Journal of Chromatography B (v.820, #1)
Chiral liquid chromatography-tandem mass spectrometric methods for stereoisomeric pharmaceutical determinations by Jiwen Chen; Walter A. Korfmacher; Yunsheng Hsieh (1-8).
The characterization of the drug metabolism and pharmacokinetic (DMPK) profiles of stereoisomers is a fundamental aspect of the drug discovery and development processes. Therefore, chiral drug bioassays are very important to pharmaceutical and biomedical researchers. The recent developments in chiral liquid chromatography coupled to atmospheric pressure ionization tandem mass spectrometry (LC-API-MS/MS) for the analysis of pharmaceuticals are reviewed. Various ionization techniques including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric photoionization (APPI) interfaced with chiral liquid chromatographic methods are described in terms of their ionization efficiencies, matrix effects and limitations. Examples were selected to demonstrate the applicability of these methods for enantioselective bioanalysis.
Keywords: Chiral separation; LC-MS/MS; Pharmaceuticals; Atmospheric pressure ionization; Tandem mass spectrometry; Drug discovery;
Association mechanism between a series of rodenticide and humic acid: A frontal analysis to support the biological data by Claire André; Catherine Guyon; Mireille Thomassin; Alexandre Barbier; Lysiane Richert; Yves-Claude Guillaume (9-14).
The binding constants (K) of a series of anticoagulant rodenticides with the main soil organic component, humic acid (HA), were determined using frontal analysis approach. The order of the binding constants was identical as the one obtained in a previous paper [J. Chromatogr. B 813 (2004) 295], i.e. bromadiolone > brodifacoum > difenacoum > chlorophacinone > diphacinone, confirming the power of this frontal analysis approach for the determination of binding constants. Moreover, and for the first time, the concentration of unbound rodenticide to HAs could be determined. Thanks this approach, we could clearly demonstrate that HA acid protected the human hepatoma cell line HepG2 against the cytotoxicity of all the rodenticides tested and that the toxicity of rodenticides was directly linked to the free rodenticide fraction in the medium (i.e. unbound rodenticide to HA).
Keywords: Frontal analysis; Anticoagulant rodenticides; Humic acid; Binding constant; Cells viability;
Gas chromatography–mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid by Frédéric Destaillats; Jean-Louis Sébédio; Olivier Berdeaux; Pierre Juanéda; Paul Angers (15-22).
Structural determination of polyunsaturated fatty acids by gas chromatography–mass spectrometry (GC–MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC–MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z = M+ − 69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z = M+ − 136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC–FID and identification by GC–MS.
Keywords: Conjugated fatty acid metabolites; Gas chromatography–mass spectrometry; Long chain polyunsaturated fatty acid; Rat liver;
Chiral chromatographic separation of β-blockers by Y.K. Agrawal; R.N. Patel (23-31).
A novel amide based chiral stationary phase m-[(+)-α-methyl benzyl carboxamide] XAD-4 has been synthesized by covalently linking R(+)-1-phenylethylamine to chloroformoyl Amberlite XAD-4 under weak alkaline conditions. The synthesized resin has been primarily characterized by m.p., elemental analysis and FT-IR and 13C NMR spectra. β-Blockers viz. atenolol, metoprolol, and propranolol were successfully separated into their enantiomers using a mixture of sodium acetate–acetic acid buffer (pH 4.1):acetonitrile (4:6, v/v) solution using the synthesized resin. Hydrogen bonding and π–π interactions are supposed to be the major analyte–chiral stationary phase interactions.
Keywords: Chiral stationary phase; XAD-4; β-Blocker;
Simultaneous determination of fluoxetine, citalopram, paroxetine, venlafaxine in plasma by high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–MS/ESI) by He Juan; Zhou Zhiling; Li Huande (33-39).
Fluoxetine, citalopram, paroxetine and venlafaxine have been widely used in the treatment of depression. However, no study has been conducted to determine the four drugs simultaneously by high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–MS/ESI).To establish a new, rapid and sensitive HPLC–MS/ESI method for simultaneous determination and screening in human plasma of the four most commonly prescribed nontricyclic antidepressants: fluoxetine, citalopram, paroxetine and venlafaxine.The analytes in plasma were extracted by solid-phase-extraction column after samples had been alkalinized. The HPLC separation of the analytes was performed on a MACHEREY-NAGEL C18 (250 mm × 4.6 mm, 5 μm, Germany) column, using water (formic acid 0.6‰, ammonium acetate: 30 mmol/l)–acetonitrile (35:65, v/v) as mobile phase, with a flow-rate of 0.85 ml/min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode.The calibration curves were linear in the 5.0–1000.0 ng/ml range for all compounds, all of them with coefficients of determination above 0.9900. The average extraction recoveries for all the four analytes were above 73.2%. The methodology recoveries were higher than 95.0%. The limits of detection (LODs) were 0.5, 0.3, 0.3 and 0.1 ng/ml for fluoxetine, citalopram, paroxetine and venlafaxine, respectively. The intra- and inter-day variation coefficients were less than 15.0%.The method is accurate, sensitive and simple for routine therapeutic drug monitoring (TDM) as well as toxicologic screening, and for the study of the pharmacokinetics and metabolism of the four drugs.
Keywords: Fluoxetine; Citalopram; Paroxetine; Venlafaxine; HPLC–MS;
Simultaneous determination of six phenolic constituents of danshen in human serum using liquid chromatography/tandem mass spectrometry by Xiaochuan Li; Chen Yu; Yongbao Cai; Gangyi Liu; Jingying Jia; Yiping Wang (41-47).
The six phenolic constituents are water-soluble components extracted from the Chinese medical herb danshen, the dried roots of Salvia miltiorrhiza Bunge (Labiatae). An liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method has been developed for the simultaneous quantification of six phenolic constituents of danshen (magnesium lithospermate B (MLB), rosmarinic acid (RA) and lithospermic acid (LA), caffeic acid (CAA), protocatechuic aldehyde (3,4-dihydroxybenzaldehyde, Pal), 3,4-dihydroxyphenyllactic acid (danshensu)) in human serum with chloramphenicol as internal standard. The serum samples were treated by special liquid–liquid extraction, and the analytes were determined using electrospray negative ionization mass spectrometry in the multiple reaction monitoring (MRM) mode, with sufficient sensitivity to allow analysis of human serum samples generated following administration of a clinically relevant dose. Good linearity over the range 8–2048 ng/mL for six phenolic constituents was observed. The intra- and inter-day precisions (CV) of analysis were <13%, and the accuracy ranged from 88 to 116%. This quantitation method was successfully applied to a pharmacokinetic study of i.v. drip infusion of Danshen injection fluid in human.
Keywords: Magnesium lithospermate B; Rosmarinic acid; Lithospermic acid; Caffeic acid; Protocatechuic aldehyde; Danshensu; Danshen; Pharmacokinetics;
Stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography–mass spectrometry for measurement of phenolic xenoestrogens in human urine samples by Migaku Kawaguchi; Norihiro Sakui; Noriya Okanouchi; Rie Ito; Koichi Saito; Shun-ichiro Izumi; Tsunehisa Makino; Hiroyuki Nakazawa (49-57).
A high-sensitivity analytical method that uses stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography–mass spectrometry (GC–MS) for the simultaneous measurement of trace amounts of phenolic xenoestrogens (PXs), such as 2,4-dichlorophenol (DCP), 4-tert-butylphenol (BP), 4-tert-octylphenol (OP), 4-nonylphenol technical isomers (NP), pentachlorophenol (PCP) and bisphenol A (BPA), in human urine samples was developed. The urine sample (1 ml) was de-conjugated by adding β-glucuronidase and sulfatase. Then, protein precipitation was performed by the addition of acetonitrile. After centrifugation, the supernatant was diluted with purified water and subjected to SBSE with in situ derivatization and TD-GC–MS. The detection limits of DCP, BP, OP, NP, PCP and BPA in the urine samples were 20, 10, 10, 50, 20 and 20 pg ml−1 (ppt), respectively. The calibration curves for PXs were linear and had correlation coefficients higher than 0.99. The average recoveries of those analytes in the urine samples were higher than 95% (RSD: <10%, n = 6) with correction using the added surrogate standards. This simple, accurate, sensitive and selective method can be used in the determination of PXs in human urine samples.
Keywords: Phenolic xenoestrogens; Stir bar sorptive extraction (SBSE); Thermal desorption (TD); In situ derivatization;
Evaluation of online extraction/mass spectrometry for in vivo cassette analysis by Nalini Sadagopan; Brandon Pabst; Lucinda Cohen (59-67).
An online extraction/mass spectrometry technique was evaluated for direct analysis of plasma samples. A simple user-friendly online extraction system that consists of two pumps, an autosampler, a six-port switching valve and a mass spectrometer is described. The system was controlled by the LC–MS software (Masslynx 3.5, Waters Corporation, Beverly, MA). Various analytical conditions such as extraction column, mobile phases, run time and wash solvent were optimized to establish an analytical method that was simple, easy to set up and generic. Sample preparation effort was minimal, which included dilution of plasma with water and centrifugation conducted in 96-well plate format. The system was used to analyze in vivo plasma samples from rat n-in-one cassette dosing studies. Concentration and pharmacokinetic (PK) data obtained from the online extraction method were comparable with data obtained from the protein precipitation extraction method. Overall, the simple, robust online extraction system provides cost savings by minimizing sample preparation and method development time. The system was used to analyze compounds from different structural classes. These studies suggest that calculated lipophilicity of a compound can be used as a tool for pre-selection of extraction column, which would save method development time for early discovery studies.
Keywords: Sample preparation; Bioanalysis; Drug discovery; TFC; Online extraction; Pharmacokinetics;
Determination of an investigational HIV integrase inhibitor in human plasma using high performance liquid chromatography with tandem mass spectrometric detection by P.T. Vallano; E.J. Woolf; B.K. Matuszewski (69-76).
An HPLC–MS/MS assay for the determination of an HIV integrase inhibitor, 5-(1,1-dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (I) in human plasma has been developed and validated. Compound I and a stable isotope labeled internal standard (II) were isolated from 0.5 mL plasma samples by solid phase extraction using an Ansys SPEC C-8 96-well plate. Extracts were separated on a Hypersil BDS C-18 HPLC column (3.0 mm × 50 mm, 3 μm) with a mobile phase consisting of 25 mM ammonium formate pH 3.0:acetonitrile (60:40) vol%/vol% pumped at 0.5 mL/min. A Sciex API 365 mass spectrometer equipped with an atmospheric pressure chemical ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 431 → 109 (I) and m/z 437 → 115 (II) used for quantitation. The assay was validated over the concentration range of 10–5000 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was 69%. The intra-day accuracy of the assay was within 4% of nominal and intra-day precision was better than 4% C.V. Following a 200 mg dose of the compound administered to human subjects, concentrations of I ranged from 21.1 to 1500 ng/mL in plasma samples collected up to 12 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 3-month period alongside clinical samples showed mean accuracies of within 6% of nominal and precision better than 3.5% C.V.
Keywords: HIV integrase inhibitor; Bioanalytical; HPLC–MS/MS;
Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography–mass spectrometry by Catarina Batista; Myftar Berisha; Matthias Karst; Kahlid Salim; Udo Schneider; Rudolf Brenneisen (77-82).
A method using gas chromatography–mass spectrometry (GC–MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before (“free AJA”) and after enzymatic hydrolysis (“total AJA”). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4 ± 37.2 ng/ml (mean ± R.S.D., n = 9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2 h was 63.8 ± 127.9 ng/ml.
Keywords: Ajulemic acid (AJA); CT-3; GC–MS; Human plasma levels;
Liquid chromatography–electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells by Olivier Nicolas; Delphine Margout; Nicolas Taudon; Michele Calas; Henri J. Vial; Françoise Bressolle (83-93).
Liquid chromatography–electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using Oasis®HLB columns with an elution solvent of 2 × 1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra® column packed with 3.5 μm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 μl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M + TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4–1282 μg/l (12.8–2564 μg/kg) for T3, 20–2000 μg/l (40–4000 μg/kg) for mTE3 and 10–2000 μg/l (40–4000 μg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 μg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 μg/l for T3, 20 μg/l for mTE3 and 10 μg/l for TE3 in plasma; 12.8 μg/kg for T3 and 40 μg/kg for mTE3 and TE3 in blood; and 12.8 μg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.
Keywords: Bis-thiazolium compound; Neutral bioprecursor; Antimalarial activity; Human plasma; Whole blood; Red blood cells; Liquid chromatography–electrospray mass spectrometry; Validation;
Determination of dichloromethane, trichloroethylene and perchloroethylene in urine samples by headspace solid phase microextraction gas chromatography–mass spectrometry by Diana Poli; Paola Manini; Roberta Andreoli; Innocente Franchini; Antonio Mutti (95-102).
A method for the determination of volatile chlorinated hydrocarbons, namely dichloromethane (DCM), trichloroethylene (TCE), and perchloroethylene (PCE), in urine samples was developed using headspace solid phase microextraction (HS-SPME) gas chromatography–mass spectrometry (GC–MS). HS-SPME was performed using a 75 μm Carboxen-polydimethylsiloxane fiber. Factors, which affect the HS-SPME process, such as adsorption and desorption times, stirring, salting-out effect, and temperature of sampling have been evaluated and optimized. The highest extraction efficiency was obtained when sampling was performed at room temperature (22 °C), from samples saturated with salt and under agitation. Linearity of the HS-SPME-GC–MS method was established over four orders of magnitude and the limit of detection was 0.005 μg/l for all the compounds. Precision, calculated as %R.S.D. at three different concentration levels, was within 1–8% for all intra- and inter-day determinations. The method was applied to the quantitative determination of TCE and PCE in human urine samples from exposed (TCE, n = 5; median, 9.32 μg/l and PCE, n = 39; median, 0.58 μg/l) and non-exposed individuals (n = 120; median concentrations, 0.64, 0.22 and 0.11 μg/l for DCM, TCE and PCE, respectively. In addition, two cases of acute accidental exposure to DCM are reported, and the elimination kinetics in blood and urine was followed up. The calculated half-lives of urinary and blood DCM were, respectively, 7.5 and 8.1 h for one subject and 3.8 and 4.3 h for the other.
Keywords: Gas chromatography; Solid phase microextraction; Dichloromethane; Trichloroethylene; Perchloroethylene; Urine analysis; Biomonitoring;
Determination of talinolol in human plasma using automated on-line solid phase extraction combined with atmospheric pressure chemical ionization tandem mass spectrometry by Emmanuel Bourgogne; Chantal Grivet; Gérard Hopfgartner (103-110).
A specific LC–MS/MS assay was developed for the automated determination of talinolol in human plasma, using on-line solid phase extraction system (prospekt 2) combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. The method involved simple precipitation of plasma proteins with perchloric acid (contained propranolol) as the internal standard (IS) and injection of the supernatant onto a C8 End Capped (10 mm × 2 mm) cartridge without any evaporation step. Using the back-flush mode, the analytes were transferred onto an analytical column (XTerra C18, 50 mm × 4.6 mm) for chromatographic separation and mass spectrometry detection. One of the particularities of the assay is that the SPE cartridge is used as a column switching device and not as an SPE cartridge. Therefore, the same SPE cartridge could be used more than 28 times, significantly reducing the analysis cost. APCI ionization was selected to overcome any potential matrix suppression effects because the analyte and IS co-eluted. The mean precision and accuracy in the concentration range 2.5–200 ng/mL was found to be 103% and 7.4%, respectively. The data was assessed from QC samples during the validation phase of the assay. The lower limit of quantification was 2.5 ng/mL, using a 250 μL plasma aliquot. The LC–MS/MS method provided the requisite selectivity, sensitivity, robustness accuracy and precision to assess pharmacokinetics of the compound in several hundred human plasma samples.
Keywords: Talinolol; Quantitative analysis; Column-switching; LC–MS/MS;
Affinity recovery of Moloney Murine Leukaemia Virus by Sharon L. Williams; Darren Nesbeth; David C. Darling; Farzin Farzaneh; Nigel K.H. Slater (111-119).
Lipid enveloped retroviruses such as Moloney Murine Leukaemia Virus (MoMuLV) are commonly used gene therapy vectors. Downstream processing protocols used for their purification are time consuming and a potentially generic, single step capture method for the recovery of retroviral particles is proposed that exploits streptavidin–biotin affinity chromatography. The ability of four conventional adsorbent solid phases, Fractogel®, Sepharose, Magnespheres® and STREAMLINE immobilised with streptavidin, to capture and recover biotinylated Moloney Murine Leukaemia Virus was studied. MoMuLV can be biotinylated whilst retaining infectivity and the biotinylated virus can be adsorbed to Streptavidin Magnespheres yielding a 2298-fold increase in titre. For optimal virus biotinylation purification using Fractogel® streptavidin can yield a 1896-fold increase in cfu/mg of protein and a 1191-fold decrease in DNA/cfu. Infectious virus can be recovered from Fractogel® streptavidin with a maximum recovery of 16.7%.
Keywords: Moloney Murine Leukaemia Virus (MoMuLV); Affinity chromatography; Gene therapy;
Rapid quantitation of plasma 2′-deoxyuridine by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry and its application to pharmacodynamic studies in cancer patients by Kong M. Li; Laurent P. Rivory; Stephen J. Clarke (121-130).
A novel method employing high-performance liquid chromatograph–mass spectrometry (LC–MS) has been developed and validated for the quantitation of plasma 2′-deoxyuridine (UdR). It involves a plasma clean-up step with strong anion-exchange solid-phase extraction (SAX-SPE) followed by HPLC separation and atmospheric pressure chemical ionization mass spectrometry detection (APCI–MS) in a selected-ion monitoring (SIM) mode. The ionization conditions were optimised in negative ion mode to give the best intensity of the dominant formate adduct [M + HCOO]− at m/z 273. Retention times were 7.5 and 12.5 min for 2′-deoxyuridine and 5-iodo-2′-deoxyuridine, an iodinated analogue internal standard (IS), respectively. Peak area ratios of 2′-deoxyuridine to IS were used for regression analysis of the calibration curve. The latter was linear from 5 to 400 nmol/l using 1 ml sample volume of plasma. The average recovery was 81.5% and 78.6% for 2′-deoxyuridine and 5-iodo-deoxyuridine, respectively. The method provides sufficient sensitivity, precision, accuracy and selectivity for routine analysis of human plasma 2′-deoxyuridine concentration with the lowest limit of quantitation (LLOQ) of 5 nmol/l. Clinical studies in cancer patients treated with the new fluoropyrimidine analogue capecitabine (N 4-pentoxycarbonyl-5′-5-fluorocytidine) have shown that plasma 2′-deoxyuridine was significantly elevated after 1 week of treatment, consistent with inhibition of thymidylate synthase (TS). These findings suggest that the mechanism of antiproliferative toxicity of capecitabine is at least partly due to TS inhibitory activity of its active metabolite 5-fluoro-2′-deoxyuridine monophosphate (FdUMP). Monitoring of plasma UdR concentrations have the potential to help clinicians to guide scheduling of capecitabine or other TS inhibitors in clinical trials. Marked differences of plasma 2′-deoxyuridine between human and rodents have also been confirmed. In conclusion, the LC–MS method developed is simple, highly selective and sensitive and permits pharmacodynamic studies of TS inhibitors in several species.
Keywords: 2′-Deoxyuridine; LC–MS; APCI; Solid-phase extraction;
Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All by Nicola Volpi; Francesca Maccari; Jens Titze (131-135).
A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 μg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Δ-disaccharides from hyaluronic acid (ΔdiHA) and dermatan sulfate/chondroitin sulfate (Δdi4s and Δdi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24 ± 0.26 μg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20 ± 0.33 μg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32 ± 2.38 and 49.66 ± 2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.
Keywords: Electrophoresis; Glycosaminoglycans; Hyaluronic acid; Dermatan; Chondroitin; Skin;
Evaluation of three methods for effective extraction of DNA from human hair by Emi Suenaga; Hiroshi Nakamura (137-141).
In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp® DNA Mini Kit method and the ISOHAIR® method. Analysis of DNA prepared from dyed hairs with the ISOHAIR® method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp® DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.
Keywords: DNA extraction; Hair analysis; Polymerase chain reaction inhibitor;
A gel filtration assay to determine glycogen synthase activity by Andreas Niederwanger; Michael Kranebitter; Andreas Ritsch; Josef R. Patsch; Michael T. Pedrini (143-145).
We developed a gel filtration assay for the determination of glycogen synthase activity in cultured cells or tissue homogenates. Compared to the commonly used filter paper assay, the gel filtration assay resulted in a more than 5-fold reduction of background levels leading to an – at least – twofold increase in precision. These benefits allow the gel filtration method to detect differences of ±5% in enzyme activity out of 300 μg total cell protein. In addition to high precision and sensitivity, the method's additional salient advantages include lesser expenditure of time and labour and reduced exposure time of the personnel to radioactivity.
Keywords: Glycogen synthase; Glycogen synthase activity; Insulin resistance; Type-2 diabetes; Gel filtration; Glycogen; UDP-glucose; Glucose-6-phosphate;
Determination of riboflavin by high-performance liquid chromatography with riboflavin-depleted urine as calibration and control matrix by Meng Chen; David M. Andrenyak; David E. Moody; Rodger L. Foltz (147-150).
A simple method, exposure to natural-light, was developed to remove riboflavin from urine to enhance its use as the biological matrix for the preparation of calibration and control samples. Riboflavin-depleted urine containing less than 1 ng/ml of riboflavin was used to validate a high-performance liquid chromatography with fluorescence detection method for the determination of urinary riboflavin. The linearity of the assay (r 2 = 0.999) was acceptable over the range of 10–5000 ng/ml. The intra-assay and inter-assay CVs were 3.3% and 9%, respectively. Subsequent stability studies found that urine riboflavin was stable for up to 6 months at 4 or −20 °C.
Keywords: Riboflavin; Urine; HPLC; Stability;
Application of a validated high-performance liquid chromatography–mass spectrometry assay to the analysis of m- and p-hydroxybenzoylecgonine in meconium by S. Pichini; E. Marchei; R. Pacifici; M. Pellegrini; Jaime Lozano; O. García-Algar (151-156).
A high-performance liquid chromatography (HPLC)–mass spectrometry (MS) assay, already validated for opiates and cocaine in meconium, has been re-applied for determination of m- and p-hydroxybenzoylecgonine, using nalorphine as the internal standard. Methodology included an initial extraction from the matrix by methanol and then a solid-phase extraction (SPE). A reversed-phase chromatography was used with a gradient of 1% acetic acid–acetonitrile coupled to atmospheric pressure ionization electrospray–mass spectrometry single ion monitoring mode. This method, validated in the range 0.005–1.00 μg analytes/g meconium, proved useful to identify and quantify these two metabolites in meconium samples, already tested for the presence of cocaine, benzoylecgonine and cocaethylene. A positivity of range of concentrations varied between 0.007 and 0.338 μg/g, confirming the importance of these two hydroxylated derivatives to monitor fetal exposure to cocaine.
Keywords: m-Hydroxybenzoylecgonine; p-Hydroxybenzoylecgonine; Meconium; LC–MS;