Journal of Chromatography B (v.1028, #C)

Simultaneous HPLC-APCI-MS/MS quantification of endogenous cannabinoids and glucocorticoids in hair by Christopher Mwanza; Zheng Chen; Quan Zhang; Shenghuo Chen; Weiwen Wang; Huihua Deng (1-10).
Hair matrix could retrospectively record association of endogenous cannabinoids (e.g. 2-arachidonoyl glycerol, 2-AG and N-arachidonoyl-ethanolamine, AEA) and glucocorticoids (e.g. cortisol and cortisone) in a myriad of physiological functions. However, depending on the extraction conditions, the spontaneous isomerization of 2-AG to 1-arachidonoylglycerol (1-AG) and the possible rearrangement of O-arachidonoyl ethanolamine (OAEA) to AEA in various sample matrices could be major obstacles encountered in the detection of both 2-AG and AEA. This study aimed to develop a novel method for simultaneous quantification of 2-AG, AEA, cortisol and cortisone in hair. Methanol was used as the incubation solution and an acidic mixture of deionized water and methanol were utilized as mobile phase in order to avert possible rearrangements of both OAEA and 2-AG. The analyses were performed on a high-performance liquid chromatography tandem mass spectrometer with atmosphere pressure chemical ionization in positive mode. The method showed good linearity in the range of 3.0–250 pg/mg for AEA, 15.0–1250 pg/mg for 2-AG and 1–250 pg/mg for cortisol and cortisone. Limit of detection was 1.5 pg/mg for AEA, 6.0 pg/mg for 2-AG and 0.5 pg/mg for cortisol and cortisone. For all four analytes, intra and inter-day coefficients of variation were less than 20% and recovery above 90%. Population analyses in 473 hair samples established that 2-AG was significantly correlated with AEA. 2-AG was significantly and positively correlated with cortisol and cortisone. There was a significant positive correlation of AEA with cortisol, but not with cortisone. Obese participants showed a significantly higher concentration of cortisone and 2-AG. Males showed significantly higher 2-AG and cortisone levels but significantly lower AEA levels than females.
Keywords: 2-Arachidonoyl glycerol; N-arachidonoyl-ethanolamine; Cortisone; Cortisol; Hair matrix;

Selective kappa opioid receptor (KOR) antagonists may have therapeutic potential as treatments for substance abuse and mood disorders. Since [D-Trp]CJ-15,208 (cyclo[Phe-d-Pro-Phe-d-Trp]) is a novel potent KOR antagonist in vivo, it is imperative to evaluate its pharmacokinetic properties to assist the development of analogs as potential therapeutic agents, necessitating the development and validation of a quantitative method for determining its plasma levels. A method for quantifying [D-Trp]CJ-15,208 was developed employing high performance liquid chromatography-tandem mass spectrometry in mouse plasma. Sample preparation was accomplished through a simple one-step protein precipitation method with acetonitrile, and [D-Trp]CJ-15,208 analyzed following HPLC separation on a Hypersil BDS C8 column. Multiple reaction monitoring (MRM), based on the transitions m/z 578.1 → 217.1 and 245.0, was specific for [D-Trp]CJ-15,208, and MRM based on the transition m/z 566.2 → 232.9 was specific for the internal standard without interference from endogenous substances in blank mouse plasma. The assay was linear over the concentration range 0.5–500 ng/mL with a mean r2  = 0.9987. The mean inter-day accuracy and precision for all calibration standards were 93–118% and 8.9%, respectively. The absolute recoveries were 85 ± 6% and 81 ± 9% for [D-Trp]CJ-15,208 and the internal standard, respectively. The analytical method had excellent sensitivity with a lower limit of quantification of 0.5 ng/mL using a sample volume of 20 μL. The method was successfully applied to an initial pharmacokinetic study of [D-Trp]CJ-15,208 following intravenous administration to mice.
Keywords: Macrocyclic peptide; Kappa opioid receptor antagonist; [D-Trp]CJ-15,208; LC–MS/MS; Multiple reaction monitoring; Peptide quantitation;

Methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs). Aberrant expression and activity of these enzymes has been reported to play an important role in the initiation and progression of tumors and its response to chemotherapy. Therefore, there is a great interest in developing strategies to detect human DNMTs activity. We propose a simple, antibody-free, label-free and non-radioactive analytical strategy in which methyltransferase activity is measured trough the determination of the 5-methylcytosine (5mC) content in DNA by a chromatographic method (HPLC-UV) previously developed. For this aim, a correlation between the enzyme activity and the concentration of 5mC obtained by HPLC-UV is previously obtained under optimized conditions using both, un-methylated and hemi-methylated DNA substrates and the prokaryotic methyltransferase M.SssI as model enzyme. The evaluation of the methylation yield in un-methylated known sequences (a 623 bp PCR-amplicon) turned to be quantitative (110%) in experiments conducted in-vitro. Methylation of hemi-methylated and low-methylated sequences could be also detected with the proposed approach. The application of the methodology to the determination of the DNMTs activity in nuclear extracts from human ovarian cancer cells has revealed the presence of matrix effects (also confirmed by standard additions) that hampered quantitative enzyme recovery. The obtained results showed the high importance of adequate sample clean-up steps.
Keywords: DNA methylation; Methyl transferases; Activity; HPLC-UV;

A rapid, sensitive, and robust aqueous normal-phase chromatography method coupled with tandem mass spectrometry was developed and validated for the quantitation of AZD1775, a Wee-1 inhibitor, in human plasma and brain tumor tissue. Sample preparation involved simple protein precipitation with acetonitrile. Chromatographic separation was achieved on ethylene bridged hybrid stationary phases (i.e., Waters XBridge Amide column) under an isocratic elution with the mobile phase consisting of acetonitrile/ammonium formate in water (10 mM, pH 3.0) (85:15,v/v) at a flow rate of 0.8 mL/min for 5 min. The lower limit of quantitation (LLOQ) was 0.2 ng/mL of AZD1775 in plasma and tissue homogenate. The calibration curve was linear over AZD1775 concentration range of 0.2–1000 ng/mL in plasma and tissue homogenate. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method (<15%). The method was successfully applied to assess the penetration of AZD1775 across the blood-brain tumor barrier, as assessed by the unbound brain-to-plasma ratio, in patients with glioblastoma.
Keywords: AZD1775; Aqueous normal-phase chromatography; Tandem mass spectrometry; LC–MS/MS; Unbound brain-to-plasma ratio; Fraction unbound;

A sensitive, reliable and accurate UHPLC–MS/MS method has been firstly established and validated for the simultaneous quantification of ginkgo flavonoids, terpene lactones and nimodipine in rat plasma after oral administration of Ginkgo biloba dispersible tablets, Nimodipine tablets and the combination of the both, respectively. The plasma samples were extracted by two step liquid–liquid extraction, nimodipine was extracted by hexane-ether (3:1, v/v) at the first step, after that ginkgo flavonoids and terpene lactones were extracted by ethyl acetate. Then the analytes were successfully separated by running gradient elution with the mobile phase consisting of 0.1% formic acid in water and methanol at a flow rate of 0.6 mL/min. The detection of the analytes was performed on a UHPLC–MS/MS system with turbo ion spray source in the negative ion and multiple reaction monitoring (MRM) mode. The calibration curves for the determination of all the analytes showed good linearity (R2  > 0.99), and the lower limits of quantification were 0.50–4.00 ng/mL. Intra-day and inter-day precisions were in the range of 3.6%–9.2% and 3.2%–13.1% for all the analytes. The mean extraction recoveries of the analytes were within 69.82%–103.5% and the matrix were within 82.8%–110.0%. The validated method had been successfully applied to compare the pharmacokinetic parameters of ginkgo flavonoids, terpene lactones and nimodipine in rat plasma after oral administration of Ginkgo biloba dispersible tablets, Nimodipine tablets with the combination of the both. There were no statistically significant differences on the pharmacokinetic behaviors of all the analytes between the combined and single administration groups. Results showed that the combination of the two agents may avoid dosage adjustments in clinic and the combination is more convenient as well as efficient on different pathogenesis of cerebral ischemia.
Keywords: Ginkgo biloba extract; Nimodipine; UHPLC–MS/MS; Pharmacokinetic;

Simultaneous determination of sulfur mustard and related oxidation products by isotope-dilution LC–MS/MS method coupled with a chemical conversion by Meiling Qi; Bin Xu; Jianfeng Wu; Yajiao Zhang; Cheng Zong; Jia Chen; Lei Guo; Jianwei Xie (42-50).
Sulfur mustard (SM) is a highly reactive alkylating vesicant with high toxicity and complicated metabolism, the in vivo profile of its oxidation metabolism is not still fully known and urgently needs to be clarified well. In this work, an isotope-dilution high performance liquid chromatography-tandem mass spectrometric method coupled with chemical conversion was developed for the simultaneous quantification of SM and its oxidation products, i.e., mustard sulfoxide (SMO) and mustard sulfone (SMO2). The accurate measurement of SM and its oxidation products with high reaction activity was achived via the method of chemical conversion of 2-(3,5-bis(mercaptomethyl)phenoxy) acetic acid into stable derivative products. Method validation was performed in whole blood matrix, the linear range of the method was between 0.2 and 1000 μg/L with correlation coefficients (r2) > 0.99, and the lower limits of quantification for SM, SMO and SMO2 were 1, 1, 0.2 μg/L, respectively. The validated method was successfully applied to a toxicokinetics research of SM and its oxidation products after SM dermal exposed rats in a single dose. All three target analytes were found in whole blood samples from poisoned rats, and significant time-dependent responses were also observed. Among them, SMO2 with relatively high toxicity was identified and quantified in vivo for the first time, while SMO was the major product in whole blood and some of them continued to be oxidized to SMO2 in vivo. These results give a direct experimental evidence to support that a large amount of SM is converted into the corresponding SMO and SMO2, and these oxidation products might cause potential combined toxic effects.
Keywords: Sulfur mustard; Mustard sulfoxide; Mustard sulfone; Chemical conversion; HPLC–MS/MS; Whole blood; Toxicokinetics;

Gout is a common metabolic disorder caused by the deposition of monosodium urate crystals within joints. A new kind of xanthine oxidase inhibitor, WSJ-537, was developed as a potential drug. In order to investigate the pharmacokinetic behavior in vivo, a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for determination the concentration of WSJ-537 in rat plasma was developed. After extraction by protein precipitation method with acetonitrile, the chromatographic separation was accomplished on a Venusil ASB C18 column(2.1 mm × 50 mm, 3 mm)at a flow rate of 0.3 mL min−1 with the mobile phase consisting of acetonitrile-ammonium acetate (33:67, v/v). An electrospray ionization (ESI) source was applied and operated in the positive ion mode. The plasma concentration was detected by multiple reactions monitoring (MRM) mode with the target fragment ions m/z 410.2 →  m/z 368.1 for WSJ-537 and m/z 244.1 →  m/z 185.0 for the IS. Good linearity was observed in the range of 20–800 ng mL−1 (r = 0.9947). The recovery of WSJ-537 in rats plasma was more than 85%. This method was suitable for pharmacokinetic studies after oral administration of 10 mg/kg WSJ-537 in rats.
Keywords: WSJ-537; Pharmacokinetics; LC–MS/MS; Rats;

A study on characteristic of different sample pretreatment methods to evaluate the entrapment efficiency of liposomes by Congcong Ran; Dan Chen; Meng Xu; Chaohui Du; Qinglian Li; Ye Jiang (56-62).
To examine how methods affect the evaluation of entrapment efficiency (EE) of liposomes, four different sample pretreatment methods were adopted in the experiment. The four sample pretreatment methods were size-exclusion chromatography (SEC), solid-phase extraction (SPE), centrifugation ultrafiltration (CF-UF) and hollow fiber centrifugal ultrafiltration (HF-CF-UF). Amphotericin B (AmB), which could self-associate to form aggregates in water is adopted as the model drugs in this paper. In the present work, it was found that the characterization results of four methods were quite different. The EE of liposome by SEC was about 93%, only 5–13% using C18 or HLB columns, and approximately 100% by CF-UF. The EE of HF-CF-UF reached up to nearly 99.0%. Further, this paper revealed the reasons making the difference of EE among four methods. Conventional SEC may distort the authentic of EE of liposomes with mainly employing some small liposomes or excessive water as eluent. For SPE, cholesterol on liposome surface could interact with the stationary phase making it hard to elute with water, and increase the risk of liposome leakage. While for CF-UF, concentration polarization was a main limitation hindering unentrapped drug to pass through membrane, making unentrapped drug undetectable in liposome. HF-CF-UF could truly reflect EE of liposomes with the concentration of unentrapped AmB lower than 25.0 μg/mL. However, when the concentration was higher than 25.0 μg/mL, AmB aggregates could be entrapped by hollow fiber. From the above analysis, this paper came to the conclusion that each method had its own feature in characterization. This study provided a reasonable guideline for choosing methods to character the EE of liposome.
Keywords: Entrapment Efficiency(EE); Hollow fiber centrifugal ultrafiltration(HF-CF-UF); Size-exclusion chromatography(SEC); Solid-phase extraction(SPE); Centrifugation ultrafiltration(CF-UF); Amphotericin B(AmB);

A rapid and sensitive LC–MS/MS method was developed, optimized and validated for quantification of sofosbuvir (SF) and ledipasvir (LD) in human plasma using eplerenone as an internal standard (IS). Analytes and IS were extracted from plasma by simple liquid–liquid extraction technique using methyl tertiary butyl ether. The prepared samples were chromatographed on Acquity UPLC BEH C18 column. Separation was done using a mobile phase formed of 0.1% formic acid and acetonitrile (50:50, v/v) in an isocratic mode at a flow rate of 0.4 ml/min. The Xevo TQD LC–MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. A full validation of the method was performed according to the FDA guidelines. Linearity was found to be in the range of 0.25–3500 ng/ml for SF and 5–2000 ng/ml for LD. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A short run time of 2 min allows analysis of more than 400 plasma samples per day. The developed method was successfully applied to both fasting and fed bioequivalence studies in healthy human volunteers.
Keywords: Sofosbuvir; Ledipasvir; UPLC–MS/MS; Plasma; Validation; Bioequivalence;

The rare N-unsubstituted glucosamine (GlcNH3 +) residues in heparan sulfate (HS) have important biological and pathophysiological roles. In this study, a high-resolution method for the separation and analysis of N-unsubstituted disaccharides of heparin/HS is described. Four N-unsubstituted disaccharides, together with eight N-substituted species, can be well-separated by ion-pair reverse-phase ultra-performance liquid chromatography. Each disaccharide can then be detected and its relative abundance quantified using electrospray ionization mass spectrometry in the negative mode. Because of its high sensitivity, without interference from proteins and other sample impurities, this method is particularly useful in the analysis of low content GlcNH3 + residues in small amounts of biological materials, eg. sera, tissue and cell culture-derived samples. This would lead to a better understanding of the biological origin of GlcNH3 + residues and their increasingly important function in human health and disease.
Keywords: Heparan sulfate; Heparin; N-unsubstituted disaccharide; LC–MS-ITTOF;

An insertion (I)/deletion (D) polymorphism in angiotensin-converting enzyme (ACE) has been associated with susceptibility to various diseases in numerous studies. Traditionally, slab gel electrophoresis (SGE) after polymerase chain reaction (PCR) has been used to genotype this ACE I/D polymorphism. In this study, single- and multi-channel microchip electrophoresis (ME) methods based on variable programmed electric field strength (PEFS) (i.e., low constant, high constant, (+)/(−) staircase, and random electric field strengths) were developed for fast high-throughput screening of this specific polymorphism. The optimum PEFS conditions were set as 470 V/cm for 0–9 s, 129 V/cm for 9–13 s, 470 V/cm for 13–13.9 s, 294 V/cm for 13.9–16 s, and 470 V/cm for 16–20 s for single-channel ME, and 615 V/cm for 0–22.5 s, 231 V/cm for 22.5–28.5 s, and 615 V/cm for 28.5–40 s for multi-channel ME, respectively. In the multi-channel PEFS-ME, target ACE I/D polymorphism DNA fragments (D = 190 bp and I = 490 bp) were identified within 25 s without loss of resolving power, which was ∼300 times faster than conventional SGE. In addition, PCR products of the ACE gene from human blood samples were detected after only 10 cycles by multi-channel PEFS-ME, but not by SGE. This parallel detection multichannel-based PEFS-ME method offers a powerful tool for fast high-throughput ACE I/D polymorphism screening with high sensitivity.
Keywords: Angiotensin-converting enzyme (ACE); Polymorphism; Microchip electrophoresis; Programmed electric field strength (PEFS); DNA;

Use of basic mobile phase to improve chromatography and boost sensitivity for quantifying tetrahydrocurcumin in human plasma by LC–MS/MS by Aimin Tan; Yanxin Wu; Molly Wong; Albert Licollari; Gordon Bolger; John C. Fanaras; George Shopp; Lawrence Helson (86-93).
Tetrahydrocurcumin (THC), a major metabolite of curcumin, is often quantified by LC–MS or LC–MS/MS using acidic mobile phases due to the concern of its instability in a basic medium. However, acidic mobile phases often lead to poor chromatography (e.g. split or double peaks) and reduced detection sensitivity in the commonly used negative ionization mode. To overcome these shortcomings, a basic mobile phase was used for the first time in the LC–MS/MS quantification of THC. In comparison with the acidic mobile phases, a single symmetrical chromatographic peak was obtained and the sensitivity increased by 7-fold or more under the equivalent conditions. The new LC–MS/MS method using the basic mobile phase has been successfully validated for the quantification of THC in human EDTA plasma over the concentration range of 5–2500 ng/ml. The within-batch accuracy (% nominal concentration) was between 88.7 and 104.9 and the between-batch accuracy ranged from 96.7 to 108.6. The CVs for within- and between-batch precisions were equal to or less than 5.5% and 9.1%, respectively. No significant matrix interference or matrix effect was observed from normal or lipemic and hemolytic plasma matrices. In addition, the common stabilities with adequate durations were established, including up to 5 days of post-preparative stability. Furthermore, when the validated method was applied to a clinical study, the passing rate of ISR samples was 83%, indicating the good reproducibility of the method. The success of the unconventional approach presented in this article demonstrates that a mobile phase could be selected based mainly on its merits to facilitate LC separation and/or MS detection. There is no need for excessive concern about the stability of the compound(s) of interest in the selected mobile phase because the run time of modern LC–MS or LC–MS/MS methods is typically only a few minutes.
Keywords: Tetrahydrocurcumin; Curcumin; Basic mobile phase; Plasma; LC–MS; Bioanalysis;

We present a simple, fast and validated method for the determination of nimodipine in plasma and cerebrospinal fluid (CSF) of patients with subarachnoid haemorrhage using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS). Plasma or CSF 250 μL aliquots were pretreated with acetonitrile spiked with lacosamide as internal standard. The chromatographic separation was performed on a Fusion (3 μm) 50 × 2.0 mm I.D. column with gradient elution of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at a flow rate of 0.35 mL/min. The MS/MS ion transitions were 419.1 → 343 for nimodipine and 251.1 → 91 for the internal standard. The linearity was determined from 2.0 to 40.0 ng/mL in plasma and 40.0–800.0 pg/mL in CSF. The lower limit of quantitation (LLOQ) of nimodipine was 0.4 ng/mL in plasma and 40 pg/mL in CSF. The mean recovery for nimodipine was ≥75% in plasma and ≥90% in CSF at all three considered concentrations. Intra- and interassay precision and accuracy were ≤15% at all quality control concentrations in plasma and CSF. The method was applied to measure plasma and CSF concentrations of nimodipine in a series of patients with subarachnoid haemorrhage treated with intravenous nimodipine. The present procedure, omitting time-consuming liquid-liquid extraction and drying steps, is faster, simpler and cheaper than published LC–MS/MS analytical methods for nimodipine in plasma and the first validated one for nimodipine in CSF.
Keywords: Nimodipine; Ultra high performance liquid chromatography tandem mass spectrometry; Plasma concentration; CSF concentration; Subarachnoid haemorrhage;

Tamoxifen (TAM) is a non-steroidal estrogen receptor antagonist that enhances erlotinib (ERL)-induced cytotoxicity in the treatment of NSCLC. ERL and TAM are metabolized by CYP3A4 enzymes. In addition, both drugs have the potential of altering the enzymatic activity through either inhibition (ERL) or induction (TAM). Thus it was expected that pharmacokinetics (PK) drug–drug interactions (DDIs) could be encountered following their co-administration. In this respect, a bioanalytical UPLC–MS/MS method has been developed and validated for the simultaneous determination of ERL and TAM in rat plasma samples, using ondansetron (OND) as an internal standard (IS). Plasma samples were prepared using mixed mode cationic solid phase extraction (SPE) STRATA™ −X-C 33 μm cartridges with good extraction recovery of both drugs from rat plasma (Er% from −13.92 to −3.32). The drugs were separated on a Waters BEH™ C18 column with an isocratic elution using a mobile phase composed of a mixture of acetonitrile and water, each with 0.15% formic acid, in the ratio of 80: 20, v/v. Quantitation was carried out using the positive ionization mode with multiple reaction monitoring (MRM) at m/z 394.20 > 278.04 (ERL), m/z 372.25 > 72.01 (TAM), and m/z 294.18 > 170.16 (OND). The method was fully validated as per the FDA guidelines over the concentration range of 0.2–50 ng/mL with very low lower limit of quantification (LLOQ) of 0.2 ng/mL for both ERL and TAM. The intra- and inter-day assay precision (in terms of relative standard deviation, RSD) and accuracy (in terms of percentage relative error, % Er) were evaluated for both drugs and the calculated values evaluated at four different concentration levels were within the acceptable limits (<15%) for concentrations other than LLOQ and 20% for LLOQ. The method was successfully applied to the study of possible PK-DDI following the oral administration of ERL and TAM in a combination, compared to their single administration.
Keywords: Erlotinib; Tamoxifen; SPE; UPLC–MS/MS; Pharmacokinetics; Rat plasma;

Multiple myeloma (MM), a malignant neoplastic serum-cell disorder, has been a serious threat to human health. The determination of 6 commonly used drug concentrations, including thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin, in MM patients was of great clinical interest. Herein, we reported a method for the rapid and simultaneous measurement of the above therapeutics by liquid chromatography-tandem mass spectroscopy (LC–MS/MS) method with solid phase extraction. Analysis was performed on a Waters XBridge® BEH C18 column (2.5 μm, 2.1 mm × 50 mm), with formic acid aqueous solution and acetonitrile as the mobile phase at flow rate 0.3 mL/min. All analytes showed good correlation coefficients (r > 0.996), and LLOQ of thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin were 4, 2, 2, 2, 2 and 2 ng/mL, respectively. The inter- and intra-day precisions and stability were expressed as variation coefficients within 15% and relative error less than 15%. Dilution effect, carryover and incurred sample reanalysis were investigated according to the 2015 edition Chinese Pharmacopoeia guidelines, as US FDA (2013, revision 1) required. The LC–MS/MS based assay described in this article may improve future clinical studies evaluating common therapeutics for MM treatment.
Keywords: Liquid chromatography-tandem mass spectroscopy; Anti-cancer drug; Human serum; Multiple myeloma patients;

Validated methods for determination of neurotransmitters and metabolites in rodent brain tissue and extracellular fluid by reversed phase UHPLC–MS/MS by Marianne Skov-Skov Bergh; Inger Lise Bogen; Elsa Lundanes; Åse Marit Leere Øiestad (120-129).
Fast and sensitive methods for simultaneous determination of dopamine (DA), the two DA-metabolites homovanillic acid (HVA) and 3-methoxytyramine (3-MT), serotonin (5-HT) and the 5-HT-metabolite 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), acetylcholine (ACh), glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rodent brain tissue (1.0–4000 nM) and extracellular fluid (ECF) (0.5–2000 nM) based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) have been developed. Of the three different sample preparation methods for brain tissue samples tested, a simple and rapid protein precipitation procedure with formic acid was found to give the best results. The neurotransmitters (NTs) and NT metabolites were separated using UHPLC with an Acquity UPLC HSS T3 C18 column (2.1 × 100 mm, 1.8 μm particle size) with acidic mobile phase. Gradient elution with methanol was used and quantification was performed using multiple reaction monitoring (MRM). The total run time was 5.2 min including equilibration time. The methods were validated by determining calibration model, intra- and inter-day precision and accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), matrix effects (ME), carry-over and stability. Surrogate analytes were used to enable determination of the recovery and ME of the endogenous analytes in brain tissue. The methods were applied for determination of NTs at basal levels in rodent brain ECF and brain tissue homogenate. The developed methods are valuable tools in the studies of mechanisms of drugs of abuse, and neurologic and psychiatric disease.
Keywords: UHPLC–MS/MS; Neurotransmitters; Metabolites; Extracellular fluid; Brain tissue; Protein precipitation;

Set-up and application of an analytical approach for the quality control of purified colostrum as food supplement by Alessandra Altomare; Luca Regazzoni; Ximena Maria Paredes Parra; Francesca Selmin; Cristiano Rumio; Marina Carini; Giancarlo Aldini (130-144).
A validated analytical procedure is here described for the quality control of the protein fraction of purified bovine colostrum used in food supplements. The proposed procedure starts with 1D and 2D-gel electrophoresis. The sample is then separated into two fractions by protein G affinity chromatography: the IgG enriched and the IgG depleted fraction (IgG-d). A size exclusion chromatography coupled to UV is then applied to the IgG and IgG-d fractions for the quantitative analysis of IgG and IgM, respectively. The IgG-d fraction is then analysed by HPLC–MS analysis for the quantitative analysis of β-lactoglobulins and α-lactoalbumin. The next step is to quantitatively measure a set of bioactive proteins selected from the bovine colostrum data bank on the basis of their claimed health benefits. The enzymatic activities of lactoperoxidase and xanthine dehydrogenase/oxidase are then tested as an index of protein functionality.
Keywords: Bovine colostrum; Quality control; Food supplement; Size exclusion chromatography; Mass spectrometry;

Chiral separation is crucial for investigating methamphetamine positive cases. While (S)-(+)-enantiomer of methamphetamine (S-MAMP) is a schedule II controlled substance, (R)-(−)-enantiomer (R-MAMP) is an active ingredient of a few over-the-counter drugs in the United States. Among biological specimen types, hair provides greater detection window than blood, urine or oral fluid, and are therefore regarded with particular interest. Herein we describe a novel non-chiral liquid chromatography-tandem mass spectrometry (LC–MS/MS) method to directly determine methamphetamine enantiomeric composition (percentage) in hair specimens.Hair samples were washed once with acetone, powdered, incubated overnight at 53 °C in 0.1 M hydrochloric acid (HCl), and subjected to a solid phase extraction (SPE). The extracts were derivatized using Marfey’s reagent at 53 °C for 60 min. The final mixture was analyzed by LC–MS/MS. Chromatographic separation was achieved using a C18 Kinetex analytical column and 60% (v/v) aqueous methanol as mobile phase (isocratic). Triple quadrupole mass spectrometer was equipped with an electro-spray ionization (ESI) source operating in negative mode and the chromatograms were acquired using a multiple-reaction monitoring (MRM) approach. The results were expressed as ratio of R- to S-MAMP and then derived to composition percentages without requiring quantitating each enantiomer.The method was precise and accurate across 0–100% S-composition at a range of 80–18,000 pg/mg. The performance of the new method was compared with an (S)-(−)-N-trifluoroacetylprolyl chloride (S-TPC) derivatization and gas chromatography-mass spectrometry (GC–MS) method on authentic methamphetamine-positive hair samples. Not only the new Marfey’s reagent approach presented satisfactory correlation with the S-TPC approach, but it also exhibited significantly improved quality (e.g., S/N) of the chromatograms.In summary, our protocol employs cost effective and minimally hazardous Marfey’s reagent to derivatize trace amounts of methamphetamine extracted from hair samples and a non-chiral LC–MS/MS approach to separate and identify the two enantiomers. The method allows determination of the methamphetamine enantiomeric composition without requiring quantitation of each enantiomer and is therefore well suited for further investigate previously determined methamphetamine positive cases. This method represents a viable tool for evaluation of long-term drug exposure.
Keywords: Marfey’s reagent; Non-chiral chromatography; Hair analysis; Enantiomer composition;

An integrated on-chip platform for negative enrichment of tumour cells by Sajay Bhuvanendran Nair Gourikutty; Chia-Pin Chang; Daniel Puiu Poenar (153-164).
The study of cancer cells in blood, popularly called circulating tumour cells (CTCs), has exceptional prospects for cancer risk assessment and analysis. Separation and enrichment of CTCs by size-based methods suffer from a well-known recovery/purity trade-off while methods targeting certain specific surface proteins can lead to risk of losing CTCs due to Epithelial to Mesenchymal Transition (EMT) and thus adversely affect the separation efficiency. A negative selection approach is thus preferred for tumour cell isolation as it does not depend on biomarker expression or defines their physical property as the separation criteria. In this work, we developed a microfluidic chip to isolate CTCs from whole blood samples without targeting any tumour specific antigen. This chip employs a two-stage cell separation: firstly, magnetophoresis depletes the white blood cells (WBCs) from a whole blood sample and is then followed by a micro-slit membrane that enables depleting the red blood cells (RBCs) and retaining only the tumour cells. By creating strong magnetic field gradients along with customized antibody complexes to target WBCs, we are able to remove >99.9% of WBCs from 1:1 diluted blood at a sample processing rate of 500 μL/min. This approach achieves an average of >80% recovery of spiked tumour cells from 2 mL of whole blood in a total assay processing time of 50 min without multiple processing steps.
Keywords: Circulating tumour cells (CTCs); Negative enrichment; White blood cell (WBC) depletion; Magnetic separation; Cell separation;

Extraction, purification, methylation and GC–MS analysis of short-chain carboxylic acids for metabolic flux analysis by Nathan D. Tivendale; Erin M. Jewett; Adrian D. Hegeman; Jerry D. Cohen (165-174).
Dynamic metabolic flux analysis requires efficient and effective methods for extraction, purification and analysis of a plethora of naturally-occurring compounds. One area of metabolism that would be highly informative to study using metabolic flux analysis is the tricarboxylic acid (TCA) cycle, which consists of short-chain carboxylic acids. Here, we describe a newly-developed method for extraction, purification, derivatization and analysis of short-chain carboxylic acids involved in the TCA cycle. The method consists of snap-freezing the plant material, followed by maceration and a 12–15 h extraction at −80 °C. The extracts are then subject to reduction (to stabilize β-keto acids), purified by strong anion exchange solid phase extraction and methylated with methanolic HCl. This method could also be readily adapted to quantify many other short-chain carboxylic acids.
Keywords: Gas chromatography-mass spectrometry; Tricarboxylic acid cycle; Methylation; Carboxylic acid; Polar metabolites; Metabolic flux;

Liquid chromatography–mass spectrometry for measuring deoxythioguanosine in DNA from thiopurine-treated patients by Sally A. Coulthard; Phil Berry; Sarah McGarrity; Azhar Ansari; Christopher P.F. Redfern (175-180).
Adverse reactions and non-response are common in patients treated with thiopurine drugs. Current monitoring of drug metabolite levels for guiding treatment are limited to analysis of thioguanine nucleotides (TGNs) in erythrocytes after chemical derivatisation. Erythrocytes are not the target tissue and TGN levels show poor correlations with clinical response. We have developed a sensitive assay to quantify deoxythioguanosine (dTG) without derivatisation in the DNA of nucleated blood cells. Using liquid chromatography and detection by tandem mass spectrometry, an intra- and inter-assay variability below 7.8% and 17.0% respectively were achieved. The assay had a detection limit of 0.0003125 ng (1.1 femtomoles) dTG and was quantified in DNA samples relative to endogenous deoxyadenosine (dA) in a small group of 20 patients with inflammatory bowel disease, all of whom had been established on azathioprine (AZA) therapy for more than 25 weeks. These patients had dTG levels of 20–1360 mol dTG/106  mol dA; three patients who had not started therapy had no detectable dTG. This method, comparable to previous methods in sensitivity, enables the direct detection of a cytotoxic thiopurine metabolite without derivatisation in an easily obtainable, stable sample and will facilitate a better understanding of the mechanisms of action of these inexpensive yet effective drugs.
Keywords: Azathioprine; Mercaptopurine thiopurine; Drug monitoring; Tandem mass spectrometry; LC–MS/MS; Thioguanine nucleotides; DNA;

Measurement of 2-nonenal and diacetyl emanating from human skin surface employing passive flux sampler—GCMS system by Keita Kimura; Yoshika Sekine; Shota Furukawa; Minami Takahashi; Daisuke Oikawa (181-185).
It is commonly said elderly persons have a characteristic body odor, and, at present, two chemical compounds have been found to vary with age in male Japanese: 2-nonenal and diacetyl. To investigate dermal emission flux of the ageing odor related compounds, we have developed a non-invasive sampling device based on a concept of passive flux sampler (PFS). The sampler was placed on the skin surface to create a headspace, and the gases emanating from skin moved toward a disk-type adsorbent. The trapped gases were then extracted with dichloromethane and determined by GCMS. The PFS was practically applied to healthy volunteers covering a wide range of age. Since emission fluxes of both compounds remarkably varied with sampling position, the nape of the neck was fixed as regular sampling position where there are dense networks of both sebaceous and eccrine glands which are potential sources of both compounds. The emission flux of 2-nonenal increased with age for both male and female volunteers, whilst the flux of diacetyl showed highest in 30s and decreased over 40s. Although diacetyl has been known as a middle-aged male odor, this study showed the odor caused by diacetyl was not specific to male.
Keywords: Human skin gas; Body odor; Passive flux sampler; Diacetyl; 2-Nonenal; Age;

In this study, biosorption based dispersive liquid liquid microextraction (Bio-DLLME) has been developed as a new method for the extraction of bisphenol A (BPA) from water samples. In this technique, the BPA is extracted into a stable cloudy phase. The colloidal phase is composed of micro-particles made from rhaminolipid biosurfactant and methanol, which dispersed in the water samples and facilitated the breakdown of analyte matrix bonds and provided high extraction yields. Rhaminolipid biosurfactants form a thin molecular interfacial film. This layer is formed between donor and recipient phase. This molecular layer, lowers the interfacial tension between immiscible phases (aqueous solution: colloidal particles) and allow dissimilar phases to mix and interact more easily. So the equilibrium state is achieved quickly and, therefore, the extraction time is very short. The attraction of the proposed method is that the extraction is fast, simple and can be done without toxic organic solvents. Also bioaggregates have several advantages such as higher environmental compatibility and biodegradability. Experimental parameters affecting the extraction efficiency were studied and optimized. Under the optimum conditions, relative recoveries of BPA were in the ranges of 98–103.3%. The calibration plot is linear in the range between 1 and 1000 μg L−1 (R2  = 0.998), and the relative standard deviation (RSD, for n = 6) is 3.24%.
Keywords: Bio-DLLME; Bioaggregates; Rhaminolipid; Bisphenol A; HPLC;

Validation of a qualitative immunochromatographic test for the noninvasive assessment of stress in dogs by F. Di Nardo; L. Anfossi; L. Ozella; A. Saccani; C. Giovannoli; G. Spano; C. Baggiani (192-198).
Salivary cortisol is regarded as a reliable parameter for the noninvasive assessment of the welfare of animals, because it is strictly related to stress levels. Several methods are available for salivary cortisol measurement in mammals, however rapid diagnostic test for detecting salivary cortisol are confined to humans. The availability of such non invasive diagnostic tools operable in situ would facilitate monitoring of animal welfare. The Cortisol stress™ test provides a simple and rapid tool to discriminate cortisol levels in canine saliva above or below 4 ng/ml, which has been suggested as the cut-off value for distinguishing unstressed dogs from those experiencing stress. The test is based on a competitive immunochromatographic assay (ICT) using gold nanoparticles as probes, in which the color intensity of the Test line is inversely correlated to the salivary cortisol level. The qualitative result is obtained by the visual observation of the color formed on the Test line compared to that of the Control lineWe evaluated the accuracy of the test by determining salivary cortisol in 85 samples of canine saliva belonging to dogs with very variable age, sex, breed, and life history, and comparing the qualitative results to those obtained by a reference ELISA kit. Agreeing results were obtained through the two methods, and the ICT showed high diagnostic sensitivity, specificity and efficiency (100%, 98.4%, and 98.8%, respectively). Furthermore, we evaluated the precision of the test by an experimental design approach, which combines errors due to within-day and between-day variation with the biological variability, and demonstrated that the test could be reliably applied for correctly classifying canine samples, according to their salivary cortisol level. Moreover, we studied the shelf-life of the device in three experimental conditions. We confirmed the stability of the ICT at 4 °C and 25 °C for at least six months and observed similar results for an accelerated stability study conducted for 7 days at 37 °C, which suggest that the stability of ICT device could be estimated by the accelerated experiment alternatively to the real-time study.
Keywords: Saliva; Cortisol; Lateral flow immunoassay; Shelf-life;

Non-destructive proton nuclear magnetic resonance (1H NMR) spectroscopy and highly sensitive ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (U-HPLC/Q-TOF-MS) coupled to data processing methods were applied to analyze the metabolic profiling changes of glycerophospholipids (GPLs) in RAW264.7 cells from inflammation to prognosis. Analysis of 1H NMR was shown that the models were grouped successfully, illustrating that all of them had significant differences. Based on the highly simple, accurate, non-targeted and non-destructively advantages of 1H NMR, it could be used as a new screening tool of anti-inflammatory drugs in the metabolic profiling of GPLs. 58 GPLs were identified by U-HPLC/Q-TOF-MS, and 19 components were firstly identified in this study compared with our previous results. In addition, ten potential biomarkers were proved, of which phosphatidylcholine (PC) (16:0/18:1) and (18:0/18:1) changed consistently in three drug-induced groups and might be the important biomarkers. Compared with 1H NMR, U-HPLC/Q-TOF-MS showed higher sensitivity and specificity and was more suitable for the determination of biomarkers apart from the deficiency of time-consuming sample preparation steps and unambiguous metabolite identification. Therefore, it is feasible to analyze the changes of GPLs during inflammation by combining 1H NMR spectroscopy with U-HPLC/Q-TOF-MS. The metabolic profiling of GPLs provides valuable evidence for inflammation diagnosis and prognosis, and might unravel the mechanisms involved in inflammation progression.
Keywords: 1H NMR; U-HPLC/Q-TOF-MS; Glycerolphospholipids; RAW264.7 cells; Chemometrics; Biomarkers;

Although the reaction mechanisms through which flavin mononucleotide works as an endogenous photosensitizer have been investigated (Baier et al., 2006; Edwards and Silva, 2001; Pajares et al., 2001; Criado et al., 2003; Massad et al., 2008) [23–27], few studies have been performed for the reactions of flavin mononucleotide with unsaturated fatty acids. To examine the reactions of flavin mononucleotide with unsaturated fatty acids bearing a double bond at different positions, an electron spin resonance, a high performance liquid chromatography-electron spin resonance and a high performance liquid chromatography-electron spin resonance-mass spectrometry were employed. The control reaction mixtures contained 25 μmol L−1 of flavin mononucleotide, 1.0 mmol L−1 of FeSO4(NH4)2SO4, 10 mmol L−1 of cholic acid, 30 mmol L−1 of phosphate buffer (pH 7.4) and 0.1 mol L−1 of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone in deuterium oxide. In addition, it also contained 4.3 mmol L−1 of one of the following: (z)-11-octadecenoic acid, (z)-6-octadecenoic acid, (z)-9-octadecenoic acid or (z, z)-9, 12-octadecadienoic acid. The control reaction mixtures without FeSO4(NH4)2SO4 and α-(4-pyridyl-1-oxide)-N-tert-butylnitrone were exposed to the visible light at 436 nm (7.8 J cm−2). After the irradiation, α-(4-pyridyl-1-oxide)-N-tert-butylnitrone was added. The reactions started from adding FeSO4(NH4)2SO4 and performed at 25 °C for 1 min. Electron spin resonance measurements of the control reaction mixtures showed prominent signals (αN  = 1.58 mT and α  = 0.26 mT). High performance liquid chromatography-electron spin resonance analyses of the control reaction mixtures showed prominent peaks at the retention times of 31.1 min {(z)-6-octadecenoic acid}, 39.6 min {(z)-9-octadecenoic acid}, 44.9 min {(z)-11-octadecenoic acid} and 40.2 min {(z, z)-9, 12-octadecadienoic acid}. High performance liquid chromatography-electron spin resonance-mass analyses of the control reaction mixtures showed that 4-carboxybutyl, 7-carboxyheptyl and 9-carboxynonyl radicals formed in the control reaction mixtures of (z)-6-octadecenoic acid, (z)-9-octadecenoic acid {or (z, z)-9,12-octadecadienoic acid} and (z)-11-octadecenoic acid, respectively. The 4-carboxybutyl, 7-carboxyheptyl and 9-carboxynonyl radicals are all generated through β-scission of alkoxy radicals formed on carboxyl ends of the double bonds of the unsaturated fatty acids. Thus, we could reveal reactive sites of unsaturated fatty acids in the photosensitized reaction of flavin mononucleotide with unsaturated fatty acids bearing a double bond at different positions.
Keywords: HPLC-ESR-MS; Visible light; 4-Carboxybutyl radical; 7-Carboxyheptyl radical; 9-Carboxynonyl radical; Singlet oxygen;

In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical detection provided limits of quantifications (LOQs) between 3.6 and 12 nM. Within the linear range, obtained recoveries were from 90.9 ± 9.9 to 120 ± 14% and intra-day and inter-day precisions found to be less than 5.5% and 12%, respectively. The analytical method was applicable for quantification of intracellular and extracellular amounts of monoamine neurotransmitters and their metabolites in guinea pig frontal cortex and hippocampal primary neuronal cell cultures. Noradrenaline, dopamine and serotonin were found to be in a range from 0.31 to 1.7 pmol per 2 million cells intracellularly, but only the biogenic metabolites could be detected extracellularly. Distinct differences in monoamine concentrations were observed when comparing concentrations in guinea pig frontal cortex and cerebellum tissue with higher amounts of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid in frontal cortex, as compared to cerebellum. The chemical turnover in frontal cortex tissue of guinea pig was for serotonin successfully predicted from the turnover observed in the frontal cortex cell culture. In conclusion, the present analytical method shows high precision, accuracy and sensitivity and is broadly applicable to monoamine measurements in cell cultures as well as brain biopsies from animal models used in preclinical neurochemistry.
Keywords: Monoamines; HPLC; Electrochemical detection; Cell culture; Guinea pig;

Kinetic and thermodynamic studies of sulforaphane adsorption on macroporous resin by Wu Yuanfeng; Zhang Lei; Mao Jianwei; Liu Shiwang; Huang Jun; You Yuru; Mei Lehe (231-236).
The adsorption equilibrium, kinetic and thermodynamic of sulforaphane (SF) adsorption onto macroporous resin in aqueous phase were studied. The SP850 resin was screened as the appropriate resin for SF purification. From the equilibrium studies, the Redlich-Peterson model was found to be the best for description of the adsorption behavior of SF onto SP850 resin, followed by the Freundlich model and the Langmuir model. Batch equilibrium experiments demonstrated that, in the examined temperature range, the equilibrium adsorption capacity of SP850 resin decreased with increasing adsorption temperature. Thermodynamics studies indicated that the adsorption of SF was a physical, exothermic, and spontaneous process. The adsorption kinetics revealed that the pseudo-second-order kinetic model was suitable to characterize the kinetics of adsorption of SF onto SP850. Finally, the intra-particle diffusion model demonstrated that SF diffused quickly into macropores, and that diffusion slowed down in the meso- and micropores.
Keywords: Sulforaphane; Macroporous resin; Adsorption; Isotherm; Kinetic; Thermodynamic;

Direct and rapid quantitation of ephedrines in human urine by paper spray ionization/high resolution mass spectrometry by Eun Sook Jeong; Ki Hun Kim; Eunju Cha; Oh-Seung Kwon; Sangwon Cha; Jaeick Lee (237-241).
A rapid and direct paper spray ionization/mass spectrometry (PSI/MS) method was developed for quantitative analysis of ephedrine, pseudoephedrine, norpseudoephedrine, and methylephedrine in human urine. This method involves the use of a triangular filter paper and high-resolution mass spectrometry, where the molecular ions of ephedrines were generated by applying high voltage after loading the spray solvent to the paper which urine sample was pre-loaded. Small amounts (2 μL) of urine spiked with an internal standard were directly analyzed for ephedrines. The PSI/MS method was validated for linearity, within- and between-run precision, accuracy, and limit of detection. The results showed good linearity (R 2  ≥ 0.9928) and acceptable precision and accuracy. Furthermore, the accuracy of the method was assessed by analyzing a blind urine sample from World Anti-Doping Agency and comparing the measured concentrations with the nominal concentrations. This test resulted in accuracies ranging from 96.4 to 106.1%, indicating that the PSI/MS method has the potential to be an alternative technique for the fast quantitation of ephedrines in doping control analysis.
Keywords: Paper spray ionization/mass spectrometry; Ephedrine; Doping control analysis;

Display OmittedFor pharmacokinetic/pharmacodynamic (PK/PD) assessment of antibiotics combinations in in vitro infection models, accurate and precise quantification of drug concentrations in bacterial growth medium is crucial for derivation of valid PK/PD relationships. We aimed to (i) develop a high-performance liquid chromatography (HPLC) assay to simultaneously quantify linezolid (LZD), vancomycin (VAN) and meropenem (MER), as typical components of broad-spectrum antibiotic combination therapy, in bacterial growth medium cation-adjusted Mueller-Hinton broth (CaMHB) and (ii) determine the stability profiles of LZD, VAN and MER under conditions in in vitro infection models. To separate sample matrix components, the final method comprised the pretreatment of 100 μL sample with 400 μL methanol, the evaporation of supernatant and its reconstitution in water. A low sample volume of 2 μL processed sample was injected onto an Accucore C-18 column (2.6 μm, 100 × 2.1 mm) coupled to a Dionex Ultimate 3000 HPLC+ system. UV detection at 251, 240 and 302 nm allowed quantification limits of 0.5, 2 and 0.5 μg/mL for LZD, VAN and MER, respectively. The assay was successfully validated according to the relevant EMA guideline. The rapid method (14 min) was successfully applied to quantify significant degradation of LZD, VAN and MER in in vitro infection models: LZD was stable, VAN degraded to 90.6% and MER to 62.9% within 24 h compared to t = 0 in CaMHB at 37 °C, which should be considered when deriving PK/PD relationships in in vitro infection models. Inclusion of further antibiotics into the flexible gradient-based HPLC assay seems promising.
Keywords: Drug degradation; Antibiotics; Mueller-Hinton broth; Time-kill studies; In vitro infection models;