A new issue of this journal has just
been published. To see abstracts of the papers it contains (with links through
to the full papers) click here:
Selected
papers from the latest issue:
Synthese and characterization of boronic acid functionalized macroporous uniform poly(4-chloromethylstyrene-co-divinylbenzene) particles and its use in the isolation of antioxidant compounds from plant extracts
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Onur Çetinkaya, Mehmet Emin Duru, Hüseyin Çiçek
Aminophenyl boronic acid (APBA) carrying uniform-macroporous poly(chloromethylstyrene-co-divinylbenzene), poly(CMS-co-DVB) particles were synthesized for selective separation of cis-diol-containing flavonoids from plant extracts. For this purpose, 2.5μm polystyrene seed particles were first swelled by a mixture of dibutyl phthalate (DBP), toluene and dodecanol, then by a monomer mixture including CMS and DVB. The repolymerization of the monomer phase in the swollen seed particles provided macroporous and uniform particles, approximately 7μm in size. Chlorine atoms on the surface of these particles were derivatized with APBA to gain affinity properties for flavonoids containing vicinal hydroxyl groups. Model adsorption studies showed that these particles selectively adsorbed quercetin and rutin containing cis-diol groups, but did not adsorb apigenin similar to quercetin and not carrying cis-diol groups. These particles were also tested in adsorption/desorption studies for ethanol and ethyl acetate extracts of the Hypericum perforatum (HP) stems to obtain high antioxidant mixtures. With ethanol extract, the antioxidant activity of the desorption solution was a bit higher than that of the post-adsorption solutions. However, the DPPH radical scavenging activity of the desorption solution decreased with respect to the original extract and post-adsorption solutions. A similar result was obtained for the antioxidant activity of the desorption solution using ethyl acetate extract. An interesting result was obtained that DPPH radical scavenging activity of the post-adsorption solution was higher than that of the original ethyl acetate extract and desorption solutions. These results were attributed to selective adsorption of antioxidant characterized cis-diol-containing apolar molecules much more rather than that radical scavenger characterized polar molecules.
Source:Journal of Chromatography B, Volume 909
Onur Çetinkaya, Mehmet Emin Duru, Hüseyin Çiçek
Aminophenyl boronic acid (APBA) carrying uniform-macroporous poly(chloromethylstyrene-co-divinylbenzene), poly(CMS-co-DVB) particles were synthesized for selective separation of cis-diol-containing flavonoids from plant extracts. For this purpose, 2.5μm polystyrene seed particles were first swelled by a mixture of dibutyl phthalate (DBP), toluene and dodecanol, then by a monomer mixture including CMS and DVB. The repolymerization of the monomer phase in the swollen seed particles provided macroporous and uniform particles, approximately 7μm in size. Chlorine atoms on the surface of these particles were derivatized with APBA to gain affinity properties for flavonoids containing vicinal hydroxyl groups. Model adsorption studies showed that these particles selectively adsorbed quercetin and rutin containing cis-diol groups, but did not adsorb apigenin similar to quercetin and not carrying cis-diol groups. These particles were also tested in adsorption/desorption studies for ethanol and ethyl acetate extracts of the Hypericum perforatum (HP) stems to obtain high antioxidant mixtures. With ethanol extract, the antioxidant activity of the desorption solution was a bit higher than that of the post-adsorption solutions. However, the DPPH radical scavenging activity of the desorption solution decreased with respect to the original extract and post-adsorption solutions. A similar result was obtained for the antioxidant activity of the desorption solution using ethyl acetate extract. An interesting result was obtained that DPPH radical scavenging activity of the post-adsorption solution was higher than that of the original ethyl acetate extract and desorption solutions. These results were attributed to selective adsorption of antioxidant characterized cis-diol-containing apolar molecules much more rather than that radical scavenger characterized polar molecules.
Highlights
► Aminophenyl boronic acid functionalized macroporous particles was synthesized. ► Selectivity of particles to cis-diol-containing flavonoids was presented. ► The adsorption behavior of particles was tested with Hypericum perforatum extracts. ► Increment of antioxidant activity in desorption solution was observed. ► The highest radical scavenging capacity was obtained with post-adsorption solution.Study of the specific interaction between l-methionine chromatography support and nucleotides
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
C. Cruz, A. Sousa, F. Sousa, J.A. Queiroz
The interaction of l-methionine-agarose with 5′-mononucleotide was investigated by saturation transfer difference (STD)-nuclear magnetic resonance (NMR) spectroscopy. Chromatographic experiments were also performed using homo-oligonucleotides of distinct molecular masses (1–30 nucleotides) to explore the effect of base hydrophobicity, temperature, pH and salt concentration on the retention of homo-oligonucleotides to l-methionine-agarose support. With STD-NMR, the results reveal that hydrophobic residues, such as the CH3 of thymine and adenine, can preferentially recognise the l-methionine side chain of the support. Also, 5′-TMP led to more contacts with the support, while 5′-UMP presented fewer STD contacts. For 5′-UMP, 5′-CMP and 5′-GMP, the main interaction with the support was through the sugar-phosphate backbone. Similar binding profiles were obtained using chromatographic experiments. Indeed, 5′-TMP had the highest retention time, followed by 5′-GMP, 3′-AMP, 5′-UMP and 5′-CMP. In general, the retention factor of homo-oligonucleotides was higher for ammonium sulphate concentration 1.5M. For the polyT3–polyT30 series, the retention time increased by about three-fold, indicating that larger homo-oligonucleotides have more hydrophobic bases, thus enhancing contact with the l-methionine support. The temperature (5, 20 and 35°C) did not influence homo-oligonucleotide retention. However, the retention time slightly increased when the pH was lower than 9. The STD-NMR technique combined with chromatographic experiments was thus successfully used to screen amino acid–nucleotide interactions.
Source:Journal of Chromatography B, Volume 909
C. Cruz, A. Sousa, F. Sousa, J.A. Queiroz
The interaction of l-methionine-agarose with 5′-mononucleotide was investigated by saturation transfer difference (STD)-nuclear magnetic resonance (NMR) spectroscopy. Chromatographic experiments were also performed using homo-oligonucleotides of distinct molecular masses (1–30 nucleotides) to explore the effect of base hydrophobicity, temperature, pH and salt concentration on the retention of homo-oligonucleotides to l-methionine-agarose support. With STD-NMR, the results reveal that hydrophobic residues, such as the CH3 of thymine and adenine, can preferentially recognise the l-methionine side chain of the support. Also, 5′-TMP led to more contacts with the support, while 5′-UMP presented fewer STD contacts. For 5′-UMP, 5′-CMP and 5′-GMP, the main interaction with the support was through the sugar-phosphate backbone. Similar binding profiles were obtained using chromatographic experiments. Indeed, 5′-TMP had the highest retention time, followed by 5′-GMP, 3′-AMP, 5′-UMP and 5′-CMP. In general, the retention factor of homo-oligonucleotides was higher for ammonium sulphate concentration 1.5M. For the polyT3–polyT30 series, the retention time increased by about three-fold, indicating that larger homo-oligonucleotides have more hydrophobic bases, thus enhancing contact with the l-methionine support. The temperature (5, 20 and 35°C) did not influence homo-oligonucleotide retention. However, the retention time slightly increased when the pH was lower than 9. The STD-NMR technique combined with chromatographic experiments was thus successfully used to screen amino acid–nucleotide interactions.
Highlights
► Interaction of l-methionine-agarose with nucleotides. ► The techniques used are STD-NMR and chromatographic experiments. ► 5′-TMP has the highest retention time on support while 5′-CMP has the lowest. ► The polynucleotide retention time increases with the salt concentration. ► 5′-TMP promoted more STD contacts with the support mainly through the base.Evaluation of a direct high-capacity target screening approach for urine drug testing using liquid chromatography–time-of-flight mass spectrometry
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Aljona Saleh, Niclas Nikolai Stephanson, Ingrid Granelli, Tomas Villén, Olof Beck
In this study a rapid liquid chromatography–time-of-flight mass spectrometry method was developed, validated and applied in order to evaluate the potential of this technique for routine urine drug testing. Approximately 800 authentic patient samples were analyzed for amphetamines (amphetamine and methamphetamine), opiates (morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine and codeine-6-glucuronide) and buprenorphines (buprenorphine and buprenorphine-glucuronide) using immunochemical screening assays and mass spectrometry confirmation methods for comparison. The chromatographic application utilized a rapid gradient with high flow and a reversed phase column with 1.8μm particles. Total analysis time was 4min. The mass spectrometer operated with an electrospray interface in positive mode with a resolution power of >10,000 at m/z 956. The applied reporting limits were 100ng/mL for amphetamines and opiates, and 5ng/mL for buprenorphines, with lower limits of quantification were 2.8–41ng/mL. Calibration curves showed a linear response with coefficients of correlation of 0.97–0.99. The intra- and interday imprecision in quantification at the reporting limits were <10% for all analytes but for buprenorphines <20%. Method validation data met performance criteria for a qualitative and quantitative method. The liquid chromatography–time-of-flight mass spectrometry method was found to be more selective than the immunochemical method by producing lower rates of false positives (0% for amphetamines and opiates; 3.2% for buprenorphines) and negatives (1.8% for amphetamines; 0.6% for opiates; 0% for buprenorphines). The overall agreement between the two screening methods was between 94.2 and 97.4%. Comparison of data with the confirmation (LC–MS) results for all individual 9 analytes showed that most deviating results were produced in samples with low levels of analytes. False negatives were mainly related to failure of detected peak to meet mass accuracy criteria (±20mDa). False positives was related to presence of interfering peaks meeting mass accuracy and retention time criteria and occurred mainly at low levels. It is concluded that liquid chromatography–time-of-flight mass spectrometry has potential both as a complement and as replacement of immunochemical screening assays.
Source:Journal of Chromatography B, Volume 909
Aljona Saleh, Niclas Nikolai Stephanson, Ingrid Granelli, Tomas Villén, Olof Beck
In this study a rapid liquid chromatography–time-of-flight mass spectrometry method was developed, validated and applied in order to evaluate the potential of this technique for routine urine drug testing. Approximately 800 authentic patient samples were analyzed for amphetamines (amphetamine and methamphetamine), opiates (morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine and codeine-6-glucuronide) and buprenorphines (buprenorphine and buprenorphine-glucuronide) using immunochemical screening assays and mass spectrometry confirmation methods for comparison. The chromatographic application utilized a rapid gradient with high flow and a reversed phase column with 1.8μm particles. Total analysis time was 4min. The mass spectrometer operated with an electrospray interface in positive mode with a resolution power of >10,000 at m/z 956. The applied reporting limits were 100ng/mL for amphetamines and opiates, and 5ng/mL for buprenorphines, with lower limits of quantification were 2.8–41ng/mL. Calibration curves showed a linear response with coefficients of correlation of 0.97–0.99. The intra- and interday imprecision in quantification at the reporting limits were <10% for all analytes but for buprenorphines <20%. Method validation data met performance criteria for a qualitative and quantitative method. The liquid chromatography–time-of-flight mass spectrometry method was found to be more selective than the immunochemical method by producing lower rates of false positives (0% for amphetamines and opiates; 3.2% for buprenorphines) and negatives (1.8% for amphetamines; 0.6% for opiates; 0% for buprenorphines). The overall agreement between the two screening methods was between 94.2 and 97.4%. Comparison of data with the confirmation (LC–MS) results for all individual 9 analytes showed that most deviating results were produced in samples with low levels of analytes. False negatives were mainly related to failure of detected peak to meet mass accuracy criteria (±20mDa). False positives was related to presence of interfering peaks meeting mass accuracy and retention time criteria and occurred mainly at low levels. It is concluded that liquid chromatography–time-of-flight mass spectrometry has potential both as a complement and as replacement of immunochemical screening assays.
Highlights
► A multi target analytical approach for clinical drugs of abuse screening was explored. ► The analysis time was short. ► The selectivity was based on high efficiency chromatography and high resolution MS. ► A method comparison was made with LC–tandem MS and immunochemistry. ► A large number (800) authentic urine specimens were used for evaluation.Determination of cyclic guanosine- and cyclic adenosine monophosphate (cGMP and cAMP) in human plasma and animal tissues by solid phase extraction on silica and liquid chromatography–triple quadrupole mass spectrometry
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Thomas Van Damme, Yanhua Zhang, Frédéric Lynen, Pat Sandra
3′,5′-Cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are essential second messenger molecules. They are involved in signal transduction within cells, in physiological functions such as neurotransmission and in the modulation of cell growth and differentiation of organisms, respectively. A quantitative solid phase extraction method (SPE) based on hydrophilic interaction on silica was developed and applied to both plasma and tissue samples. The stable isotope-labeled internal standards 2D1,15N3-3′,5′-cGMP and 13C10,15N5-3′,5′-cAMP were added prior to the sample preparation to ensure high precision and accuracy. The samples were analyzed by reversed-phase liquid chromatography (RP-LC). Negative electrospray (ESI)-MS/MS was used to selectively monitor several transitions of each metabolite. The method for the analysis of 3′,5′-cAMP and 3′,5′-cGMP in plasma was validated in the range of 0.15–20ng/mL (R 2 =0.9996 and 0.9994 for 3′,5′-cAMP and 3′,5′-cGMP, respectively). Basal plasma concentrations for fifteen healthy human patients determined with this method varied between 4.66–9.20ng/mL for 3′,5′-cAMP and between 0.30–1.20ng/mL for 3′,5′-cGMP, with precisions better than 9.1%. 3′,5′-cGMP and 3′,5′-cAMP together with their 2′,3′-isomers were also determined in a semi quantitative way in animal tissues. The structures of the isomers were confirmed by analysis with LC-high resolution time-of-flight MS and subsequently by comparison of retention times with standards.
Source:Journal of Chromatography B, Volume 909
Thomas Van Damme, Yanhua Zhang, Frédéric Lynen, Pat Sandra
3′,5′-Cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are essential second messenger molecules. They are involved in signal transduction within cells, in physiological functions such as neurotransmission and in the modulation of cell growth and differentiation of organisms, respectively. A quantitative solid phase extraction method (SPE) based on hydrophilic interaction on silica was developed and applied to both plasma and tissue samples. The stable isotope-labeled internal standards 2D1,15N3-3′,5′-cGMP and 13C10,15N5-3′,5′-cAMP were added prior to the sample preparation to ensure high precision and accuracy. The samples were analyzed by reversed-phase liquid chromatography (RP-LC). Negative electrospray (ESI)-MS/MS was used to selectively monitor several transitions of each metabolite. The method for the analysis of 3′,5′-cAMP and 3′,5′-cGMP in plasma was validated in the range of 0.15–20ng/mL (R 2 =0.9996 and 0.9994 for 3′,5′-cAMP and 3′,5′-cGMP, respectively). Basal plasma concentrations for fifteen healthy human patients determined with this method varied between 4.66–9.20ng/mL for 3′,5′-cAMP and between 0.30–1.20ng/mL for 3′,5′-cGMP, with precisions better than 9.1%. 3′,5′-cGMP and 3′,5′-cAMP together with their 2′,3′-isomers were also determined in a semi quantitative way in animal tissues. The structures of the isomers were confirmed by analysis with LC-high resolution time-of-flight MS and subsequently by comparison of retention times with standards.
Highlights
► HILIC SPE was developed for cGMP and cAMP analysis in biological samples. ► Low ng/mL quantification limits were obtained by LC–MS/MS. ► Plasma volumes as low as 200μL were thereby used. ► 2′,3′ and 3′,5′ isomers could simultaneously be monitored. ► The four isomers were monitored in tissue samples.Analysis of new potential anticonvulsant compounds in mice brain tissue by SPE/HPLC/DAD
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Jolanta Flieger, Magdalena Pizoń, Tomasz Plech, Jarogniew J. Łuszczki
This paper describes a novel reversed-phase high performance liquid chromatography (RP-HPLC) with photo-diode array detection (DAD) method for the determination of three new derivatives of 4-alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with different antiepileptic activity in the brains of mice treated with the doses of 300mgkg−1 of body weight. Samples were prepared by solid-phase extraction (SPE) method using BAKERBOND™ spe Octadecyl (C18) and analyzed by the use of an isocratic elution mode over an Zorbax Extend-C18 column (150mm×4.6mm I.D., 5-μm, Agilent Technologies). The mobile phase consisted of 80% methanol (for compound TP-315) and 85% acetonitrile (for compound TP-321) for 80% 2-propanol (for TP-323) at a flow rate of 1.0mLmin−1 and 0.5mLmin−1 in the last case. Gradient elution mode was also proposed for all examined analytes in mixture with common antiepileptic drugs: carbamazepine, phenobarbital and phenytoin in view of possible synergistic activity. Photodiode-array investigations of the peaks after degradation studies indicate the stability of the compounds under conditions proposed for sample preparation procedure. Linear coefficients of correlation (r 2) were >0.995 for all analytes. The proposed strategy gives extraction yields higher than 95% with the intra- and inter-day relative standard deviation lower than 3% and 5%, respectively. This method was applied to the analysis of brain tissue of mice treated with investigated compounds. Obtained results enable to explain the differences in their pharmacological activity.
Source:Journal of Chromatography B, Volume 909
Jolanta Flieger, Magdalena Pizoń, Tomasz Plech, Jarogniew J. Łuszczki
This paper describes a novel reversed-phase high performance liquid chromatography (RP-HPLC) with photo-diode array detection (DAD) method for the determination of three new derivatives of 4-alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with different antiepileptic activity in the brains of mice treated with the doses of 300mgkg−1 of body weight. Samples were prepared by solid-phase extraction (SPE) method using BAKERBOND™ spe Octadecyl (C18) and analyzed by the use of an isocratic elution mode over an Zorbax Extend-C18 column (150mm×4.6mm I.D., 5-μm, Agilent Technologies). The mobile phase consisted of 80% methanol (for compound TP-315) and 85% acetonitrile (for compound TP-321) for 80% 2-propanol (for TP-323) at a flow rate of 1.0mLmin−1 and 0.5mLmin−1 in the last case. Gradient elution mode was also proposed for all examined analytes in mixture with common antiepileptic drugs: carbamazepine, phenobarbital and phenytoin in view of possible synergistic activity. Photodiode-array investigations of the peaks after degradation studies indicate the stability of the compounds under conditions proposed for sample preparation procedure. Linear coefficients of correlation (r 2) were >0.995 for all analytes. The proposed strategy gives extraction yields higher than 95% with the intra- and inter-day relative standard deviation lower than 3% and 5%, respectively. This method was applied to the analysis of brain tissue of mice treated with investigated compounds. Obtained results enable to explain the differences in their pharmacological activity.
Highlights
► Separation method in sample together with standard antiepileptic drugs taking into account observed their synergic action. ► Quantitative determination of new ant-epileptic compounds in mouse brain tissue by HPLC–DAD after sample clean-up step by the use of SPE procedure. ► Explanation of the different anti-epileptic activity.Investigation of bioaccumulation profile of oestrogens in zebrafish liver by hollow fibre protected liquid phase microextraction with gas chromatography–mass spectrometric detection
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Sivarajan Kanimozhi, Chanbasha Basheer, Shanmugam Neveliappan, Kelvin Ang, Feng Xue, Hian Kee Lee
The applicability of hollow fibre protected liquid phase microextraction (HF-LPME) for the determination of three oestrogens, namely estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) from individual zebrafish liver samples, in a bioaccumulation study on these organisms, is reported. The oestrogens were extracted from single, mechanically crushed and minced livers from fish that were heaved in tubes containing water spiked at low concentration of the analytes. Extraction was performed with ∼3μL of toluene contained in the hollow fibre. In order to achieve high extraction efficiency, the parameters that could affect the effectiveness of HF-LPME were optimized, i.e. the extracting organic solvent, extraction time, stirring speed and pH of the aqueous phase. For gas chromatography/mass spectrometry (GC/MS) analysis, injection port derivatization of the oestrogens with bis(trimethylsilyl)trifluoroacetamide was conducted. Under the most favourable extraction and derivatization conditions, enrichment factors of 158–279 were obtained. Linearity of the HF-LPME–GC/MS method was evaluated from 1 to 50μg/L and the coefficient of determination (r 2 ) ranged from 0.9687 to 0.9926. The LODs were between 0.017 and 0.033μg/L (at a signal to noise ratio of 3) with relative standard deviations (RSDs, analytes spiked at 5μg/L) of between 15 and 17% (n =3).
Source:Journal of Chromatography B, Volume 909
Sivarajan Kanimozhi, Chanbasha Basheer, Shanmugam Neveliappan, Kelvin Ang, Feng Xue, Hian Kee Lee
The applicability of hollow fibre protected liquid phase microextraction (HF-LPME) for the determination of three oestrogens, namely estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) from individual zebrafish liver samples, in a bioaccumulation study on these organisms, is reported. The oestrogens were extracted from single, mechanically crushed and minced livers from fish that were heaved in tubes containing water spiked at low concentration of the analytes. Extraction was performed with ∼3μL of toluene contained in the hollow fibre. In order to achieve high extraction efficiency, the parameters that could affect the effectiveness of HF-LPME were optimized, i.e. the extracting organic solvent, extraction time, stirring speed and pH of the aqueous phase. For gas chromatography/mass spectrometry (GC/MS) analysis, injection port derivatization of the oestrogens with bis(trimethylsilyl)trifluoroacetamide was conducted. Under the most favourable extraction and derivatization conditions, enrichment factors of 158–279 were obtained. Linearity of the HF-LPME–GC/MS method was evaluated from 1 to 50μg/L and the coefficient of determination (r 2 ) ranged from 0.9687 to 0.9926. The LODs were between 0.017 and 0.033μg/L (at a signal to noise ratio of 3) with relative standard deviations (RSDs, analytes spiked at 5μg/L) of between 15 and 17% (n =3).
Highlights
► HF-LPME was used to quantitate the oestrogens from individual zebrafish liver. ► Injection port derivatization of the oestrogens was performed. ► Bioaccumulation of zebrafish liver was investigated using tank experiments.High-throughput bioanalytical method for analysis of synthetic cannabinoid metabolites in urine using salting-out sample preparation and LC–MS/MS
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Enrique G. Yanes, Dennis P. Lovett
Herbal smoking mixtures which are sold as incense or potpourri and often referred to as ‘Spice’ are actually inactive plant matter adulterated with alkylamino indole based synthetic cannabinoids such as JWH-018 and JWH-073. Due to the inclusion of five synthetic cannabinoids, including JWH-018 and JWH-073, as Schedule I drugs by the Drug Enforcement Agency (DEA) in March 2011, it has become necessary for forensic laboratories to develop analytical methods to test for the presence of metabolites of synthetic cannabinoids. When a new analyte of interest emerges, most laboratories strive to develop a sample preparation procedure and validate an analytical method as quickly as possible and therefore, rely on effective but time consuming traditional protocols such as solid phase and liquid–liquid extraction. This research focuses on the examination of all aspects of sample preparation and analytical method development to streamline the analysis of four urinary metabolites of JWH-018 and JWH-073. A detailed evaluation of the β-glucuronide hydrolysis step lead to the reduction of time required for hydrolysis from 1h at 50°C to only 10min at room temperature. By utilizing a salting-out assisted liquid–liquid extraction (SALLE) in place of traditional liquid–liquid extraction with a volatile solvent, processing time was saved and waste was reduced. The analysis run time was also shortened to one-third of a typical published run time by utilizing UPLC with isocratic conditions in place of conventional HPLC running a gradient method.
Source:Journal of Chromatography B, Volume 909
Enrique G. Yanes, Dennis P. Lovett
Herbal smoking mixtures which are sold as incense or potpourri and often referred to as ‘Spice’ are actually inactive plant matter adulterated with alkylamino indole based synthetic cannabinoids such as JWH-018 and JWH-073. Due to the inclusion of five synthetic cannabinoids, including JWH-018 and JWH-073, as Schedule I drugs by the Drug Enforcement Agency (DEA) in March 2011, it has become necessary for forensic laboratories to develop analytical methods to test for the presence of metabolites of synthetic cannabinoids. When a new analyte of interest emerges, most laboratories strive to develop a sample preparation procedure and validate an analytical method as quickly as possible and therefore, rely on effective but time consuming traditional protocols such as solid phase and liquid–liquid extraction. This research focuses on the examination of all aspects of sample preparation and analytical method development to streamline the analysis of four urinary metabolites of JWH-018 and JWH-073. A detailed evaluation of the β-glucuronide hydrolysis step lead to the reduction of time required for hydrolysis from 1h at 50°C to only 10min at room temperature. By utilizing a salting-out assisted liquid–liquid extraction (SALLE) in place of traditional liquid–liquid extraction with a volatile solvent, processing time was saved and waste was reduced. The analysis run time was also shortened to one-third of a typical published run time by utilizing UPLC with isocratic conditions in place of conventional HPLC running a gradient method.
Highlights
► Analysis of four urinary metabolites of JWH-018 and JWH-073 was streamlined. ► β-Glucuronide hydrolysis step was reduced to only 10min at room temperature. ► A salting-out assisted liquid–liquid extraction reduced processing time and waste. ► The analysis run time was shortened to 3.2min by utilizing UPLC.Molecularly imprinted solid phase extraction of urinary diethyl thiophosphate and diethyl dithiophosphate and their analysis by gas chromatography–mass spectrometry
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Mariane Gonçalves Santos, Ricardo Vilela Vitor, Felipe Lopes Andrade, Isarita Martins, Eduardo Costa Figueiredo
An analytical method involving molecularly imprinted solid phase extraction (MISPE) and gas chromatography–mass spectrometry (GC–MS) was developed for the analysis of organophosphates metabolites (diethyl thiophosphate – DETP and diethyl dithiophosphate – DEDTP) in human urine samples. A DETP molecularly imprinted polymer (MIP) was synthesized using 4-vinylpiridine as the functional monomer and ethylene glycol dimethacrylate as the cross-linker. The conditioning step of the MISPE was conducted by running 3mL of acetonitrile, 3mL of 0.1molL−1 dibasic phosphate buffer at pH 11 and 2mL of water through the molecularly imprinted polymer (MIP) cartridge. The extraction step was executed using 1.0mL of a urine sample, with the pH previously adjusted to 3.0. Finally, the analytes were eluted with 3mL of acetonitrile and derivatized with 3% 2,3,4,5,6-pentafluorobenzyl bromide solution at room temperature for 1h. The sample was analyzed by GC–MS in the SIM (selected ion monitoring) mode. Analytical calibration curves for DETP and DEDTP were constructed using a pool of urine samples and six levels of concentration. The method was found to be linear from 10 to 500μgL−1 (r >0.99) with limits of quantification of 10μgL−1 for both analytes. The within-day and between-day precisions were evaluated (as %RSD) and all the results were <15% for both analytes. The method was accurate (relative error<±15%), with good robustness.
Source:Journal of Chromatography B, Volume 909
Mariane Gonçalves Santos, Ricardo Vilela Vitor, Felipe Lopes Andrade, Isarita Martins, Eduardo Costa Figueiredo
An analytical method involving molecularly imprinted solid phase extraction (MISPE) and gas chromatography–mass spectrometry (GC–MS) was developed for the analysis of organophosphates metabolites (diethyl thiophosphate – DETP and diethyl dithiophosphate – DEDTP) in human urine samples. A DETP molecularly imprinted polymer (MIP) was synthesized using 4-vinylpiridine as the functional monomer and ethylene glycol dimethacrylate as the cross-linker. The conditioning step of the MISPE was conducted by running 3mL of acetonitrile, 3mL of 0.1molL−1 dibasic phosphate buffer at pH 11 and 2mL of water through the molecularly imprinted polymer (MIP) cartridge. The extraction step was executed using 1.0mL of a urine sample, with the pH previously adjusted to 3.0. Finally, the analytes were eluted with 3mL of acetonitrile and derivatized with 3% 2,3,4,5,6-pentafluorobenzyl bromide solution at room temperature for 1h. The sample was analyzed by GC–MS in the SIM (selected ion monitoring) mode. Analytical calibration curves for DETP and DEDTP were constructed using a pool of urine samples and six levels of concentration. The method was found to be linear from 10 to 500μgL−1 (r >0.99) with limits of quantification of 10μgL−1 for both analytes. The within-day and between-day precisions were evaluated (as %RSD) and all the results were <15% for both analytes. The method was accurate (relative error<±15%), with good robustness.
Highlights
► A MIP was developed for selective extraction of dialkyl phosphates in urine samples. ► The eluate was efficiently analyzed by gas chromatography–mass spectrometry. ► Good figures of merit were attained. ► The method is an interesting alternative to analyze dialkyl phosphates in urine.On-line capillary electrophoresis for enhanced detection sensitivity of feline panleukopenia virus
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Ahram Shin, Mijin Lee, Sangduk Kim, Seong Ho Kang
A rapid on-line capillary electrophoresis (CE) method for highly sensitive detection of DNA molecules with specific lengths was developed based on the combination of base stacking (BS) and programmed field strength gradients (PFSG). The BS method has been performed for on-column concentration to improve detection sensitivity without any modification of the CE system. PFSG increased the electrophoretic velocity of DNA molecules, which effectively decreased analysis time. Using the BS and PFSG combination, the amplified PCR product (340-bp DNA) of cats infected with feline panleukopenia virus was detected within 6.5min. Detection sensitivity (∼10-fold) was enhanced compared to conventional CE analysis. The combined on-line CE/BS-PFSG methodology could be an effectively rapid analysis technique for the highly sensitive detection of disease-related specific DNA molecules.
Source:Journal of Chromatography B, Volume 909
Ahram Shin, Mijin Lee, Sangduk Kim, Seong Ho Kang
A rapid on-line capillary electrophoresis (CE) method for highly sensitive detection of DNA molecules with specific lengths was developed based on the combination of base stacking (BS) and programmed field strength gradients (PFSG). The BS method has been performed for on-column concentration to improve detection sensitivity without any modification of the CE system. PFSG increased the electrophoretic velocity of DNA molecules, which effectively decreased analysis time. Using the BS and PFSG combination, the amplified PCR product (340-bp DNA) of cats infected with feline panleukopenia virus was detected within 6.5min. Detection sensitivity (∼10-fold) was enhanced compared to conventional CE analysis. The combined on-line CE/BS-PFSG methodology could be an effectively rapid analysis technique for the highly sensitive detection of disease-related specific DNA molecules.
Highlights
► On-line combination of BS and PFSG methods with CE. ► Enhance sensitivity and decrease analysis time without any modification. ► 10-fold higher detection sensitivity and ∼2-fold faster analysis of FP virus. ► Rapid and highly sensitive analysis technique for disease-related specific DNA.Comparison of methanol and acetonitrile eluents for the quantitation of chelators specific to soft-metal ions by HPLC
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Shinya Ogawa, Etsuro Yoshimura
HPLC eluent systems employing acetonitrile and methanol were evaluated for the quantitation of glutathione (GSH) and phytochelatin (PC n ), a family of peptides implicated in heavy-metal detoxification in higher plants. The detection system is based on the dequenching of copper(I)–bathocuproine disulfonate and is specific for soft-metal chelators. Although both elution systems yielded comparable analytical performance for each PC n , the acetonitrile system had a lower sensitivity for GSH and a steadily increasing baseline. The inferior properties of the acetonitrile system may be due to complex formation between acetonitrile and Cu(I) ions. Both methods were applied to measure peptide levels in the primitive red alga Cyanidioschyzon merolae. Coefficients of variation (CVs) were less than 5%, except for GSH and PC4 determinations in the acetonitrile system, in cases when CV values were found to be 8.8% and 6.3%, respectively. Recoveries were greater than 96%, except for GSH determination in the acetonitrile system, with a recovery of 84.4%; however, the concentration measured in the acetonitrile system did not differ from that measured in the methanol system at a significance level of 0.05.
Source:Journal of Chromatography B, Volume 909
Shinya Ogawa, Etsuro Yoshimura
HPLC eluent systems employing acetonitrile and methanol were evaluated for the quantitation of glutathione (GSH) and phytochelatin (PC n ), a family of peptides implicated in heavy-metal detoxification in higher plants. The detection system is based on the dequenching of copper(I)–bathocuproine disulfonate and is specific for soft-metal chelators. Although both elution systems yielded comparable analytical performance for each PC n , the acetonitrile system had a lower sensitivity for GSH and a steadily increasing baseline. The inferior properties of the acetonitrile system may be due to complex formation between acetonitrile and Cu(I) ions. Both methods were applied to measure peptide levels in the primitive red alga Cyanidioschyzon merolae. Coefficients of variation (CVs) were less than 5%, except for GSH and PC4 determinations in the acetonitrile system, in cases when CV values were found to be 8.8% and 6.3%, respectively. Recoveries were greater than 96%, except for GSH determination in the acetonitrile system, with a recovery of 84.4%; however, the concentration measured in the acetonitrile system did not differ from that measured in the methanol system at a significance level of 0.05.
Highlights
► HPLC detection system was established to quantify chelators specific to soft-metal ions. ► The system employed dequenching of Cu(I)–BCS complexes as a detection system. ► GSH and PC2–PC4 were successfully determined. ► Methanol was superior over acetonitrile as an eluent.Improving the imprinting effect by optimizing template:monomer:cross-linker ratios in a molecularly imprinted polymer for sulfadimethoxine
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Lou Ann Tom, Nicole A. Schneck, Carla Walter
Four non-covalently prepared molecularly imprinted polymers (MIPs) for sulfadimethoxine (SDM) were prepared using different ratios of SDM template, methacrylic acid monomer, and ethylene glycol dimethacrylate cross-linker. The imprinting factor (IF) was calculated by comparing the retention of SDM on the imprinted polymer with a comparable non-imprinted polymer. The template:monomer:cross-linker ratio of 1:6:20 resulted in an IF of 3.94 which is higher than found in previous studies. A significant decrease in IF to 0.89 when template:cross-linker ratio was 1:40 contradicts most literature where higher cross-linker concentration improves selectivity. IF was 4.36 when 20% water was added to the acetonitrile HPLC mobile phase during evaluation. Retention of SDM increased as water concentration changed as: 20, 40, 0, 60 and 70%, indicating a combination of shape recognition, hydrogen bonding and hydrophobic interactions contributing to retention of analyte. The MIP has the potential for use in SPE for purification and concentration of SDM and with further optimization, possibly direct HPLC analysis.
Source:Journal of Chromatography B, Volume 909
Lou Ann Tom, Nicole A. Schneck, Carla Walter
Four non-covalently prepared molecularly imprinted polymers (MIPs) for sulfadimethoxine (SDM) were prepared using different ratios of SDM template, methacrylic acid monomer, and ethylene glycol dimethacrylate cross-linker. The imprinting factor (IF) was calculated by comparing the retention of SDM on the imprinted polymer with a comparable non-imprinted polymer. The template:monomer:cross-linker ratio of 1:6:20 resulted in an IF of 3.94 which is higher than found in previous studies. A significant decrease in IF to 0.89 when template:cross-linker ratio was 1:40 contradicts most literature where higher cross-linker concentration improves selectivity. IF was 4.36 when 20% water was added to the acetonitrile HPLC mobile phase during evaluation. Retention of SDM increased as water concentration changed as: 20, 40, 0, 60 and 70%, indicating a combination of shape recognition, hydrogen bonding and hydrophobic interactions contributing to retention of analyte. The MIP has the potential for use in SPE for purification and concentration of SDM and with further optimization, possibly direct HPLC analysis.
Highlights
► MIPs for sulfadimethoxine were prepared non-covalently. ► Imprinting factors for four template:monomer:cross-linker ratios were evaluated. ► Monomer ratio should at least equal the hydrogen bonding sites on the template. ► Excess monomer or cross-linker can decrease the imprinting factor. ► Retention of SDM on MIP by HPLC increases with higher water in mobile phase.Determination of tamsulosin in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B, Volume 909
Chang-Ik Choi, Hye-In Lee, Jung-Woo Bae, Yun-Jeong Lee, Ji-Yeong Byeon, Choon-Gon Jang, Seok-Yong Lee
Tamsulosin, a selective α1-adrenoceptor antagonist, is used for the treatment of benign prostatic hyperplasia (BPH). We developed and validated a rapid, sensitive, and simplified liquid chromatography analytical method utilizing tandem mass spectrometry (LC–MS/MS) for the determination of tamsulosin in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tamsulosin was achieved using a reversed-phase Luna C18 column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5)–methanol (25:75, v/v) and quantified by MS/MS detection in ESI positive ion mode. The flow rate of the mobile phase was 200μL/min and the retention times of tamsulosin and the internal standard (IS, diphenhydramine) were 0.8 and 0.9min, respectively. The calibration curves were linear over a range of 0.01–20ng/mL (r >0.999). The lower limit of quantification using 500μL of human plasma was 0.01ng/mL. The mean accuracy and precision for intra- and inter-day validation of tamsulosin were both within acceptable limits. The present LC–MS/MS method showed improved sensitivity for quantification of tamsulosin in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.
Source:Journal of Chromatography B, Volume 909
Chang-Ik Choi, Hye-In Lee, Jung-Woo Bae, Yun-Jeong Lee, Ji-Yeong Byeon, Choon-Gon Jang, Seok-Yong Lee
Tamsulosin, a selective α1-adrenoceptor antagonist, is used for the treatment of benign prostatic hyperplasia (BPH). We developed and validated a rapid, sensitive, and simplified liquid chromatography analytical method utilizing tandem mass spectrometry (LC–MS/MS) for the determination of tamsulosin in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tamsulosin was achieved using a reversed-phase Luna C18 column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5)–methanol (25:75, v/v) and quantified by MS/MS detection in ESI positive ion mode. The flow rate of the mobile phase was 200μL/min and the retention times of tamsulosin and the internal standard (IS, diphenhydramine) were 0.8 and 0.9min, respectively. The calibration curves were linear over a range of 0.01–20ng/mL (r >0.999). The lower limit of quantification using 500μL of human plasma was 0.01ng/mL. The mean accuracy and precision for intra- and inter-day validation of tamsulosin were both within acceptable limits. The present LC–MS/MS method showed improved sensitivity for quantification of tamsulosin in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.
Highlights
► The more rapid, sensitive and simplified LC–MS/MS method for human plasma tamsulosin. ► A liquid–liquid sample extraction procedure without a sample-alkalinizing step. ► The lower limit of quantification using 500μL of human plasma was 0.01ng/mL.A highly sensitive capillary electrophoresis method using p-nitrophenyl 5′-thymidine monophosphate as a substrate for the monitoring of nucleotide pyrophosphatase/phosphodiesterase activities
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Sang-Yong Lee, Sébastien A. Lévesque, Jean Sévigny, Christa E. Müller
A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20cm effective length x 75.5μm (id)), electrokinetic injection for 60 s, 70mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of -80μA, constant capillary temperature of 15°C and detection at 400nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (Km values for NPP1 and NPP3, Ki values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.
Source:Journal of Chromatography B
Sang-Yong Lee, Sébastien A. Lévesque, Jean Sévigny, Christa E. Müller
A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20cm effective length x 75.5μm (id)), electrokinetic injection for 60 s, 70mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of -80μA, constant capillary temperature of 15°C and detection at 400nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (Km values for NPP1 and NPP3, Ki values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.
Highlights
► A sensitive nucleotide pyrophosphatase (NPP) assay was developed. ► p-Nitrophenyl 5′-thymidine monophosphate was used as an artificial substrate. ► A CE-VIS method coupled with dynamically polybrene-coated capillaries was used. ► A combination of phosphate-HEPES buffer notably increased the method sensitivity. ► The obtained LOD and LOQ for p-nitrophenolate are in the nanomolar range.Comprehensive Approach to the Quantitative Analysis of Mitochondrial Phospholipids by HPLC-MS
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Junhwan Kim, Charles L. Hoppel
A normal-phase HPLC-MS method was established to analyze mitochondrial phospholipids quantitatively as well as qualitatively. An efficient extraction procedure and chromatographic conditions were developed using twelve standardized phospholipids and lysophospholipids. The chromatographic conditions provided physical separation of phospholipids by class, and efficient ionization allowed detection of low abundance phospholipids such as phosphatidylglycerol and monolysocardiolipin. The chromatographic separation of each class of phospholipid permitted qualitative identification of molecular species without interference from other classes. This is advantageous for mitochondrial lipidomics because the composition of mitochondrial phospholipids varies depending on tissue source, pathological condition, and nutrition. Using the method, seven classes of phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, and monolysocardiolipin) were detected in rat heart and skeletal muscle mitochondria and all but phosphatidylserine were quantified. The concentration was calculated using standard curves with an internal standard generated for each class of phospholipid. The method was validated for intraday and interday variation and showed excellent reproducibility and accuracy. This new method, with each step documented, provides a powerful tool for accurate quantitation of phospholipids, a basic structural component of mitochondrial membranes.
Source:Journal of Chromatography B
Junhwan Kim, Charles L. Hoppel
A normal-phase HPLC-MS method was established to analyze mitochondrial phospholipids quantitatively as well as qualitatively. An efficient extraction procedure and chromatographic conditions were developed using twelve standardized phospholipids and lysophospholipids. The chromatographic conditions provided physical separation of phospholipids by class, and efficient ionization allowed detection of low abundance phospholipids such as phosphatidylglycerol and monolysocardiolipin. The chromatographic separation of each class of phospholipid permitted qualitative identification of molecular species without interference from other classes. This is advantageous for mitochondrial lipidomics because the composition of mitochondrial phospholipids varies depending on tissue source, pathological condition, and nutrition. Using the method, seven classes of phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, and monolysocardiolipin) were detected in rat heart and skeletal muscle mitochondria and all but phosphatidylserine were quantified. The concentration was calculated using standard curves with an internal standard generated for each class of phospholipid. The method was validated for intraday and interday variation and showed excellent reproducibility and accuracy. This new method, with each step documented, provides a powerful tool for accurate quantitation of phospholipids, a basic structural component of mitochondrial membranes.
Highlights
► We developed a comprehensive analytical method based on HPLC-MS to analyze mitochondrial phospholipids. ► We optimized extraction and simplification procedures. ► We developed normal phase HPLC conditions for a rigorous quantitation with an internal standard and standard curves. ► We detected and quantitated low abundance phospholipids such as monolysocardiolipin and phosphatidylglycerol.LC-MS/MS method for determination of geldanamycin derivative GM-AMPL in rat plasma to support preclinical development
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Yan-ping Li, Lin-yan Gao, Kai-tong Li, Shuai Meng, Jian-hua Zhu, Dong Li, Jie Jin, Guang-zhi Shan, Zhuo-rong Li
A LC-MS/MS method for determining the concentration of the small molecule Hsp90 inhibitor, GM-AMPL, has been developed and validated in rat plasma to support preclinical development. 17-[2-(Morpholin-4-yl)ethyl]amino-17-demethoxygeldanamycin (GM-AMPL) and the internal standard 17-allylamino-17-demethoxygeldanamycin (17-AAG) were sufficiently separated on a Venusil MP C18 column that was eluted with 80% methanol in water at 40°C. Quantification studies were performed with a multiple reaction monitoring (MRM) transition of m/z 657.3→614.3 and 584.3→541.3 for GM-AMPL and IS, respectively, in the negative ion mode. The present method exhibited good linearity (R>0.999) over the concentration range of 2-600ng/mL for GM-AMPL in rat plasma with a lower limit of quantification (LLOQ) of 2ng/mL. The intra-batch and inter-batch assay coefficients of variation (CV) was in range of 1.56%-6.84% and 1.62%-6.98%, respectively. The plasma samples were extracted with methanol to precipitate protein with extraction recovery in range of 84.09%-95.25%. The matrix effect was determined as internal substance (IS) normalized matrix factor of 1.09, 1.18 and 1.05 for samples with three concentration levels of 4, 40 and 400ng/mL, respectively. This validated method was further applied to successfully determine the pharmacokinetic parameters and oral availability of GM-AMPL in Sprague–Dawley rats following intravenous injection and oral administration.
Source:Journal of Chromatography B
Yan-ping Li, Lin-yan Gao, Kai-tong Li, Shuai Meng, Jian-hua Zhu, Dong Li, Jie Jin, Guang-zhi Shan, Zhuo-rong Li
A LC-MS/MS method for determining the concentration of the small molecule Hsp90 inhibitor, GM-AMPL, has been developed and validated in rat plasma to support preclinical development. 17-[2-(Morpholin-4-yl)ethyl]amino-17-demethoxygeldanamycin (GM-AMPL) and the internal standard 17-allylamino-17-demethoxygeldanamycin (17-AAG) were sufficiently separated on a Venusil MP C18 column that was eluted with 80% methanol in water at 40°C. Quantification studies were performed with a multiple reaction monitoring (MRM) transition of m/z 657.3→614.3 and 584.3→541.3 for GM-AMPL and IS, respectively, in the negative ion mode. The present method exhibited good linearity (R>0.999) over the concentration range of 2-600ng/mL for GM-AMPL in rat plasma with a lower limit of quantification (LLOQ) of 2ng/mL. The intra-batch and inter-batch assay coefficients of variation (CV) was in range of 1.56%-6.84% and 1.62%-6.98%, respectively. The plasma samples were extracted with methanol to precipitate protein with extraction recovery in range of 84.09%-95.25%. The matrix effect was determined as internal substance (IS) normalized matrix factor of 1.09, 1.18 and 1.05 for samples with three concentration levels of 4, 40 and 400ng/mL, respectively. This validated method was further applied to successfully determine the pharmacokinetic parameters and oral availability of GM-AMPL in Sprague–Dawley rats following intravenous injection and oral administration.
Highlights
► First report on oral bioavailability of novel Hsp90 inhibitor GM-AMPL. ► The sensitive and reliable LC-MS/MS method for determination of GM-AMPL in rat plasma. ► Simple and stable extraction procedure of analytes from plasma samples. ► Validated method feasible to future pharmacokinetic study.Determination of very low stable isotope enrichments of [2H5]-phenylalanine in chicken liver using liquid chromatography-tandem mass spectrometry (LC-MS/MS)
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Katrin Wilkerling, Hana Valenta, Susanne Kersten, Sven Dänicke
Stable isotope labeled amino acids are frequently used to examine nutritive effects on protein synthesis. This technique is characterized by tracing the incorporation of the label into newly synthesized proteins. In the present investigation, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of very low enrichment of protein-bound L-[2H5]-phenylalanine ([2H5]-phe) in chicken liver. The LC-MS/MS measurements were carried out in positive atmospheric pressure chemical ionization (APCI) mode. Two mass transitions each for [2H5]-phe (171.1/125.1 and 171.1/106.1) and phe (166.1/91.1 and 166.1/93.1) were chosen for quantification and qualification. Due to the high excesses of phe, less sensitive transitions were chosen in the case of phe. The separation was carried out on a phenyl-hexyl column using 0.1% formic acid as eluent A and methanol as eluent B. The method was calibrated with calibration standard solutions in the range of 0.01 - 0.5 mole percent excess (MPE). Linear regression analysis led to coefficients of determination (r2) greater than 0.9995. The method was applied on liver samples from experiments investigating nutritive effects on tissue protein synthesis in broiler chickens. These samples were analyzed with a gas chromatography-mass spectrometry (GC-MS) method and reanalyzed with the developed LC-MS/MS method one year later. Compared to GC-MS, the main advantages of the LC-MS/MS method are its higher selectivity as well as the elimination of the need to convert and derivatize the samples prior to measuring.
Source:Journal of Chromatography B
Katrin Wilkerling, Hana Valenta, Susanne Kersten, Sven Dänicke
Stable isotope labeled amino acids are frequently used to examine nutritive effects on protein synthesis. This technique is characterized by tracing the incorporation of the label into newly synthesized proteins. In the present investigation, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of very low enrichment of protein-bound L-[2H5]-phenylalanine ([2H5]-phe) in chicken liver. The LC-MS/MS measurements were carried out in positive atmospheric pressure chemical ionization (APCI) mode. Two mass transitions each for [2H5]-phe (171.1/125.1 and 171.1/106.1) and phe (166.1/91.1 and 166.1/93.1) were chosen for quantification and qualification. Due to the high excesses of phe, less sensitive transitions were chosen in the case of phe. The separation was carried out on a phenyl-hexyl column using 0.1% formic acid as eluent A and methanol as eluent B. The method was calibrated with calibration standard solutions in the range of 0.01 - 0.5 mole percent excess (MPE). Linear regression analysis led to coefficients of determination (r2) greater than 0.9995. The method was applied on liver samples from experiments investigating nutritive effects on tissue protein synthesis in broiler chickens. These samples were analyzed with a gas chromatography-mass spectrometry (GC-MS) method and reanalyzed with the developed LC-MS/MS method one year later. Compared to GC-MS, the main advantages of the LC-MS/MS method are its higher selectivity as well as the elimination of the need to convert and derivatize the samples prior to measuring.
Highlights
► LC-MS/MS method for measurement of stable isotope enrichment of L-[2H5]-phenylalanine. ► Method is suited for investigations of nutritive effects on tissue protein synthesis. ► No need for derivatization.A liquid chromatography mass spectrometry method for the measurement of cystathionine β-synthase activity in cell extracts
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Desirée E.C. Smith, Marisa I.S. Mendes, Leo A.J. Kluijtmans, Mirian C.H. Janssen, Yvo M. Smulders, Henk J. Blom
Background In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine ß-synthase (CBS) a sensitive analytical method is necessary. Methods An electrospray LC-MS/MS method preceded by a solid phase extraction step was developed for the measurement of CBS activity in cell extracts. Nonafluoropentanoic acid was used as an ionpair to provide the underivatized cystathionine the desired retention on a C18 column. Results A detection limit of 50pmol cystathionine/h/mg protein was achieved. In fibroblasts, intra- and inter-assay CVs for the CBS activity were 5.2% and 14.7%, respectively. A Km value of 8μmol/L for homocysteine, and 2.5μmol/L for serine was calculated. In fibroblasts wildtype, heterozygous, and homozygous CBS activity ranges measured were 8.5-27.0, 4.2-13.4, 0.0-0.7 nmol/h x mg protein, respectively. The method was applied to a study were rats were fed 2 diets. Increase of dietary methionine (7.7 versus 3.8mg/kg methionine) significantly increased the CBS activity in rat liver lysates from a median of 58.0 to a median of 71.5 (P=0.037) nmol/h x mg protein. In a lymphoblasts cell culture experiment, the addition of Hcy to the culture media increased the activity of CBS 3 fold. Conclusion This LC-MS/MS is able to diagnose CBS deficiency at the enzyme level, and can accurately measure the effect diets or therapy might have on the CBS activity in a variety of cell types.
Source:Journal of Chromatography B
Desirée E.C. Smith, Marisa I.S. Mendes, Leo A.J. Kluijtmans, Mirian C.H. Janssen, Yvo M. Smulders, Henk J. Blom
Background In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine ß-synthase (CBS) a sensitive analytical method is necessary. Methods An electrospray LC-MS/MS method preceded by a solid phase extraction step was developed for the measurement of CBS activity in cell extracts. Nonafluoropentanoic acid was used as an ionpair to provide the underivatized cystathionine the desired retention on a C18 column. Results A detection limit of 50pmol cystathionine/h/mg protein was achieved. In fibroblasts, intra- and inter-assay CVs for the CBS activity were 5.2% and 14.7%, respectively. A Km value of 8μmol/L for homocysteine, and 2.5μmol/L for serine was calculated. In fibroblasts wildtype, heterozygous, and homozygous CBS activity ranges measured were 8.5-27.0, 4.2-13.4, 0.0-0.7 nmol/h x mg protein, respectively. The method was applied to a study were rats were fed 2 diets. Increase of dietary methionine (7.7 versus 3.8mg/kg methionine) significantly increased the CBS activity in rat liver lysates from a median of 58.0 to a median of 71.5 (P=0.037) nmol/h x mg protein. In a lymphoblasts cell culture experiment, the addition of Hcy to the culture media increased the activity of CBS 3 fold. Conclusion This LC-MS/MS is able to diagnose CBS deficiency at the enzyme level, and can accurately measure the effect diets or therapy might have on the CBS activity in a variety of cell types.
Highlights
► A straightforward LC-MS/MS method for the determination of CBS enzyme activity. ► The method can be applied to different cell types. ► The method can be used for diagnosis of cystathionine ß-synthase deficiency. ► The method can be used to study effects of treatment of homocystinuria. ► The method can be used to study effects of diet on mild hyperhomocystenemia.Validation of an LC-MS/MS method for the quantification of choline-related compounds and phospholipids in foods and tissues
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Yeping Xiong, Yuan-Yuan Zhao, Sue Goruk, Kirsten Oilund, Catherine J. Field, René L. Jacobs, Jonathan M. Curtis
A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method was developed and validated to simultaneously quantify six aqueous choline-related compounds and eight major phospholipids classes in a single run. HILIC chromatography was coupled to positive ion electrospray mass spectrometry. A combination of multiple scan modes including precursor ion scan, neutral loss scan and multiple reaction monitoring was optimized for the determination of each compound or class in a single LC/MS run. This work developed a simplified extraction scheme in which both free choline and related compounds along with phospholipids were extracted into a homogenised phase using chloroform/methanol/water (1:2:0.8) and diluted into methanol for analysis of target compounds in a variety of sample matrices. The analyte recoveries were evaluated by spiking tissues and food samples with two isotope-labeled internal standards, PC-d3 and Cho-d3. Recoveries of between 90% and 115% were obtained by spiking a range of sample matrices with authentic standards containing all 14 of the target analytes. The precision of the analysis ranged from 1.6 to 13%. Accuracy and precision was comparable to that obtained by quantification of selected phospholipid classes using 31P NMR. A variety of sample matrices including egg yolks, human diets and animal tissues were analyzed using the validated method. The measurements of total choline in selected foods were found to be in good agreement with values obtained from the USDA choline database.
Source:Journal of Chromatography B
Yeping Xiong, Yuan-Yuan Zhao, Sue Goruk, Kirsten Oilund, Catherine J. Field, René L. Jacobs, Jonathan M. Curtis
A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method was developed and validated to simultaneously quantify six aqueous choline-related compounds and eight major phospholipids classes in a single run. HILIC chromatography was coupled to positive ion electrospray mass spectrometry. A combination of multiple scan modes including precursor ion scan, neutral loss scan and multiple reaction monitoring was optimized for the determination of each compound or class in a single LC/MS run. This work developed a simplified extraction scheme in which both free choline and related compounds along with phospholipids were extracted into a homogenised phase using chloroform/methanol/water (1:2:0.8) and diluted into methanol for analysis of target compounds in a variety of sample matrices. The analyte recoveries were evaluated by spiking tissues and food samples with two isotope-labeled internal standards, PC-d3 and Cho-d3. Recoveries of between 90% and 115% were obtained by spiking a range of sample matrices with authentic standards containing all 14 of the target analytes. The precision of the analysis ranged from 1.6 to 13%. Accuracy and precision was comparable to that obtained by quantification of selected phospholipid classes using 31P NMR. A variety of sample matrices including egg yolks, human diets and animal tissues were analyzed using the validated method. The measurements of total choline in selected foods were found to be in good agreement with values obtained from the USDA choline database.
Highlights
► Quantification of 14 choline-related compounds or phospholipid classes is achieved. ► The validated method uses a single hydrophilic interaction (HILIC) LC-MS/MS run. ► Choline, betaine and phospholipid classes are extracted by a single phase solvent. ► Phospholipid quantification is consistent with 31P NMR data. ► Total choline measurements in foods consistent with USDA choline database.Electrospun Polyvinyl Alcohol Ultra-Thin Layer Chromatography of Amino Acids
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Tian Lu, Susan V. Olesik
Electrospun polyvinyl alcohol (PVA) ultrathin layer chromatographic (UTLC) plates were fabricated using in-situ crosslinking electrospinning technique. The value of these ULTC plates were characterized using the separation of fluorescein isothiocyanate (FITC) labeled amino acids and the separation of amino acids followed visualization using ninhydrin. The in-situ crosslinked electrospun PVA plates showed enhanced stability in water and were stable when used for the UTLC study. The selectivity of FITC labeled amino acids on PVA plate was compared with that on commercial Si-Gel plate. The efficiency of the separation varied with analyte concentration, size of capillary analyte applicator, analyte volume, and mat thickness. The concentration of 7mM or less, 50μm i.d. capillary applicator, minimum volume of analyte solution and three-layered mat provides the best efficiency of FITC-labeled amino acids on PVA UTLC plate. The efficiency on PVA plate was greatly improved compared to the efficiency on Si-Gel HPTLC plate. The hydrolysis products of aspartame in diet coke, aspartic acid and phenylalanine, were also successfully analyzed using PVA-UTLC plate.
Source:Journal of Chromatography B
Tian Lu, Susan V. Olesik
Electrospun polyvinyl alcohol (PVA) ultrathin layer chromatographic (UTLC) plates were fabricated using in-situ crosslinking electrospinning technique. The value of these ULTC plates were characterized using the separation of fluorescein isothiocyanate (FITC) labeled amino acids and the separation of amino acids followed visualization using ninhydrin. The in-situ crosslinked electrospun PVA plates showed enhanced stability in water and were stable when used for the UTLC study. The selectivity of FITC labeled amino acids on PVA plate was compared with that on commercial Si-Gel plate. The efficiency of the separation varied with analyte concentration, size of capillary analyte applicator, analyte volume, and mat thickness. The concentration of 7mM or less, 50μm i.d. capillary applicator, minimum volume of analyte solution and three-layered mat provides the best efficiency of FITC-labeled amino acids on PVA UTLC plate. The efficiency on PVA plate was greatly improved compared to the efficiency on Si-Gel HPTLC plate. The hydrolysis products of aspartame in diet coke, aspartic acid and phenylalanine, were also successfully analyzed using PVA-UTLC plate.
Highlights
► Electrospun crosslinked PVA as UTLC stationary phase. ► Eleven laser dye labeled amino acids were tested on the PVA UTLC plate. ► The separation efficiency on PVA UTLC plate was much higher than on Si-gel HPTLC plate. ► Unlabeled amino acids, alanine, methionine, arginine, and phenylalanine were baseline separated on PVA UTLC plate using ninhydrin as visualization reagent.The experimental study of Astragalus membranaceus on meridian tropsim: The distribution study of astragaloside IV in rat tissues
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Yan-xu chang, Yu-gang Sun, Jin Li, Qiu-Hong Zhang, Xin-Rong Guo, Bo-li Zhang, Hua Jin, Xiu-mei gao
According to Traditional Chinese medicine (TCM) theories, TCM with different meridian tropism have different therapeutic effects. In view of the meridian tropism of Astragalus membranaceus (Huangqi), astragaloside IV, one of the effective phytochemicals of Huangqi, was appointed and observed its distribution in rat tissues following a single intravenous (i.v.) dose. A simple and accurate LC-ESI-MS/MS method was developed and validated for astragaloside IV quantification in heart, liver, spleen, lung and kidney using warfarin as an internal standard (IS). Chromatographic separation was performed on a Eclipse plus C18 (4.6 mm×100mm, 1.8μm) when the flow rate was set at 0.300mLmin−1 and ammonium acetate aqueous solution - acetonitrile was used as mobile phase. The intra- and inter-day precisions of the quality control samples were within 15% and accuracies were within 90.0-110%. The recoveries were more than 90.0% at high, medium and low concentrations, respectively. This method was successfully applied for distribution of astragaloside IV after intravenous (i.v.) dose of 4mgkg−1 astragaloside IV in rats. Astragaloside IV concentration was highest in liver and kidney and remained much higher than that in other tissues over the experiment course. Lung, heart and spleen were also detected to contain astragaloside IV. The results clearly demonstrated that astragaloside IV was one of the material bases of the meridian tropism of Huangqi.
Source:Journal of Chromatography B
Yan-xu chang, Yu-gang Sun, Jin Li, Qiu-Hong Zhang, Xin-Rong Guo, Bo-li Zhang, Hua Jin, Xiu-mei gao
According to Traditional Chinese medicine (TCM) theories, TCM with different meridian tropism have different therapeutic effects. In view of the meridian tropism of Astragalus membranaceus (Huangqi), astragaloside IV, one of the effective phytochemicals of Huangqi, was appointed and observed its distribution in rat tissues following a single intravenous (i.v.) dose. A simple and accurate LC-ESI-MS/MS method was developed and validated for astragaloside IV quantification in heart, liver, spleen, lung and kidney using warfarin as an internal standard (IS). Chromatographic separation was performed on a Eclipse plus C18 (4.6 mm×100mm, 1.8μm) when the flow rate was set at 0.300mLmin−1 and ammonium acetate aqueous solution - acetonitrile was used as mobile phase. The intra- and inter-day precisions of the quality control samples were within 15% and accuracies were within 90.0-110%. The recoveries were more than 90.0% at high, medium and low concentrations, respectively. This method was successfully applied for distribution of astragaloside IV after intravenous (i.v.) dose of 4mgkg−1 astragaloside IV in rats. Astragaloside IV concentration was highest in liver and kidney and remained much higher than that in other tissues over the experiment course. Lung, heart and spleen were also detected to contain astragaloside IV. The results clearly demonstrated that astragaloside IV was one of the material bases of the meridian tropism of Huangqi.
Highlights
► A simple and accurate LC-ESI-MS/MS method was developed and validated for. ► astragaloside IV quantification in heart, liver, spleen, lung and kidney in rat. ► The developed LC-MS/MS method was sensitive enough for distribution study of astragaloside IV in rat tissues following following a single intravenous dose of. ► 4mgkg−1 astragaloside IV to rats. ► Liver, kidney, lung, heart and spleen were detected to contain astragaloside IV, and astragaloside IV concentration was highest in liver and kidney. ► These results clearly demonstrated that Astragaloside IV was one of the material bases. ► of the meridian tropism of Huangqi.A Fast Liquid Chromatography Mass Spectrometry (LC-MS) Method for Quantification of Major Polar Metabolites in Plants
08 November 2012,
09:35:30
Publication year:
2012
Source:Journal of Chromatography B
Zhiqian Liu, Simone Rochfort
Current liquid chromatography (LC) based methods for the analysis of polar plant metabolites require multiple runs using complex mobile phases and a combination of different columns. Here we describe a fast liquid chromatography mass spectrometry (LC-MS) method for the determination of major polar metabolites in plants that requires only a single run using a single column. The method takes advantage of the ability to acquire both positive and negative data in an ion trap mass spectrometer (MS) and also the accurate mass capability of the orbitrap MS. The separation of polar compounds is achieved with a polar, reversed-phase column (Synergi Hydro-RP). A single analysis with a 25min runtime is able to reliably determine the level of nearly all essential amino acids, several major organic acids and several major sugars in plant materials, as exemplified by analysis of a perennial ryegrass extract. The level of detection on column was below 0.1ng (average 0.03ng) for most amino acids, below 5ng (average 2.3ng) for organics acids and below 1ng (average 0.64ng) for sugars. The levels of quantified metabolites in ryegrass varied from 22μg/g dry weight for histidine to 41mg/g dry weight for sucrose.
Source:Journal of Chromatography B
Zhiqian Liu, Simone Rochfort
Current liquid chromatography (LC) based methods for the analysis of polar plant metabolites require multiple runs using complex mobile phases and a combination of different columns. Here we describe a fast liquid chromatography mass spectrometry (LC-MS) method for the determination of major polar metabolites in plants that requires only a single run using a single column. The method takes advantage of the ability to acquire both positive and negative data in an ion trap mass spectrometer (MS) and also the accurate mass capability of the orbitrap MS. The separation of polar compounds is achieved with a polar, reversed-phase column (Synergi Hydro-RP). A single analysis with a 25min runtime is able to reliably determine the level of nearly all essential amino acids, several major organic acids and several major sugars in plant materials, as exemplified by analysis of a perennial ryegrass extract. The level of detection on column was below 0.1ng (average 0.03ng) for most amino acids, below 5ng (average 2.3ng) for organics acids and below 1ng (average 0.64ng) for sugars. The levels of quantified metabolites in ryegrass varied from 22μg/g dry weight for histidine to 41mg/g dry weight for sucrose.
No comments:
Post a Comment