammalian cell lysate was applied and more than 3000 proteins were identified through a typical run. ► Good separation degree and wide protein coverage were achieved. ► Protein retention on mixed-bed IEX was related to extreme pI and percentage of charged residues in proteins.The fractionation of complex samples at the protein level prior to shotgun proteomics analysis is an efficient means to more comprehensive analysis of samples. A mixed-bed ion-exchange (IEX) column, packed with both weak anion exchange (WAX) and weak cation exchange (WCX) materials, was used for the first dimensional separation of complex samples at the protein level using volatile solvents. The peptides from digestion of each fraction were then identified by 2D SCX-RP-LC–MS/MS. We applied this 3D strategy to mouse mammary tumor 4T1 cell lysate and identified a total of 3084 proteins in a typical experiment. The moderate separation performance of the mixed-bed IEX column facilitated the in-depth identification of the proteins in the complex sample. There were some acceptable inter-fraction overlaps. Nearly half (45.8%) of the proteins were only identified in single fractions, while 82.3% were identified in no more than 3 fractions. The identified proteins covered a broad range of pI, size and grand average hydrophobicity (GRAVY) values. Detailed analysis of proteins identified in each fraction elucidated the separation characteristics of mixed-bed IEX. Retention on mixed-bed IEX was associated, but not restricted to the extreme pI values (pI  < 5, pI  > 10) and to the percentage of charged residues of both signs. In conclusion, we have exploited the mixed-bed IEX column to establish an efficient and comprehensive identification method for complex samples.
Keywords: 3D LC–MS/MS; Mixed-bed IEX chromatography; Protein pre-fractionation;

Magnetic molecularly imprinted nanoparticles based on dendritic-grafting modification for determination of estrogens in plasma samples by Shu Wang; Ruoyu Wang; Xiaoli Wu; Yang Wang; Cheng Xue; Jinhua Wu; Junli Hong; Jie Liu; Xuemin Zhou (105-112).
► The dendritic-grafting method was proposed to introduce more functional groups. ► EE2 was selected as the pseudo template to synthesize magnetic MIPs (MMIPs). ► MMIPs have good evaluation (high selectivity, good adsorbance and fast kinetics). ► MMIPs were first used as MDSPE materials for complicated samples pretreatment. ► MMIPs-MDSPE-HPLC was effectively enriched and detected trace estrogens in samples.In order to resolve the low imprinting efficiency of surface molecularly imprinted polymers (MIPs), a dendritic-grafting method introducing more functional groups was proposed to modify the SiO2-coated magnetic nanoparticles (SiO2-coated MNPs). And then magnetic MIPs (MMIPs) were obtained using 17-ethyl estradiol (EE2) as a pseudo template with dendronized SiO2-coated MNPs as the supporter, aiming to avoid residual template leakage and to increase the imprinting efficiency. The resulting MMIPs showed high adsorption capacity, quick binding kinetics and good selectivity for trace estrogens. Meanwhile, MMIPs were used as magnetic dispersive solid-phase extraction (MDSPE) materials coupled with HPLC-UV for the detection of trace estrogens. The amounts of three estrogens which were detected from the plasma samples of pregnant women were 5.28, 5.31 and 4.17 ng mL−1, and the average recoveries were 87.8%, 93.1% and 90.6% for the spiked samples with RSDs in the range of 1.4–6.3%, respectively. All these results reveal that MMIPs as MDSPE materials has good applicability to selective extraction and separation of trace estrogens from complex samples.
Keywords: Magnetic molecularly imprinted polymers; Estrogens; Magnetic dispersive solid-phase extraction; Dendritic-grafting modification; Plasma samples;

Liquid chromatography–tandem mass spectrometry to determine the stability of collagen pentapeptide (KTTKS) in rat skin by Eun Ji Park; Myung Sun Kim; Yun Lim Choi; Young-Hee Shin; Hye Suk Lee; Dong Hee Na (113-117).
► Sensitive LC–MS/MS method for determining the stability of KTTKS peptide in skin. ► LC–MS/MS was successfully applied to investigate the stability of KTTKS in rat skin. ► KTTKS was very susceptible to the peptide bond cleavage by aminopeptidases present in the skin.The objective of this study was to develop a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine the stability of collagen pentapeptide (KTTKS), which is a subfragment of collagen and has been proved to promote the extracellular release of collagen in skin fibroblast, in rat skin. The chromatographic condition was optimized on an Acclaim C-18 column (2.1 mm × 150 mm, 3 μm) under isocratic elution using a mobile phase consisting of deionized water and acetonitrile (87:13, v/v) mixture containing 5 mM pentafluoropropionic acid as an ion-pairing reagent. The quantitation of KTTKS was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The calibration curve showed good linearity in the concentration range of 0.05–10.0 μg/mL (r 2  > 0.999). The intra- and inter-day precisions were 0.8–6.5% and 2.4–5.8%, respectively, and the intra- and inter-day accuracies were 96.3–102.7% and 92.8–98.5%, respectively. The developed LC–MS/MS method was successfully applied to investigate the degradation rate and sites of KTTKS in rat skin homogenate. KTTKS was found to be very susceptible to the peptide bond cleavage by aminopeptidases present in the skin.
Keywords: Collagen pentapeptide; LC–MS/MS; Skin; Stability;

Quantitative analysis of an anti-viral immune escape compound ML-7 in feline plasma using ultra performance liquid chromatography/electrospray ionization mass spectrometry by Marc Cherlet; Sabine Gleich; Hannah Dewerchin; Hans Nauwynck; Sylvie Daminet; Patrick De Backer; Siska Croubels (118-126).
► An analysis method for ML-7 in feline plasma by UPLC–ESI-MS/MS was optimized. ► Solid-phase extraction clean-up on MCX column was used to minimize matrix effect. ► Validation was performed according to EC guidelines, with an LOQ at 1 ng ml−1. ► Method useful for the analysis of ML-7 in plasma of other animal species or human. ► Method allows for monitoring of ML-7 in treated cats for pharmacokinetic analysis.An analytical method for the quantitative measurement of ML-7, a product with possible anti-immune escape activity for feline infectious peritonitis virus (FIPV), in feline plasma was developed and validated. The sample preparation consists of a solid-phase extraction step on an MCX cartridge. ML-7 and ML-9, used as the internal standard for the analysis, were separated on an ACQUITY UPLC™ BEH C18 reversed-phase column (1.7 μm, 50 mm × 2.1 mm I.D.), using isocratic elution with acetonitrile and 0.1% formic acid in water as the mobile phase. Both compounds were subsequently quantified in MRM mode on a Micromass® Quattro Premier™ XE triple quadrupole mass spectrometer. The use of a Thermo Scientific® Exactive™ orbitrap mass spectrometer made it possible to confirm the proposed fragmentation pattern of both ML-7 and ML-9. A validation study according to EC requirements was carried out, in which the method showed good performance. Linear behaviour was observed in the 1–2500 ng ml−1 range, which is relevant for real sample analysis. Accuracy and precision were within the criteria requested by the EC requirements throughout this concentration range. Extraction recovery of ML-7 was 72%. Matrix effect for ML-7 was not higher than 8%. The method was successfully used for the monitoring of ML-7 in feline plasma after intravenous, subcutaneous or oral administration of an ML-7 formulation, for the determination of pharmacokinetic parameters, with a limit of quantification of 1 ng ml−1 and a limit of detection of 0.4 ng ml−1. The proposed method also shows good characteristics for the analysis of ML-7 in plasma of other animal species and human plasma.
Keywords: ML-7/ML-9; Anti-FIPV immune escape activity; Feline plasma; SPE extraction; Quantification; UPLC–ESI-MS/MS;

Quantitative analysis of olanzapine in rat brain microdialysates by HPLC–MS/MS coupled with column-switching technique by Qiaoling Zheng; Feng Wang; Huande Li; Ping Xu; Huaibo Tang; Lanfang Li; Rihua Cheng (127-132).
► A rapid and sensitive method for determination of olanzapine was developed. ► This method used HPLC–MS/MS with column-switching technique. ► It needed no complicated sample processing. ► We studied the pharmacokinetics of olanzapine in rat brain with microdialysis.A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method coupled with column-switching technique was developed for the determination of olanzapine in rat brain microdialysates. A C8 guard column was used to desalt the samples before analytical separation on a C18 column and detection with tandem mass spectrometry. The mobile phase consisted of methanol/acetonitrile/water (v/v/v, 22.5/22.2/55) was used for desalting and the mobile phase consisted of methanol/acetonitrile/water (v/v/v, 43/43/14) was for analytical separation, water in both mobile phases contained 0.1% ammonium acetate. The lower limit of quantification (LLOQ) for olanzapine was 0.085 ng/ml. The method was linear from LLOQ to 34 ng/ml with a coefficient of determination >0.998. Intra- and inter-day accuracy and precision were determined with variability less than 13.24% (R.S.D). This sensitive method was successfully applied to quantify the concentration of olanzapine in rat brain microdialysates. With this study, the effect of the alcohol extract of Schisandra sphenanthera Rehd. et Wils on the concentration of olanzapine in brain was investigated.
Keywords: Olanzapine; HPLC–MS/MS; Microdialysis; Schisandra sphenanthera Rehd. et Wils;

A gas chromatography–mass spectrometry assay to quantify camphor extracted from goat serum by Kyung-Min Lee; Susie Y. Dai; Timothy J. Herrman; Jeffrey M.B. Musser (133-136).
► A reliable GC–MS method was developed for detection of camphor in goat blood. ► The method was sensitive and specific enough for quantification of camphor. ► The method offers a lower LOD and LOQ for camphor than conventional methods. ► The method yields a good precision and accuracy for monitoring camphor in animal. ► The validation study with two goats showed the good practicability of the method.A sensitive gas chromatography–mass spectrometry (GC–MS) method was developed and validated for quantification and pharmacokinetics of camphor, a major monoterpene of juniper plant, in goat serum. Camphor and internal standard (terpinolene) eluates from solid phase extraction (SPE) with ethyl acetate yielded well resolved peaks and were clearly identified in total and selected ion chromatograms. The elution and injection volumes were optimized for improved detection and quantification of camphor based on peak shape, signal to noise ratio, recoveries, and repeatability. The matrix calibration curve with the good linearity (R 2  = 0.998) and response in the range of 0.005–10.0 μg/mL was used to determine camphor concentration in goat serum. The GC–MS method offered sufficiently low limits of detection (1 ng/mL) and quantitation (3 ng/mL) for camphor concentration in goat serum for the pharmacokinetic study. The proposed method showed good intra- and inter-day variation with relative standard deviation (RSD) of 0.2–7.7% and produced good recovery (96.0–111.6%) and reproducibility (1.6–6.1%) at all spiked levels. Using this method on serum samples obtained from two goats orally dosed with camphor confirmed that the method is suitable for camphor studies in animals.
Keywords: Camphor; Gas chromatography–mass spectrometry; Goat serum; Juniper; Monoterpene;

Liquid chromatography–tandem mass spectrometric assay for therapeutic drug monitoring of the tyrosine kinase inhibitor pazopanib in human plasma by Rolf W. Sparidans; Tahani T.A. Ahmed; Eline W. Muilwijk; Marieke E.B. Welzen; Jan H.M. Schellens; Jos H. Beijnen (137-140).
► The first validated bioanalytical assay for pazopanib has been reported. ► The assay has successfully been validated in the 0.1–100 μg/ml range. ► The drug is sufficiently stable under all conditions relevant for the assay. ► The assay could be used for therapeutic drug monitoring.A quantitative bioanalytical liquid chromatography–tandem mass spectrometric assay for the tyrosine kinase inhibitor pazopanib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing pazopanib-d4 as internal standard. The extract was injected into the chromatographic system after dilution with water (1:9, v/v). The system consisted of a sub-2 μm particle, trifunctional bonded octadecyl silica column with isocratic elution using 0.005% (v/v) of formic acid in a mixture of water (76%, v/v) and acetonitrile (24%, v/v). The analyte was quantified using the selected reaction monitoring mode of a triple quadrupole mass spectrometer with a heated electrospray interface. The assay was validated in a 0.1–100 μg/ml calibration range. Within day precisions were 3.6–5.2%, between day precisions 4.0–8.3% and accuracies between 106% and 113% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. The assay has successfully been used to assess drug levels for therapeutic drug monitoring in patients treated with pazopanib.
Keywords: Pazopanib; VEGFR; LC–MS/MS; Human plasma; Therapeutic drug monitoring;

Development and validation of sensitive liquid chromatography/tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue by Lei He; John C. Grecula; Yonghua Ling; Michael D. Enzerra; Mario Ammirati; Kari Kendra; Robert Cavaliere; Nina Mayr; John McGregor; Thomas Olencki; Ewa Mrozek; Mani Matharbootham; Chima Oluigbo; Mitch A. Phelps (141-144).
► Bendamustine is FDA approved and is under evaluation for use in multiple cancers, including brain malignancies. ► This is the first report of a fully validated LC–MS/MS assay for bendamustine quantification in mouse brain tissue as a surrogate for human brain tissue. ► This assay is being applied to measure plasma and brain tumor concentrations of bendamustine in an ongoing clinical trial in patients with metastatic brain lesions.A liquid chromatography–tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue was developed and fully validated. Methanol was used to precipitate proteins in brain tissue. Bendamustine and internal standard (chlorambucil) were separated with reverse-phase chromatography on a C-18 column with a gradient of water and 95% methanol in 0.1% formic acid. Positive mode electrospray ionization was applied with selected reaction monitoring to achieve 5 ng/ml lower limits of quantitation in mouse brain tissue. The calibration curve for bendamustine in mouse brain was linear between 5 and 2000 ng/ml. The within- and between-batch accuracy and precision of the assay were within 15% at 10, 100 and 1000 ng/ml. The recovery and matrix effect of bendamustine in mouse brain tissue ranged from 41.1% to 51.6% and 107.4% to 110.3%, respectively. The validated method was then applied to quantitate bendamustine in an animal study. Results indicate the assay can be applied to evaluate bendamustine disposition in mouse brain tissue. This assay will be applied in the future to detect and quantify bendamustine in human brain tissue samples.
Keywords: Bendamustine; Liquid chromatography–mass spectrometry; LC–MS/MS, Brain;

► To extract and purify lignans from Dysosna versipellis. ► Ultrahigh pressure extraction combined with HSCCC was applied. ► Two compounds with high purities were obtained with high yield. ► The method was very useful for the extraction and purification of podophyllotoxins.Ultrahigh pressure extraction (UPE) was employed to extract podophyllotoxin and 4′-demethylpodophyllotoxin from Dysosma versipellis. The effects of extraction parameters including extraction solvents, pressure, time and solid/liquid ratio were investigated using a High Hydrostatic Pressure Processor. The optimal condition for UPE of the target compounds was 80% methanol, 200 MPa of pressure, 1 min of extraction time and 1:12 (g/mL) of solid/liquid ratio. Podophyllotoxin and 4′-demethylpodophyllotoxin in the crude extract were purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (10:10:8:12, v/v), and the fractions were analyzed by HPLC, ESI-MS and 1H NMR. As a result, 73.7 mg podophyllotoxin and 16.5 mg 4′-demethylpodophyllotoxin with purities over 96% were obtained from 260 mg crude sample in one-step separation.
Keywords: Ultrahigh pressure extraction (UPE); High-speed counter-current chromatography (HSCCC); Dysosma versipellis; Podophyllotoxin; 4′-Demethylpodophyllotoxin;

Liquid chromatography–tandem mass spectrometric assay for the ALK inhibitor crizotinib in mouse plasma by Rolf W. Sparidans; Seng Chuan Tang; Luan N. Nguyen; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (150-154).
► The first validated bioanalytical assay for crizotinib has been reported. ► The assay has successfully been validated in the 10–10,000 ng/ml range. ► The drug is stable under all conditions relevant for the assay. ► Oxidative metabolites can simultaneously be monitored. ► First pharmacokinetics in male wild type FVB mice have reported.A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for the ALK inhibitor crizotinib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing crizotinib-13C2-2H5 as internal standard. The extract was directly injected into the chromatographic system after dilution with water. This system consisted of a sub-2 μm particle, trifunctional bonded octadecyl silica column with a gradient using 0.1% (v/v) of ammonium hydroxide in water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 10–10,000 ng/ml calibration range with r 2  = 0.99980 ± 0.00014 for double logarithmic linear regression (n  = 5). Within day precisions (n  = 6) were 3.4–4.8%, between day (3 days; n  = 18) precisions 3.6–4.9%. Accuracies were between 107% and 112% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Oxidative metabolites of crizotinib were monitored semi-quantitatively. Finally, the assay was successfully used to assess drug pharmacokinetics in mice.
Keywords: Crizotinib; ALK inhibitor; LC–MS/MS; Oxidative metabolites; Mouse plasma;

Comments on “Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support” by Krishnan Sushma; Chuvappumkal Joseph Bilgimol; Mookambeswaran A. Vijayalakshmi; Padikara Kutty Satheeshkumar (155).