ABSTRACT
Research on polymer impurities has always been important in the quality control of cephalosporins. Research on polymers in cephalosporins that lack active amino groups on the C-7 side chain has not been reported. Therefore, our study used cefazolin sodium, which is widely used in the clinic, as an example. The polymer in cefazolin sodium and its product "cefazolin sodium pentahydrate for injection" was analyzed by column switching liquid chromatography-high resolution mass spectrometry. Two polymer impurity peaks were detected and the possible structures of these polymers were suggested. Through two-dimensional liquid chromatography, the chromatographic peaks following Sephadex gel chromatography and high-performance gel chromatography were compared to those obtained by reverse high-performance liquid chromatography (HPLC) for cefazolin sodium as reported in the Chinese Pharmacopoeia. The HPLC method proves more suitable for polymer detection than Sephadex gel chromatography and high-performance gel chromatography. The method of polymer detection for cefazolin sodium was established using the method of related substances HPLC as described in the Chinese Pharmacopoeia.
ABSTRACT
Abstract Amino acids (AA) composition in cocoa beans can predict the synthesis of compounds which affect cocoa flavor. Thus, their determination is of great interest for the community implied in the commercialization and production of cocoa. In consequence, in this work, the analysis of AA produced during cocoa beans fermentation and roasting was carried out. A high-performance liquid chromatographic method with DAD detection at 254 nm was optimized and validated for their selective determination in six varieties of cocoa beans with different genotypes, all of them grown in Venezuela. AA were extracted by defatted milled cocoa powder ultrasonication using purified water at 70 °C. Then, they were derivatized with phenyl isothiocyanate, and their derivatives were separated, using a reversed-phase column with gradient elution, achieving a satisfactory resolution among the peaks (greater than 1.0) in less than 29 min. 110 cocoa samples were analyzed. Results showed a significant content of free AA, ranging from 3.87 to 5.97 g/kg in absence of fermentation with a predominance of acidic AA. Moreover, there is a progressive increase in the AA content while fermentation process occurs, with a predominance of hydrophobic AA such as alanine, valine, isoleucine, leucine, phenylalanine, and tyrosine. On the other hand, all cocoa types showed a partial degradation of free AA during the roasting step, especially the hydrophobic ones.
Resumen La determinación de aminoácidos (AA) en granos de cacao es de gran interés ya que estos son considerados como unos de los precursores de su sabor y aroma. Por esta razón, el presente trabajo tuvo como objetivo optimizar y validar un método por cromatografía líquida con detección DAD a 254 nm para la determinación selectiva de AA durante la fermentación y tostado en seis variedades de granos de cacao con diferentes genotipos, todos estos cultivados en Venezuela. Los AA se extrajeron del polvo de cacao molido y desgrasado con agua pura a 70 ºC, utilizando la técnica de ultrasonido. Luego, se derivatizaron con fenilotiocianato para separar sus derivados con buena resolución en menos de 29 min en una columna de fase reversa, utilizando gradiente de elución. Se analizaron 110 muestras de cacao. Los resultados mostraron un contenido significativo de AA libres, entre 3,87 y 5,97 g/kg, en ausencia de fermentación con predominio de AA ácidos, y un aumento progresivo en el contenido de AA, mientras ocurre el proceso de fermentación, con un predominio de AA hidrófobos como alanina, valina, isoleucina, leucina, fenilalanina y tirosina. Además, todos los tipos de cacao mostraron una degradación parcial de AA libres durante la etapa de tostado, especialmente los AA hidrófobos.
Resumo A determinação dos aminoácidos (AA) nos grãos de cacau é importante, pois são considerados um dos precursores de seu sabor e aroma. Neste trabalho, um método foi otimizado e validado por cromatografia líquida com detecção DAD a 254 nm para a determinação seletiva de AA durante a fermentação e torrefação em seis variedades de grãos de cacau com diferentes genótipos, todos cultivados na Venezuela. Os AAs foram extraídos do pó de cacau moído e desengordurados com água pura a 70 ºC usando a técnica de ultrassom. Em seguida, foram derivatizados com feniltiocianato, e os derivados foram separados com boa resolução em menos de 29 minutos em uma coluna de fase invertida usando eluição em gradiente. Foram analisadas 110 amostras de cacau. Os resultados mostraram um conteúdo significativo de AA livre entre 3,87 e 5,97 g/kg na ausência de fermentação com predominância de AA ácidos e um aumento progressivo no conteúdo de AA enquanto o processo de fermentação ocorre com predominância de AA hidrófobos como alanina, valina, isoleucina, leucina, fenilalanina e tirosina. Além disso, todos os tipos de cacau apresentaram uma degradação parcial do AA livre durante a fase de torrefação, principalmente o AA hidrofóbico.
ABSTRACT
To establish a method for the determination of polymer impurities in cefixime raw materials and preparations, a cefixime degradation solution containing polymer impurities was prepared by forced polymerization. Polymer impurities in the degradation solution were separated and identified by high performance gel chromatography and the column switching-LC-MSn method. A new RP-HPLC method for cefixime polymer was established and validated with a Phenomenex Gemini-C18 column using a mobile phase gradient elution of 0.5% formic acid-water solution and 0.5% formic acid-acetonitrile solution. The results showed that when using this high performance gel chromatography method some small molecular weight impurities were co-eluted with the polymers, resulting in a poor specificity and poor quantitative accuracy. But when using the RP-HPLC method, three polymer impurities were detected with good specificity, sensitivity and robustness, including two cefixime dimers, and dehydrate dimer. Therefore, the described RP-HPLC method is suitable for the quality control of polymer impurities in cefixime, and cefixime degradation solution can be used as suitable solution for analysis of cefixime polymers.
ABSTRACT
OBJECTIVE: To characterize the impurity structures in vancomycin raw material. METHODS: Impurities were eluted gradiently on a Chromasil 100-5 C18(4.6 mm×250 mm, 5 μm) column, with triethylamine buffer solution (pH 3.2)-acetonitrile-tetrahydrofuran (92∶7∶1, V/V/V) as mobile phase A, and triethylamine buffer solution (pH 3.2)-acetonitrile-tetrahydrofuran (70∶29∶1, V/V/V) as mobile phase B. Stress tests were performed on the raw material to specify those impurities. By application of on-line LC/MSn method, the impurities were analyzed in positive mode and their structures were characterized based on the degradation mechanism and mass fragmentation regularity of sugar-lipopeptides. RESULTS: Totally 14 impurities were characterized in the raw material, seven of which were reported for the first time. The relationships between the chemical structures and chromatographic behaviors of vancomycin, demethylvancomycin and methylated vancomycin with their two β-isomers were summarized. CONCLUSION: The structures of related impurities in vancomycin raw material can be rapidly identified by on-line LC/MSn method together with stress degradation experiments.
ABSTRACT
OBJECTIVE:To establish the method for the determination of doxofylline in human serum. METHODS:Online two-dimensional column switching-HPLC was adopted to determine the concentration of doxofylline in human serum. First-dimensional chromatographic column was Waters C18column,and middle column was SC2. First-dimensional and second-dimensional column mobile phrase were methanol-water(70 : 30,V/V). The detection wavelength was 273 nm,and column temperature was 40 ℃. Sample volume was 10 μL. RESULTS:The linear range of doxofylline were 0.5-50.00 μg/mL(r=0.999 9), and the detection limit was 0.01 μg/mL. The quantitative limit was 0.5 μg/mL. RSDs of intra-day and inter-day were 1.51%-1.89%and 1.52%-1.92%(n=5). The accuracy were 97.91%-104.19%(n=5). Extraction recoveries rate were 91.63%-93.44%(RSD<2.00%,n=3). RSD of matrix effect were lower than or equal to 3.01%(n=6),and RSD of stability test was lower than 5.00%(n=6). After 3 patients were given intravenous injection of Doxofylline and glucose injection(0.3 g/d)up to steady state,the serum concentrations of doxofylline were 3.23,3.35,3.68 μg/mL before medication on the next day(RSD=2.28%,2.34%, 2.14%,n=5). CONCLUSIONS:The method is simple,rapid,accurate and suitable for the determination of plasma concentration of doxofylline.
ABSTRACT
OBJECTIVE: To study the impurity profile of cefradine dihydrate. METHODS: The impurity peaks of cefradine dihydrate were identified using column-switching technique and LC-MS, and the origination of the impurities were investigated by the forced degradation study. RESULTS: The impurity profile of cefradine dihydrate included fourteen impurities, nine of them were structure known impurities controlled by EP, two of them were newly identified impurities and three of them were structure unknown impurities. CONCLUSION: The origination and influence factors of the impurities are investigated though the impurity profile study of cefradine dihydrate, and it can provide reference for the quality control of cefradine dihydrate.
ABSTRACT
A new analytical method has been developed for the simultaneous determination of CrⅢand CrⅥusing on-line sample pretreatment valve-switching ion chromatography. The organic matrix in leather was removed by using a reverse-phase column as the pretreatment column. Before injection, EDTA was added into sample solution to react with the CrⅢto form anion which could absorb visible light strongly. After injection, the ions separated by the pretreatment column were received in a collection loop. Then the ions were delivered into an analytical column and separated. CrⅥ then was derived with the derivatization reagent 1, 5-diphenylcarbazide ( DPC) , and detected together with CrⅢ-EDTA complex by a UV-Vis detector. Under the optimum conditions, the linear range of the method for CrⅢ and CrⅥ was 0. 3-10 mg/L (r=0. 9991) and 0. 05-2 mg/L ( r = 0. 9992 ), whereas detection limits ( S/N = 3 ) were 80. 78 μg/L and 6. 67 μg/L, respectively. The recoveries were in the range of 88. 7%-108. 5% with the relative standard deviations for retention time and peak area less than 3%. The method could be applied to determine CrⅢ and CrⅥ in leather and cloth effectively and quickly.
ABSTRACT
OBJECTIVE: To establish a rapid column-switching-ultra high performance liquid chromatography method for determining meropenem concentration in human plasma. METHODS: Meropenem plasma samples were diluted by internal standard aqueous solution (doxofylline), filtrated by microporous membrane, and then extracted by on-line column-switching method. The samples were chromatographed on Waters Acquity UPLC@BEH-C18 column using gradient elution method (mobile phase A and extracting mobile phase C: methanol -0.05 mol·L-1 K2HPO4 (pH 7.0) 5:95; mobile phase B: methanol). The maximum absorption wavelength of meropenem was 299 nm. The column temperature were 35°C. The meropenem concentration was calculated by internal standard method and external standard method, respectively. The results of the two methods were compared by paired t-test using SPSS statistical software. RESULTS: The liner range of the calibration curve for meropenem was 0.67-86.00 mg·L-1 (r=0.9999). The method recovery was (93.59±1.15)%-(99.21±4.06)% and extraction recovery was above 85.0%, relative standard deviation was below 5.0%. The SPSS statistic results indicated there was no significant difference between the concentrations calculated by the two methods and existed good correlation. The transformed equation is ρInternal=1.039ρExternal-0.468 (r=0.9970). CONCLUSION: On-line column-switching method is used and it is proved to be rapid, sensitive and suitable for therapeutic drug monitoring and pharmacokinetic investigation of meropenem in human plasma.
ABSTRACT
AIM: To establish a method using HPLC with column switching for determination of Danshensu in human blood serums. RESULTS: A linearity range of 0.0248～0.3720?g was obtained. The recovery was 99.2%. CONCLUSION: The method is simple, sensitive and suitable for pharmacoknetics studies and clinical detection of Compound Danshen Dripping Pills