Journal of Chromatography B (v.937, #C)

A sensitive and simple method for the methotrexate quantification was developed using aminopterin as internal standard. Methotrexate is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma. The compound was quantified by liquid-chromatography coupled to electrospray ionization (positive ion-mode) low-energy collision dissociation-tandem mass spectrometry. Quantitative detection was by multiple reaction monitoring of the transitions of the [M + H]+ ion of MTX to its common product ion at m/z 308.4 and of aminopterin at m/z 441.2 →  m/z 294.0. The method demonstrated linearity over at least three orders of magnitude and had a detection limit of 1 ng/ml for methotrexate. A run time of less than 8.0 min for each sample made it possible to analyze a large number of human saliva samples per day. Application of this procedure was demonstrated to a saliva excretion study of methotrexate on the samples obtained after an intravenously administration of 1 mg/kg/dose of methotrexate to six patients with acute lymphoblastic leukemia
Keywords: Methotrexate; Liquid chromatography; Tandem mass spectrometry; Saliva excretion study;

Centrifugal partition extraction (CPE) was developed for the first time in the pH-zone-refining mode to fractionate a crude bark extract of the African tree Anogeissus leiocarpus Guill. & Perr. (Combretaceae). The fractionation process was performed at a flow rate of 20 mL/min using a biphasic solvent system composed of methyl tert-butyl ether/acetonitrile/water (4:1:5, v/v/v) in the ascending mode. Sodium hydroxide (40 mM) and trifluoroacetic acid (30 mM) were used as retainer and displacer agents, respectively. In a single run of 67 min, 3 g of the initial crude extract were successfully separated into fractions selectively enriched in ionizable triterpenes, ellagic acid derivatives and flavonoids. The antioxidant potential of the initial crude extract, isolated compounds and fraction pools was also evaluated by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) stable free radical scavenging assay, providing an interesting view about the effect of the degree of substitution of ellagic acid derivatives on their radical scavenging activity. This study will demonstrate that centrifugal partition extraction used in the pH-zone-refining mode can be proposed as an efficient strategy for the rapid screening of natural phenolic compounds.
Keywords: Centrifugal partition extraction; Centrifugal partition chromatography; pH-zone-refining; Anogeissus leiocarpus; Ellagic acid; DPPH;

2-Methyltetrahydrofuran and cyclopentylmethylether: Two green solvents for efficient purification of membrane proteins like FhuA by Stefanie-Joana Tenne; Julia Kinzel; Marcus Arlt; Fabrizio Sibilla; Marco Bocola; Ulrich Schwaneberg (13-17).
β-Barrel shaped membrane proteins are attractive hosts for hybrid catalysts in which reactions are controlled through space. Production and extraction of β-barrel shaped membrane proteins in gram scale is challenging due to their hydrophobicity. Solvent mixtures such as chloroform/methanol (CM) are widely used for membrane protein extraction but toxicity and mutagenicity were reported in several cases. 2-Methyltetrahydrofuran (2-MeTHF) and cyclopentylmethylether (CPME) are two green (reduction of solvent-related environmental damage in chemical production) and potentially efficient solvents for membrane protein purification. On the example of the ferric hydroxamate uptake protein component A (FhuA) a 4-Step method was developed to provide gram amounts of highly purified FhuA: cell disruption (Step 1), removal of membrane protein impurities with n-octyl-poly-oxyethylene (oPOE) (Step 2), dissolution of membranes and FhuA precipitation (Step 3), and refolding using urea and dialysis with polyethylene-polyethyleneglycol (PE-PEG; Step 4) resulted in high FhuA purity (95% 2-MeTHF, 80% CPME; 70 mg FhuA per liter fermenter broth). Structural integrity of FhuA protein was confirmed by circular dichroism (CD) and a translocation functionality assay.
Keywords: Membrane protein; FhuA; Organic solvent; Extraction; Green solvent;

A rapid and specific liquid chromatography-electrospray ionization–tandem mass spectrometry (LC-ESI–MS/MS) method was developed for the simultaneous determination of two active diterpenoids: Kirenol and ent-16β,17-dihydroxy-kauran-19-oic acid (DHKA) from Herba Siegesbeckiae in rat plasma using osthole as an internal standard (IS). Plasma sample pretreatment involved a one-step liquid–liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters Symmetry C18 column (2.1 mm × 100 mm, 3.5 μm) with isocratic elution using methanol–5 mmol/L aqueous ammonium acetate (80:20, v/v) as the mobile phase at a flow rate of 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode under positive and negative electrospray ionization. The calibration curves were linear over the range of 50.0–25,000 ng/mL for Kirenol, and 25.0–12,500 ng/mL for DHKA. The extraction recoveries of the two analytes and the IS were all over 85%. The intra- and inter-day precision (relative standard deviation) values were less than 16.8% and the accuracy (relative error) ranged from −10.7 to 10.6% at four quality control levels. The validated method was successfully applied to a comparative pharmacokinetic study of the two diterpenoids in rat plasma after intragastric administration of Kirenol, DHKA and Herba Siegesbeckiae extract. The results showed that there were obvious differences between the pharmacokinetic behaviors after oral administration of Herba Siegesbeckiae extract compared with each of the substances alone.
Keywords: Herba Siegesbeckiae; Kirenol; ent-16β,17-dihydroxy-kauran-19-oic acid; Pharmacokinetics; LC-ESI–MS/MS;

Rapid separation and quantitation of curcuminoids combining pseudo two-dimensional liquid flash chromatography and NMR spectroscopy by G.K. Jayaprakasha; G.A. Nagana Gowda; Sixto Marquez; Bhimanagouda S. Patil (25-32).
Rapid separation, characterization and quantitation of curcuminoids are important owing to their numerous pharmacological properties including antimicrobial, antiviral, antifungal, anticancer, and anti-inflammatory activities. In the present study, pseudo two-dimensional liquid flash chromatography was used for the separation of four curcuminoids (curcumin, demethoxy curcumin, bisdemethoxy curcumin and dihydro bisdemethoxy curcumin) for the first time. Silica and diol columns were used for separation of curcuminoids using gradient mobile phase. The separated peaks were monitored at 244, 360 nm to obtain four compounds. The purity of compounds were determined by rapid quantitative 1H NMR (qNMR) using 3-(trimethylsilyl) propionic-(2,2,3,3-d4) acid sodium salt (TSP-d4) (0.012%) in D2O. These results were compared with those obtained by HPLC method. The purity of isolated curcuminoids using pseudo 2D chromatography was found to be in the range of 92.4–95.45%. The structures of these compounds were characterized unambiguously using 13C (APT) NMR spectra. The developed pseudo 2D separation technique has the advantage of simplified automation with shorter run time compared to conventional separation techniques. The method that combines rapid pseudo 2D separation and simple quantitation using qNMR reported herein can be of wide utility for routine analysis of curcuminoids in complex mixtures.
Keywords: Curcuma longa; Turmeric; Two-dimensional separation; Preparative chromatography; qNMR; LC–MS;

A sensitive, sophisticated and practical bioanalytical assay for the simultaneous determination of six tyrosine kinase inhibitors (imatinib, sunitinib, nilotinib, dasatinib, pazopanib, regorafenib) and two active metabolites (N-desmethyl imatinib and N-desethyl sunitinib) was developed and validated. For the quantitative assay, a mixture of three stable isotopes as internal standards was added to human serum, standards and controls. Thereafter, samples were pre-treated using protein precipitation with methanol. The supernatant was diluted with water and injected into an ultra pressure liquid chromatographic system with an Acquity TQ tandem mass spectrometry detector. The compounds were separated on an Acquity BEH C18 analytical column (100 mm × 2.1 mm ID, 1.7 μm particle size) and eluted with a linear gradient system. The ions were detected in the multiple reaction monitoring mode. The lower limit of quantification and the linearity of all compounds generously met with the concentrations that are to be expected in clinical practice. The developed bioanalytical assay can be used for guiding TKI therapy in daily clinical practice as well as for investigator-initiated research.
Keywords: LC–MS/MS; Tyrosine kinase inhibitors; Metabolites;

An LC–MS assay for the screening of cardiovascular medications in human samples by Eduardo Dias; Brian Hachey; Candace McNaughton; Hui Nian; Chang Yu; Brittany Straka; Nancy J. Brown; Richard M. Caprioli (44-53).
Cardiovascular drugs are the most commonly prescribed medications. Some prior assays successfully detect cardiovascular drugs among multiple classes using a single sample. Here, we develop an assay to detect a broad range of cardiovascular drug classes to include commonly used cardiovascular drugs and evaluate the assay's analytical and statistical properties in a clinical setting. We describe a protocol for drug detection that encompasses 34 commonly prescribed cardiovascular drugs or drug metabolites with a single LC–MS/MS method using 100 μL of serum or plasma. Drug classes monitored by this assay include: anticoagulants, angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB), beta blockers, calcium channel blockers, diuretics, statins, and vasodilators, as well as digoxin, fenofibrate, and niacin. Analytical accuracy and precision for each drug were evaluated by repeating the assay on spiked samples at low, medium, and high concentrations. In 294 clinical samples obtained from hospitalized patients for whom medication administration was recorded, we evaluated the assay's statistical sensitivity, specificity, and accuracy. For the 34 drugs or drug metabolites, the assay was statistically sensitive (>0.90) for all drugs except captopril (0.25), isosorbide (0.81), and niacin (0.89). The assay was statistically specific for all drugs, with a minimum specificity of 0.94 (aspirin). To our knowledge, this method is the first method of simultaneous analysis of 34 cardiovascular drugs or drug metabolites from nine drug classes evaluated using clinical samples from hospitalized patients
Keywords: Cardiovascular drugs; Drug monitoring; Selectivity; Mass spectrometry; Liquid chromatography; Clinical samples;

A novel UPLC–ESI-MS/MS method for the quantitation of disulfiram, its role in stabilized plasma and its application by Ling Zhang; Ying Jiang; Guanghui Jing; Yilin Tang; Xi Chen; Dan Yang; Yu Zhang; Xing Tang (54-59).
Disulfiram (DSF) has been used to treat alcoholism for many years and it has been suggested to play a key role in combatting many kinds of tumors. However, disulfiram has complex pharmacokinetics and is rapidly eliminated which limits its use as a tumor treatment. Therefore, a rapid and sensitive analytical method based on ultra performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UPLC–ESI-MS/MS) was developed and validated for the determination of disulfiram in rat plasma. Blood samples were pre-stabilized with a stabilizing agent and then plasma was obtained and subjected to solid phase extraction (SPE), and chromatographed on a Phenomenex Kinetex® XB C18 column with gradient elution using a mobile phase consisting of acetonitrile–water (containing 0.1% formic acid and 1 mM ammonium acetate) at a flow rate of 0.2 mL/min for 3 min. Multiple reactions monitoring in positive mode was carried out with disulfiram at 296.95/115.94 and diphenhydramine (internal standard, IS) at 256.14/167.02 over a linear range from 0.6 to 1200 ng/mL. The extraction recovery of disulfiram for different concentrations ranged from 75.7% to 78.3%. The intra- and inter-day precision was less than 8.93% and 12.39%, respectively, and the accuracy was within ±7.75%. The validated method was successfully applied to a pharmacokinetic study of disulfiram in rat plasma after oral administration of a dose of 180 mg/kg.
Keywords: Disulfiram; Ultra performance liquid chromatography; Electrospray ionization; Mass spectrometry; Pharmacokinetics;

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol and its two metabolites in human plasma and urine. Separation of metoprolol, α-hydroxymetoprolol, O-desmethylmetoprolol and esmolol (internal standard) was achieved on an Agilent XDB-C18 column (150 mm × 4.6 mm, 5 μm) using fluorescence detection with Ex 216 nm and Em 312 nm. The mobile phase consisted of ACN–H2O-0.1%TFA. The analysis was performed in less than 16 min with a flow rate of 0.8 mL/min. The assay was linear over the concentration range of 5–600 ng/mL and 2.5–300 ng/mL for metoprolol and its metabolites, respectively. The LOQ were 5.0 and 2.5 ng/mL for plasma and urine, respectively. Good precision and accuracy for metoprolol and its two metabolites were obtained. The extraction recoveries were found to be more than 86.91% both in plasma and urine. At the same time, the method was successfully applied to nine healthy volunteers who had been given an oral tablet of 100 mg metoprolol.
Keywords: HPLC-FLD detection; Metoprolol; α-Hydroxymetoprolol; O-desmethylmetoprolol; Human plasma and urine; Pharmacokinetics;

The use of chromatographic techniques for the separation and the identification of insect lipids by Magdalena Cerkowniak; Alan Puckowski; Piotr Stepnowski; Marek Gołębiowski (67-78).
The paper presents the state of knowledge on the chromatographic techniques used in the analysis of insect lipids. Lipids are one of the forms of protection against entomopathogenic fungi. Their composition may include a number of different compounds, such as long chain hydrocarbons, waxes, alcohols, aldehydes and free fatty acids. These compounds may be presented in different amounts depending on the species of insects, living environment, lifestyle, seasons, etc. The most commonly used techniques in the analysis of lipids of insects are: gas chromatography, high performance liquid chromatography, and combined techniques such as gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS). Compounds present in the cuticular lipids of insects may serve diverse functions such as minimizing transpiration. It was also found that extracts of insects possess antifungal effects. Determination of lipid profiles and biological activity of the identified compounds can effectively contribute to the knowledge of the defense mechanisms of insects, and thus, impact the development of new methods of combating harmful insects.
Keywords: Lipids; Entomopathogenic fungi; Insects; Combined analytical techniques; Chromatography; Extraction;

Quantification of the 5-lipoxygenase inhibitor zileuton in human plasma using high performance liquid chromatography–tandem mass spectrometry by Phillip Pian; Edward Labovitz; Keith Hoffman; Claudia F. Clavijo; Rachael Rzasa Lynn; Jeffrey L. Galinkin; Alexander A. Vinks; Punam Malik; Uwe Christians (79-83).
Zileuton is an orally active, selective inhibitor of 5-lipoxygenase, which catalyzes the first step in the conversion of arachadonic acid into leukotrienes. Given the important role of leukotrienes in inflammation and cell signaling, multiple studies have investigated the efficacy of zileuton in the treatment of human disease. Examples of disease targets include asthma, ulcerative colitis, rheumatoid arthritis, and more recently, acne, ischemic/reperfusion injury, inflammatory pain, and sickle cell anemia. Zileuton is currently approved for the prophylaxis and chronic treatment of asthma. We report the development and validation of a sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the quantification of zileuton in human EDTA plasma. The range of reliable response was 3.05–20,000 ng/mL in human plasma. The calibration curves had a correlation coefficient of r 2  > 0.99. The intra-day precision was 3.4–5.3%. The inter-day precision ranged from 4.5% to 7.3% and inter-day accuracy from 100% to 107%. No matrix interferences, ion suppression/enhancement, or carry-over was observed. The assay met all predefined acceptance criteria and was subsequently employed to measure plasma zileuton concentrations in a clinical trial.
Keywords: Zileuton; 5-Lipoxygenase inhibitor; LC–MS/MS; Human plasma;

A simple and efficient chromatographic method for large-scale preparative separation of phenylethanoid glycosides (mainly contain echinacoside and acteoside) from Cistanche deserticola was developed. The adsorption properties of eight macroporous resins were evaluated. Three selected resins were further screened depending on the adsorption kinetics curves, in which HPD300 resin showed the best separation efficiency. The adsorption isotherm data on HPD300 resin were fitted to the Freundlich equation in certain concentration range. The dynamic adsorption and desorption experiments were carried out on columns packed with HPD300 resin to optimize the separation process. The breakthrough curves showed that acteoside had a higher affinity to the resin than echinacoside. The contents of echinacoside and acteoside in the product increased from 1.79% and 1.43% in the crude extracts to 16.66% and 15.17%, with recovery yields of 80.41% and 90.17%, respectively. The purity of total phenylethanoid glycosides in the product was 76.58%.
Keywords: Macroporous resin; Phenylethanoid glycosides; Echinacoside; Acteoside; Cistanche deserticola Y. C. Ma; Separation;

In order to elucidate the role of organic anion transporters (OATs) in the renal elimination of gallic acid and gentisic acid, a new, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous determination of gallic acid and gentisic acid in cell lysate, using Danshensu as the internal standard (IS). After a simple liquid–liquid extraction, the analytes were detected in negative ESI mode using selected reaction monitoring. The precursor-to-product ion transitions (m/z) were 169.0 → 125.0, 153.1 → 108.0, and 196.8 → 135.2 for gallic acid, gentisic acid, and the IS, respectively. Chromatographic separation was achieved on a C18 column using mobile phases consisting of water with 0.1% acetic acid (A) and acetonitrile with 0.05% formic acid. (B) The total run time was 3 min and calibration curves were linear over the concentrations of 0.33–2400 ng/mL for both compounds (r 2  > 0.995). Good precision (between 3.11% and 14.1% RSD) and accuracy (between −12.7% and 11% bias) was observed for quality controls at concentrations of 0.33 (lower limit of quantification), 1, 50, and 2000 ng/mL. The mean extraction recovery of gallic acid and gentisic acid was 80.7% and 83.5%, respectively. Results from post-column infusion and post-extraction methods indicated that the analytical method exhibited negligible matrix effects. Finally, this validated assay was successfully applied in a cellular uptake study to determine the intracellular concentrations of gallic acid and gentisic acid in OAT expressing cells.
Keywords: Cell lysate; LC–MS/MS; Matrix effects; Organic solute carrier; Phenolic acids; SLC22;

The determination of Prostaglandin (PG) E1 in plasma is challenged by its low concentration (pg/mL) and endogenous interference. An LC–MS/MS method for the determination of PGE1 in dog plasma has been developed and validated. Plasma being sampled at 4 °C and treated with indomethacin effectively inhibited interferents synthesized post-sampling. Samples were subjected to one-step extraction and separated by reversed phase HPLC with a short cycle time of 3 min. An LLOQ of 10 pg/mL was achieved with 500 μl plasma. The method was applied to a pharmacokinetic study in beagle dogs involving an intravenous infusion of 3.2 μg/kg PGE1. The half-life was recovered at 7 min. The simple, sensitive and rapid method was suitable to be applied to pharmacokinetic studies of PGE1 at clinically relevant doses.
Keywords: Prostaglandin E1; LC–MS/MS; Pharmacokinetics; Beagle dog; Plasma;

Global gas chromatography/time-of-flight mass spectrometry (GC/TOFMS)-based metabonomic profiling of lyophilized human feces by Lee Cheng Phua; Poh Koon Koh; Peh Yean Cheah; Han Kiat Ho; Eric Chun Yong Chan (103-113).
Gas chromatography mass spectrometry (GC/MS)-based fecal metabonomics represents a powerful systems biology approach for elucidating metabolic biomarkers of lower gastrointestinal tract (GIT) diseases. Unlike metabolic profiling of fecal water, the profiling of complete fecal material remains under-explored. Here, a gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) method was developed and validated for the global metabonomic profiling of human feces. Fecal and fecal water metabotypes were also profiled and compared. Additionally, the unclear influence of blood in stool on the fecal metabotype was investigated unprecedentedly. Eighty milligram of lyophilized feces was ultrasonicated with 1 mL of methanol:water (8:2) for 30 min, followed by centrifugation, drying of supernatant, oximation and trimethylsilylation for 45 min. Lyophilized feces demonstrated a more comprehensive metabolic coverage than fecal water, based on the number of chromatographic peaks. Principal component analysis (PCA) indicated occult blood (1 mgHb/g feces) exerted a negligible effect on the fecal metabotype. Conversely, a unique metabotype related to feces spiked with gross blood (100 mgHb/g feces) was revealed (PCA, R 2 X  = 0.837, Q 2  = 0.794), confirming the potential confounding effect of gross GIT bleeding on the fecal metabotype. This pertinent finding highlights the importance of prudent interpretation of fecal metabonomic data, particularly in GIT diseases where bleeding is prevalent.
Keywords: Feces; Metabonomics; Metabolomics; Metabolic profiling; Gas chromatography mass spectrometry; Colorectal cancer;