ABSTRACT
@#TA method for the content determination of methionine sulfoxide and methionine sulfone in compound amino acid injection (18AA-II) was established in order to investigate their level in 155 batches of this product, and to explore the reason for the generation of these two impurities.The determination was performed on an Agilent Poroshell 120 EC-C18 column with mobile phases of sodium acetate/tetrahydrofuran solution (A) and sodium acetate solution -acetonitrile-methanol (B, 200∶400∶400) (gradient elution) at the flow rate of 0.5 mL/min.The excitation wavelength and the emission wavelength of the fluorescence detector were 233 nm and 441 nm, respectively.The column temperature was 40 °C, and the injection volume was 8 μL.The contents of methionine sulfoxide and methionine sulfone from 155 batches of compound amino acid injection (18AA-II) was determined using this method, and the residual oxygen content was detected by headspace gas analyzer.The results showed that the linear range of methionine sulfoxide and methionine sulfone were 0.128 1-10.250 0 μg/mL (r = 0.999 9) and 0.261 0-10.440 0 μg/mL (r = 0.999 8), respectively.The limits of quantitation were 0.13 μg/mL and 0.26 μg/mL, respectively; the limits of detection were 0.04 μg/mL and 0.09 μg/mL, respectively.RSDs of precision, stability and repetitive test were all lower than 1.3%.The recoveries ranged 98.00%-100.79% (RSD = 1.15%, n = 9) and 98.19%-102.31% (RSD = 1.33%, n = 9).The content level of oxidized related substances from different manufacturers showed significant difference, showing relevance with the residual oxygen content to some extent, yet no significant correlation with the added amount of antioxygen (sodium pyrosulfite).The method is validated to be useful for the content control of methionine sulfoxide and methionine sulfone in compound amino acid injection (18AA-II).It is quite necessary to include the determination of oxidized related substance into the quality specification.Manufacturers should strengthen the control of remaining oxygen in their products.
ABSTRACT
Objective To establish a method for HPLC to determine content of acetyl tyrosine in pediatric compound amino acid injection (19AA-Ⅰ).Methods The chromatographic separation was achieved on a Agilent Hypersil ODS column (250 mm ×4.0 mm,5 μm) with Agilent 1200 liquid chromatography system.The mobile phases consisted of 20 mmol/L sodium dihydrogen phosphate solution ( adjusting pH to 2.5 with phosphoric acid)-acetonitrile (90:10) at a flow rate of 1.0 mL/min, the detection wavelength was 210 nm, the column temperature was 25℃.Results Acetyl tyrosine was completely separated from other amino acids.The calibration curves for acetyl tyrosine revealed good linearity in the range of 12.062-120.62μg/mL (r=0.9999).The average recoveries (n=9) of acetyl tyrosine was 100.0%, RSD% (n=9) was 0.9.The limits of quantification (S/N=10) was 0.15μg/mL.Conclusion The methodological validation results indicate that the established method can be applied to quality control of acetyl tyrosine in pediatric compound amino acid injection (19AA-Ⅰ).
ABSTRACT
An efficient and sensitive analytical method for the simultaneous content determination of 18 amino acids in compound amino acid injection was developed using high performance liquid chromatography (HPLC) with pre-column derivatization. Phenyl isothiocyanate (PITC) was used as derivatization reagent. The target compounds were separated on a Unitary-C18 column (250 mm í 4.6 mm, 5 μm) in gradient elution mode using sodium acetate and the mixture of acetonitrile, methanol and water as mobile phases. The detection wavelength was 254 nm. The derivatization reagent dosage, derivatization time, salt concentration, the pH and the column temperature of mobile phase were investigated. Finally, 18 amino acids were separated within 40 minutes. The method showed that the good linearity (r2 ≥ 0.997 7) was at a range from 9 μg·mL-1 to 1 021 μg·mL-1. The recoveries ranged from 92.6% to 110.7%. And the relative standard deviations (RSD) ranged from 0.01% to 5.68%. The limits of quantification (LOQ, S/N = 10) ranged from 0.02 μg·mL-1 to 13.41 μg·mL-1. This method, which was simple, sensitive and accurate, can be applied for the content determination of amino acids in compound amino acid injections.