Journal of Chromatography B (v.863, #2)

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously determine berberine, palmatine and jatrorrhizine in rat plasma. After mixing with the internal standard (IS) tetrahydropalmatine, plasma samples were pretreated by protein precipitation with acetonitrile–methanol (1:2, v/v). Chromatographic separation was carried out on a C18 column using a mixture of water (containing 0.1% formic acid) and acetonitrile (30:70, v/v) as mobile phase. The detection was performed by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source operating in the positive ionization mode. The method was linear over the concentration range of 1.0–250.0 ng/mL for all components. The intra- and inter-day precision values were less than 14.6% and the deviations were within ±4.0%. The fully validated LC–MS/MS method has been successfully applied to the pharmacokinetic study of berberine, palmatine and jatrorrhizine in rat plasma after oral administration of coptis–evodia herb couple. Three peaks were observed in both individual and mean plasma–concentration curves of berberine, palmatine and jatrorrhizine, which may be attributed to distribution re-absorption and enterohepatic circulation.
Keywords: Berberine; Palmatine; Jatrorrhizine; LC–MS/MS; Coptis–evodia herb couple; Pharmacokinetics;

Quantitative chiral analysis of salsolinol in different brain regions of rats genetically predisposed to alcoholism by Tatiana Rojkovicova; Yehia Mechref; Jason A. Starkey; Guangxiang Wu; Richard L. Bell; William J. McBride; Milos V. Novotny (206-214).
A method to determine the catecholamine content in putamen (CPU) and midbrain (MB) regions of the brain of alcohol-preferring rats (P) is presented with a focus on the low-level detection of S,R-salsolinol, a metabolite of dopamine and a putative alcoholism marker. The developed strategy allows both quantitative profiling of related catecholamines and the enantiomeric separation and quantification of the S- and R-salsolinol isomers and their ratios. The described LC/MS strategy simplifies the current methodology that typically employs GC–MS by eliminating the need for derivatization. The data also suggest an increase in the non-enzymatic formation of salsolinol as a consequence of ethanol exposure.
Keywords: Catecholamines; Salsolinol; Alcoholism; Tandem mass spectrometry; Multiple reaction monitoring (MRM); Chiral separations;

The aim of this paper is to investigate the characteristics of atractylenolide I (AO-I) in the body by a GC–MS method. All bio-samples were cleared up with a liquid–liquid extraction procedure. The calibration curves were linear within a range of 5–1000 ng/mL for plasma samples, 0.06–16.00 μg/g for cerebellum samples, and 0.03–8.00 μg/g for other tissue samples. The limit of quantification (LOQ) for AO-I was 1.0 ng/mL or 1.0 ng/g (S/N ≥ 10) in the bio-samples. In the applications, the main pharmacokinetic parameters were firstly obtained as follows: T max  = 0.37 ± 0.19 h, C max  = 0.26 ± 0.05 μg/mL, AUC = 1.95 ± 0.30 μg h/mL and k a  = 10.08 ± 5.60 h−1. The tissue distribution of AO-I in rats after the oral administration of 50.0 mg/kg was from 0.225 to 0.031 μg/g with a decreasing tendency in different tissues like liver > kidney > spleen > cerebellum > heart > cerebrum > lung. The protein binding in rat plasma, human plasma and bovine serum albumin was 80.8 ± 3.9, 90.6 ± 3.1 and 60.9 ± 5.1%, respectively.
Keywords: Atractylenolide I; Pharmacokinetics; Tissue distribution; Protein binding; GC–MS;

Determination of ecabet in human plasma by high-performance liquid chromatography–tandem mass spectrometry by Dan Zhang; Xiaolin Du; Mingyuan Liu; Hao Li; Yao Jiang; Limei Zhao; Jingkai Gu (223-228).
This paper describes a simple, robust and cost-effective assay for the determination of ecabet in human plasma. After a simple step of protein precipitation using methanol, plasma samples were analyzed by reverse phase high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) with valsartan as the internal standard (I.S.). Ecabet and the I.S. valsartan were separated on a Venusil MP C18 analytical column using methanol–10 mM ammonium acetate (75:25, v/v, pH 3.0) as mobile phase at a flow rate of 1.0 mL/min. Ecabet and I.S. were eluted at 0.91 and 0.92 min, respectively, ionized in negative mode, and then detected by multiple reaction monitoring (MRM) essay. The MRM transitions of m/z 379.1 →  m/z 277.1 and m/z 434.3 →  m/z 350.1 were used to quantify ecabet and I.S., respectively. The assay was linear over the concentration range of 10–6000 ng/mL and was successfully applied to a pharmacokinetic study in healthy volunteers.
Keywords: Ecabet; LC–MS/MS;

Liquid phase microextraction by back extraction (LPME–BE) combined with high performance liquid chromatography (HPLC)-fluorescence detection was developed for the determination of tramadol in human plasma. Tramadol was extracted from 2 mL of basic sample solution (donor phase) with pH 11.5 through a micro liter-size organic solvent phase (100 μL n-octane) for 25 min and finally into a 3.5 μL acidic aqueous acceptor microdrop with pH 2.5 suspended in the organic phase from the tip of a HPLC microsyringe needle for 15 min with the stirring rate of 1250 rpm. After extraction for a period of time, the microdrop was taken back into the syringe and injected into HPLC. Effected the experimental parameters such as the nature of the extracting solvent and its volume, sample temperature, stirring rate, volume of the acceptor phase, pH and extraction time on LPME–BE efficiency was investigated. At the optimized condition, enrichment factor of 366 and detection limit (LOD) of 0.12 μg L−1 were obtained. The calibration curve was linear (r  = 0.999) in the concentration range of 0.3–130 μg L−1. Within-day relative standard deviation RSD (S/N = 3) and between-day RSD were 3.16% and 6.29%, respectively. The method was successfully applied to determine the concentration of tramadol in the plasma and urine samples and satisfactory results were obtained.
Keywords: Liquid phase microextraction with back extraction; Tramadol; HPLC; Urine;

Hybrid hybridomas (quadromas) are derived by fusing at least two hybridomas, each producing a different antibody of predefined specificity. The resulting cell secretes not only the immunoglobulins of both parents but also hybrid molecules manifesting the binding characteristics of the individual fusion partners. Purification of the desired bispecific immunoprobe with high specific activity from a mixture of bispecific and monospecific monoclonal antibodies requires special strategies. Using a dual, sequential affinity chromatography (Protein-G chromatography followed by m-aminophenyleboronic acid agarose column), we have purified bispecific monoclonal antibodies (BsMAb) as a preformed HRPO (Horseradish Peroxidase) complex (BsMAb–HRPO). The quadroma culture supernatant was initially processed on a Protein-G column to isolate all the species of immunoglobulins. This pre-enriched fraction was subsequently passed through the aminophenyleboronic acid column super saturated with HRPO. The column matrix has the ability to bind to proteins such as HRPO with vicinal diols. The enzyme loaded column captures the desired bispecific anti-SARS-CoV × anti-HRPO species with the elimination of the monospecific anti-SARS-CoV MAb to result in a high specific activity diagnostic probe. The presence of anti-HRPO MAb is an acceptable impurity as it will not bind to the target SARS-CoV NP antigen and will get washed out during the ELISA procedure.
Keywords: SARS-CoV; Bispecific monoclonal antibodies; m-Aminophenylboronic acid;

Selective isolation of in vitro phase II conjugates using a lipophilic anionic exchange solid phase extraction method by Sébastien Gagné; Julie Laterreur; Louiza Mahrouche; Dan Sørensen; Jacques Yves Gauthier; Vouy Linh Truong; Nathalie Chauret; Jean-François Lévesque (242-248).
Identification, characterization and structure elucidation of human metabolites of drug candidates is crucial for the pharmaceutical industry to assess their activity against the therapeutic target of interest and potential toxicological effects. It often requires in vitro synthesis of microgram quantities of metabolites of interest with enzymatic preparations, pre-concentration of the reaction mixture by solid phase extraction (SPE), metabolite isolation using HPLC systems coupled to fraction collectors prior to nuclear magnetic resonance characterization. The method reported herein is a rapid and simple technique using solely off-line mixed phase anionic exchange lipophilic SPE cartridges to selectively isolate glucuronide and sulfate metabolites from their parent compound. This approach capitalizes on the pKa differences between the parent compound, devoided of acidic moieties, and the negatively charged glucuronide and/or sulfate metabolites. Once loaded on the SPE cartridge, the incubation mixture is washed successively with a basic aqueous solution, methanol to elute the non-anionic parent compounds, and then with an acidic methanolic solution to protonate and recover the phase II conjugates. Over 100 μg (>95% purity) of 17α-ethynylestradiol-3-glucuronide and 6-gingerol-4′-glucuronide were successfully isolated using this technique, as well as glucuronide and a sulfate conjugates of 1-{4′-[(1R)-2,2-difluoro-1-hydroxyethyl]biphenyl-4-yl}cyclopropanecarboxamide (DHBC) synthesized in-house. Their structures were confirmed by Ultra Performance Liquid Chromatography coupled to Quadrupole-Time of flight (UPLC-QTof) and nuclear magnetic resonance analysis.
Keywords: Phase II metabolite; Glucuronide conjugate; Sulfate conjugate; SPE; NMR; Accurate mass;

Determination of abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine concentration in human plasma by MALDI-TOF/TOF by Stefania Notari; Carmine Mancone; Tonino Alonzi; Marco Tripodi; Pasquale Narciso; Paolo Ascenzi (249-257).
The interest in therapeutic drug monitoring (TDM) of antiretroviral drugs has grown significantly since highly active antiretroviral therapy (HAART) became a standard of care in clinical practice. TDM is useful to determine the best dosage regimen adapted to each patient. Here, we apply MALDI-TOF/TOF technology to quantify abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine in the plasma of HIV-infected patients, by standard additions analysis. Regression of standard additions was linear over the whole anti-HIV concentration range explored (1.00 × 10−2–1.00 pmol/μL). The absolute recovery ranged between 80% and 110%. Values of the drug concentration determined by MALDI-TOF/TOF were in the range of 1.00 × 10−2–1.00 pmol/μL. The limit of quantification value was 1.00 × 10−2  pmol/μL for abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine.
Keywords: Human plasma; Anti-HIV drug determination; MALDI-TOF/TOF;

SR141716 (rimonabant) is an endocannabinoid receptor antagonist. Endocannabinoids are a class of chemicals that affect neurotransmission via G-protein coupled CB1 (brain) and CB2 (peripheral tissue) receptors. Numerous animal studies have shown that SR141716 binds with the CB1 receptor in the brain, resulting in several biological consequences including reduced alcohol intake and reward as well as reduced food consumption. In this work, an analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) has been developed and validated for the quantitative measurement of SR141716 in both human and rat plasma to support the investigation of this compound. A suitable internal standard (AM251) has been chosen and the experimental conditions have been optimized for the separation and detection of singly charged positive ions of SR141716 and the internal standard. A protein precipitation protocol has been developed for extraction of SR141716 and the internal standard from plasma samples. Quantitation was achieved using multiple-reaction-monitoring (MRM) mode for SR141716 (m/z 463 →  m/z 363) and the internal standard (m/z 555 →  m/z 455) and calibration curve over the concentration range of 5.00–1000 ng/ml was plotted using the peak-area ratio versus the concentration of SR141716 with a LOD and LLOQ of 1.09 and 3.62 ng/ml, respectively. The method developed has been used to analyze SR141716 in rat plasma samples from an animal study.
Keywords: SR141716; Rimonabant; Endocannabinoid receptor antagonist; LC–ESI-MS/MS; Human and rat plasma;

Efficient purification of small unsaturated oligoglucuronides by reversed-phase chromatography by Marie-Laure Tavernier; Emmanuel Petit; David Lesur; Anne Wadouachi; Bernard Courtois; Josiane Courtois; Philippe Michaud (266-272).
Ion-exchange chromatography has been applied to purification of unsaturated oligoglucuronans. After an isocratic elution on a strong anion-exchange column, the collected fractions were desalted by low pressure size exclusion chromatography. However, this efficient separation was limited by the time required to desalt. So, we developed a reversed-phase chromatography method using back ionization of oligomers. Two C18 columns were tested with trifluoroacetic acid (TFA 0.7%) as eluent. Different selectivities and column stabilities were observed in this acidic condition. The scale up for semi-preparative applications enabled us to recover pure unsaturated oligoglucuronans without desalting step.
Keywords: Oligouronides; Oligoglucuronan; Reversed-phase chromatography;

Quantification of cationic anti-malaria agent methylene blue in different human biological matrices using cation exchange chromatography coupled to tandem mass spectrometry by Jürgen Burhenne; Klaus-Dieter Riedel; Jens Rengelshausen; Peter Meissner; Olaf Müller; Gerd Mikus; Walter E. Haefeli; Ingeborg Walter-Sack (273-282).
Selective and sensitive methods for the determination of the cationic dye and anti-malarial methylene blue in human liquid whole blood, dried whole blood (paper spot), and plasma depending on protein precipitation and cation exchange chromatography coupled to electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) have been developed, validated according to FDA standards, and applied to samples of healthy individuals and malaria patients within clinical studies. Acidic protein precipitation with acetonitrile and trifluoroacetic acid was used for liquid whole blood and plasma. For the extraction of methylene blue from paper spots aqueous acetonitrile was used. Sample extracts were chromatographed on a mixed mode column (cation exchange/reversed phase, Uptisphere MM1) using an aqueous ammonium acetate/acetonitrile gradient. Methylene blue was quantified with MS/MS in the selected reaction monitoring mode using ESI and methylene violet 3RAX as internal standard. Depending on the sample volume (whole blood and plasma 250 μL, and 100 μL on paper spots) the method was linear at least within 75 and 10,000 ng/mL and the limit of quantification in all matrices was 75 ng/mL. Batch-to-batch accuracies of the whole blood, plasma, and paper spot methods varied between −4.5 and +6.6%, −3.7 and +7.5%, and −5.8 and +11.1%, respectively, with corresponding precision ranging from 3.8 to 11.8% CV. After a single oral dose (500 mg) methylene blue concentrations were detectable for 72 h in plasma. The methods were applied within clinical studies to samples from healthy individuals and malaria patients from Burkina Faso.
Keywords: Methylene blue; Cation exchange chromatography; Tandem mass spectrometry; Whole blood; Plasma; Paper spots;

Benzene and toluene are important industrial chemicals and ubiquitous environmental pollutants. The urinary mercapturic acids of benzene and toluene, S-phenylmercapturic acid (S-PMA) and S-benzylmercapturic acids (S-BMA) are specific biomarkers for the determination of low-level exposures. We have developed and validated a fast, specific and very sensitive method for the simultaneous determination of S-PMA and S-BMA in human urine using an automated multidimensional LC–MS–MS-method that requires no additional sample preparation. Analytes are stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry using isotopically labelled S-PMA as internal standard. The lower limit of quantification (LLOQ) for both analytes was 0.05 μg/L urine and sufficient to quantify the background exposure of the general population. Precision within series and between series for S-PMA and S-BMA ranged from 1.0% to 12.2%, accuracy was 108% and 100%, respectively. We applied the method on spot urine samples of 30 subjects of the general population with no known exposure to benzene or toluene. Median levels (range) for S-PMA and S-BMA in non-smokers (n  = 15) were 0.14 μg/L (<0.05–0.26 μg/L) and 8.2 (1.6–77.4 μg/L), respectively. In smokers (n  = 15), median levels for S-PMA and S-BMA were 1.22 μg/L (0.17–5.75 μg/L) and 11.5 μg/L (0.9–51.2 μg/L), respectively. Due to its automation, our method is well suited for application in large environmental studies.
Keywords: Biological monitoring; Benzene; Toluene; Mercapturic acids; Environmental exposure;

HPLC–MS analysis of tryptic protein digests in combination with fluorescence detection is presented as a convenient and quantitative method to gain insight into the relative reactivity of lysine side chains. In this scheme (tandem) mass spectrometry was used for identification of the modified residue, whereas fluorescence detection allowed determination of their relative abundance. Our method identified ‘labeling hot-spots’ at two flexible parts of the collagen-binding protein CNA35, positions that were consistent with all available structural and biochemical data on the collagen-binding properties of CNA35.
Keywords: Fluorescence; Labeling; LC–MS; Collagen-binding protein; Tryptic digestion;