A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation o...A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.展开更多
A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromato...A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromatography was performed on a Purospher? Star, C18 (5 mm, 250 × 4.6 mm) column using acetonitrile: buffer (0.01 mM) (40:60, v/v, pH adjusted to 3.0), as a mobile phase. The mobile phase was pumped at a flow rate of 1.0 mL·min-1 and UV detection was performed at 240 nm. The method was validated for linearity, accuracy, precision and specificity. The method was applied to study the interaction between amlodipine and H1-receptor antagonists. These interactions were carried out in simulated gastric juice (pH 1), simulated full stomach (pH 4), blood pH (pH 7.4) and simulating GI (pH 9). The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques. In most cases the in vitro availability of amlodipine was decreased. It was observed that the change in in vitro availability was pH dependent.展开更多
AIM:To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography(RP-HPLC),and to evaluate the retinal neuroprotective effect after topical administration with...AIM:To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography(RP-HPLC),and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure(IOP)model.METHODS:The eyes of albino rabbits were administered topically and ipsilaterally with 0.2%moxonidine.A RPHPLC method was employed for the identification and quantification of moxonidine between 2 and 480 min,which presented in the aqueous humor and iris-ciliary body.Flash electroretinography(F-ERG)amplitude and superoxide dismutase(SOD)level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP.Histological and ultrastructural observation underwent to analyze the changes of retinal morphology,the inner retinal layers(IRL)thickness,and retinal ganglion cell(RGC)counting.RESULTS:Moxonidine was detectable between 2 and 480 min after administration,and the peak concentration developed both in the two tissues at 30 min,0.51μg/m Lin aqueous humor and 1.03μg/g in iris-ciliary body.In comparison to control,F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15(P<0.01)in the high IOP model;SOD levels were significantly higher at all time-points(P<0.01)with a maximum level of 20.29 U/mgprot at day 15;and RGCs were significantly higher(P<0.05).CONCLUSION:Moxonidine is a viable neuroprotective agent with application to high IOP model.All layers of retina,including RGC layer,retinal nerve fiber layer and INL,are more preserved after moxonidine administration.SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.展开更多
Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compar...Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.展开更多
A new, rapid and efficient reverse phase Liquid Chromatography (RP-LC) method was developed for determination of Gibberellin A4 (GA4) in samples of flower stalk of Dasylirion cedrosanum and vegetative tissue of Epithe...A new, rapid and efficient reverse phase Liquid Chromatography (RP-LC) method was developed for determination of Gibberellin A4 (GA4) in samples of flower stalk of Dasylirion cedrosanum and vegetative tissue of Epithelantha micromeris. Purification of GA4 was carried out by solid phase extraction (SPE), in Epithelantha micromeris. In the chromatography method was obtaining a retention time of 2.1 min, using Hypersil GOLD C-18 column (100 × 4.6 mm dim and size particle 5 μ), mobile phase 50/50 acetonitrile/water and a flow 1.0 ml/min. Detection was carried out by a UV detector set at 205 nm, and a quantization limit of 0.4 mg/L. The obtained correlation coefficient was 0.995.展开更多
A reversed phase (RP)/hydrophilic interaction (HILIC)/ion exchange (IEX) mixed tri-mode stationary phase (TMSP) has been prepared via a divergent synthesis scheme starting from propylamine on silica then by amine-epox...A reversed phase (RP)/hydrophilic interaction (HILIC)/ion exchange (IEX) mixed tri-mode stationary phase (TMSP) has been prepared via a divergent synthesis scheme starting from propylamine on silica then by amine-epoxy reactions with 1,4-butanedioldiglycidyl ether and tertiary amines (N,Ndimethyldecylamine, DMDA). Its retention mechanism was found to follow RP/HILIC/IEX mixed-mode.The stop-flow test revealed that TMSP had good compatibility with 100% aqueous mobile phase. It demonstrated effective separation towards several kinds of compounds or drug molecules and their counterions within a single run.展开更多
The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode arr...The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode array detector. The separation was done by using symmetry C-18 ODS (25 cm × 0.46 cm internal diameter) 5 μm analytical column containing mobile phase of Phosphate buffer (0.02 M) and methanol [65:35% v/v] adjusted pH to 4.8 by adding dilute ortho phosphoric acid. The method was run at 1 ml·min<sup>-1</sup> at 286 nm detection. The drug was eluted at 5.484 min. After developing the method, it was assured for the intended use by validation which was done according to ICH Q2B guidelines. The analytical parameters checked were linearity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, ruggedness and robustness. It was observed that the response of the detector was linear in the range of 6 - 14 μg/ml with correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability indicating assay method was established by using the samples generated by forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative and photolytic degradation and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the analysis of quality of the rucaparib drug.展开更多
The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmi...The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmic solution method development was determined on Waters HPLC instrument using a UV Detector. The separation was done by using L11, Zorbex SB phenyl (4.6 mm × 250 mm internal diameter) 5 μm analytical column, containing mobile phase of Phosphate buffer (0.02 M), methanol, and acetonitrile [50:30:20 % v/v]. The method was run at 1 ml·min<sup>-1</sup> at 210 nm for Bimatoprost and 295 nm for Timolol for detection. The drug was eluted at 10.81 min for Bimatoprost and 3.77 min for Timolol. After developing the method, it was assured for the intended use by validation, which was done according to ICH Q2B guidelines. The analytical parameters checked were Specificity/Selectivity, linearity, Range, accuracy, ruggedness, and robustness. It was observed that the response of the detector was linear in the range of 6 - 18 μg/ml with a correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability-indicating assay method was established by using the samples generated by the forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative, and photolytic degradation, and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the quantitative analysis of Bimatoprost 0.3% + Timolol 0.5% Ophthalmic Solution drugs for pharmaceutical use. Currently, there is no official method for Bimatoprost & Timolol combination products in USP or BP. Available research work related to single Bimatoprost or Timolol products was not suitable for testing Bimatoprost and Timolol combination drugs. Additionally, there is no stability-indicating method to test Bimatoprost & Timolol combination products which insist us to do research and develop a new reverse phase-HPLC indicating method which will be faster and more accurate.展开更多
In this work, we developed a highly sensitive method to detect melamine based on reversed phase liquid chromatography mass spectrometry. A mass spectrometry compatible ion pair, heptafluorobu-tyric acid(HFBA), was use...In this work, we developed a highly sensitive method to detect melamine based on reversed phase liquid chromatography mass spectrometry. A mass spectrometry compatible ion pair, heptafluorobu-tyric acid(HFBA), was used to separate melamine by reversed phase liquid chromatography prior to electrospray mass spectrometry. The incorporation of isotope internal standard and multiple reaction monitoring improved the accuracy and linearity of quantification. Based on this strategy, the method limit of quantification was 0.1 ng/g. The limits of quantification were 8 ng/g for liquid milk and 15 ng/g for dry milk powder. This method provided a reproducible and stable approach to sensitive detection and quantification of melamine.展开更多
An on-line UV spectrometric method for the quantitative determination ofmethanol increment of methanol-water in the mobile phase (i.e., of greater concentration than thatof the mobile phase) by frontal analysis (FA) o...An on-line UV spectrometric method for the quantitative determination ofmethanol increment of methanol-water in the mobile phase (i.e., of greater concentration than thatof the mobile phase) by frontal analysis (FA) of insulin in reversed phase liquid chromatography(RPLC) was presented. When the methanol increment concentration ranged from 0.05% to 0.50%,V(CH_3OH)/V(H_2O), a set of elution curves could be obtained at 198 nm by a diode-array detector inthe presence of 47% methanol, V(CH_3OH)/V(H_2O) containing 0.03% hydrochloric acid,V(CH_3OH-H_2O)/V(HCl) in the mobile phase. The plateau height of the elution curves of the methanolincrement was found to be proportional to the methanol increment in the mobile phase. The methanolincrement could be determined on a quantitative basis. When the method was used to investigate theelution curve of insulin by FA in RPLC, a small plateau, being the methanol increment, was detectedbefore the usual insulin plateau of each elution curve. In this case the methanol increment wasfound to vary with insulin concentration in the mobile phase. When that concentration was between0.025 mg/mL and 0.30 mg/mL, the methanol increment could be determined in the range from 0.03% to0.19% with a deviation of ±10%. A nuclear magnetic resonance spectrometer (NMR) was also employedto confirm the obtained result. A methodology with a very rigorous experimental procedure forobtaining results of such accuracy is also included. The presented result may be used to prove thata displacement process definitely occurs as insulin is adsorbed by the RPLC stationary phase inFA.展开更多
Ⅰ. INTRODUCTION Recently, reversed phase liquid chromatography (RPLC) has become a very efficient standard technique for the modern analyses and is widely used for the analysis of many types of compounds. Retention p...Ⅰ. INTRODUCTION Recently, reversed phase liquid chromatography (RPLC) has become a very efficient standard technique for the modern analyses and is widely used for the analysis of many types of compounds. Retention prediction plays an important part in the thermodynamic studies in RPLC. Many attempts have been made to establish Kováts-like retention展开更多
A rapid, sensitive, and robust reversed-phase liquid chromatography with tandem mass spectrometry method was developed and validated for the determination of total and unbound ceritinib, a secondgeneration ALK inhibit...A rapid, sensitive, and robust reversed-phase liquid chromatography with tandem mass spectrometry method was developed and validated for the determination of total and unbound ceritinib, a secondgeneration ALK inhibitor, in patient plasma and brain tumor tissue samples. Sample preparation involved simple protein precipitation with acetonitrile. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C_(18) column using a 4-min gradient elution consisting of mobile phase A(0.1% formic acid in water) and mobile phase B(0.1% formic acid in acetonitrile), at a flow rate of 0.4 m L/min. Ceritinib and the internal standard([^(13)C_6]ceritinib) were monitored using multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantitation(LLOQ) was 1 n M of ceritinib in plasma. The calibration curve was linear over ceritinib concentration range of 1–2000 n M in plasma. The intra-and interday precision and accuracy were within the generally accepted criteria for bioanalytical method( o15%).The method was successfully applied to assess ceritinib brain tumor penetration, as assessed by the unbound drug brain concentration to unbound drug plasma concentration ratio, in patients with brain tumors.展开更多
With frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to em...With frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to employ the stoichiometric displacement theory (SDT) for investigating solute adsorption in physical chemistry. Six kinds of mobile phase in RPLC were employed to study the effects on the elution curves and adsorption isotherms of insulin. The key points of this paper are: (1) The stability of insulin due to delay time after preparing, the organic solvent concentration, the kind and the concentration of ion pairing agent in mobile phase were found to affect both elution curve and adsorption isotherm very seriously. (2) To obtain a valid and comparable result, the composition of the mobile phase employed in FA must be as same as possible to that in usual RPLC of either analytical scale or preparative purpose. (3) Langmuir Equation and the SDT were employed to imitate these obtained adsorption isotherms. The expression for solute adsorption from solution of the SDT was found to have a better elucidation to the insulin adsorption from mobile phase in RPLC.展开更多
Ⅰ. INTRODUCTIONReversed-phase ion-pair liquid chromatography is widely used in the separation of ionized organic compounds. In recent years, the separation of inorganic ions by the reversedphase ion-pair liquid chrom...Ⅰ. INTRODUCTIONReversed-phase ion-pair liquid chromatography is widely used in the separation of ionized organic compounds. In recent years, the separation of inorganic ions by the reversedphase ion-pair liquid chromatography with indirect UV detection or conductivity展开更多
Thr extract of E. containing recombinant human interferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride (Gu . HCl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and p...Thr extract of E. containing recombinant human interferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride (Gu . HCl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and purify rhIFN- with acetic acid-water as mobile phase. Gu I-ICI and most impure proteins can be separated by this way. Compared with the usual dilution method, the bioactivity recovery of the purified rhIFN- was found to be over 500%. In addition, compared to common organic solvents employed ill RPLC, acetic acid has higher freezing point, and therefore, it is easy to concentrate the aim-protein by freeze-drying when acetic acid-water is used as mobile phase ill RPLC.展开更多
The interaction index c which was derived from the fundamental retention equationlogk’ =a + cC_B in reversed--phase high--performancc liquid chromatography (RP-HPLC) quan-titatively describes the difference in the in...The interaction index c which was derived from the fundamental retention equationlogk’ =a + cC_B in reversed--phase high--performancc liquid chromatography (RP-HPLC) quan-titatively describes the difference in the interaction between solute--strong. solvent andsolute--weak solvent; it has shown to be a constant for a specific solute even when columnsystems with different C18 packings are used. The theoretical basis for peak identificationby using interaction index has been proposed, which was based on the a,c values on stan-dard C18 column by utilizing linear a-a plots on column pairs and the linear relationship be-tween parameters a and c for the structural related compounds. Through the establishmentof parameters a,c data based on the standard C18 column for a certain type of compounds,the retention of thesc compounds on various C18 columns can be predicted. Typical exam-ples have been given to verify the correctness of this method.展开更多
With insulin methanol water, and the ion pairing agent, hydrochloric acid and trifluroacetic acid (TFA), the character of the first plateau (FP) on the elution curve of frontal analysis in reversed phase liquid chro...With insulin methanol water, and the ion pairing agent, hydrochloric acid and trifluroacetic acid (TFA), the character of the first plateau (FP) on the elution curve of frontal analysis in reversed phase liquid chromatography (RPLC) was investigated by on line UV spectrometry and identified with nuclear magnetic resonance (NMR) spectrometry and mass spectrometry. The profile of the FP is the same as that of a usual elution curve of methanol in frontal analysis (FA). When the insulin concentration was limited to a certain range, the height of the FP was found to be proportional to the insulin concentration in mobile phase and its length companying to shorten. The FP profile on the intersection of two tangents reflects the components of the microstructure in the depth direction of the bonded stationary phase layer and the desorption dynamics of the displaced components. The displaced methanol was quantitatively determined by NMR and on line UV spectrometries. TFA with high UV absorbance can not be used as an ion pairing agent for the investigation of the FP in RPLC, but it can be used as a good marker to investigate the complicated transfer process of components in the stationary phase in RPLC. A stoichiometric displacement process between solute and solvent was proved to be valid in both usual and FA in RPLC. From the point of view of dynamics of mass transfer, the solutes can only contact to the surface of stationary phase in usual RPLC, while solute can penetrate into it in FA of RPLC. The solvation of insulin in methanol and water solution as an example indicating the usage of the FP in the FA was also investigated in this paper.展开更多
In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identi?ed peptides in reverse phase liquid chromatogra- phy and electrospray tandem mass spectrometry (RPLC...In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identi?ed peptides in reverse phase liquid chromatogra- phy and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Fol- lowing protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identi?ed peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol. Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state (+2, +3) peptides are likely to be identi?ed, and the hydropathicity of the peptides might not be related to whether it is more likely to be identi?ed or not. The number of identi?ed peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identi?ed peptides present three types of redundancy, namely iden- ti?cation, charge, and sequence redundancy, which may repress low abundance protein identi?cation.展开更多
文摘A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.
文摘A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromatography was performed on a Purospher? Star, C18 (5 mm, 250 × 4.6 mm) column using acetonitrile: buffer (0.01 mM) (40:60, v/v, pH adjusted to 3.0), as a mobile phase. The mobile phase was pumped at a flow rate of 1.0 mL·min-1 and UV detection was performed at 240 nm. The method was validated for linearity, accuracy, precision and specificity. The method was applied to study the interaction between amlodipine and H1-receptor antagonists. These interactions were carried out in simulated gastric juice (pH 1), simulated full stomach (pH 4), blood pH (pH 7.4) and simulating GI (pH 9). The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques. In most cases the in vitro availability of amlodipine was decreased. It was observed that the change in in vitro availability was pH dependent.
基金Supported by the Key Science and Technology Program of Shaanxi Province,China(No.2015SF146).
文摘AIM:To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography(RP-HPLC),and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure(IOP)model.METHODS:The eyes of albino rabbits were administered topically and ipsilaterally with 0.2%moxonidine.A RPHPLC method was employed for the identification and quantification of moxonidine between 2 and 480 min,which presented in the aqueous humor and iris-ciliary body.Flash electroretinography(F-ERG)amplitude and superoxide dismutase(SOD)level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP.Histological and ultrastructural observation underwent to analyze the changes of retinal morphology,the inner retinal layers(IRL)thickness,and retinal ganglion cell(RGC)counting.RESULTS:Moxonidine was detectable between 2 and 480 min after administration,and the peak concentration developed both in the two tissues at 30 min,0.51μg/m Lin aqueous humor and 1.03μg/g in iris-ciliary body.In comparison to control,F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15(P<0.01)in the high IOP model;SOD levels were significantly higher at all time-points(P<0.01)with a maximum level of 20.29 U/mgprot at day 15;and RGCs were significantly higher(P<0.05).CONCLUSION:Moxonidine is a viable neuroprotective agent with application to high IOP model.All layers of retina,including RGC layer,retinal nerve fiber layer and INL,are more preserved after moxonidine administration.SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.
基金the National Natural Science Foundation in China(No.20705028)the Foundation of Key Laboratory of Modem Separation Science in Shaanxi Province(No.05JS61).
文摘Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.
文摘A new, rapid and efficient reverse phase Liquid Chromatography (RP-LC) method was developed for determination of Gibberellin A4 (GA4) in samples of flower stalk of Dasylirion cedrosanum and vegetative tissue of Epithelantha micromeris. Purification of GA4 was carried out by solid phase extraction (SPE), in Epithelantha micromeris. In the chromatography method was obtaining a retention time of 2.1 min, using Hypersil GOLD C-18 column (100 × 4.6 mm dim and size particle 5 μ), mobile phase 50/50 acetonitrile/water and a flow 1.0 ml/min. Detection was carried out by a UV detector set at 205 nm, and a quantization limit of 0.4 mg/L. The obtained correlation coefficient was 0.995.
基金financially supported by the National Natural Science Foundation of China(Nos. 21477037, 21322502)the Outstanding Young Talent Cultivation Fund of East China University of Science and Technology
文摘A reversed phase (RP)/hydrophilic interaction (HILIC)/ion exchange (IEX) mixed tri-mode stationary phase (TMSP) has been prepared via a divergent synthesis scheme starting from propylamine on silica then by amine-epoxy reactions with 1,4-butanedioldiglycidyl ether and tertiary amines (N,Ndimethyldecylamine, DMDA). Its retention mechanism was found to follow RP/HILIC/IEX mixed-mode.The stop-flow test revealed that TMSP had good compatibility with 100% aqueous mobile phase. It demonstrated effective separation towards several kinds of compounds or drug molecules and their counterions within a single run.
文摘The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode array detector. The separation was done by using symmetry C-18 ODS (25 cm × 0.46 cm internal diameter) 5 μm analytical column containing mobile phase of Phosphate buffer (0.02 M) and methanol [65:35% v/v] adjusted pH to 4.8 by adding dilute ortho phosphoric acid. The method was run at 1 ml·min<sup>-1</sup> at 286 nm detection. The drug was eluted at 5.484 min. After developing the method, it was assured for the intended use by validation which was done according to ICH Q2B guidelines. The analytical parameters checked were linearity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, ruggedness and robustness. It was observed that the response of the detector was linear in the range of 6 - 14 μg/ml with correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability indicating assay method was established by using the samples generated by forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative and photolytic degradation and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the analysis of quality of the rucaparib drug.
文摘The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmic solution method development was determined on Waters HPLC instrument using a UV Detector. The separation was done by using L11, Zorbex SB phenyl (4.6 mm × 250 mm internal diameter) 5 μm analytical column, containing mobile phase of Phosphate buffer (0.02 M), methanol, and acetonitrile [50:30:20 % v/v]. The method was run at 1 ml·min<sup>-1</sup> at 210 nm for Bimatoprost and 295 nm for Timolol for detection. The drug was eluted at 10.81 min for Bimatoprost and 3.77 min for Timolol. After developing the method, it was assured for the intended use by validation, which was done according to ICH Q2B guidelines. The analytical parameters checked were Specificity/Selectivity, linearity, Range, accuracy, ruggedness, and robustness. It was observed that the response of the detector was linear in the range of 6 - 18 μg/ml with a correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability-indicating assay method was established by using the samples generated by the forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative, and photolytic degradation, and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the quantitative analysis of Bimatoprost 0.3% + Timolol 0.5% Ophthalmic Solution drugs for pharmaceutical use. Currently, there is no official method for Bimatoprost & Timolol combination products in USP or BP. Available research work related to single Bimatoprost or Timolol products was not suitable for testing Bimatoprost and Timolol combination drugs. Additionally, there is no stability-indicating method to test Bimatoprost & Timolol combination products which insist us to do research and develop a new reverse phase-HPLC indicating method which will be faster and more accurate.
基金Supported by the National Natural Science Foundation of China (Grant No. 30425021)National High Technology Research and Development Program of China (Grant No. 2007AA02Z334)
文摘In this work, we developed a highly sensitive method to detect melamine based on reversed phase liquid chromatography mass spectrometry. A mass spectrometry compatible ion pair, heptafluorobu-tyric acid(HFBA), was used to separate melamine by reversed phase liquid chromatography prior to electrospray mass spectrometry. The incorporation of isotope internal standard and multiple reaction monitoring improved the accuracy and linearity of quantification. Based on this strategy, the method limit of quantification was 0.1 ng/g. The limits of quantification were 8 ng/g for liquid milk and 15 ng/g for dry milk powder. This method provided a reproducible and stable approach to sensitive detection and quantification of melamine.
文摘An on-line UV spectrometric method for the quantitative determination ofmethanol increment of methanol-water in the mobile phase (i.e., of greater concentration than thatof the mobile phase) by frontal analysis (FA) of insulin in reversed phase liquid chromatography(RPLC) was presented. When the methanol increment concentration ranged from 0.05% to 0.50%,V(CH_3OH)/V(H_2O), a set of elution curves could be obtained at 198 nm by a diode-array detector inthe presence of 47% methanol, V(CH_3OH)/V(H_2O) containing 0.03% hydrochloric acid,V(CH_3OH-H_2O)/V(HCl) in the mobile phase. The plateau height of the elution curves of the methanolincrement was found to be proportional to the methanol increment in the mobile phase. The methanolincrement could be determined on a quantitative basis. When the method was used to investigate theelution curve of insulin by FA in RPLC, a small plateau, being the methanol increment, was detectedbefore the usual insulin plateau of each elution curve. In this case the methanol increment wasfound to vary with insulin concentration in the mobile phase. When that concentration was between0.025 mg/mL and 0.30 mg/mL, the methanol increment could be determined in the range from 0.03% to0.19% with a deviation of ±10%. A nuclear magnetic resonance spectrometer (NMR) was also employedto confirm the obtained result. A methodology with a very rigorous experimental procedure forobtaining results of such accuracy is also included. The presented result may be used to prove thata displacement process definitely occurs as insulin is adsorbed by the RPLC stationary phase inFA.
文摘Ⅰ. INTRODUCTION Recently, reversed phase liquid chromatography (RPLC) has become a very efficient standard technique for the modern analyses and is widely used for the analysis of many types of compounds. Retention prediction plays an important part in the thermodynamic studies in RPLC. Many attempts have been made to establish Kováts-like retention
基金supported by the United States Public Health Service Cancer Center Support Grant P30 CA022453Novartis for providing the study drug and isotope-labeled internal standard and providing financial support for the clinical study
文摘A rapid, sensitive, and robust reversed-phase liquid chromatography with tandem mass spectrometry method was developed and validated for the determination of total and unbound ceritinib, a secondgeneration ALK inhibitor, in patient plasma and brain tumor tissue samples. Sample preparation involved simple protein precipitation with acetonitrile. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C_(18) column using a 4-min gradient elution consisting of mobile phase A(0.1% formic acid in water) and mobile phase B(0.1% formic acid in acetonitrile), at a flow rate of 0.4 m L/min. Ceritinib and the internal standard([^(13)C_6]ceritinib) were monitored using multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantitation(LLOQ) was 1 n M of ceritinib in plasma. The calibration curve was linear over ceritinib concentration range of 1–2000 n M in plasma. The intra-and interday precision and accuracy were within the generally accepted criteria for bioanalytical method( o15%).The method was successfully applied to assess ceritinib brain tumor penetration, as assessed by the unbound drug brain concentration to unbound drug plasma concentration ratio, in patients with brain tumors.
文摘With frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to employ the stoichiometric displacement theory (SDT) for investigating solute adsorption in physical chemistry. Six kinds of mobile phase in RPLC were employed to study the effects on the elution curves and adsorption isotherms of insulin. The key points of this paper are: (1) The stability of insulin due to delay time after preparing, the organic solvent concentration, the kind and the concentration of ion pairing agent in mobile phase were found to affect both elution curve and adsorption isotherm very seriously. (2) To obtain a valid and comparable result, the composition of the mobile phase employed in FA must be as same as possible to that in usual RPLC of either analytical scale or preparative purpose. (3) Langmuir Equation and the SDT were employed to imitate these obtained adsorption isotherms. The expression for solute adsorption from solution of the SDT was found to have a better elucidation to the insulin adsorption from mobile phase in RPLC.
文摘Ⅰ. INTRODUCTIONReversed-phase ion-pair liquid chromatography is widely used in the separation of ionized organic compounds. In recent years, the separation of inorganic ions by the reversedphase ion-pair liquid chromatography with indirect UV detection or conductivity
基金This work was supported by the National Natural Science Foundation of China!(29675017,39880003)
文摘Thr extract of E. containing recombinant human interferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride (Gu . HCl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and purify rhIFN- with acetic acid-water as mobile phase. Gu I-ICI and most impure proteins can be separated by this way. Compared with the usual dilution method, the bioactivity recovery of the purified rhIFN- was found to be over 500%. In addition, compared to common organic solvents employed ill RPLC, acetic acid has higher freezing point, and therefore, it is easy to concentrate the aim-protein by freeze-drying when acetic acid-water is used as mobile phase ill RPLC.
文摘The interaction index c which was derived from the fundamental retention equationlogk’ =a + cC_B in reversed--phase high--performancc liquid chromatography (RP-HPLC) quan-titatively describes the difference in the interaction between solute--strong. solvent andsolute--weak solvent; it has shown to be a constant for a specific solute even when columnsystems with different C18 packings are used. The theoretical basis for peak identificationby using interaction index has been proposed, which was based on the a,c values on stan-dard C18 column by utilizing linear a-a plots on column pairs and the linear relationship be-tween parameters a and c for the structural related compounds. Through the establishmentof parameters a,c data based on the standard C18 column for a certain type of compounds,the retention of thesc compounds on various C18 columns can be predicted. Typical exam-ples have been given to verify the correctness of this method.
文摘With insulin methanol water, and the ion pairing agent, hydrochloric acid and trifluroacetic acid (TFA), the character of the first plateau (FP) on the elution curve of frontal analysis in reversed phase liquid chromatography (RPLC) was investigated by on line UV spectrometry and identified with nuclear magnetic resonance (NMR) spectrometry and mass spectrometry. The profile of the FP is the same as that of a usual elution curve of methanol in frontal analysis (FA). When the insulin concentration was limited to a certain range, the height of the FP was found to be proportional to the insulin concentration in mobile phase and its length companying to shorten. The FP profile on the intersection of two tangents reflects the components of the microstructure in the depth direction of the bonded stationary phase layer and the desorption dynamics of the displaced components. The displaced methanol was quantitatively determined by NMR and on line UV spectrometries. TFA with high UV absorbance can not be used as an ion pairing agent for the investigation of the FP in RPLC, but it can be used as a good marker to investigate the complicated transfer process of components in the stationary phase in RPLC. A stoichiometric displacement process between solute and solvent was proved to be valid in both usual and FA in RPLC. From the point of view of dynamics of mass transfer, the solutes can only contact to the surface of stationary phase in usual RPLC, while solute can penetrate into it in FA of RPLC. The solvation of insulin in methanol and water solution as an example indicating the usage of the FP in the FA was also investigated in this paper.
基金This work was partially supported by grants fromthe National Basic Research Program (973) of China(2004CB520804), and National Natural Science Foun-dation of China (No. 30270657, 30230150, 3037030).
文摘In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identi?ed peptides in reverse phase liquid chromatogra- phy and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Fol- lowing protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identi?ed peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol. Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state (+2, +3) peptides are likely to be identi?ed, and the hydropathicity of the peptides might not be related to whether it is more likely to be identi?ed or not. The number of identi?ed peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identi?ed peptides present three types of redundancy, namely iden- ti?cation, charge, and sequence redundancy, which may repress low abundance protein identi?cation.
