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Rapid evaluation for heterogeneities in monoclonal antibodies by liquid chromatography/mass spectrometry with a column-switching system
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Ryosuke Kuribayashi, Noritaka Hashii, Akira Harazono, Nana Kawasaki
The development of therapeutic antibodies has grown over the last several years. Most of the recombinant monoclonal antibodies (mAbs) produced by mammalian cells are glycoproteins. Glycosylation of the mAbs can be associated with effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, as well as immunogenicity and clearance. Thus, mAb glycan heterogeneity is a significant characteristic associated with the safety and efficacy of the products. Therefore, glycan heterogeneity should be evaluated during research and development (R&D) and during development of mAbs manufacturing processes to identify the process parameters that affect glycan heterogeneity and to enhance understanding of the manufacturing process. There is an increasing need for a rapid, easy, and automated evaluation method for glycan heterogeneity. Liquid chromatography/mass spectrometry (LC/MS) is a method that can be used to analyze glycoforms. LC/MS is marked by the ability to measure the oligosaccharide composition of each glycoform, whereas other general methods, such as capillary electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ion-exchange chromatography, cannot. However, a laborious off-line purification of mAbs is required to evaluate glycan heterogeneities. In this study, we demonstrate the use of a rapid, easy, and automated evaluation system for mAb glycoforms by LC/MS. This LC/MS system uses a column-switching system equipped with 2 columns, a protein A affinity column and a reversed-phase column (desalting column). We devised 2 column-switching systems: one that targeted intact mAbs (system 1) and one that targeted the light and heavy chains of the mAbs (system 2). Our results show that the proposed systems are applicable as a tool to evaluate the glycoforms in several situations, including the research, development, and production processes of mAbs. Additionally, we hope that our systems are useful as process analytical technology (PAT) for molecular heterogeneities containing glycoforms of mAbs in implementation of quality by design (QbD).
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Ryosuke Kuribayashi, Noritaka Hashii, Akira Harazono, Nana Kawasaki
The development of therapeutic antibodies has grown over the last several years. Most of the recombinant monoclonal antibodies (mAbs) produced by mammalian cells are glycoproteins. Glycosylation of the mAbs can be associated with effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, as well as immunogenicity and clearance. Thus, mAb glycan heterogeneity is a significant characteristic associated with the safety and efficacy of the products. Therefore, glycan heterogeneity should be evaluated during research and development (R&D) and during development of mAbs manufacturing processes to identify the process parameters that affect glycan heterogeneity and to enhance understanding of the manufacturing process. There is an increasing need for a rapid, easy, and automated evaluation method for glycan heterogeneity. Liquid chromatography/mass spectrometry (LC/MS) is a method that can be used to analyze glycoforms. LC/MS is marked by the ability to measure the oligosaccharide composition of each glycoform, whereas other general methods, such as capillary electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ion-exchange chromatography, cannot. However, a laborious off-line purification of mAbs is required to evaluate glycan heterogeneities. In this study, we demonstrate the use of a rapid, easy, and automated evaluation system for mAb glycoforms by LC/MS. This LC/MS system uses a column-switching system equipped with 2 columns, a protein A affinity column and a reversed-phase column (desalting column). We devised 2 column-switching systems: one that targeted intact mAbs (system 1) and one that targeted the light and heavy chains of the mAbs (system 2). Our results show that the proposed systems are applicable as a tool to evaluate the glycoforms in several situations, including the research, development, and production processes of mAbs. Additionally, we hope that our systems are useful as process analytical technology (PAT) for molecular heterogeneities containing glycoforms of mAbs in implementation of quality by design (QbD).
Highlights
► We developed evaluation systems for heterogeneities in monoclonal antibodies (mAbs). ► The systems using LC/MS are applicable in the R&D, and production processes of mAbs. ► Our systems are useful as process analytical technology for heterogeneities of mAbs.Development of NIRS method for quality control of drug combination artesunate–azithromycin for the treatment of severe malaria
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Chantal Boyer, Karen Gaudin, Tina Kauss, Alexandra Gaubert, Abdelhakim Boudis, Justine Verschelden, Mickaël Franc, Julie Roussille, Jacques Boucher, Piero Olliaro, Nicholas J. White, Pascal Millet, Jean-Pierre Dubost
Near infrared spectroscopy (NIRS) methods were developed for the determination of analytical content of an antimalarial-antibiotic (artesunate and azithromycin) co-formulation in hard gelatin capsule (HGC). The NIRS consists of pre-processing treatment of spectra (raw spectra and first-derivation of two spectral zones), a unique principal component analysis model to ensure the specificity and then two partial least-squares regression models for the determination content of each active pharmaceutical ingredient. The NIRS methods were developed and validated with no reference method, since the manufacturing process of HGC is basically mixed excipients with active pharmaceutical ingredients. The accuracy profiles showed β-expectation tolerance limits within the acceptance limits (±5%). The analytical control approach performed by reversed phase (HPLC) required two different methods involving two different preparation and chromatographic methods. NIRS offers advantages in terms of lower costs of equipment and procedures, time saving, environmentally friendly.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Chantal Boyer, Karen Gaudin, Tina Kauss, Alexandra Gaubert, Abdelhakim Boudis, Justine Verschelden, Mickaël Franc, Julie Roussille, Jacques Boucher, Piero Olliaro, Nicholas J. White, Pascal Millet, Jean-Pierre Dubost
Near infrared spectroscopy (NIRS) methods were developed for the determination of analytical content of an antimalarial-antibiotic (artesunate and azithromycin) co-formulation in hard gelatin capsule (HGC). The NIRS consists of pre-processing treatment of spectra (raw spectra and first-derivation of two spectral zones), a unique principal component analysis model to ensure the specificity and then two partial least-squares regression models for the determination content of each active pharmaceutical ingredient. The NIRS methods were developed and validated with no reference method, since the manufacturing process of HGC is basically mixed excipients with active pharmaceutical ingredients. The accuracy profiles showed β-expectation tolerance limits within the acceptance limits (±5%). The analytical control approach performed by reversed phase (HPLC) required two different methods involving two different preparation and chromatographic methods. NIRS offers advantages in terms of lower costs of equipment and procedures, time saving, environmentally friendly.
Comparative analysis of quinolizidine alkaloids from different parts of Sophora alopecuroides seeds by UPLC–MS/MS
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Hanqing Wang, Sheng Guo, Dawei Qian, Yefei Qian, Jin-ao Duan
The seeds of Sophora alopecuroides L. have been utilized as a crude drug in China for thousands of years. Quinolizidine alkaloids are the main bioactive components of this plant. To determine the distribution and content of quinolizidine alkaloids in different seed organs (seed coat and cotyledon), a reliable method has been established using ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UPLC–MS/MS). Seven constituents, namely cytisine, oxymatrine, oxysophocarpine, sophoridine, sophoramine, matrine, and sophocarpine, were simultaneously determined in 10min. The proposed method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. The analysis results showed there were remarkable differences in the distribution and contents of the chemical markers between seed coat and cotyledon. The established approach could be helpful for the quality control of S. alopecuroides seeds, and also for the determination of this type class of alkaloids in other medicinal herbs. The present study can provide necessary information for the rational utilization of S. alopecuroides resources.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Hanqing Wang, Sheng Guo, Dawei Qian, Yefei Qian, Jin-ao Duan
The seeds of Sophora alopecuroides L. have been utilized as a crude drug in China for thousands of years. Quinolizidine alkaloids are the main bioactive components of this plant. To determine the distribution and content of quinolizidine alkaloids in different seed organs (seed coat and cotyledon), a reliable method has been established using ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UPLC–MS/MS). Seven constituents, namely cytisine, oxymatrine, oxysophocarpine, sophoridine, sophoramine, matrine, and sophocarpine, were simultaneously determined in 10min. The proposed method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. The analysis results showed there were remarkable differences in the distribution and contents of the chemical markers between seed coat and cotyledon. The established approach could be helpful for the quality control of S. alopecuroides seeds, and also for the determination of this type class of alkaloids in other medicinal herbs. The present study can provide necessary information for the rational utilization of S. alopecuroides resources.
A multidisciplinary approach for the analysis of an adulterated dietary supplement where the active pharmaceutical ingredient was embedded in the capsule shell
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Adam Lanzarotta, John B. Crowe, Mark Witkowski, Bryan M. Gamble
Fourier transform infrared (FT-IR) microspectroscopic imaging, Raman microspectroscopy, optical microscopy and high performance liquid chromatography with mass spectrometric (LC/MS) detection were employed to examine a dietary supplement adulterated with an undeclared active pharmaceutical ingredient (API). While a trace level of the API was detected in the capsule contents, a higher concentration of API was found in the capsule shell, which indicated the use of an unconventional manufacturing process to hide the API and thus avoid detection. This study demonstrates the need for a multidisciplinary approach to provide a complete assessment of a suspect adulterated dietary supplement.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Adam Lanzarotta, John B. Crowe, Mark Witkowski, Bryan M. Gamble
Fourier transform infrared (FT-IR) microspectroscopic imaging, Raman microspectroscopy, optical microscopy and high performance liquid chromatography with mass spectrometric (LC/MS) detection were employed to examine a dietary supplement adulterated with an undeclared active pharmaceutical ingredient (API). While a trace level of the API was detected in the capsule contents, a higher concentration of API was found in the capsule shell, which indicated the use of an unconventional manufacturing process to hide the API and thus avoid detection. This study demonstrates the need for a multidisciplinary approach to provide a complete assessment of a suspect adulterated dietary supplement.
Characterization of currently marketed heparin products: Adverse event relevant bioassays
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Cynthia D. Sommers, Nicolas Montpas, Albert Adam, David A. Keire
The polyanion oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in heparin products and was associated with severe hypotensive responses and other symptoms in patients receiving the drug. The OSCS associated adverse reactions were attributed to activation of the contact system via the plasma mediator, activated factor XII (FXIIa), which triggers kallikrein (KK) activity. Unlike heparin alone, OSCS, is able to activate FXII in plasma and stably bind to FXIIa enhancing plasma KK activity and the induction of vasoactive mediators such as bradykinin (BK), C3a and C5a. Similarly OSCS can interfere with heparin neutralization by the polycationic drug protamine. Here, we assess heparin (heparin sodium, dalteparin, tinzaparin or enoxaparin)–protamine complex formation and plasma based bioassays of KK, BK and C5a in a 96-well plate format. We establish the normal range of variation in the optimized bioassays across multiple lots from 9 manufacturers. In addition, because other oversulfated (OS) glycosaminoglycans (GAGs) besides OSCS could also serve as possible economically motivated adulterants (EMAs) to heparin, we characterize OS-dermatan sulfate (OSDS), OS-heparan sulfate (OSHS) and their native forms in the same assays. For the protamine test, OS-GAGs could be distinguished from heparin. For the KK assay, OSCS and OSDS were most potent followed by OSHS, and all had similar efficacies. Finally, OSDS had a greater efficacy in the C5a and BK assays followed by OSCS then OSHS. These data established the normal range of response of heparin products in these assays and the alteration in the responses in the presence of possible EMAs.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Cynthia D. Sommers, Nicolas Montpas, Albert Adam, David A. Keire
The polyanion oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in heparin products and was associated with severe hypotensive responses and other symptoms in patients receiving the drug. The OSCS associated adverse reactions were attributed to activation of the contact system via the plasma mediator, activated factor XII (FXIIa), which triggers kallikrein (KK) activity. Unlike heparin alone, OSCS, is able to activate FXII in plasma and stably bind to FXIIa enhancing plasma KK activity and the induction of vasoactive mediators such as bradykinin (BK), C3a and C5a. Similarly OSCS can interfere with heparin neutralization by the polycationic drug protamine. Here, we assess heparin (heparin sodium, dalteparin, tinzaparin or enoxaparin)–protamine complex formation and plasma based bioassays of KK, BK and C5a in a 96-well plate format. We establish the normal range of variation in the optimized bioassays across multiple lots from 9 manufacturers. In addition, because other oversulfated (OS) glycosaminoglycans (GAGs) besides OSCS could also serve as possible economically motivated adulterants (EMAs) to heparin, we characterize OS-dermatan sulfate (OSDS), OS-heparan sulfate (OSHS) and their native forms in the same assays. For the protamine test, OS-GAGs could be distinguished from heparin. For the KK assay, OSCS and OSDS were most potent followed by OSHS, and all had similar efficacies. Finally, OSDS had a greater efficacy in the C5a and BK assays followed by OSCS then OSHS. These data established the normal range of response of heparin products in these assays and the alteration in the responses in the presence of possible EMAs.
Isolation and structure characterization of related impurities in Sodium Tanshinone IIA Sulfonate by LC/ESI-MSn and NMR
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Baolin Wang, Hao Wang, En Wang, Wenjing Yan, Wencai Ye, Ping Li
Sodium Tanshinone IIA Sulfonate (STS) is a water-soluble derivative of tanshinone IIA, an important lipophilic component isolated from the roots of Salvia miltiorrhiza. STS, as injection drug, is widely and successfully used in China for treating cardiovascular disease. Eight impurities were detected in STS bulk drug by LC-UV and LC–ESI-MS n . Low pressure C18 column chromatography and preparative HPLC were used for the isolation of three principal impurities. Based on LC/ESI-MS n and NMR analysis, related impurities were characterized as sodium (±)-hydroxyl tanshinone sulfonate (1), sodium tanshinone I sulfonate (2), sodium salvianolic acid sulfonate (3), sodium 1,2-dehydrotanshinone IIA sulfonate (4), sodium tanshinone IIB sulfonate (5), sodium 3-hydroxyl salvianolic acid sulfonate (6), sodium 1,16-dihydroxyl-15,16-dihydrotanshinone IIA sulfonate (7), and sodium methyl 1,2-dehydrotanshinonate sulfonate (8). The 1H and 13C NMR data of impurity 1 were reported in this paper for the first time. Also, to the best of our knowledge, impurity 4 had not been isolated as pure substance until now. The possible mechanism for the formation of impurities is also discussed.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Baolin Wang, Hao Wang, En Wang, Wenjing Yan, Wencai Ye, Ping Li
Sodium Tanshinone IIA Sulfonate (STS) is a water-soluble derivative of tanshinone IIA, an important lipophilic component isolated from the roots of Salvia miltiorrhiza. STS, as injection drug, is widely and successfully used in China for treating cardiovascular disease. Eight impurities were detected in STS bulk drug by LC-UV and LC–ESI-MS n . Low pressure C18 column chromatography and preparative HPLC were used for the isolation of three principal impurities. Based on LC/ESI-MS n and NMR analysis, related impurities were characterized as sodium (±)-hydroxyl tanshinone sulfonate (1), sodium tanshinone I sulfonate (2), sodium salvianolic acid sulfonate (3), sodium 1,2-dehydrotanshinone IIA sulfonate (4), sodium tanshinone IIB sulfonate (5), sodium 3-hydroxyl salvianolic acid sulfonate (6), sodium 1,16-dihydroxyl-15,16-dihydrotanshinone IIA sulfonate (7), and sodium methyl 1,2-dehydrotanshinonate sulfonate (8). The 1H and 13C NMR data of impurity 1 were reported in this paper for the first time. Also, to the best of our knowledge, impurity 4 had not been isolated as pure substance until now. The possible mechanism for the formation of impurities is also discussed.
Characterization of currently marketed heparin products: Analysis of heparin digests by RPIP-UHPLC–QTOF-MS
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Bo Wang, Lucinda F. Buhse, Ali Al-Hakim, Michael T. Boyne Ii, David A. Keire
Previously, the FDA validated a method to assess the structure and composition of heparin products by separating and quantifying disaccharide level digests by reverse-phase-ion-pairing liquid chromatography (RPIP-HPLC) coupled to a low resolution and low sensitivity ion trap mass-spectrometer. Here, improved separation, information content and sensitivity were obtained through the use of reverse phase ion-pairing ultra-high pressure liquid chromatography (RPIP-UHPLC) coupled with a quadrupole time-of-flight (Q-TOF) mass spectrometer. Thus, with the new method, improved structural characterization of the same 20 lots of heparin sodium active pharmaceutical ingredients (APIs) as were analyzed in the previous work were obtained. In addition, for the first time, 10 low molecular weight heparin (LMWH) lots were characterized representing multiple lots manufactured by three different processes (dalteparin, tinzaparin or enoxaparin). In this study, UHPLC separation conditions and the enzymatic digesting protocol were optimized for analysis of disaccharide level digests of heparin and positive and negative electrospray ionization (ESI) modes were tested. The negative ion mode ESI analysis was found to be superior to the positive ion mode for these measurements, and a combination of heparin lyase II and III were optimal for heparin digestion. The data obtained establishes the normal variation in the composition of heparin sodium or LMWHs in this assay. These values are useful as possible product benchmarks and for surveillance of the heparin products being imported into the US market.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Bo Wang, Lucinda F. Buhse, Ali Al-Hakim, Michael T. Boyne Ii, David A. Keire
Previously, the FDA validated a method to assess the structure and composition of heparin products by separating and quantifying disaccharide level digests by reverse-phase-ion-pairing liquid chromatography (RPIP-HPLC) coupled to a low resolution and low sensitivity ion trap mass-spectrometer. Here, improved separation, information content and sensitivity were obtained through the use of reverse phase ion-pairing ultra-high pressure liquid chromatography (RPIP-UHPLC) coupled with a quadrupole time-of-flight (Q-TOF) mass spectrometer. Thus, with the new method, improved structural characterization of the same 20 lots of heparin sodium active pharmaceutical ingredients (APIs) as were analyzed in the previous work were obtained. In addition, for the first time, 10 low molecular weight heparin (LMWH) lots were characterized representing multiple lots manufactured by three different processes (dalteparin, tinzaparin or enoxaparin). In this study, UHPLC separation conditions and the enzymatic digesting protocol were optimized for analysis of disaccharide level digests of heparin and positive and negative electrospray ionization (ESI) modes were tested. The negative ion mode ESI analysis was found to be superior to the positive ion mode for these measurements, and a combination of heparin lyase II and III were optimal for heparin digestion. The data obtained establishes the normal variation in the composition of heparin sodium or LMWHs in this assay. These values are useful as possible product benchmarks and for surveillance of the heparin products being imported into the US market.
Screening and analyzing potential hepatotoxic compounds in the ethanol extract of Asteris Radix by HPLC/DAD/ESI-MSn technique
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Xiaodong Liu, Peipei Cao, Chaofeng Zhang, Xianghong Xu, Mian Zhang
A refined toxic fraction, Fr-2, from the ethanol extract of Aster tataricus roots was obtained by a toxic tracing isolation. The hepatotoxic effects of Fr-2 were proved by the level elevation of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in mice serum and further confirmed by the histopathological examination on mice liver. A simple method was developed for qualitative analysis of constituents in Fr-2 fraction by high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (ESI-MS n ). As a result, 35 compounds were detected and identified in Fr-2. Among them, 28 compounds are pentapeptides, including 18 cyclic and 10 linear ones, and the sum of their peak area occupied about 87% (71.2% for cyclic and 15.8% for linear) of total area of chromatographic peak. Other 7 compounds are phenols, occupied about 5.9% of total area of chromatographic peak. Moreover, the structures of 10 cyclic and 2 linear peptides were reported for the first time. The results suggested that the peptides, especially the cyclic ones, are probably the principal toxic substances in Fr-2 also in A. tataricus roots.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Xiaodong Liu, Peipei Cao, Chaofeng Zhang, Xianghong Xu, Mian Zhang
A refined toxic fraction, Fr-2, from the ethanol extract of Aster tataricus roots was obtained by a toxic tracing isolation. The hepatotoxic effects of Fr-2 were proved by the level elevation of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in mice serum and further confirmed by the histopathological examination on mice liver. A simple method was developed for qualitative analysis of constituents in Fr-2 fraction by high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (ESI-MS n ). As a result, 35 compounds were detected and identified in Fr-2. Among them, 28 compounds are pentapeptides, including 18 cyclic and 10 linear ones, and the sum of their peak area occupied about 87% (71.2% for cyclic and 15.8% for linear) of total area of chromatographic peak. Other 7 compounds are phenols, occupied about 5.9% of total area of chromatographic peak. Moreover, the structures of 10 cyclic and 2 linear peptides were reported for the first time. The results suggested that the peptides, especially the cyclic ones, are probably the principal toxic substances in Fr-2 also in A. tataricus roots.
Comparative evaluation of pKa prediction tools on a drug discovery dataset
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
György T. Balogh, Ákos Tarcsay, György M. Keserű
Due to their impact on pharmacokinetic and pharmacodynamic properties the accurate prediction of dissociation constants is of outmost importance in drug discovery settings. The prediction accuracy, however, is typically assessed on public datasets most likely included in the training sets of the available tools. In this work we therefore tested five pK a prediction softwares such as ACD, Epik, Marvin, PharmaAlgorithm and Pallas on novel, never-published compounds. Our dataset consists of 177pK a values of 95 structurally diverse in-house compounds prepared for real-life drug discovery programs. The thorough analysis of prediction accuracy allowed us identifying the best practice and exploring the limitations of the current methods. Mean absolute errors (0.86–1.28) obtained for this set of discovery compounds indicates the potential in the improvement of the available pK a prediction approaches. Limitations were further characterized by measuring and evaluating 39pK a values of additional 28 commercially available compounds representing the most challenging chemotypes. We believe that these results would facilitate further developments and hopefully contribute to the necessary improvement of the prediction accuracy.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
György T. Balogh, Ákos Tarcsay, György M. Keserű
Due to their impact on pharmacokinetic and pharmacodynamic properties the accurate prediction of dissociation constants is of outmost importance in drug discovery settings. The prediction accuracy, however, is typically assessed on public datasets most likely included in the training sets of the available tools. In this work we therefore tested five pK a prediction softwares such as ACD, Epik, Marvin, PharmaAlgorithm and Pallas on novel, never-published compounds. Our dataset consists of 177pK a values of 95 structurally diverse in-house compounds prepared for real-life drug discovery programs. The thorough analysis of prediction accuracy allowed us identifying the best practice and exploring the limitations of the current methods. Mean absolute errors (0.86–1.28) obtained for this set of discovery compounds indicates the potential in the improvement of the available pK a prediction approaches. Limitations were further characterized by measuring and evaluating 39pK a values of additional 28 commercially available compounds representing the most challenging chemotypes. We believe that these results would facilitate further developments and hopefully contribute to the necessary improvement of the prediction accuracy.
Simultaneous determination of nine model compounds in permeability samples using RP-HPLC: Application to prove the cassette administration principle in single pass intestinal perfusion study in rats
19 June 2012,
16:47:32
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Wahajuddin, Sheelendra Pratap Singh, K.S.R. Raju, Asad Nafis, Girish Kumar Jain
A simple, sensitive and specific reversed phase high performance liquid chromatographic (RP-HPLC) method for simultaneous determination of atenolol, paracetamol, hydrochlorothiazide, caffeine, cephalexin, metoprolol, propranolol, ketoprofen along with phenol red (a non-absorbable compound) in samples obtained from intestinal in situ single-pass perfusion studies, was developed and validated. Chromatography was carried out on RP18 column with mobile phase comprising of 10mM phosphate buffer (pH 2.5) and methanol in gradient mode. The calibration curves were linear for all nine permeability model compounds (r 2 >0.999) across the concentration range of 1.25–40μg/ml. The coefficient of variation for intra and inter-day assay precision was between 0.04 and 3.08% and the accuracy was between 98.39 and 109.45%. Stability studies were carried out at different storage conditions and all the analytes were found to be stable. The method was successfully applied for analysing the permeability samples obtained from in situ single pass perfusion studies. The effective permeability (P eff) values obtained upon cassette administration were in close proximity to the permeability values obtained upon single administration of model compounds. In conclusion, the developed RP-HPLC method can be used for high throughput cassette validation of rat in situ perfusion model for intestinal permeability assessment.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volumes 67–68
Wahajuddin, Sheelendra Pratap Singh, K.S.R. Raju, Asad Nafis, Girish Kumar Jain
A simple, sensitive and specific reversed phase high performance liquid chromatographic (RP-HPLC) method for simultaneous determination of atenolol, paracetamol, hydrochlorothiazide, caffeine, cephalexin, metoprolol, propranolol, ketoprofen along with phenol red (a non-absorbable compound) in samples obtained from intestinal in situ single-pass perfusion studies, was developed and validated. Chromatography was carried out on RP18 column with mobile phase comprising of 10mM phosphate buffer (pH 2.5) and methanol in gradient mode. The calibration curves were linear for all nine permeability model compounds (r 2 >0.999) across the concentration range of 1.25–40μg/ml. The coefficient of variation for intra and inter-day assay precision was between 0.04 and 3.08% and the accuracy was between 98.39 and 109.45%. Stability studies were carried out at different storage conditions and all the analytes were found to be stable. The method was successfully applied for analysing the permeability samples obtained from in situ single pass perfusion studies. The effective permeability (P eff) values obtained upon cassette administration were in close proximity to the permeability values obtained upon single administration of model compounds. In conclusion, the developed RP-HPLC method can be used for high throughput cassette validation of rat in situ perfusion model for intestinal permeability assessment.
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