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papers from the latest issue:
Improving the efficiency of quantitative 1H NMR: An innovative external standard–internal reference approach
08 September 2013,
16:40:54
Publication date: 25 January
2014
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Yande Huang , Bao-Ning Su , Qingmei Ye , Venkatapuram A. Palaniswamy , Mark S. Bolgar , Thomas V. Raglione
The classical internal standard quantitative NMR (qNMR) method determines the purity of an analyte by the determination of a solution containing the analyte and a standard. Therefore, the standard must meet the requirements of chemical compatibility and lack of resonance interference with the analyte as well as a known purity. The identification of such a standard can be time consuming and must be repeated for each analyte. In contrast, the external standard qNMR method utilizes a standard with a known purity to calibrate the NMR instrument. The external standard and the analyte are measured separately, thereby eliminating the matter of chemical compatibility and resonance interference between the standard and the analyte. However, the instrumental factors, including the quality of NMR tubes, must be kept the same. Any deviations will compromise the accuracy of the results. An innovative qNMR method reported herein utilizes an internal reference substance along with an external standard to assume the role of the standard used in the traditional internal standard qNMR method. In this new method, the internal reference substance must only be chemically compatible and be free of resonance-interference with the analyte or external standard whereas the external standard must only be of a known purity. The exact purity or concentration of the internal reference substance is not required as long as the same quantity is added to the external standard and the analyte. The new method reduces the burden of searching for an appropriate standard for each analyte significantly. Therefore the efficiency of the qNMR purity assay increases while the precision of the internal standard method is retained.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Yande Huang , Bao-Ning Su , Qingmei Ye , Venkatapuram A. Palaniswamy , Mark S. Bolgar , Thomas V. Raglione
The classical internal standard quantitative NMR (qNMR) method determines the purity of an analyte by the determination of a solution containing the analyte and a standard. Therefore, the standard must meet the requirements of chemical compatibility and lack of resonance interference with the analyte as well as a known purity. The identification of such a standard can be time consuming and must be repeated for each analyte. In contrast, the external standard qNMR method utilizes a standard with a known purity to calibrate the NMR instrument. The external standard and the analyte are measured separately, thereby eliminating the matter of chemical compatibility and resonance interference between the standard and the analyte. However, the instrumental factors, including the quality of NMR tubes, must be kept the same. Any deviations will compromise the accuracy of the results. An innovative qNMR method reported herein utilizes an internal reference substance along with an external standard to assume the role of the standard used in the traditional internal standard qNMR method. In this new method, the internal reference substance must only be chemically compatible and be free of resonance-interference with the analyte or external standard whereas the external standard must only be of a known purity. The exact purity or concentration of the internal reference substance is not required as long as the same quantity is added to the external standard and the analyte. The new method reduces the burden of searching for an appropriate standard for each analyte significantly. Therefore the efficiency of the qNMR purity assay increases while the precision of the internal standard method is retained.
Graphical abstract
Combined HPLC-DAD–MS, HPLC–MSn and NMR spectroscopy for quality control of plant extracts: The case of a commercial blend sold as dietary supplement
08 September 2013,
16:40:54
Publication date: 25 January
2014
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): A. Karioti , E. Giocaliere , C. Guccione , G. Pieraccini , E. Gallo , A. Vannacci , A.R. Bilia
The efficiency of 1D and 2D NMR spectroscopy along with HPLC-DAD–MS analyses in characterising the content of a dietary supplement is demonstrated. Experiments directly performed on a lyophilised sample of a commercial product gave details on the quality control of the product. The lack of the marker constituents of some of the declared plant species (Crataegus oxyacantha, Olea europea, Capsella bursa-pastoris and Fumaria officinalis) and the presence of banned adulterants, responsible for the strong antihypertensive effect of the supplement were established. The analyses proved the presence of indole alkaloids belonging to the group of Rauwolfia sp., such as ajmaline, reserpine and yohimbine. Quantitative HPLC analysis showed that the content of reserpine in the product was in the therapeutic range and therefore responsible for the collapses of the patients.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): A. Karioti , E. Giocaliere , C. Guccione , G. Pieraccini , E. Gallo , A. Vannacci , A.R. Bilia
The efficiency of 1D and 2D NMR spectroscopy along with HPLC-DAD–MS analyses in characterising the content of a dietary supplement is demonstrated. Experiments directly performed on a lyophilised sample of a commercial product gave details on the quality control of the product. The lack of the marker constituents of some of the declared plant species (Crataegus oxyacantha, Olea europea, Capsella bursa-pastoris and Fumaria officinalis) and the presence of banned adulterants, responsible for the strong antihypertensive effect of the supplement were established. The analyses proved the presence of indole alkaloids belonging to the group of Rauwolfia sp., such as ajmaline, reserpine and yohimbine. Quantitative HPLC analysis showed that the content of reserpine in the product was in the therapeutic range and therefore responsible for the collapses of the patients.
Graphical abstract
Determination of pKa values of a triptolide derivative and its impurities by pressure-assisted capillary electrophoresis
08 September 2013,
16:40:54
Publication date: 25 January
2014
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Jian-Ling Wang , Xue-Jiao Xu , Dong-Ying Chen
A pressure-assisted capillary zone electrophoresis method was developed to determine the pK a values of a triptolide derivative (LLDT-246) and its impurities. The method was performed in an uncoated fused-silica capillary under the electric voltage of 18kV and 50mbar of external pressure applied simultaneously. A series of running electrolyte buffers were used with pH ranging between 2.2 and 10.0 with the constant ionic strength of 0.05M. The values of pK a of LLDT-246 and two impurities were calculated based on the pH dependence of effective mobilities (μ eff). The pK a value of LLDT-246 was in good agreement with that of determined by potentiometric titration method.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Jian-Ling Wang , Xue-Jiao Xu , Dong-Ying Chen
A pressure-assisted capillary zone electrophoresis method was developed to determine the pK a values of a triptolide derivative (LLDT-246) and its impurities. The method was performed in an uncoated fused-silica capillary under the electric voltage of 18kV and 50mbar of external pressure applied simultaneously. A series of running electrolyte buffers were used with pH ranging between 2.2 and 10.0 with the constant ionic strength of 0.05M. The values of pK a of LLDT-246 and two impurities were calculated based on the pH dependence of effective mobilities (μ eff). The pK a value of LLDT-246 was in good agreement with that of determined by potentiometric titration method.
Confirmatory analysis of stanozolol metabolites in bovine, pig and sheep urines using an optimized clean-up and liquid chromatography–tandem mass spectrometry
08 September 2013,
16:40:54
Publication date: 25 January
2014
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Ádám Tölgyesi , Virender K. Sharma , Jenő Fekete
This paper describes a new liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the analysis of three stanozolol metabolites (16β-hydroxystanozolol, 3′-hydroxystanozolol, and 4β-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC–MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core–shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15μg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Ádám Tölgyesi , Virender K. Sharma , Jenő Fekete
This paper describes a new liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the analysis of three stanozolol metabolites (16β-hydroxystanozolol, 3′-hydroxystanozolol, and 4β-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC–MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core–shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15μg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples.
Graphical abstract
Pharmacokinetic study of ginsenoside Rc and simultaneous determination of its metabolites in rats using RRLC-Q-TOF-MS
08 September 2013,
16:40:54
Publication date: 25 January
2014
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Jinghui Sun , Wei Wu , Yingying Guo , Qiujie Qin , Shuying Liu
A rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q-TOF-MS) method was developed for pharmacokinetic study of ginsenoside Rc and applied in the simultaneous determination of ginsenoside Rc metabolites in rats. The experimental results indicate that the concentration versus time profile of ginsenoside Rc shows a two-compartment pharmacokinetic model after intravenous administration of ginsenoside Rc at a dosage of 0.4mg/kg for rats. In the metabolic study, prototype ginsenoside Rc and its deglycosylated metabolites Mb, Mc, and compound K were characterized by comparing the retention time (t R), accurate mass, and characteristic MS/MS fragment ions with standard compounds. The experiments show that part of the ginsenoside Rc was excreted through urine as prototype and part was metabolized into metabolites Mb and Mc after intravenous administration. In contrast, most of ginsenoside Rc were transformed into Mc and CK in feces after oral administration. The in vivo metabolic pathway of ginsenoside Rc was summarized.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Jinghui Sun , Wei Wu , Yingying Guo , Qiujie Qin , Shuying Liu
A rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q-TOF-MS) method was developed for pharmacokinetic study of ginsenoside Rc and applied in the simultaneous determination of ginsenoside Rc metabolites in rats. The experimental results indicate that the concentration versus time profile of ginsenoside Rc shows a two-compartment pharmacokinetic model after intravenous administration of ginsenoside Rc at a dosage of 0.4mg/kg for rats. In the metabolic study, prototype ginsenoside Rc and its deglycosylated metabolites Mb, Mc, and compound K were characterized by comparing the retention time (t R), accurate mass, and characteristic MS/MS fragment ions with standard compounds. The experiments show that part of the ginsenoside Rc was excreted through urine as prototype and part was metabolized into metabolites Mb and Mc after intravenous administration. In contrast, most of ginsenoside Rc were transformed into Mc and CK in feces after oral administration. The in vivo metabolic pathway of ginsenoside Rc was summarized.
Graphical abstract
A comparison of methods for classifying samples as truly specific with confirmatory immunoassays
08 September 2013,
16:40:54
Publication date: 25 January
2014
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Thomas Jaki , John-Philip Lawo , Martin J. Wolfsegger , Peter Allacher , Frank Horling
Biotechnology-derived therapeutics may induce an unwanted immune response leading to the formation of anti-drug antibodies (ADAs) which can result in altered efficacy and safety of the therapeutic protein. Anti-drug antibodies may, for example, affect pharmacokinetics of the therapeutic protein or induce autoimmunity. It is therefore crucial to have assays available for the detection and characterization of ADAs. Commonly, a screening assay is initially used to classify samples as either ADA positive or negative. A confirmatory assay, typically based on antigen competition, is subsequently employed to separate false positive samples from truly positive samples. In this manuscript we investigate the performance of different statistical methods classifying samples in competition assays through simulation and analysis of real data. In our evaluations we do not find a uniformly best method although a simple t-test does provide good results throughout. More crucially we find that very large differences between uninhibited and inhibited measurements relative to the assay variability are required in order to obtain useful classification results questioning the usefulness of competition assays with high variability.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 88
Author(s): Thomas Jaki , John-Philip Lawo , Martin J. Wolfsegger , Peter Allacher , Frank Horling
Biotechnology-derived therapeutics may induce an unwanted immune response leading to the formation of anti-drug antibodies (ADAs) which can result in altered efficacy and safety of the therapeutic protein. Anti-drug antibodies may, for example, affect pharmacokinetics of the therapeutic protein or induce autoimmunity. It is therefore crucial to have assays available for the detection and characterization of ADAs. Commonly, a screening assay is initially used to classify samples as either ADA positive or negative. A confirmatory assay, typically based on antigen competition, is subsequently employed to separate false positive samples from truly positive samples. In this manuscript we investigate the performance of different statistical methods classifying samples in competition assays through simulation and analysis of real data. In our evaluations we do not find a uniformly best method although a simple t-test does provide good results throughout. More crucially we find that very large differences between uninhibited and inhibited measurements relative to the assay variability are required in order to obtain useful classification results questioning the usefulness of competition assays with high variability.
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