Journal of Chromatography B (v.878, #31)
Editorial Board (i).
Review on the analysis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its phase I and phase II metabolites in biological matrices, foodstuff and beverages by S.F. Teunissen; H. Rosing; A.H. Schinkel; J.H.M. Schellens; J.H. Beijnen (3199-3216).
The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), has been shown to be carcinogenic in rodents, mice and rats. Following phase I N-hydroxylation and phase II esterification PhIP exerts its carcinogenic effect by binding to DNA purines. Quantitative and qualitative analysis of its bioactivated metabolites as well as it detoxification products is important in studying its biological effects and inter- and intra-individual exposures. A review is presented with an extensive coverage of publications specifically reporting on the analysis of PhIP and its phase I and II metabolites in biological matrices, foodstuff and beverages. Analytical techniques such as liquid and gas chromatography coupled with various detection techniques (mass spectrometry, ultraviolet or fluorescence detection) were mostly applied. We conclude that since the initial identification of PhIP in 1986 a large set of assays has been developed for the analysis of PhIP and its phase I and phase II metabolites in a wide range of matrices, these included food products and biological samples such as plasma, urine and faeces. In addition, it was shown that numerous metabolites were recovered and identified. Thus, we conclude that liquid chromatography coupled to mass spectrometry is clearly the method of choice for sensitive qualitative as well as quantitative analysis with high selectivity and reaching lower quantification levels in the sub pg/mL range. The main aim of this review is that it can be used by other researchers as a resource for method development and optimization of analytical methods of PhIP and its carcinogenic or detoxification products.
Keywords: PhIP; Metabolites; Review; Chromatography; Mass spectrometry;
A highly sensitive ultra performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) technique for quantitation of protein free and bound efavirenz (EFV) in human seminal and blood plasma by Lindsay B. Avery; Teresa L. Parsons; David J. Meyers; Walter C. Hubbard (3217-3224).
A combined UPLC–tandem mass spectrometric (UPLC–MS/MS) technique has been validated for quantitation of protein free efavirenz (EFV) as well as total concentrations of EFV in human blood and seminal plasma. The analytical method possesses capabilities for concentration measurements of EFV ranging from 0.5 to 10,000 ng/ml with an accuracy (%dev) of −5.2–8.0% and precision (%CV) of <8%. Standard curves were linear with coefficients of variation (r 2) >0.98. The method employs a racemic fluorinated analog of EFV (F-EFV) as the internal standard. EFV and F-EFV were eluted from a reverse-phase UPLC column via gradient elution with detection via negative ion multiple reaction monitoring (MRM). EFV and F-EFV, respectively, were detected via the following MRM transitions: m/z 314.0 > 244.1 and m/z 298.0 > 227.9. The time required for the analysis of each sample was 8.0 min. The analytical technique is capable of a reliable detection limit of ∼15–20 fmol of EFV injected on column.
Keywords: Seminal plasma; Efavirenz; UPLC–MS/MS; Blood plasma; Electrospray ionization (ESI); HIV/AIDS;
Protein profile analysis of cellular samples from the cervix for the objective diagnosis of cervical cancer using HPLC-LIF by Sujatha Bhat; Ajeetkumar Patil; Lavanya Rai; V.B. Kartha; C. Santhosh (3225-3230).
Protein profiles of cytologic samples from the cervix were studied using High Performance Liquid Chromatographic (HPLC) separation combined with ultra-sensitive laser induced fluorescence (LIF) detection. HPLC-LIF protein profiles of samples from clinically normal subjects, individuals suffering from cervical cancer (different stages), and subjects who had other gynecological problems related to cervix, like erosion of cervix and Nabothian cyst, but no malignancy, were subjected to Principal Component Analysis (PCA). The application of HPLC-LIF protein profiling combined with PCA was found to be a highly efficient method for discrimination of different classes of samples with high sensitivity and specificity. Diagnostic accuracy and optimal threshold – decision criterion – for objective discrimination were estimated using sensitivity–specificity pairs and Youden's index (J) plots.
Keywords: Cellular samples; HPLC-LIF; Principal Component Analysis; Youden's index;
Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry by Akwasi A. Amoako; Timothy H. Marczylo; Patricia M.W. Lam; Jonathon M. Willets; Amanda Derry; Janine Elson; Justin C. Konje (3231-3237).
The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6–17.7% and 6.3–12.5%, respectively. Analyses were linear over the range 0.237–19 nM for anandamide and oleoylethanolamide and 0.9–76 nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100 fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200 fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at −80 °C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21 nM (0.09–0.27), 1.785 nM (0.48–2.32) and 15.54 nM (7.05–16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.
Keywords: Endocannabinoids; Anandamide; Oleoylethanolamide; Palmitoylethanolamide; Seminal plasma; Mass spectrometry;
Direct quantification of dimethylsulfoniopropionate (DMSP) with hydrophilic interaction liquid chromatography/mass spectrometry by Astrid Spielmeyer; Georg Pohnert (3238-3242).
A simple, derivatization free method for the direct determination of dimethylsulfoniopropionate (DMSP) using hydrophilic interaction liquid chromatography (HILIC)/mass spectrometry is introduced. DMSP is a zwitterionic osmolyte which is produced from marine plankton, macro algae and higher plants. Due to its central role in climate relevant geochemical processes as well as in plant physiology and chemical ecology there is a great interest in methods for its quantification. Since DMSP is labile and difficult to extract currently most protocols for quantification are based on indirect methods. Here we show that ultra performance liquid chromatography/mass spectrometry using a HILIC stationary phase is suitable for the direct quantification of DMSP from aqueous samples and microalgal extracts. The protocol requires minimal sample preparation and phytoplankton samples can be investigated after filtration of small volumes. The limit of detection is 20 nM and the calibration curve is linear in the range of 60 nM to 50 μM. The use of [2H6]-DMSP as internal standard allows prolonged sample storage since it is transformed with the same kinetics as natural DMSP. This makes the method suitable for both laboratory and field studies.
Keywords: Dimethylsulfide (DMS); Dimethylsulfoniopropionate (DMSP); HILIC; Mass spectrometry; Quantification; UPLC/MS;
Electrochemical characterization of repaglinide and its determination in human plasma using liquid chromatography with dual-channel coulometric detection by David Jirovský; Zdenka Bartošová; Jana Skopalová; Vítezslav Maier (3243-3248).
A simple, fast and sensitive HPLC method employing dual-channel coulometric detection for the determination of repaglinide in human plasma is presented. The assay involved extraction of repaglinide by ethyl acetate and isocratic reversed-phase liquid chromatography with dual-channel coulometric detection. The mobile phase composition was 50 mM disodium hydrogen phosphate/acetonitrile (60:40, v/v), pH of the mobile phase 7.5 set up with phosphoric acid. For all analyses, the first cell working potential was +380 mV, the second was +750 mV (vs. Pd/H2). Calibration curve was linear over the concentration range of 5–500 nmol L−1. Rosiglitazone was used as an internal standard. The limit of detection (LOD) was established at 2.8 nmol L−1, and the lower limit of quantification (LLOQ) at 8.5 nmol L−1. The developed method was applied to human plasma samples spiked with repaglinide at therapeutical concentrations. It was confirmed that the method is suitable for pharmacokinetic studies or therapeutic monitoring.
Keywords: Repaglinide; Rosiglitazone; HPLC; Coulometric detection; Peroral antidiabetics; Plasma;
Enantiomeric composition analysis of pranoprofen in equine plasma and urine by chiral liquid chromatography–tandem mass spectrometry in selected reaction monitoring mode by J. Yu; K.S. Han; G. Lee; M.J. Paik; K.R. Kim (3249-3254).
The enantioseparation of pranoprofen after its addition in racemic form into equine plasma and urine was conducted by chiral liquid chromatography–tandem mass spectrometry in selected reaction monitoring mode. The methods for the assay of both enantiomers were linear (r ≥ 0.9943) in the low range from 0.001 to 0.1 μg/mL and high range from 0.01 to 1.0 μg/mL with good precision (% RSD ≤ 5.6) and accuracy (% RE = −5.3 to 1.9). When racemic pranoprofen was orally administered to four horses at a single dose of 3.1 mg/kg, the median plasma concentrations of (R)-pranoprofen were lower than the levels of (S)-pranoprofen from start to finish. In contrast, the urinary level of (R)-pranoprofen was 2.5 fold higher than (S)-pranoprofen level for the first 6 h, followed by its rapid decrease down below (S)-pranoprofen concentration. Monitoring of the R/S ratios in equine urine may be useful for the prevention of false positive in horse doping test.
Keywords: Pranoprofen; Chiral liquid chromatography–tandem mass spectrometry in selected reaction monitoring mode; Enantiomeric composition; Equine plasma and urine; Horse doping;
A fast and simple assay for busulfan in serum or plasma by liquid chromatography–tandem mass spectrometry using turbulent flow online extraction technology by Dustin R. Bunch; Courtney Heideloff; James C. Ritchie; Sihe Wang (3255-3258).
Busulfan is used in myeloablative preparation regimens for hematopoietic bone marrow transplantation. Due to its narrow therapeutic range therapeutic drug monitoring of busulfan is recommended. In this study a fast and simple method for measuring busulfan in serum or plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed utilizing turbulent flow online extraction technology. Serum or plasma was mixed with acetonitrile containing d8-busulfan. After centrifugation the supernatant was injected onto a turbulent flow preparatory column then transferred to a C18 analytical column monitored by a tandem mass spectrometer set at positive electrospray ionization. The analytical cycle time was 4.0 min. The method was linear from 0.15 to 41.90 μmol/L with an accuracy of 87.9–103.0%. Inter- and intra-assay CVs across four concentration levels were 2.1–7.8%. No significant carryover or ion suppression was observed. No interference was observed from commercial control materials containing more than 100 compounds. Comparison with a well established LC–MS/MS method using patient specimens (n = 45) showed a mean bias 1.3% with Deming regression of slope 1.02, intercept −0.02 μmol/L, and a linear correlation coefficient 0.9883. The LC–MS/MS method coupled with turbulent flow online sample cleaning technology described here offers reliable busulfan quantitation in serum or plasma with minimum manual sample preparation and was fully validated for clinical use.
Keywords: Busulfan; Liquid chromatography; Tandem mass spectrometry; Turbulent flow;
Analysis of mouse liver membrane proteins using multidimensional separations and tandem mass spectrometry by Zhuowei Wang; Min Wang; Wei Tong (3259-3266).
In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6 M guanidine hydrochloride, 20 mM Tris–HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0–1.0 M) gradient, the bound proteins were eluted out at 0.1–1.0 M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.
Keywords: Mouse liver; Membrane protein; Tandem liquid chromatography; SDS-PAGE; Reverse-phase HPLC; Ion-trap mass spectrometry;
Potential bias and mitigations when using stable isotope labeled parent drug as internal standard for LC–MS/MS quantitation of metabolites by Wenying Jian; Richard W. Edom; Yaodong Xu; Joseph Gallagher; Naidong Weng (3267-3276).
In recent years, increasing emphasis has been placed on quantitative characterization of drug metabolites for better insight into the correlation between metabolite exposure and toxicological observations or pharmacological efficacy. One common strategy for metabolite quantitation is to adopt the stable isotope labeled (STIL) parent drug as the internal standard in an isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay. In the current work, we demonstrate this strategy could have a potential pitfall resulting in quantitation bias if the internal standard is subject to ion suppression from the co-eluting parent drug in the incurred samples. Propranolol and its metabolite 4-hydroxypropranolol were used as model compounds to demonstrate this phenomenon and to systematically evaluate different approaches to mitigate the issue, including atmospheric pressure chemical ionization (APCI) mode of ionization, increased internal standard concentration, quantitation without internal standard, the use of a structural analog as internal standard, and dilution of the samples. Case studies of metabolite quantitation in nonclinical and clinical studies in drug development were also included to demonstrate the importance of using an appropriate bioanalytical strategy for metabolite quantitation in the real world. We present that bias of metabolite concentrations could pose a potential for poor estimation of safety risk. A strategy for quantitation of metabolites in support of drug safety assessment is proposed.
Keywords: Stable isotope labeled internal standard; Metabolite; Quantitation; LC–MS/MS; Ion suppression; Safety assessment;
Dexamethasone quantification in dried blood spot samples using LC–MS: The potential for application to neonatal pharmacokinetic studies by Parul Patel; Sangeeta Tanna; Hussain Mulla; Venkatesh Kairamkonda; Hitesh Pandya; Graham Lawson (3277-3282).
A high-performance liquid chromatography (LC–MS) method has been developed and validated for the determination of dexamethasone in dried blood spot (DBS) samples. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30 μl blood spots on specimen collection cards. An 8 mm disc was cut from the DBS sample and extracted using a combination of methanol: water (70:30, v/v) containing the internal standard, triamcinolone acetonide. Extracts were centrifuged and chromatographic separation was achieved using a Zorbax Eclipse Plus C18 column using gradient elution with a mobile phase of acetonitrile and water with formic acid at a flow rate of 0.2 ml/min. LC–MS detection was conducted with single ion monitoring using target ions at m/z 393.1 for dexamethasone and 435.1 for the internal standard. The developed method was linear within the tested calibration range of 15–800 ng/ml. The overall extraction recovery of dexamethasone from DBS samples was 99.3% (94.3–105.7%). The accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤15% at all concentrations. Factors with potential to affect drug quantification measurements such as blood haematocrit, the volume of blood applied onto the collection card and spotting device were investigated. Although a haematocrit related effect was apparent, the assay accuracy and precision values remained within the 15% variability limit with fluctuations in haematocrit of ±5%. Variations in the volume of blood spotted did not appear to affect the performance of the developed assay. Similar observations were made regarding the spotting device used. The methodology has been applied to determine levels of dexamethasone in DBS samples collected from premature neonates. The measured concentrations were successfully evaluated using a simple 1-compartment pharmacokinetic model. Requiring only a microvolume (30 μl) blood sample for analysis, the developed assay is particularly suited to pharmacokinetic studies involving paediatric populations.
Keywords: Dried blood spot (DBS); Dexamethasone; Chronic lung disease; Guthrie card; LC–MS;
Validated hydrophilic interaction LC–MS/MS method for determination of N-methyl-2-pyrrolidinone residue: Applied to a depletion study of N-methyl-2-pyrrolidinone in swine liver following intramuscular administration of drug N-methyl-2-pyrrolidinone formulation by Yanhong Liu; Yanhong Ji; Jiangtao Chen; Ximing Chen; Guangxiang Wang; Youjun Shang; Xiangtao Liu; Jack Pan (3283-3289).
A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry method for determination of N-methyl-2-pyrrolidinone in swine liver was developed and validated. After the fortification of N-methyl-d3-2-pyrrolidinone-d6 as the deuterium-labeled internal standard, N-methyl-2-pyrrolidinone in swine liver was extracted by acetonitrile and the supernatant was led through a C18 + WAX mixed-mode SPE cartridge for removal of the matrix interferences. The final eluate was acidified by formic acid and then injected onto a 3 μm 15 cm × 2.1 mm TX column for hydrophilic interaction chromatographic analysis. Mass spectrometry detection was carried on a PE Sciex API 4000 triple quadrupole mass spectrometer using positive turbo-ion spray ionization mode. The MRM transitions were 100 → 58 for N-methyl-2-pyrrolidinone and 109 → 62 for N-methyl-d3-2-pyrrolidinone-d6. Solvent calibration standards could be readily used for quantitative analysis of N-methyl-2-pyrrolidinone with excellent precision and accuracy, although there are endogenous levels of N-methyl-2-pyrrolidinone in many blank matrices. The true recovery was nearly 100% and the MRM signal of N-methyl-2-pyrrolidinone was suppressed about 30% because of the matrix effect. Nevertheless, N-methyl-d3-2-pyrrolidinone-d6 completely compensated the ion-suppression effect and the injection-to-injection variation. The detection limit was 5 ng g−1 swine liver. The validated method was applied to a depletion study of N-methyl-2-pyrrolidinone in swine liver following intramuscular administration of a drug N-methyl-2-pyrrolidinone formulation.
Keywords: N-methyl-2-pyrrolidinone; C18 + WAX; LC–MS/MS; Matrix effect;
Development and validation of a liquid chromatography–fluorescence–mass spectrometry method to measure glyphosate and aminomethylphosphonic acid in rat plasma by J. Bernal; J.L. Bernal; M.T. Martin; M.J. Nozal; A. Anadón; M.R. Martínez-Larrañaga; M.A. Martínez (3290-3296).
A simple and fast method has been developed and validated to measure glyphosate (GLYP) and aminomethylphosphonic acid (AMPA) in rat plasma based on reversed-phase high performance liquid chromatography (RP-HPLC) coupled to fluorescence (FLD) and electrospray ionization mass spectrometry (ESI-MS) detection. After protein precipitation with acetonitrile, GLYP and AMPA were derivatized with 9-fluorenylmethylchloroformate (FMOC-Cl) and then separated on a C12 column (250 mm × 4.60 mm i.d.) using a gradient of an ammonium formate (20 mM, pH 8.5) and acetonitrile mobile phase. Selected ion monitoring (SIM) mode of the MS was used to obtain maximum sensitivity when quantifying GLYP and AMPA. The validation shows the method to be consistent and reliable, with an intra- and inter-day precision for GLYP and AMPA > 9% for both detectors. For both compounds the accuracy ranged from 2.1% to 7.8% for the intra-day readings, and from 4.1% to 8.6% for the inter-day values. The efficacy of GLYP extraction ranged from 87% to 93% and it was between 76% and 88% for AMPA. Moreover, the limits of quantification (LOQ) for GLYP and AMPA were 5 and 10 ng/mL, respectively with FLD, and 0.4 and 2 ng/mL with ESI-MS. The method was successfully applied to simultaneously measure both compounds in rat plasma samples several days after oral administration of glyphosate.
Keywords: Glyphosate; AMPA; Rat plasma; HPLC–FLD–ESI-MS;
A HPLC-UV method for the determination of puerarin in rat plasma after intravenous administration of PEGylated puerarin conjugate by Xinyi Liu; Hongying Zhi; Feng Du; Zuguang Ye; Naijie Wang; Wenjie Qin; Jianrong Li (3297-3302).
A sensitive and reproducible HPLC method for quantitative determination of puerarin (PUE) in rat plasma was developed and validated using 4-hydroxybenzaldehyde as an internal standard. The separation of PUE was performed on a CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The method was validated and found to be linear in the range of 80–12,000 ng/mL. The limit of quantification was 80 ng/mL based on 100 μL of plasma. The variations for intra- and inter-day precision were less than 8.3%, and the accuracy values were between 98% and 105.2%. The extraction recoveries were more than 85%. The method was successfully applied in the comparative study of pharmacokinetics of PEGylated puerarin (PEG-PUE) versus PUE in rats. Compared with PUE, PEG-PUE showed a 5.2-fold increase in half-life of PUE and a 4.7-fold increase in mean residence time. In addition, this method was also successfully applied to determine the low plasma concentration of PUE regenerated from PEG-PUE in vitro.
Keywords: PEGylated puerarin; Puerarin; Pharmacokinetics; Rat plasma; HPLC-UV;
Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry by Omnia A. Ismaiel; Tianyi Zhang; Rand G. Jenkins; H. Thomas Karnes (3303-3316).
Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid–liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.
Keywords: Matrix effect; Lipids; Quantitative analysis; Selectivity;
Quantitative determination of the diastereoisomers of hexabromocyclododecane in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry by Caiming Tang (3317-3322).
A sensitive, simple and feasible method has been developed and validated for the simultaneous determination of three diastereoisomers of hexabromocyclododecane (HBCD) in human plasma using liquid chromatography tandem mass spectrometry (LC–MS/MS). The simple pretreatment generally involved protein precipitation with methanol (MeOH). The separation was performed with a C18 reverse phase column. The mobile phases were 5 mM ammonium acetate (NH4AC) in water and acetonitrile (ACN). The mass spectrometer was operated using negative electrospray ionization (ESI) source and the data acquisition was carried out with multiple reaction monitoring (MRM) mode. The analyte quantifications were performed by external standard method with matrix-matched calibration curves. The method was partially validated with the evaluations of accuracy, precision, linearity, limit of quantification (LOQ), limit of detection (LOD), recovery, matrix effect and carryover effect. With the present method, the intra-batch accuracies were 94.7–104.3%, 91.9–109.3% and 89.8–105.0% for α-, β- and γ-HBCD, respectively. And the inter-batch accuracies were ranged from 94.2% to 109.7%. Both intra-batch and inter-batch precisions (relative standard deviation, RSD, %) of the analytes were no more than 11.2%. The recoveries were from 79.0% to 108.9% and the LOQ was 10 pg/mL for each diastereoisomer. The linear range was 10–10,000 pg/mL with the linear correlation coefficient R 2 > 0.996. No significant matrix effect and carryover effect of the analytes were observed in this study. This method is in possession of sufficient resolution, high sensitivity as well as selectivity and convenient to be applied to the trace determination of HBCDs in human plasma.
Keywords: Hexabromocyclododecane; Diastereoisomers; Human plasma; Brominated flame retardants; LC–MS/MS;
High-throughput characterization of lipopolysaccharide-binding proteins using mass spectrometry by Yun-Gon Kim; Yung-Hun Yang; Byung-Gee Kim (3323-3326).
Lipopolysaccharide (LPS)-binding proteins interact with LPS in human serum and mediate various immune responses. We describe a high-throughput LPS-binding protein profiling platform for discovering unknown LPS-binding proteins and potential inflammatory mediators. As a pull-down method, the LPS molecules were immobilized onto epoxy beads and then directly incubated with human serum to screen LPS-binding proteins. Through the “untargeted” mass spectrometric approach, potential LPS-binding proteins which elicit various immune responses in human serum were identified by a highly sensitive LTQ Orbitrap Hybrid Fourier Transform Mass Spectrometer (LTQ Orbitrap FT MS). Therefore, this mass spectrometry (MS)-based profiling method is straightforward for screening unknown LPS-binding proteins and provides physiologically relevant binding partners in human serum.
Keywords: Lipopolysaccharide (LPS); LPS-binding proteins; Mass spectrometry (MS); Human serum;
Determination of solifenacin in human plasma by liquid chromatography–tandem mass spectrometry by Jan Macek; Pavel Ptáček; Josef Klíma (3327-3330).
A liquid chromatography–electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50 × 4 mm, 3 μm particles), the mobile phase consisted of methanol – 100 mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363 → 193 and 368 → 198 for solifenacin and the internal standard solifenacin-D5, respectively. The lower limit of quantitation was 0.47 ng/ml using 0.25 ml of plasma and linearity was demonstrated up to 42 ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.
Keywords: Solifenacin; HPLC; LC–MS/MS; Plasma; Pharmacokinetics;