Journal of Chromatography B (v.865, #1-2)
Editorial Board (iii).
Size-exclusion chromatography with evaporative light scattering detection: Method for determination of polydimethylsiloxanes by Krystyna Mojsiewicz-Pieńkowska (1-6).
In recent years the evaporative light scattering detector has become a promising device in the analysis of variable chemical compounds using liquid chromatography. Due to the detection specificity, based on the scattering of the laser light on non-volatile analyte particles, this detector is considered a most universal one. Many authors consider detector signal as a mass signal and subsequently, evaporative light scattering detector has been regarded as a mass detector. Although the scientists pinpoint to many advantages of this device, many of its drawbacks were also noticed. Due to variable examinations carried out some scientist characterised the detector response as a non-linear, seeing in fact a significant limitation of this detector for the purposes of quantitative tests. The author of the present study researched, in many ways, for the solution to this problem, by carrying out tests on polydimethylsiloxanes (PDMS) of a linear structure. The aim of this study was to test the dependence of the evaporative light scattering detector signal upon the molecular weight of PDMS of a linear structure and viscosity ranging from 10 to 60 000 cSt and the injected mass. The evaluation of function monotonicity of the detector response and determination of the function for particular analytes referred to the mass ranges of 8.9–149.0 μg. In order to find the dependence of the integrated signal value of the detector signal intensity, expressed as a surface area in μg, upon analyte mass for particular PDMS, several analytical functions and formulas were used. Parameters of regression equations were calculated for linear and non-linear functions as well as their logarithmic transformations. The aim of the research for the optimal regression equation could mean increased reliability of results obtained from analyses of PDMS.
Keywords: Polydimethylsiloxanes; Evaporative light scattering detection; Linearity;
Size exclusion chromatography with evaporative light scattering detection: Method for the determination of polydimethylsiloxanes by Krystyna Mojsiewicz-Pieńkowska (7-12).
Issues concerned with molecular weight distribution analysis of linear polydimethylsiloxanes have not been extensively investigated and mastered, yet. Current publications do not provide detailed research data on the evaluation of the polymerization degree of polydimethylsiloxanes (PDMS) present in variable matrices: e.g. pharmaceuticals, cosmetics, foodstuffs nor indicate molecular weights of the polymer used. However, the information on molecular weight, i.e. viscosity, is of primary importance as it directly affects PDMS toxicity, absorption and migration in the living organism. The vast majority of currently applied methods prove to be insufficiently specific for PDMS of a particular molecular weight and therefore alternative analytical methods have to be further researched. In this paper the results of determination of molecular weights in linear polydimethylsiloxanes, using size exclusion chromatography with the evaporative light scattering detector are described. The column calibration curve obtained from low-dispersion standard polystyrene of molecular weights ranging 376–2,570,000 Da was used to determine PDMS molecular weights. Precision and accuracy of determination was obtained. For the mobile phase flow-rate of 0.3 ml/min relative standard deviation RSD ranged to 0.45% and the accuracy of measurement amounted to −0.42%, whereas for flow-rate of 1.0 ml/min RSD ranged to 0.38% and accuracy to +2.15%.
Keywords: Polydimethylsiloxanes; Size exclusion chromatography; Gel permeation chromatography; Evaporative light scattering detection;
Enantiomeric analysis of anatabine, nornicotine and anabasine in commercial tobacco by multi-dimensional gas chromatography and mass spectrometry by Baizhan Liu; Chaoying Chen; Da Wu; Qingde Su (13-17).
A fully automated multi-dimensional gas chromatography (MDGC) system with a megabore precolumn and cyclodextrin-based analytical column was developed to analyze the enantiomeric compositions of anatabine, nornicotine and anabasine in commercial tobacco. The enantiomer abundances of anatabine and nornicotine varied among different tobacco. S-(−)-anatabine, as a proportion of total anatabine, was 86.6% for flue-cured, 86.0% for burley and 77.5% for oriental tobacco. S-(−)-nornicotine, as a proportion of total nornicotine, was 90.8% in oriental tobacco and higher than in burley (69.4%) and flue-cured (58.7%) tobacco. S-(−)-anabasine, as a proportion of total anabasine, was relatively constant for flue-cured (60.1%), burley (65.1%) and oriental (61.7%) tobacco. A simple solvent extraction with dichloromethane followed by derivatisation with trifluoroacetic anhydride gave relative standard deviations of less than 1.5% for the determination of the S-(−)-isomers of all three alkaloids. The study also indicated that, a higher proportion of S-(−)-nornicotine is related to the more active nicotine demethylation in the leaf.
Keywords: Anabasine; Anatabine; Enantiomeric composition; GC/MS; MDGC; Nornicotine; Tobacco;
Quantitative analysis of 17 amino acids in the connective tissue of patients with pelvic organ prolapse using capillary electrophoresis–tandem mass spectrometry by Naz Shama; Sang Wook Bai; Bong Chul Chung; Byung Hwa Jung (18-24).
The simultaneous determination of 17 amino acids in connective tissue using capillary electrophoresis is described in this study. Separation was carried out on a fused silica capillary column (80 cm × 50 mm i.d.) with 1 M formic acid as the running electrolyte. The detection was conducted on a mass spectrometer by selective reaction monitoring (SRM) mode via an electrospray ionization source. Tissue samples were prepared by reduction and acid hydrolysis to extract amino acids; over 84.3% recovery was seen for all compounds. The method allowed for sensitive, reproducible, and reliable quantification, and all 17 amino acids were separated using this method. Good linearity over the investigated concentration ranges was observed, with values of R higher than 0.993 for all the analytes. Precision and accuracy examined at three concentration levels ranged from 0.2% to 19.5% and 84.1% to 120.0%, respectively. Matrix effects were also tested and ranged from −9.1% to 15.4%. The validated method was applied to the quantitation of 17 amino acids in pelvic connective tissue of pelvic organ prolapsed patients. Methionine, glutamine, and histidine were significantly higher in the experimental patients compared to the controls. This suggests that changes in the amino acid concentrations within the connective tissue could be a factor in the genesis of pelvic organ prolapse. Therefore, this method is potentially applicable for amino acid analysis in tissue, providing a more complete understanding of pelvic organ prolapse.
Keywords: Amino acid; CE/MS; Connective tissue; Quantitative analysis;
Development of a two-step injector for GC–MS with on-column derivatization, and its application to the determination of amphetamine-type stimulants (ATS) in biological specimens by Akihiro Miki; Munehiro Katagi; Kei Zaitsu; Hiroshi Nishioka; Hitoshi Tsuchihashi (25-32).
A two-step auto-injector has been developed for the automated on-column derivatization and subsequent GC–MS of amine-type drugs and metabolites. To effectively derivatize such analytes, this injector has been designed to inject the derivatization reagent several seconds after the sample has been injected. Eleven kinds of amphetamine-type stimulants (ATS) and their typical metabolites were examined, using the trifluoroacetylation reagent N-methyl bis(trifluoroacetamide) (MBTFA). Although the quantitative derivatization of the hydroxyl groups was difficult, this technique was successfully applied to the determination of ATS in urine, blood, and hair specimens. The detection limits of methamphetamine and amphetamine in hair were 0.2 and 0.1 ng/mg hair, respectively, in the full-scan mode, when a 10 mg hair sample is analyzed.
Keywords: On-column derivatization; MBTFA; Trifluoroacetylation; ATS; Methamphetamine; MDMA; GC–MS; Sample injector; Automation;
Development of a reference material using methamphetamine abusers’ hair samples for the determination of methamphetamine and amphetamine in hair by Sooyeun Lee; Yonghoon Park; Wonkyung Yang; Eunyoung Han; Sanggil Choe; Sangwhan In; Miae Lim; Heesun Chung (33-39).
In the present study, we developed a reference material (RM) using authentic hair samples for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) in human hair. MA abusers’ hair samples were collected, homogenized and finally bottled. The concentration of each bottle was determined using two extraction methods, agitation with 1% HCl in methanol at 38 °C and ultrasonication with methanol/5 M HCl (20:1), followed by gas chromatography/mass spectrometry (GC–MS) after derivatization with trifluoroacetic anhydride (TFAA). Both analytical procedures were fully validated and their extraction efficiency was compared. The homogeneity of analytes was evaluated and their property values were determined with their uncertainties. The two methods were acceptable to analyze MA and AP in human hair through the validation and comparative studies using spiked and authentic hair samples as well as NIST SRM 2379 certified reference material. Satisfying homogeneity was reached for MA and AP in the prepared RM. Finally, a human hair RM containing MA and AP is prepared at the level of 7.64 ± 1.24 and 0.54 ± 0.07 ng/mg, respectively. This material can be useful in forensic laboratories for internal quality control and external quality assurance.
Keywords: Reference material; Methamphetamine; Amphetamine; Hair analysis;
Determination of citrulline in human plasma, red blood cells and urine by electron impact (EI) ionization gas chromatography–mass spectrometry by Carole Rougé; Clotilde Des Robert; Alexander Robins; Olivier Le Bacquer; Marie-France De La Cochetière; Dominique Darmaun (40-47).
A method was developed by using gas chromatography–mass spectrometry in the electron impact ionization mode to quantify citrulline in plasma, red blood cells (RBC) and urine. For all three fluids, citrulline was extracted on ion exchange resins, before derivatization to its propyl-heptaflorobutyryl-ester. Assay precision (coefficient of variation, CV) was <5%, recovery% was >90% and the within- and between-day CV were <10% on 200 μL of plasma and RBC, and 400 μL of urine. The current method allows for the detection of 20 pmol of natural citrulline in aqueous standards, and small volumes (<100 μL) of biological fluids.
Keywords: Nutrition; Protein; Amino acids; Stable isotopes;
Development and validation of a stereoselective liquid chromatography–tandem mass spectrometry assay for quantification of S- and R-metoprolol in human plasma by Berit P. Jensen; Caroline F. Sharp; Sharon J. Gardiner; Evan J. Begg (48-54).
A stereoselective liquid chromatography–tandem mass spectrometry assay was developed and validated for quantification of S- and R-metoprolol at concentrations of 0.5–50 μg/L in human plasma. Metoprolol was extracted from plasma by liquid–liquid extraction with ethyl acetate (82% recovery). Chromatographic separation of the enantiomers was achieved on a chiral Chirobiotic T column using an isocratic mobile phase consisting of methanol/acetic acid/ammonia (100/0.15/0.15, v/v/v). An ion trap mass spectrometer with an electrospray interface was used for detection in the positive mode, monitoring the m/z transition 268 → 191 for metoprolol. Standard curves for S- and R-metoprolol fitted quadratic functions (r 2 ≥ 0.9995) over the range 0.5–50 μg/L in plasma, with 0.5 μg/L representing the limit of quantification. In this range, relative standard deviations were <6% for intra-day precision and <10% for inter-day precision. The accuracy was within the range of 92–105%.
Keywords: Metoprolol; Enantiomers; LC–MS/MS; Plasma;
Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography–tandem mass spectrometry by Thomas P.C. Dorlo; Michel J.X. Hillebrand; Hilde Rosing; Teunis A. Eggelte; Peter J. de Vries; Jos H. Beijnen (55-62).
A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the quantification of miltefosine is presented. A 250 μL human EDTA plasma aliquot was spiked with miltefosine and extracted by a solid-phase extraction method. Separation was performed on a Gemini C18 column (150 mm × 2.0 mm I.D., 5 μm) using an alkaline eluent. Detection was performed by positive ion electrospray ionization followed by triple-quadrupole mass spectrometry. The assay has been validated for miltefosine from 4 to 2000 ng/mL using 250 μL human EDTA plasma samples. Results from the validation demonstrate that miltefosine can be accurately and precisely quantified in human plasma. At the lowest level, the intra-assay precision was lower than 10.7%, the inter-assay precision was 10.6% and accuracies were between 95.1 and 109%. This assay is successfully used in a clinical pharmacokinetic study with miltefosine.
Keywords: Miltefosine; Hexadecylphosphocholine; Leishmaniasis; Cutaneous leishmaniasis; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics;
Differential liquid phase proteomic analysis of the effect of selenium supplementation in LNCaP cells by Antonella Roveri; Maria Pia Vitale; Elena Serain; Mattia Zaccarin; Pierluigi Mauri; Dario Di Silvestre; Antonella De Palma; Massimo Gion; Stefano Toppo; Matilde Maiorino; Fulvio Ursini (63-73).
The effect of 100 nM sodium selenite supplementation was studied on LNCaP cells by a proteomic approach, on ProteomeLab™ PF 2D platform. Proteins were separated by liquid phase bi-dimensional chromatography and analyzed by pair-wise alignment of peaks to detect those differentially expressed. Differential expression threshold was set at a twice difference level and proteins matching this criterion were identified by MALDI-TOF and confirmed by ESI-ion trap MS/MS. Not all differentially expressed proteins found by PF 2D could be identified by MS analysis, the sensitivity of which emerging as the limiting factor. Thus, only the most abundant proteins, differently expressed following selenium supplementation, were identified. We positively showed an increase of expression of thioredoxin reductase 1, enolase 1, phosphoglycerate mutase 1, glyceraldehyde-3-phosphate dehydrogenase, heterogeneous nuclear ribonucleoprotein A2/B1, isoform A2, Ras-GTPase-activating protein SH3-domain-binding protein and Keratin 18 and a decrease of expression of peroxiredoxin 1 and heat shock protein 70, protein 8, isoform 1. Results are consistent, at least in part, with the less oxidant environment brought about by the synthesis of Se-dependent peroxidases, keeping low the steady-state concentration of hydrogen peroxide.
Keywords: Bi-dimensional chromatography; Selenium; Glycolytic enzymes; PF 2D;
Determination of voriconazole in human plasma and saliva using high-performance liquid chromatography with fluorescence detection by Claudia Michael; Jens Teichert; Rainer Preiss (74-80).
Voriconazole is a widely used triazole antifungal agent with a broad spectrum including Aspergillus species. A simple, sensitive and selective high-performance liquid chromatography method for the determination of voriconazole in human plasma and saliva was developed. Drug and internal standard (UK-115 794) were extracted from alkaline plasma and saliva with n-hexane–ethyl acetate (3:1, v/v) and analyzed on a Luna C 18 column with fluorimetric detection set at excitation and emission wavelengths of 254 and 372 nm, respectively. The calibration curve was linear through the range of 0.1–10 μg/ml using a 0.3 ml sample volume. The intra- and inter-day precisions were all below 6.1% for plasma and below 9.1% for saliva. Accuracies ranged from 94 to 109% for both matrices. Mean recovery was 86 ± 4% for voriconazole. The method showed acceptable values for precision, recovery and sensitivity and is well suited for routine analysis work and for pharmacokinetic studies.
Keywords: Voriconazole; HPLC; Fluorescence detector; Plasma; Saliva;
Quantification of seven nucleoside/nucleotide reverse transcriptase inhibitors in human plasma by high-performance liquid chromatography with tandem mass-spectrometry by Thomas Le Saux; Stéphanie Chhun; Elisabeth Rey; Odile Launay; Laurence Weiss; Jean-Paul Viard; Gérard Pons; Vincent Jullien (81-90).
A simple analytical method was developed in 100 μL of plasma for the simultaneous assay of the 7 nucleoside/nucleotide reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, and zidovudine) currently used for the treatment of HIV-infected patients. After adding the internal standard, 6-beta-hydroxy-theophyline, plasma samples were precipitated with 500 μL acetonitrile and the supernatants were evaporated to dryness. The residues were reconstituted with 500 μL of water and 10 μL of the extracts were injected in the chromatographic system. The chromatographic separation was performed with a C-18 column and a gradient mobile phase consisting of a mixture of water and acetonitrile, both containing 0.05% formic acid. Analytes quantification was performed by electrospray ionisation triple quadrupole mass-spectrometry in the positive mode using selected reaction monitoring (SRM). Intra- and inter-assay precision and accuracy were lower than 20% for the limit of quantification, and 15% for higher concentrations. The method has been implemented to assess plasma concentrations of patients infected by HIV and was found suitable for therapeutic drug monitoring.
Keywords: Tandem mass-spectrometry; Antiretroviral drugs; HIV; Liquid chromatography;
Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of nateglinide, cilostazol and its active metabolite 3,4-dehydro-cilostazol in Wistar rat plasma and its application to pharmacokinetic study by Kanthi Kiran V.S. Varanasi; V. Sridhar; Suresh Potharaju; R. Shraddha; S.P.N. Sivakumar; S. Kanaga Sabapathi; T.R.S. Satheeshmanikandan; V.V.S. Swaroop Kumar (91-98).
Nateglinide (NTG), an insulin secretogogue, has been studied in rats for drug–drug interaction with cilostazol (CLZ), an antiplatelet agent commonly used in diabetics. We developed a liquid chromatography tandem mass spectrometry (LC–MS/MS) based method that is capable of simultaneous monitoring plasma levels of nateglinide, cilostazol, and its active metabolite 3,4-dehydro-cilostazol (DCLZ). All analytes including the internal standard (Repaglinide) were chromatographed on reverse phase C18 column (50 mm × 4.6 mm i.d., 5 μm) using acetonitrile: 2 mM ammonium acetate buffer, pH 3.4 (90:10, v/v) as mobile phase at a flow rate 0.4 ml/min in an isocratic mode. The detection of analyte was performed on LC–MS/MS system in the multiple reaction monitoring (MRM) mode. The quantitations for analytes were based on relative concentration. The method was validated over the concentration range of 20–2000 ng/ml and the lower limit of quantitation was 20 ng/ml. The recoveries from spiked control samples were >79% for all analytes and internal standard. Intra- and inter-day accuracy and precision of validated method were with in the acceptable limits of <15% at all concentration. The quantitation method was successfully applied for simultaneous estimation of NTG, CLZ and DCLZ in a pharmacokinetic drug–drug interaction study in Wistar rats.
Keywords: LC–MS/MS; Bioanalytical; Cilostazol; Nateglinide; 3,4-Dehydro-cilostazol; Rat plasma;
Simultaneous determination of calycosin-7-O-β-d-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid in rat plasma after oral administration of Danggui Buxue Tang extract for their pharmacokinetic studies by liquid chromatography–mass spectrometry by Xiao-Dong Wen; Lian-Wen Qi; Ping Li; Kang-De Bao; Xiao-Wei Yan; Ling Yi; Chang-Yin Li (99-105).
A sensitive and reliable high-performance liquid chromatography–mass spectrometry (HPLC–MS) was developed and validated for simultaneous quantification of five main bioactive components, i.e., calycosin-7-O-β-d-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid in rat plasma after oral administration of Danggui Buxue Tang (DBT) extract. Plasma samples were extracted with solid-phase extraction (SPE) separated on an Inertsil ZORBAX C18 column and detected by MS with electrospray ionization (ESI) interface in negative selective ion monitoring (SIM) mode. Calibration curves offered linear ranges of two orders of magnitude with r 2 > 0.99. The method had the lower limit quantification of 0.55, 0.46, 1.07, 1.12 and 4.6 ng/mL for calycosin-7-O-β-d-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid, respectively, with precision less than 10%. The RSD of intra- and inter-day variations ranged from 2.10% to 6.19% and 2.37% to 6.72%. This developed method was subsequently applied to pharmacokinetic studies of the five compounds in rats successfully.
Keywords: HPLC–MS; Calycosin-7-O-β-d-glucoside; Ononin; Astragaloside IV; Astragaloside I; Ferulic acid; Danggui Buxue Tang extract; Pharmacokinetic;
Quantification methods of folpet degradation products in plasma with HPLC-UV/DAD: Application to an in vivo toxicokinetic study in rats by Mireille Canal-Raffin; Mathilde Receveur; Béatrice Martinez; Karine Titier; Celine Ohayon; Isabelle Baldi; Mathieu Molimard; Nicholas Moore; Patrick Brochard (106-113).
Solid-phase extractions followed by HPLC-UV/DAD methods were developed for occupational biological monitoring or forensic investigations of the fungicide folpet using its degradation products, phthalimide and phthalamic acid as plasma biomarkers. These methods show good linearity (r > 0.9955), precision (CV < 15%) and accuracy (bias < 14.8%). The lower limits of quantification for phthalimide and phthalamic acid were 10 and 20 ng/ml and the absolute recoveries were higher than 86% and 68%, respectively. Applying these methods, a plasma toxicokinetic study of folpet in rats after intratracheal administration of Folpan 80WG® showed that inhalation of folpet could be a route of exposure with an important systemic absorption.
Keywords: Fungicide; Folpet; Phthalimide; Phthalamic acid; HPLC; Rat; Intratracheal toxicokinetic study;
Simultaneous quantification of four active schisandra lignans from a traditional Chinese medicine Schisandra chinensis(Wuweizi) in rat plasma using liquid chromatography/mass spectrometry by Bao-Lian Wang; Jin-Ping Hu; Wei Tan; Li Sheng; Hui Chen; Yan Li (114-120).
A simple, rapid and sensitive method was developed for the simultaneous quantification of four active schisandra lignans (schisandrin, schisantherin A, deoxyshisandrin and γ-schisandrin) from a traditional Chinese medicine Schisandra chinensis(Wuweizi) in rat plasma using a high-performance liquid chromatography system coupled to a positive ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of three volumes of methanol followed by centrifugation. The analytes and internal standard (IS) bicyclol were separated on a Zorbax SB-C18 column (3.5 μm, 2.1 mm × 100 mm) with mobile phase of methanol/water (70:30, v/v) containing 0.1% formic acid at a flow rate of 0.2 mL/min with an operating temperature of 25 °C. Detection was performed on a Trap XCT mass spectrometer equipped with an electrospray ionization (ESI) source operated in selected ion monitoring (SIM) mode. Positive ion ESI was used to form sodium adduct molecular ions at m/z 455 for schisandrin, m/z 559 for schisantherin A, m/z 439 for deoxyshisandrin, m/z 423 for γ-schisandrin, and m/z 413 for the internal standard bicyclol. Linear detection responses were obtained for the four test compounds ranging from 0.010 to 2.0 μg/mL and the lower limits of quantitation (LLOQs) for four lignans were 0.010 μg/mL. The intra- and inter-day precisions (R.S.D.%) were within 12.5% for all analytes, while the deviation of assay accuracies was within ±13.0%. The average recoveries of analytes were greater than 80.0%. All analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of the four lignans after oral administration of Schisandra chinensis extraction to rats.
Keywords: Schisandra chinensis; Wuweizi; Schisandrin; Schisantherin A; Deoxyshisandrin; γ-Schisandrin; Schisandra lignans; HPLC–MS; Quantification; Pharmacokinetics;
Determination of toxaphene specific congeners in fish liver oil and feedingstuff using gas chromatography coupled to high resolution mass spectrometry by Bruno Veyrand; Anaïs Venisseau; Philippe Marchand; Jean-Philippe Antignac; Bruno Le Bizec (121-126).
A new method for the determination of nine toxaphene specific congeners in fish liver oil and feedingstuff has been developed. The samples were extracted using pressurized liquid extraction followed by a purification on silica and florisil columns. Identification and quantification were conducted using GC–(EI)-HRMS, and comparison with MS/MS detection was performed, using electron ionization and negative chemical ionization. Limits of detection were ranged from 0.01 to 0.22 μg kg−1 (12% moisture) as required for feed samples. The calibration curves showed a good linearity for all congeners (R 2 > 0.99). Repeatability was below 9% for all the congeners and recoveries were in-between 73 and 86%. This analytical method was applied to the quantification of thirteen real samples collected within national monitoring plans for further risk assessment.
Keywords: Toxaphene; GC–HRMS; GC–MS/MS; Fish oil;
Development and validation of a liquid chromatography–tandem mass spectrometric assay for pitavastatin and its lactone in human plasma and urine by Lei Tian; Yiling Huang; Youhong Jia; Lu Hua; Yishi Li (127-132).
A rapid, selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method with electrospray ionization (ESI) was developed and validated for the simultaneous determination of pitavastatin and its lactone in human plasma and urine. Following a liquid–liquid extraction, both the analytes and internal standard racemic i-prolact were separated on a BDS Hypersil C8 column, using methanol–0.2% acetic acid in water (70: 30, v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 422.4 → m/z 290.3 for pitavastatin, m/z 404.3 → m/z 290.3 for pitavastatin lactone and m/z 406.3 → m/z 318.3 for the internal standard, respectively. Linear calibration curves of pitavastatin and its lactone were obtained in the concentration range of 1–200 ng/ml, with a lower limit of quantitation of 1 ng/ml. The intra- and inter-day precision values were less than 4.2%, and accuracies were between −8.1 and 3.5% for both analytes. The proposed method was utilized to support clinical pharmacokinetic studies of pitavastatin in healthy subjects following oral administration.
Keywords: Pitavastatin; Pitavastatin lactone; Liquid chromatography; Tandem mass spectrometry;
Purification of five azaspiracids from mussel samples contaminated with DSP toxins and azaspiracids by Carmen Alfonso; Amparo Alfonso; Paz Otero; Paula Rodríguez; Mercedes R. Vieytes; Chris Elliot; Cowan Higgins; Luis M. Botana (133-140).
Human intoxications during toxic episodes in shellfish are a very important concern for public health, as well as for economic interests of producer regions. Although initially each toxin appeared in a determined geographical zone, nowadays many of them are found in multiple places worldwide. In addition, more toxic compounds (new toxins or new analogs of known toxins) are being isolated and identified, which bring about new risks for public health. An example of this situation is the group of azaspiracids (AZAs). Initially these toxins were concentrated in Irish coasts but today appear in many different geographic locations; in the first toxic episode only three analogs were isolated, but now it is known that the group is comprised of at least eleven identified compounds. A substantial problem associated with all these new toxins is the extreme difficulty associated with the study of their toxic effects and mechanisms of action due to the very small quantities of purified toxin available. Therefore, the study of procedures to isolate them from contaminated shellfish or to synthesize them is of tremendous importance. In this paper we design a complete procedure to obtain AZAs analogs from mussels contaminated with DSP toxins and azaspiracids by means of three consecutive steps: an extraction procedure to remove toxins from shellfish, a solid phase extraction (SPE) to clean the samples and separate DSP toxins and AZAs, and a preparative HPLC to isolate each analog. In all the steps LC/MS is used to detect and quantify the toxins. Large amounts of AZA1, AZA2, AZA3, AZA4 and AZA5 were obtained by use of this procedure, which can be utilized in future studies relating to the toxins such as the production of certified materials and standards.
Keywords: Azaspiracids; OA; DTX-2; DSP toxins; Purification; Preparative HPLC; LC/MS; SPE; AZA1; AZA2; AZA3; AZA4; AZA5;
An optimized method for determination of benzene in exhaled air by gas chromatography–mass spectrometry using solid phase microextraction as a sampling technique by Leiliane C.A. Amorim; Joana P. Carneiro; Zenilda L. Cardeal (141-146).
The determination of benzene in exhaled air has contributed for the increase in the use of breath analysis in biological monitoring. This paper describes SPME as a sampling technique for determining benzene in exhaled air by GC–MS. A system was developed to generate a gaseous benzene standard by a permeation method to accomplish the breath analyses. The method presented good resolution, repeatability (the mean of %RSD values for intra-day measurements was 6.3), sensitivity (2.4 and 3.1 ppb for LOD and LOQ, respectively), and linearity of response (R 2 = 0.994). After optimizing the conditions, analyses of real samples were performed on two groups (exposed and not exposed to benzene). The results presented an average of 8.2 ppb for the control group and 25.3 ppb for the exposed group.
Keywords: Benzene; Breath analysis; SPME; GC–MS;
Quantitative 2-D gel electrophoresis-based expression proteomics of albumin and IgG immunodepleted plasma by Maxim D. Seferovic; Violet Krughkov; Devanand Pinto; Victor K.M. Han; Madhulika B. Gupta (147-152).
Proteomic analysis of plasma is challenging because of its large dynamic range, which prevents the detection of low abundance proteins. Immunodepletion of high abundance proteins, such as albumin and IgG, has emerged as a favored technology to overcome this problem; however its suitability in quantitative expression proteomics has not yet been adequately addressed. In this study, albumin and IgG immunodepletion was evaluated by ELISAs and the reproducibility of depletion was tested with 2-DGE. Depletion of plasma resulted in removal of 62 ± 1.2% of the total protein, 93 ± 1.4% of the albumin (0.43 μg/μL, residual), and 94 ± 1.5% of the IgG (0.21 μg/μL, residual). These results were confirmed by immunoblotting. Computerized image analysis of 2-D gels using Progenesis SameSpots software revealed an enhancement in the number of visible spots (675–1325), with 10 ± 6% inter-gel variability in spot density. LC–ESI-MS/MS identification of newly resolved protein spots further validated the procedure. An innovative application of the software employed led to identification of 11 proteins lost non-specifically during depletion. This study demonstrates the effectiveness of immunodepletion of albumin and IgG in quantitative 2-DGE-based differential analysis of plasma proteins.
Keywords: Plasma proteome; Albumin; IgG; Immunodepletion; ELISA; 2-DGE software; Expression proteomics;
Determination of the cyclic depsipeptide FK228 in human and mouse plasma by liquid chromatography with mass-spectrometric detection by Xiaohong Chen; Erin R. Gardner; William D. Figg (153-158).
An analytical method was developed and validated for the quantitative determination of the cyclic depsipeptide FK228 (romidepsin, formerly FR901228; NSC 630176), a histone deacetylase inhibitor, in human and mouse plasma. Calibration curves were linear in the concentration range of 2–1000 ng/mL. Sample pretreatment involved a liquid–liquid extraction of 0.1 mL aliquots of plasma with ethyl acetate. FK228 and the internal standard, harmine, were separated on a Zorbax SB C18 column (75 mm × 2.1 mm, 3.5 μm), using a mobile phase composed of methanol and 0.2% formic acid. The column eluent was monitored by mass spectrometry with electrospray ionization. Accuracy and precision of four concentrations of quality control samples ranged from 101.5 to 106.4% and 0.7 to 3.5% in human plasma and 93.6 to 100.6% and 0.6 to 6.5%, in mouse plasma, respectively. This method represents a significant improvement over our previously published analytical assay for this agent, decreasing the sample volume requirements, increasing the accuracy and precision (through addition of a suitable internal standard), expanding the analytical range and validating in additional biological matrices. The developed method was applied to study the pharmacokinetics of FK228 in over 1000 clinical and preclinical samples.
Keywords: Depsipeptide; Human plasma; Mouse plasma; LC/MS;
Simultaneous determination of two Amadori compounds in Korean red ginseng (Panax ginseng) extracts and rat plasma by high-performance anion-exchange chromatography with pulsed amperometric detection by Kyung-Mi Joo; Chan-Woong Park; Hye-Jin Jeong; Sang Jun Lee; Ih Seop Chang (159-166).
A new simple, rapid and sensitive high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAEC–PAD) was developed and validated for the simultaneous determination of two Amadori compounds, arginyl-fructose and arginyl-fructosyl-glucose in Korean red ginseng (Panax ginseng) extracts, rat plasma. Separation of the two target analytes was efficiently undertaken on CarboPac PA1 anion-exchange column with isocratic elution (400 mM sodium hydroxide and deionized water (90:10, v/v)) at flow rate 0.7 mL/min within 15 min of single chromatographic run. Under optimized conditions, the detection limits (signal-to-noise ratio equal to 3) were 20 and 25 ng/mL for arginyl-fructose and arginyl-fructosyl-glucose, respectively. Calibration curves of peak area for the two analytes were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 94.15–102.62%. This method was successfully applied to the quantification of arginyl-fructose and arginyl-fructosyl-glucose in herbal extracts and in the plasma samples from rat.
Keywords: High-performance anion-exchange chromatography; Pulsed amperometric detection; Amadori compounds; Red ginseng; Rat plasma;