Journal of Chromatography B (v.879, #31)

► We developed and validated a robust LC–MS/MS method for urinary metanephrines. ► Specificity was enhanced and interferences resolved using multiple mass transitions. ► This sensitive, high-throughput LC–MS/MS method is suitable for clinical use.Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC–MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L. The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC–MS method. Injection-to-injection time was 6 min. The validated LC–MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well.
Keywords: Liquid chromatography; Mass spectrometry; Metanephrine; Normetanephrine; Pheochromocytoma; Solid phase extraction;

Development of proteomic tools to study protein adsorption on a biomaterial, titanium grafted with poly(sodium styrene sulfonate) by S. Oughlis; S. Lessim; S. Changotade; F. Bollotte; F. Poirier; G. Helary; J.J. Lataillade; V. Migonney; D. Lutomski (3681-3687).
► Chromatography enables study of protein adsorption on polyNaSS/titanium biomaterial. ► Chromatography and proteomics lead to identify proteins adsorbed on biomaterial. ► PolyNaSS/titanium surface modulates adsorption of proteins.It is known that protein adsorption is the initial interaction between implanted biomaterials and biological environment. Generally, a complex protein layer will be formed on material surfaces within a few minutes and the composition of this layer at the interface determines the biological response to the implanted material, and therefore the long-term compatibility of the biomaterial. Despite different techniques exist to observe protein adsorption on biomaterials, none of them led to the identification of adsorbed proteins. In this paper, we report a chromatographic technique coupled to proteomics to analyse and identify proteins from complex biological samples adsorbed on biomaterial surfaces. This approach is based on (1) elaboration of the chromatographic support containing the biomaterial (2) a chromatography step involving adsorption of proteins on the biomaterial (3) the high-resolution separation of eluted proteins by 2-DE gel and (4) the identification of proteins by mass spectrometry. Experiments were performed with proteins from platelets rich plasma (PRP) adsorbed on a biomaterial which consist in titanium bioactivated with PolyNaSS. Our results show that chromatographic approach combined to 2-DE gels and mass spectrometry provides a powerful tool for the analysis and identification of proteins adsorbed on various surfaces.
Keywords: Liquid chromatography; Biomaterial; Titanium; Proteomic; Platelets rich plasma;

A sensitive and specific HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of isoforskolin in beagle dogs by Lulu Tian; Yaqin Wang; Yi Ling; Jiajun Yin; Jun Chen; Jianming Huang (3688-3693).
► The first study to develop an HPLC–MS/MS method for the determination of isoforskolin in plasma. ► The first time to study the pharmacokinetic characterization of isoforskolin in beagle dogs.A sensitive and specific high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) method has been developed and validated for the determination of isoforskolin in canine plasma. Liquid–liquid extraction was used to extract isoforskolin and the internal standard (I.S.) eplerenone from canine plasma. The chromatographic separation was performed on an Agela Venusil XBP Phenyl column with an isocratic mobile phase consisting of methanol–2 mM ammonium acetate–formic acid (62:38:0.1, v/v/v), pumped at 0.35 mL/min. Isoforskolin and I.S. were detected at m/z 433.4 → 373.3 and m/z 415.3 → 163.5 in positive ion and multiple reaction monitoring (MRM) mode, respectively. The standard curves were linear over the concentration range of 0.1–200 ng/mL (r  > 0.99). The intra- and inter-batch accuracy values for isoforskolin at four concentrations were 90.2–108.3% and 97.8–106.6%, respectively. The RSDs were less than 6.0%. The mean extraction recoveries of isoforskolin and I.S. were 97.0 and 88.4%, respectively. The method was successfully applied to the pharmacokinetic study after an intravenous administration of isoforskolin in beagle dogs.
Keywords: Isoforskolin; HPLC–ESI-MS/MS; Liquid–liquid extraction; Pharmacokinetics;

Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method by Abdellah Tebani; Dimitri Schlemmer; Apolline Imbard; Odile Rigal; Dominique Porquet; Jean-François Benoist (3694-3699).
► When sialic acid is assayed by reversed-phase LC its short retention time leads to important ion suppression effects. ► We butylated sialic acid to improve its analytical separation. ► Butylated sialic acid allowed a more sensitive detection in positive mode. ► Stable isotope of sialic acid was used as an internal standard to minimize matrix effect.The measurement of urine sialic acid (N Acetylneuraminic Acid: Neu5Ac) is useful for screening sialic acid storage disorders. We developed a new LC MS/MS method for the determination of a sialic acid. Urine samples were analyzed, after an HCl n-Butanol derivatization step, by a reverse phase based high-performance liquid chromatography method using 1,2,3-13C3 N-Acetyl-d-neuraminic Acid (13C-Neu5Ac) as an internal standard. Selective detection was performed by tandem mass spectrometry using an electrospray source operating in positive ionization mode employing multiple reactions monitoring to monitor N-Acetylneuraminic Acid and the internal standard. The transitions m/z 366 → 330 and 369 → 333 for Neu5Ac and 13C-Neu5Ac were respectively monitored. The limit of the method quantification was 1.40 μM of N-Acetylneuraminic Acid and the calibration curve showed a good linearity up to 1000 μM. The within assay precision and accuracy of the method ranged from 3.22 to 5.95% and 98.69 to 109.18%, respectively and the between assay precision and accuracy ranged, respectively, from 5.15 to 7.65% and 96.14 to 102.30%. The method can be applied for the determination of N-Acetylneuraminic Acid concentrations in urine and other biological fluids (e.g., amniotic and peritoneal fluids).
Keywords: Tandem mass spectrometry; N-Acetylneuraminic Acid; Sialic acid storage disease;

► α-Lipoic acid and coenzyme Q10 decrease the glucose level in plasma samples. ► α-Lipoic acid and coenzyme Q10 have positive influence on the lactulose/mannitol index. ► Validation data prove the complementarity of the IC-ECD and IC–MS/MS methods. ► Newly developed 2PAD method has been proven to be equivalent to standard 4PAD.The present paper demonstrates that electrochemical detection (ECD) coupled to ion chromatography and electrospray ionization tandem mass spectrometry (IC-ECD–ESI/MS/MS) can be used to rapidly estimate some indications of the health status of organisms. The lactulose to mannitol ratio (L/M) is used as a non-invasive assay to investigate small intestinal absorption pathways and mucosal integrity. In the present study, an evaluation of the negative effects of nonsteroidal anti-inflammatory drug meloxicam perorally administrated to a group of dogs was carried out by determining the lactulose/mannitol index using the IC-ECD–ESI/MS/MS hyphenated technique. According to the results of the study, meloxicam altered gastrointestinal permeability. Coenzyme Q10 (CoQ10) was tested to determine if it could prevent meloxicam induced gastrointestinal damage and it was found that CoQ10 could be an effective preventive treatment. Furthermore, plasma glucose concentration level was determined to be an indirect indicator of the oxidative state in the blood. To find out the beneficial effects of a double antioxidant combination (α-lipoic acid (ALA) and CoQ10) on the total glucose level in chickens, ALA and CoQ10 were provided as food additives in factory farm raised chicken. The results of the pilot study indicate that the glucose level in the plasma of chickens group fed with CoQ10 and ALA was significantly decreased compared to the control group. Ion chromatography (IC) utilizing pulsed amperometric detection (PAD) was compared to ion chromatography coupled with tandem mass spectrometry (MS/MS) as an analytical tool for monitoring the carbohydrate level in biological fluids. In electrochemical detection, the newly developed two-pulse waveform successfully withstands matrix effects in biological samples. Continuous on-line desalting of the high salt concentrations used as the eluent for carbohydrate separation from the anion-exchange column allows coupling of IC and MS techniques. A make-up solution (0.5 mM LiCl) was delivered prior to MS detection for efficient ionization of eluted carbohydrates. Method validation showed that both used techniques are practically comparable and some advantages of each are presented.
Keywords: Carbohydrates; High-performance anion-exchange chromatography; Pulsed amperometric detection; Mass spectrometry; Lactulose–mannitol index;

Determination of the new anthelmintic monepantel and its sulfone metabolite in milk and muscle using a UHPLC–MS/MS and QuEChERS method by Brian Kinsella; Patrick Byrne; Helen Cantwell; Martin McCormack; Ambrose Furey; Martin Danaher (3707-3713).
► An analytical method was developed for the new anthelmintic monepantel. ► Monepantel and sulfone metabolite were analyzed in goat's milk and ovine muscle. ► We used a modified QuEChERS extraction method and UHPLC–MS/MS analysis. ► Successfully single-laboratory validated according to the 2002/657/EC guidelines.This is the first paper to report a method for the detection of the new anthelmintic monepantel and its sulfone metabolite in goat's milk and ovine muscle. Samples were extracted and purified using a modified QuEChERS method. A concentration step was included when analyzing in the low μg kg−1 range. Analysis was carried out by ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) in a 13 min run time using atmospheric pressure electrospray ionisation in the negative mode (ESI) and multiple reaction monitoring (MRM) scanning. Monepantel (m/z 472) and monepantel-sulfone (m/z 504) both had product ions at m/z 186 and m/z 166. The method has been single-laboratory validated according to the 2002/657/EC guidelines. The mean recovery in milk was 108 and 106% for monepantel and monepantel-sulfone, respectively. The mean recovery in muscle was 109 and 108% for monepantel and monepantel-sulfone, respectively. The coefficients of variation for the within laboratory repeatability and reproducibility were ≤6.4% in milk and ≤14.2% in muscle. The decision limits (CCα) in milk were 2.20 and 2.08 μg kg−1 for monepantel and monepantel-sulfone, respectively. The decision limits (CCα) in muscle were 771 and 746 μg kg−1 for monepantel and monepantel-sulfone, respectively.
Keywords: Monepantel; Monepantel-sulfone; Amino-acetonitrile derivatives; Goat's milk; Ovine muscle; Ultra high performance liquid chromatography–tandem mass spectrometry; QuEChERS;

In situ derivatization-DLLME-GC/MS method was validated and developed. ► TCAs in human urine sample were determined by that suitable method. ► Proposed method has many practical advantages, and was achieved high sensitivity.A simple, rapid and sensitive method termed dispersive liquid–liquid microextraction (DLLME) combined with gas chromatography–mass spectrometry (GC/MS) was developed for the determination of tricyclic antidepressants (TCAs) in human urine sample. An appropriate mixture of methanol (disperser solvent), carbon tetrachloride (extraction solvent), and acetic anhydride (derivatization reagent) was injected rapidly into human urine sample. After extraction, the sedimented phase was analyzed by GC/MS. The calibration curves obtained with human urine were linear with a correlation coefficient of over 0.99 in the range of 2.0/5.0–100 ng mL−1. Under the optimum conditions (carbon tetrachloride: 10 μL, methanol: 150 μL), the detection limits and the quantification limits of the tricyclic antidepressants were 0.5–2.0 ng mL−1 and 2.0–5.0 ng mL−1, respectively. The average recoveries of TCAs were 88.2–104.3%. Moreover, the inter- and intra-day precision and accuracy was acceptable at all concentrations. The results showed that DLLME is applicable to the determination of trace amounts of TCAs in human urine sample.
Keywords: Dispersive liquid–liquid microextraction (DLLME); In situ derivatization GC–MS; Tricyclic antidepressant; Human urine;

► Docetaxel-loaded NLC was modified by a novel amphiphilic copolymer FA-PEG-PCHL. ► The modified formulation was defined as FA-DTX-NLC. ► An LC–MS method was developed and validated for in vivo study. ► FA-DTX-NLC may lead a long blood circulating effect and targeting ability. ► FA-DTX-NLC could be one of the promising suspensions for docetaxel in cancer.A novel amphiphilic copolymer, folate-poly(PEG-cyanoacrylate-co-cholesteryl cyanoacrylate) (FA-PEG-PCHL) was synthesized to modify docetaxel-loaded nanostructured lipid carrier to lead to a long blood circulating effect and targeting ability for the delivery of antitumor drug in cancer. To investigate the characteristics of modified docetaxel-loaded nanostructured lipid carrier in vivo, a liquid chromatography–mass spectrometry method was developed and validated for the determination of docetaxel in rat plasma and tumor-bearing mouse tissue samples. The biosamples were extracted by liquid–liquid extraction method with ether and separated on a C18 column (150 mm × 4.6 mm, 5 μm) using a mobile phase consisting of methanol–0.01% formic acid water (82:18, v/v). The standard curves were linear over the ranges of 0.01–4.0 μg/mL for plasma and 0.02–8.0 μg/g for tissue samples, respectively. The validated method was successfully applied to the pharmacokinetic study in rat plasma and tissue distribution study in mouse tissues of docetaxel after an intravenous administration of docetaxel injection (DTX injection), docetaxel-loaded nanostructured lipid carrier (DTX-NLC) and FA-PEG-PCHL-modified docetaxel-loaded nanostructured lipid carrier (FA-DTX-NLC), respectively. The results indicated that the FA-DTX-NLC led to significant differences in pharmacokinetic profile and tissue distribution. Nanostructured lipid carrier modified by FA-PEG-PCHL could be one of the promising suspensions for the delivery of docetaxel in cancer.
Keywords: Docetaxel; FA-PEG-PCHL; Nanostructured lipid carrier; Pharmacokinetics; Tissue distribution; LC–MS;

Chlorpromazine quantification in human plasma by UPLC–electrospray ionization tandem mass spectrometry. Application to a comparative pharmacokinetic study by Ney Carter Borges; Vinicius Marcondes Rezende; Jose Marcos Santana; Ricardo Pereira Moreira; Roberto Fernandes Moreira; Patrícia Moreno; Diego Carter Borges; José Luiz Donato; Ronilson Agnaldo Moreno (3728-3734).
► UPLC–APCI-MS method to quantify chlorpromazine in human plasma. ► Method was applied in a relative bioavailability study in order to compare a test chlorpromazine 100 mg simple dose formulation. ► The chlorpromazine was extracted from human plasma by a simple liquid–liquid extraction. ► This method agrees with the requirements proposed by the US Food and Drug Administration of high sensitivity, specificity and high sample throughput in comparative pharmacokinetic assays such as bioequivalence.In the present study a method to quantify chlorpromazine in human plasma using cyclobenzaprine as the internal standard (IS) is described. The analyte and the IS were extracted from human plasma by a liquid–liquid extraction with diethyl ether/dichloromethane (70/30, v/v) and analyzed by an ultra performance liquid chromatography (UPLC) coupled to an electrospray tandem triple quadrupole mass spectrometer in positive mode (UPLC–ES+-MS/MS). Chromatography was performed isocratically on an Aquity UPLC BEH C18 1.7 μm (50 mm × 2.1 mm i.d.) operating at 40 °C. The mobile phase was a mixture of 65% water + 1% formic acid and 35% of acetonitrile at a flow-rate of 0.5 mL/min. The lowest concentration quantified was 0.5 ng/mL and a linear calibration curve over the range 0.5–200 ng/mL was obtained, showing intra-assay precisions from 2.4 to 5.8%, and inter-assay precisions from 3.6 to 9.9%. The intra-assay accuracies ranged from 96.9 to 102.5%, while the inter-assay accuracies ranged from 94.1 to 100.3%. This analytical method was applied in a relative bioavailability study in order to compare a test chlorpromazine 100 mg simple dose formulation versus a reference in 57 volunteers of both sexes. The study was conducted in an open randomized two-period crossover design and with a fourteen days washout period. Plasma samples were obtained over a 144-h interval. Since the 90% CI for both C max, AUClast and AUC0–inf were within the 80–125% interval proposed by the Food and Drug Administration and ANVISA, it was concluded that chlorpromazine 100 mg/dose was bioequivalent to the reference formulation, according to both the rate and extent of absorption.
Keywords: Chlorpromazine; UPLC–MS/MS; Bioavailability; Pharmacokinetics;

► We developed a sensitive and selective UHPLC–MS/MS method. ► We simultaneously determine three compounds in rat plasma and urine. ► This method was successfully applied to pharmacokinetic studies in rats. ► The method is useful for further studies on Rhizoma Belamcandae in clinical research.A sensitive and reliable ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC–ESI–MS/MS) has been developed and validated for the simultaneous determination of three active components, i.e., tectorigenin, irigenin and irisflorentin, in rat plasma and urine after oral administration of Rhizoma Belamcandae extract. Chromatographic separation was achieved on a Zorbax SB-C18 column (50 mm × 2.1 mm, 1.8 μm; Agilent, USA) with gradient elution using a mobile phase that consisted of acetonitrile – 0.1% formic acid in water at a flow rate of 0.4 mL/min. Detection was performed by a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between the negative (for tectorigenin and irigenin) and positive (for irisflorentin) ionization modes. The calibration curve was linear over a range of 50–50,000 ng/mL for tectorigenin, 10–5000 ng/mL for irigenin and 0.1–200 ng/mL for irisflorentin, respectively. The intra- and inter-day precisions (RSD %) were within 11.3% for all analytes, whereas the deviation of assay accuracies ranged from −8.7 to +11.1%. All analytes were proven to be stable during all sample storage and analysis procedures. This method was successfully applied to a pharmacokinetic study of the three isoflavones after oral administration of Rhizoma Belamcandae extract to rats.
Keywords: Rhizoma Belamcandae; Pharmacokinetic; Excretion; Quantitative analysis; UHPLC–ESI–MS/MS;

Extractive ethoxycarbonylation in high-temperature gas chromatography–mass spectrometry based analysis of serum estrogens by Ju-Yeon Moon; Se Mi Kang; Myeong Hee Moon; Jongki Hong; Ki Tae Kim; Dae Hoon Jeong; Young Nam Kim; Bong Chul Chung; Man Ho Choi (3742-3748).
► Estrogen metabolism is associated with many hormonal-dependent diseases. ► Estrogens are derivatized with ethoxycarbonylation followed by pentafluoropropionylation. ► Derivatized estrogens are effectively chromatographed by high-temperature GC–MS. ► The two-step derivatization gives better selectivity as well as sensitivity in human serum.A comprehensive gas chromatography–mass spectrometry (GC–MS)-based profiling was developed as a practical assay for quantification of 18 endogenous estrogens in serum samples. The present GC–MS method was conducted with the two-phase extractive ethoxycarbonlyation (EOC) of the phenolic hydroxy groups of estrogen with ethyl chlorformate combined with the non-polar n-hexane extraction. The subsequent perfluoroacylation of aliphatic hydroxy groups with pentafluoropropionyl anhydride (PFPA) was conducted. The serum samples were separated through a high temperature GC column (MXT-1) within an 8-min run and analyzed in selected-ion monitoring mode with good chromatographic properties for 18 estrogens as their EOC-PFP derivatives. The limit of quantification (LOQ) was 0.025–0.10 ng/mL for most estrogens analyzed except for E3 and 2-OH-E3 (0.5 ng/mL each). The devised method was found to be linear over a 103-fold concentration range with a correlation coefficient (r 2  > 0.992), whereas the precision (% CV) and accuracy (% bias) ranged from 3.1 to 16.3% and from 93.5 to 111.1%, respectively. Decreased 2-methoxy-17β-estradiol levels were confirmed in patients with preeclampsia than healthy pregnant women. This technique can be used for a clinical diagnosis as well as understanding the pathogenesis in estrogen-related disorders.
Keywords: Estrogen; Serum; GC–MS; Extractive ethoxycarbonylation; Preeclampsia;

The relationship between Candida species charge density and chitosan activity evaluated by ion-exchange chromatography by A. Palmeira-de-Oliveira; L.A. Passarinha; C. Gaspar; R. Palmeira-de-Oliveira; B. Sarmento; J. Martinez-de-Oliveira; C. Pina-Vaz; A.G. Rodrigues; J.A. Queiroz (3749-3751).
Chitosan, a natural biopolymer presents antifungal activity that seems to be dependent on the interaction of its cationic amino groups and yeast cell surface. In this work we used ion-exchange chromatography to assess the surface charge density of Candida species and subsequently to relate this with their sensitivity profile to chitosan. The ability of several strains from distinct Candida species to interact with strong anionic and cationic exchangers was tested and the yeasts charge surface was assessed by measuring the zeta potential. Our results showed that all the yeast cells tested presented no interaction with the cationic resin and a species-related pattern of interaction was observed with the anionic resin. Specifically, regarding the Q-Sepharose support, Candida glabrata showed the lower retention affinity, followed by Candida albicans, presenting Candida tropicalis an intermediate profile; Candida parapsilosis and Candida guilliermondii revealed a stronger ionic interaction. The yeasts retention synergy in the anionic resin corroborates with the zeta potential outcomes. The behavior observed fit with sensitivity patterns to chitosan as the most susceptible species to chitosan presented higher affinity to the anionic resin in contrast to the less sensitive ones (C. albicans and C. glabrata). This data confirms and reinforces that chitosan activity is probably mediated by an ionic reaction between its amino free groups and ionic charges at the cell surface.
Keywords: Ion-exchange chromatography; Cells charge density; Antifungal activity; Candida species; Chitosan;

In this study, the extraction of γ-hydroxybutyrate (GHB) from urine using solid-phase extraction (SPE) is described. SPE was performed on anion exchange columns after samples of urine had been diluted with de-ionized water. After application of the diluted samples containing GHB-d6 as an internal standard, the sorbent was washed with deionized water and methanol and dried. The GHB was eluted from the SPE column with a solvent consisting of methanol containing 6% glacial acetic acid. The eluent was collected, evaporated to dryness, and dissolved in mobile phase (100 μL) for analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in negative multiple reaction monitoring (MRM) mode. Liquid chromatography was performed in gradient mode employing a biphenyl column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 50 and 100 ng/mL, respectively. The method was found to be linear from 500 ng/mL to 10,000 ng/mL (r 2  > 0.995). The recovery of GHB was found to be greater than 75%. In this report, results of authentic urine samples analyzed for GHB by this method are presented. GHB concentrations in these samples were found to be range from less than 500 ng/mL to 5110 ng/mL.
Keywords: GHB; SPE; Forensic toxicology;

Quantification of lactose content in human and cow's milk using UPLC–tandem mass spectrometry by Gerhard Fusch; Arum Choi; Niels Rochow; Christoph Fusch (3759-3762).
► Quantification of lactose content in cow's and human milk with high precision and accuracy. ► Dilute and shoot method: new method requires only dilution of cow's and human milk for simple preparation. ► Can be used for a routine purpose. ► Method is not affected by oligosaccharides such as enzymatic lactose assays.A sensitive, accurate, and specific quantitative UPLC–MS/MS method was developed for lactose measurement of cow's and human milk and validated with cow's milk samples certified by an external laboratory. The new method employs only a dilution of raw cow's and human milk for simple preparation with no need to remove protein and fat prior to analysis with UPLC–MS/MS. It was operated in negative mode to detect lactose molecules and labeled 13C12-lactose with the highest sensitivity. The principle advantages of the new LC–MS/MS method were: completed lactose determination in 5 min, absolute recovery of 97–107%, lower limit of detection <5 ng/L, and 99% linearity over the concentration range of 0.7–4.4 mg/L for both cow's and human milk. The mean lactose concentration of 51 human milk samples was measured as 56.8 ± 5.5 g/L ranging from 43 to 65 g/L. The described method represents validated lactose analysis with high accuracy and precision for a routine lactose determination in raw human milk.
Keywords: LC–MS/MS; Carbohydrate; Lactose; Human milk; Cow's milk; Nutrition;

Quantification of a novel natural antioxidant (UP302) in rat plasma using ultra-high performance liquid chromatography–tandem mass spectrometry by Shuang-Qing Zhang; Ling Zhu; Nie Wen; Min Yu; Yi-Zheng Shen; Qi Jia; Zuo-Gang Li; Bo Li (3763-3766).
► A LC–MS/MS was firstly established for the measurement of UP302 in plasma. ► The method was fully validated. ► The method was applied to stability and pharmacokinetics.UP302 is a novel natural antioxidant isolated from Dianella ensifolia (Liliaceae). In the investigation, a specific and sensitive ultra-high performance liquid chromatography–tandem mass spectrometry for quantitative determination of UP302 in rat plasma was developed and validated. UP302 and the internal standard daidzein were extracted from 100 μL aliquots of rat plasma using methanol. Detection of UP302 and IS was done by tandem mass spectrometry, operating in negative ion and selected reaction monitoring acquisition mode. The precursor–product ion transitions monitored for UP302 and daidzein were m/z 301.1 → 135.2 and 252.9 → 132.0, respectively. The linearity of the method was observed within the concentration range of 5–2000 ng/mL. Intra- and inter-day assay variations were less than 15%, and the accuracy values were between 99.2% and 107.3%. The method was successfully applied to stability investigation of UP302 incubated in rat plasma at 37 °C and measurement of UP302 in plasma after intravenous administration of UP302 to rats at a single dose of 5 mg/kg. Incubation stability revealed that within first one hour, UP302 was rapidly declined approximately 35% and remained stable after 4 h. Pharmacokinetic values of half-life, volume of distribution, systemic clearance and mean residence time were 0.87 ± 0.58 h, 6.90 ± 3.35 L/kg, 5.89 ± 1.21 L/h kg and 0.34 ± 0.13 h, respectively.
Keywords: UP302; UHPLC–MS/MS; Stability; Pharmacokinetics;

► Separation of alkaloids from Dactylicapnos scandens by pH-zone-refining CCC. ► Apparatus with an adjustable length of the separation column was used. ► The two-phase solvent system was petroleum ether–ethyl acetate–methanol–water (3:7:1:9, v/v). ► Changing the length of separation column, resolution of the compounds can be improved. ► Protopin, (+) corydine, (+) isocorydine, (+) glaucine were obtained with high purity.pH-Zone-refining counter-current chromatography was successfully applied for the preparative separation of alkaloids from Dactylicapnos scandens. The two-phase solvent system was composed of petroleum ether–ethyl acetate–methanol–water (3:7:1:9, v/v), where 20 mM of triethylamine (TEA) was added to the upper phase as a retainer and 5 mM of hydrochloric acid (HCl) to the aqueous phase as an eluter. In this experiment, the apparatus with an adjustable length of the separation column was used for the separation of alkaloids from D. scandens and the resolution of the compounds can be remarkably improved by increasing the length of the separation column. As a result, 70 mg protopin, 30 mg (+) corydine, 120 mg (+) isocorydine and 40 mg (+) glaucine were obtained from 1.0 g of the crude extracts and each with 99.2%, 96.5%, 99.3%, 99.5% purity as determined by HPLC. The chemical structures of these compounds were confirmed by positive ESI-MS and 1H NMR.
Keywords: Dactylicapnos scandens; Alkaloids; pH-Zone-refining counter-current chromatography; Preparative separation;

► 2-(Diphenylmethyl)pyrrolidine (desoxy-D2PM) is sold on the recreational drug market. ► Adverse effects associated with its use are increasingly reported. ► A quantitative method was validated in whole unpreserved human blood.The increased availability of new psychoactive substances (“legal highs”) from retail shops or internet sources has caught the imagination of consumers, law enforcement and scientific communities. The present study describes the identification of 2-(diphenylmethyl)pyrrolidine (DPMP, desoxy-D2PM) as the key constituent found in an internet product called “A3A New Generation”. Analytical characterization of this new generation “legal high” product was based on gas chromatography (EI/CI) ion trap mass spectrometry and liquid chromatography (HPLC) using electrospray ionization tandem mass spectrometry and diode array detection. A quantitative method was also developed and validated for the detection of DPMP in human whole blood using HPLC single wavelength ultraviolet detection (210 nm). Evaluation of standard method validation parameters was found to be satisfactory. The circulation of DPMP on the market is another example of psychoactive substances described decades ago in the patent literature which are beginning to be rediscovered by recreational drug communities. The ability to unambiguously identify and detect this psychoactive compound should therefore be of interest to those who are exposed to the recreational drugs field.
Keywords: Legal highs; DPMP; Desoxy-D2PM; Stimulant; LC-UV; GC–MS; LC–MS/MS; Blood;

Isolation and purification of seven lignans from Magnolia sprengeri by high-speed counter-current chromatography by Yu Sun; Zongyuan Yu; Wenjuan Duan; Lei Fang; Shuangshuang Xu; Xiao Wang (3775-3779).
► To purify lignans from Magnolia sprengeri Pamp. by HSCCC. ► A two-step separation was established. ► In the first separation, four compounds were isolated using stepwise elution. ► The residues were used for further separation, three compounds were isolated. ► This method was efficient and rapid for the separation of lignans.Seven lignans including (−)-maglifloenone, futoenone, magnoline, cylohexadienone, fargesone C, fargesone A and fargesone B were isolated and purified from Magnolia sprengeri Pamp. using high-speed counter-current chromatography (HSCCC) with two-step separation. In the first step, a stepwise elution mode with the two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (1:0.8:0.6:1.2, 1:0.8:0.8:1, v/v) was used and 15.6 mg of (−)-maglifloenone, 19.2 mg of futoenone, 10.8 mg of magnoline, 14.7 mg of cylohexadienone and 217 mg residues were obtained from 370 mg crude extract. In the second step, the residues were successfully separated by HSCCC with the solvent system composed of petroleum ether–ethyl acetate–methanol–water (1:0.8:1.2:0.6, v/v), yielding 33.2 mg of fargesone C, 47.5 mg of fargesone A and 17.7 mg of fargesone B. The purities of the separated compounds were all over 95% determined by HPLC. The chemical structures of these compounds were confirmed by 1H NMR, 13C NMR and ESI-MS.
Keywords: Magnolia sprengeri Pamp.; Lignans; HSCCC;