ABSTRACT
@#Objective To develop and verify a high performance liquid chromatography(HPLC) method for the determination of ethylenediaminetetraacetic acid disodium salt(EDTA-2Na) residues in the bulk of NMM tumor DNA vaccine for the quality control of DNA vaccine.Methods After NMM tumor DNA vaccine bulk was complexed with copper sulfate,a HPLC method for the determination of EDTA-2Na residues was developed with Agilent ZORBA XSB-C18(150 mm × 4.6 mm,5 μm) as the chromatographic column,water,tetrabutylammonium hydroxide 10% and acetonitrile solution(74.5:0.5:25)as the mobile phase.The detection method was as follows:the detection wavelength was 254 nm,the flow rate was 0.8 mL/min,the column temperature was 20 ℃ and the injection volume was 20 μL.The method was verified for the specificity,linear range,limit of detection(LOD),limit of quantification(LOQ),solution stability,durability,accuracy and precision,and used to detect the EDTA-2Na residues in several batches of DNA vaccine bulk.Results When EDTA-2Na control and DNA vaccine bulk with EDTA-2Na reacted with copper sulfate,the absorption peak appeared at around 5.3 min,while no absorption peak was observed when DNA vaccine bulk reacted with copper sulfate;In the range of 4～400 μg/mL,the control solution concentration showed a good linear relationship with the peak area,R~2=0.999 9;The LOD of the method was 10 ng/mL,and the LOQ was 40 ng/mL;The solution of control and sample was stable after placed for 12 h;When the detection conditions changed slightly(different mobile phase ratio,flow rate and column temperature),the influence on the detection results was within acceptable range;The average recovery rate of EDTA-2Na in low,medium and high concentration standard added samples was 101.38% with the RSD of 0.39%;0.1 mg/mL control solution was injected continuously for 6 times,and the peak area RSD was 0.04%.EDTA-2Na was not detected in 6 sample solution,and the peak area RSD of DNA vaccine bulk with EDTA-2Na solution was 0.02%,indicating a good intermediate precision.EDTA-2Na residue was not detected in these batches of DNA vaccine bulk.Conclusion The developed method is simple,accurate,reliable with good specificity,which can be used for the determi-nation of EDTA-2Na residues in DNA vaccine bulk.
ABSTRACT
Objective:To establish a headspace GC method for the determination of residual formaldehyde in EDTA-2Na. Meth-ods:A headspace GC was used to separate the residual solvents on an Agilent DB-WAX (30 m×0.32 mm,0.5 μm) capillary col-umn with an FID detector. The carrier gas was nitrogen at the flow rate of 1.0 ml·min-1. The temperature of the injector was 230 ℃and that of the FID was 260 ℃. The programmed column temperature was set as follows: maintained at 40 ℃ for 6 min, and then raised to 230 ℃ at the rate of 40 ℃·min-1and maintained for 10 min. The injection volume was 1μl with the split ratio of 2:1. The reference solvent and sample were filled into the containers of headspace injector and the containers were in equilibrium at 95℃ for 25 min. The amount of residual formaldehyde was calculated by an external standard method.Results:There was a good linear relationship of formaldehyde reference solvent within the concentration range of 5.10-102.00 μg·ml-1(r=0.999 7). The average recovery was 84.60%(RSD=3.82%,n=9). Conclusion: The method is simple,rapid and accurate for the determination of residual formalde-hyde in EDTA-2Na.
ABSTRACT
Objective:To establish an HPLC method for the determination of EDTA-2Na in amphotericin B. Methods: A Waters C18 column(50 mm × 4. 6mm, 5 μm) was used. The mobile phase A was acetic acid solution (1. 5 ml acetic acid was added into 1000ml water, and 41 ml 10% tetrabutylammonium hydroxide solution was added), and the mobile B was acetonitrile with gradient e-lution. The flow rate was 0. 8 ml·min-1 , the column temperature was 30℃, the detection wavelength was 260 nm and the injection volume was 25μl. Results:The results showed that EDTA-2Na in amphotericin B could be detected without any interference. The cal-ibration curve of EDTA-2Na was linear within the range of 0. 92-7. 37μg·ml-1(r=0. 999 9), the LOD was 1. 93 ng·ml-1 and the LOQ was 6. 45 ng·ml-1. The average recovery was 102. 5% (RSD=2. 8%, n=9). Conclusion: The method is simple, selective and accurate. It can be used for the quality control of EDTA-2Na in amphotericin B.
ABSTRACT
Objective: Due to the limited resource and the large demand, many kinds of Bovis Calculus (BC) including artificial Bovis Calculus (ABC), in vivo cultured Bovis Calculus (in vivo CBC), and in vitro cultured Bovis Calculus (in vitro CBC) were used in Chinese patent medicines (CPMs). Previous studies have shown that the chemical constituents of ABC and their properties were different from other BC. The two types of CBC with much higher price than ABC were approximately equivalent with natural Bovis Calculus in quality and clinical effect. The aim of the study is to establish a rapid and effective method for the identification of BC in CPMs. Methods: An HPLC method with the higher specificity for analyzing bilirubin was established to distinguish ABC from other three kinds of BC by comparing the change of bilirubin content with the addition of EDTA-2Na as the extraction solvent and stabilizer. Results: The bilirubin content in CPMs containing ABC was basically unchanged, while that in CPMs containing other kinds of BC showed significant difference. The proposed method was employed to analyze a variety of CPMs containing Bovis Calculus (CPMBCs) and proven to be universal. Conclusion: An effective analytical method is established for the quality control of CPMBCs and further ensures the safety and efficacy of these drugs in clinical practice. © 2013 Tianjin Press of Chinese Herbal Medicines.