亲水作用色谱法测定表阿霉素及其杂质

建立了一种以亲水作用色谱分离测定表阿霉素的新方法。采用硅胶色谱柱及高极性有机溶剂水相缓冲溶液流动相。对流动相的pH、缓冲溶液的浓度及流速进行了优化,确定了以乙腈甲酸钠缓冲溶液(pH2.9)(90∶10,V/V)作为流动相的最佳条件。对优化的分离条件进行系统适应性实验,结果表明表阿霉素与有关杂质之间的分离度和拖尾因子均达到药典要求。该法具有良好的线性(相关系数0.9971～0.9991)和重复性(峰面积RSD<1.0%),方法简便实用,用于实际样品分析,结果满意。     

Development and validation of an HPLC method for the determination of process-related impurities in pridinol mesylate, employing experimental designs

A simple high performance liquid chromatographic method for the determination of process-related impurities in bulk drug of the central anticholinergic compound pridinol mesylate, has been developed and validated. Spectroscopically characterized synthetic impurities were used as standards. The chromatographic separation was optimized employing an experimental design strategy, and was achieved on a C₁₈ column with a mobile phase containing 50 mM potassium phosphate buffer (pH 6.4), MeOH and 2-propanol (20:69:11, v/v/v), delivered at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 245 nm. The optimized method was thoroughly validated, demonstrating to be selective, when the chromatogram was recorded with a diode-array detector and peak purities were evaluated (>0.9995). The method is robust and linear (r² > 0.99) over the range 0.05-2.5% (5-250% with regards to the 1% specification limit for both process-related impurities); it is also precise, regarding repeatability (RSD ≤ 1.5% for all of the analytes) and intermediate precision aspects and LOQ values for the impurities are below 0.01%. Method accuracy, evidenced by low bias of the results and analyte recoveries in the range of 99.1-102.7%, was assessed at five analyte concentration levels. The usefulness of the determination was also demonstrated through the analysis of different lots of pridinol mesylate bulk substance. The results indicate that the method is suitable for the quality control of the bulk manufacturing of pridinol mesylate drug substance.     

Capillary electrophoresis determination of diatrizoic acid and its impurities in diatrizoate radiopaque solutions

A capillary electrophoresis method has been developed for the separation and determination of diatrizoic acid (DTZA) and its four mono- and diiodo degradation products (2-iodo,4-iodo, 2,4-diiodo, and 2,6-diiodo-3,5-diacetamidobenzoic acid) in radio-paque solution for injection (RSI). DTZA and its degradants were assayed in suitable dilutions without pretreatment. Optimum conditions included the use of low-pressure sample injection (6 s), sample and standard solutions with a molarity less than that of the separation buffer, and adjustment of the buffer molarity to obtain a current of 50 A at a constant 15kV separation voltage. After each six runs or less, the inlet and outlet buffer were replaced with more buffer from the same batch. When the method was applied to a sample of SI levels of 5–10 mg/ml were found for each of the mono and diiodo impurities. The optimum method showed a precision (peak area measurement) in the range 1.7–7.2% RSD, depending on the concentration. A linear correlation coefficient of 0.997 was obtained over a DTZA concentration range of 5–60 mg/mL.     

A Validated, Specific, Stability-Indicating RP-LC Method for Analysis of Gatifloxacin in the Presence of Degradation Products and Process-Related Impurities

A validated, specific, stability-indicating reversed-phase liquid chromatographic method has been developed for quantitative analysis of gatifloxacin, its degradation products, and its process-related impurities in bulk samples and in pharmaceutical dosage forms. Forced degradation of gatifloxacin bulk sample was conducted in accordance with ICH guidelines. Acidic, basic, neutral, and oxidative hydrolysis, thermal stress, and photolytic degradation were used to assess the stability-indicating power of the method. Substantial degradation was observed during oxidative hydrolysis. No degradation was observed under the other stress conditions. The method was optimized using samples generated by forced degradation and sample solution spiked with impurities. Good resolution of the analyte peak from peaks corresponding to process-related impurities and degradation products was achieved on a C₁₈ column by use of a simple linear mobile-phase gradient prepared from mixtures of acetonitrile and an aqueous solution of sodium dihydrogen orthophosphate dihydrate and triethylamine adjusted to pH 6.5 with orthophosphoric acid. Detection was performed at 240 nm. Limits of detection and quantification were established for gatifloxacin and its process-related impurities. When the stressed test solutions were assayed by comparison with gatifloxacin working standard the mass balance was always close to 99.3%, indicating the method was stability-indicating. Validation of the method was performed in accordance with ICH requirements. The method was found to be suitable for checking the quality of bulk samples of gatifloxacin at the time of batch release and also during storage.     

Capillary zone electrophoresis method for the determination of famotidine and related impurities in pharmaceuticals

A simple and rapid capillary zone electrophoresis (CZE) method with UV detection has been developed for the determination of famotidine and its potential impurities in pharmaceutical formulations. The electrophoretic separation of these compounds was performed using a fused silica capillary and 37.5mmolL(-1) phosphate buffer pH 3.5 as the electrolyte. Under the optimised conditions, six impurities could be resolved from the famotidine peak in less than 7min. The calibration curves obtained for the seven compounds were linear over the concentration range investigated (from 1.5 to 78.5microg mL(-1)). The intra- and inter-day relative standard deviations for the migration times and corrected peak areas were less than 2% and 5%, respectively. The detection limits were found to be 0.09microg mL(-1) for famotidine, and from 0.1 to 0.62microg mL(-1) depending on the impurities. The method has been successfully applied to the determination of famotidine in commercial dosage forms.     

Reversed phase ion-pair high performance liquid chromatographic gradient separation of related impurities in 2,4-disulfonic acid benzaldehyde di-sodium salt

A reversed phase ion-pair gradient liquid chromatographic method has been developed and validated for purity determination of the hydrophilic compound 2,4-disulfonic acid benzaldehyde di-sodium salt (2,4-DSAD) containing both hydrophilic and more lipophilic related impurities. Mixtures of acetonitrile-phosphate buffer containing tetrahexylammonium hydrogen sulfate as the ion-pairing reagent were used as the mobile phase. A linear gradient, which generated simultaneous change in the concentration of organic modifier, buffer concentration and the concentration of ion-pairing reagent, was applied. The method allows detection of impurities at low levels (0.01% w/w). Excellent repeatability for both retention time (RSD< or =0.3%, n = 6) and detector response (RSD = 0.03%, n = 6 for the main peak and RSD = 6%, n = 6, for an impurity at 0.01 area% level) was obtained. The method was shown to be robust for routine analysis and has been successfully transferred to the quality control laboratory.     

A Validated, Specific Stability-Indicating RP-LC Method for Moxifloxacin and Its Related Substances

A validated, specific, stability-indicating reversed-phase liquid chromatographic method has been developed for quantitative analysis of moxifloxacin and its related substances in bulk samples and pharmaceutical dosage forms in the presence of degradation products and process-related impurities. Forced degradation studies were performed on bulk samples of moxifloxacin, in accordance with ICH guidelines, using acidic, basic, and oxidizing conditions, and thermal and photolytic stress, to show the stability-indicating power of the method. Significant degradation was caused by oxidative stress and by basic conditions; no degradation was observed under the other stress conditions. The method was optimized by analysis of the samples generated during the forced degradation studies and sample solutions spiked with the impurities. Good resolution between the analyte peak and peaks corresponding to process-related impurities and degradation products was achieved on a C₁₈ column with a simple linear mobile phase gradient prepared from aqueous sodium dihydrogen orthophosphate dihydrate containing triethylamine, pH adjusted to 3.0 with orthophosphoric acid, and methanol. Detection was performed at 240 nm. Limits of detection and quantification were established for moxifloxacin and its process related impurities. When the stressed test solutions were assayed against moxifloxacin working standard solution the mass balance was always between 99.3 and 100.1%, indicating the method was stability-indicating. The method was validated in accordance with ICH guidelines, and found to be suitable for checking the quality of bulk samples of moxifloxacin at the time of release of a batch and during storage (long term and accelerated stability testing was conducted).     

Synergistic extraction and spectrophotometric determination of plutonium(VI)

A rapid method for the synergistic extraction and spectrophotometric determination of plutonium(VI) in milligram amounts using a mixture of thenoyltrifluoroacetone (TTA) and tributyl phosphate (TBP) in benzene has been developed. Quantitative extraction is obtained from aqueous solutions of pH=2 affording separation from many commonly occurring impurities, viz., thorium, fission products and cladding materials. The precision and accuracy of the method has been found to be∼1%. The study of the solid product isolated from the extraction system indicates the stoichiometry of the extracted species as PuO₂(TTA)₂ TBP.     

A simple isocratic LC method for quantification of trace-level inorganic degradation impurities (ferricyanide,ferrocyanide, nitrite,and nitrate) in sodium nitroprusside injection and robustness by quality using design approach

This study developed and validated a trace-level quantification inorganic impurities method using reversed-phase HPLC and performed the robustness check using quality-by-design approach by varying the multiple factors simultaneously. This method is economical and simple and exhibits its stability-indicating nature [for the determination of ferrocyanide ([Fe(CN)₆]^4–), ferricyanide ([Fe(CN)₆]³⁻), nitrate (NO₃^–), and nitrite (NO₂^–)] in sodium nitroprusside (SNP) drug substance and liquid dosage form. Chromatographic separation was achieved using a USP L43 column (ACE PFP, 150 × 4.6 mm, 3 μm) with a simple isocratic elution. The buffer consists of potassium dihydrogen phosphate (50 mM), tetrabutylammonium hydrogen sulfate (9 mM), and tetrabutylammonium hydroxide (25 mM). The buffer pH was adjusted to 7.2 with tetrabutylammonium hydroxide. The mobile phase was mixed with the buffer and acetonitrile (68:32 v/v). The flow rate was 0.8 mL/min, column temperature was maintained at 30°C, and injection volume was 5.0 μL. The SNP impurities were monitored at 225 nm using a UV detector. Further, the method was validated per the International Council for Harmonisation (ICH) guidelines, and forced degradation studies were carried out under different stress conditions. The detector responses were plotted against concentrations, and correlation was linear (r > 0.999) over the range of 0.8–7.5 μg/mL for ferricyanide; 1.0–37.5 μg/mL for SNP; and 0.2–7.5 μg/mL for ferrocyanide, nitrite, and nitrate. The method repeatability was established for all the impurities with relative standard deviation (%), and the results were found to be less than 2.0.     

Quantification of immunoglobulin G and characterization of process related impurities using coupled Protein A and size exclusion high performance liquid chromatography

The present work describes two HPLC-UV methods for multi-protein quantification using (i) only a Protein A sensor cartridge (Protein A HPLC) and (ii) the same Protein A cartridge in combination with a size exclusion HPLC column (PSEC-HPLC). The possibility to simultaneously quantify immunoglobulin G (IgG) besides a non-binding protein such as bovine serum albumin (BSA) increases the applicability of Protein A HPLC. Its most pronounced feature is its independence of the buffer system, pH-value and salt content of the investigated sample solvent, which includes cell media. A comparison with the state-of-the-art, the photometrical Bradford method, shows that Protein A HPLC is as sensitive as Bradford, but that it comes with an extended linear range of 4 orders of magnitude, ranging from 0.15 [μg abs] to 1 [mg abs] absolute injected protein amount. The applicability of the PSEC-HPLC method is demonstrated for the analysis of real cell culture feed samples. While Protein A binds IgG, the SEC-column distributes the feed impurities by their molecular weight. The peak area ratios of IgG and the feed impurities of interest are then plotted against the collected sample fraction. These Protein A-Size-Exclusion-Chromatographic diagrams (PSEC-plot) combine the performance information of feed impurities and IgG in a single plot. Further it is shown that both methods are suitable for the performance evaluation of antibody purification media using static as well as dynamic binding experiments performed on DEAE-Fractogel and Capto Adhere. The investigated test samples were “mock” protein solutions with increasing complexity ranging from simple PBS buffer to serum free cell media and “real” cell culture feed solutions.     

A new capillary electrophoresis buffer for determining organic and inorganic anions in electroplating bath with surfactant additives

Monitoring of trace impurities in electroplating bath is needed to meet EU requirements for WEEE and RoHS and for quality control of electrodeposits. Methods using IC and 100% aqueous CE buffer were found producing non-repeatable results attributed to interference of surfactants and major methanesulphonate anion. A new CE buffer containing 1.5 mM tetraethylenepentaamine, 3 mM 1,3,5-benzenetricarboxylic acid and 15 mM Tris in 20% (v/v) methanol at pH = 8.4 was shown to enhance the separation window, reduce interaction between buffer and bath constituents, and give satisfactory repeatability with baseline separation for 14 organic and inorganic anions within 14 min, good repeatability for migration time (0.32–0.57% RSD), satisfactory peak area and peak height (2.9–4.5 and 3–4.7% respectively), low detection limit (S/N = 2, 20–150 ppb), and wide working ranges (0.1–100 ppm). The CE buffer with 20% (v/v) methanol has demonstrated its capability for identifying anion impurities causing problem in aged tin bath and the use of only 10-fold dilution to produce reliable results for quality assessment in plating bath containing high surfactant additives.     

Analysis of process impurities in the basic drug SB-253149 using capillary electrophoresis and on-line mass spectrometric detection

Capillary electrophoresis with on-line electrospray ionisation mass spectrometry (CE/ESI-MS) has been used to identify process impurities in a batch of the anti-atherosclerotic drug, SB-253149. The impurities were separated from the main drug compound by capillary electrophoresis (CE) using an ammonium formate buffer at low pH in an untreated fused silica capillary. The CE method was initially developed using UV as the detection mode and then later structural elucidation work was achieved using an ion trap mass spectrometer. To maintain peak resolution and peak shape when the CE system was coupled to the mass spectrometer, a modified capillary cassette linked to a coaxial sheath flow electrospray ionisation (ESI) interface was used. By performing MS/MS experiments in conjunction with chemical knowledge of the reactivities of SB-253149, it was possible to propose molecular structures for impurities detected in the batch of SB-253149. The results from this study revealed that most of the process impurities in SB-253149 were dimeric derivatives of the parent molecule as well as trace levels of the starting material. This type of information was vital in process control and optimisation for the synthetic route for this drug.     

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C₁₈ 250 × 4.6 mm column, 5 μm (1.0 ml min⁻¹, 280 nm, 50 μl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.     

Neutron activation analysis of PbxSn1-xTe using extraction chromatography

A simple procedure of neutron activation analysis for the determination of 16 impurities in Pb_xSn_1–xTe with detection limits from 1×10^–4% for Ni and Zr to 2×10^–9% for Sc has been developed. The procedure is based on extraction chromatographic separation of impurities from the irradiated sample.     

Simultaneous Determination of Metformin Hydrochloride, Cyanoguanidine and Melamine in Tablets by Mixed-Mode HILIC

A simple and specific hydrophilic interaction liquid chromatography (HILIC) procedure for the quantification of metformin hydrochloride (MFH) and its impurities in bulk pharmaceuticals and finished dosage forms has been developed. The method is based on hydrophilic interaction of the analytes with silica. The influence of the weaker solvent, acetonitrile, pH and the nature and ionic strength of the buffer was studied. Linearity range and percent recoveries for MFH were 100–400 μg mL⁻¹ and 100.62%, respectively. Good validation data were obtained for all compounds. The method separates impurities cyanoguanidine (CGD), melamine (MLN) and other degradation products with a run time of less than 13 min. Degradation studies involved thermal stress, hydrolysis at various pHs and chemical and photolytic oxidation.     

A Stability Indicating LC Method for Deferasirox in Bulk Drugs and Pharmaceutical Dosage Forms

A novel, sensitive, stability indicating RP-LC method has been developed for the quantitative determination of deferasirox, its related impurities in both bulk drugs and pharmaceutical dosage forms. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in an isocratic mode and quantitation was by ultraviolet detection at 245 nm. The mobile phase consisted of buffer, acetonitrile and methanol (50:45:5, v/v) delivered at a flow rate of 1.0 mL min^?1. Buffer consisted of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 3.0 by using orthophosphoric acid. In the developed LC method the resolution (R _s ) between deferasirox and its four potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.999 for deferasirox and its four impurities. This method was capable to detect all four impurities of deferasirox at a level of 0.002% with respect to test concentration of 0.5 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precision values for all four impurities and for deferasirox was found to be within 2.0% RSD. The method showed good and consistent recoveries for deferasirox in bulk drugs (98.3–101.1%), pharmaceutical dosage forms (100.2–103.1%) and for its all the four impurities (99.7–102.1%). The test solution was found to be stable in methanol for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid stress hydrolysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.95%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

On-line extraction and determination of carbofuran in raw milk by direct HPLC injection on an ISRP column

Summary A method has been developed for extraction and determination of carbofuran in milk. The method involved direct injection of raw milk on to a human serum albumin dimethyloctyl-silica gel (HSA-C₈) column and the use of 80:20 (v/v) 0.01 M phosphate buffer pH 5.5-acetonitrile as mobile phase. UV spectrophotometric detection was performed at 220 nm. Identification was based on retention time. Quantification was performed by automatic peak-area determination and was calibrated by use of an external standard.     

Stress Degradation Behavior of Entacapone and Development of LC Stability-Indicating Related Substances and Assay Method

A new, sensitive, stability indicating gradient RP-LC related substances and assay method has been developed for the quantitative determination of entacapone in bulk drugs. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination of buffer and acetonitrile. Buffer consisted of 0.1% orthophosphoric acid, delivered in a gradient mode and quantitation was carried out using ultraviolet detection at 220 nm with a flow rate of 1.5 mL min⁻¹. In the developed LC method the resolution (R _s ) between entacapone and its three potential process impurities were found to be >2.0. Regression analysis showed an r ² value (correlation coefficient) >0.99 for entacapone and its three potential impurities. This method was capable to detect all three process impurities of entacapone at a level of 0.003% with respect to test concentration of 0.5 mg mL⁻¹ for a 20 μL injection volume. The inter- and intra-day precision values for all three impurities and for entacapone was found to be within 2.0% RSD. The method has shown good and consistent recoveries for entacapone in bulk drugs (99.2–101.5%) and its three impurities (99.5–102.2%). The test solution was found to be stable in diluent for 48 h. The drug substances were subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid stress, base stress and oxidative conditions. The stressed test solutions were assayed against the qualified working standard of entacapone and the mass balance in each case was close to 99.7% indicating that the developed method was stability-indicating. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

     

Stress Degradation Behavior of Entacapone and Development of LC Stability-Indicating Related Substances and Assay Method

A new, sensitive, stability indicating gradient RP-LC related substances and assay method has been developed for the quantitative determination of entacapone in bulk drugs. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination of buffer and acetonitrile. Buffer consisted of 0.1% orthophosphoric acid, delivered in a gradient mode and quantitation was carried out using ultraviolet detection at 220 nm with a flow rate of 1.5 mL min^?1. In the developed LC method the resolution (R _s ) between entacapone and its three potential process impurities were found to be >2.0. Regression analysis showed an r ² value (correlation coefficient) >0.99 for entacapone and its three potential impurities. This method was capable to detect all three process impurities of entacapone at a level of 0.003% with respect to test concentration of 0.5 mg mL^?1 for a 20 μL injection volume. The inter- and intra-day precision values for all three impurities and for entacapone was found to be within 2.0% RSD. The method has shown good and consistent recoveries for entacapone in bulk drugs (99.2–101.5%) and its three impurities (99.5–102.2%). The test solution was found to be stable in diluent for 48 h. The drug substances were subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid stress, base stress and oxidative conditions. The stressed test solutions were assayed against the qualified working standard of entacapone and the mass balance in each case was close to 99.7% indicating that the developed method was stability-indicating. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

A Stability-Indicating LC Method for Analysis of Balsalazide Disodium in the Bulk Drug and in Pharmaceutical Dosage Forms

A novel, sensitive, stability-indicating gradient RP-LC method has been developed for quantitative analysis of balsalazide disodium and its related impurities both in the bulk drug and in pharmaceutical dosage forms. Efficient chromatographic separation was achieved on a C₁₈ stationary phase with a simple mobile-phase gradient prepared from methanol and phosphate buffer (10 mm potassium dihydrogen orthophosphate monohydrate, adjusted to pH 2.5 by addition of orthophosphoric acid). The mobile-phase flow rate was 1.0 mL min^?1. Quantification was achieved by use of ultraviolet detection at 240 nm. Under these conditions resolution of balsalazide disodium from its three potential impurities was greater than 2.0. Regression analysis resulted in a correlation coefficient greater than 0.99 for balsalazide disodium and all three impurities. This method was capable of detecting the three impurities at 0.003% of the test concentration of 0.3 mg mL^?1, using an injection volume of 10 μL. Inter-day and intra-day precision for all three impurities and for balsalazide disodium was within 2.0% RSD. Recovery of balsalazide disodium from the bulk drug (99.2–101.5%) and from pharmaceutical dosage forms (99.8–101.3%), and recovery of the three impurities (99.1–102.1%) was consistently good. The test solution was found to be stable in 70:30 (v/v) methanol–water for 48 h. When the drug was subjected to hydrolytic, oxidative, photolytic, and thermal stress, acidic and alkaline hydrolysis and oxidizing conditions led to substantial degradation. The RP-LC method was validated for linearity, accuracy, precision, and robustness.