ABSTRACT
Objective To establish a method for simulataneous determination of 18 free amino acids in snake venom. Methods Preparation of free amino acid samples by membrane. HPLC analysis was performed after derivatization by using phenyl isothiocyanate (PITC) as a derivative reagent, samples were analyzed on Ultimate LP-C18 column with gradient elution column of 0.05 mol/L sodium acetate buffer and methanol-acetonitrile- water (40:40:20), and current speed was1.0 mL/min, and the column temperature was set at 35℃, and detection wavelength was 254nm. Results The 18 kinds of amino acids showed a good linearity with the correlation coefficients ≥0.99. The recovery rate was 74.59%~110.62%. Snake venom contained 17 kinds of amino acids, the total content of amino acids was 0.2%. Conclusion The method was accurate, reproducible and reliable, and can be used for the determination of amino acids in snake venom and related products.
ABSTRACT
Objective To develop a HPLC method for determining 14 hydrolyzed amino acids in Pfaf fia .Methods The sample was derivatized with phenyl isothiocyanate (PITC) .Amino acids were separated on Waters XBridge Shield RP18 (4.6 mm × 250 mm ,5 μm) column at the flow rate of 0.8 ml/min ,detected at 254 nm .The column temperature was 25 ℃ . Results The response was linear for 14 amino acids with a correlation coefficient r>0 .9990 .The average recoveries (n=6) were 90 .2%-105 .1% .Amino acids derivative solution remained stable in 24 hours .Conclusion This well-established method is very reliable .It can be used as a quantitative determination method for 14 amino acids in Pfaf fia .
ABSTRACT
Aim: The aim of the present investigation was to perform the precolumn derivatization of Amantadine Hydrochloride (AMT) with phenylisothiocyanate and to develop a RP-HPLCPDA method for the quantification of Amantadine Hydrochloride-phenylisothiocyanate (AMT-PITC) complex in bulk and dosage forms which is rapid, sensitive and economical. Study Design: Method development and Validation study. Methodology: A Phenomenex C18 RP column of 250 x 4.6mm dimensions and 5μm particle size with mobile phase containing water and acetonitrile (40:60% v/v) was used at isocratic mode binary pump and eluent was monitored at 273nm. Results & Discussion: The retention time of AMT-PITC complex was 6.3 min. The developed method showed a good linearity in the concentration range of 10-50μg/mL with a correlation coefficient >0.998. The recoveries ranged between 95-105% with a Relative Standard Deviation of (RSD) < 2%. Conclusion: The developed method was validated as per ICH guidelines and successfully used for quantification of AMT by derivatization with PITC. The method was found to be rapid, specific and accurate.
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.
ABSTRACT
Objective: To establish a RP-HPLC method for the simultaneous determination of 13 amino acids in Asini Corii Colla. Methods: Asini Corii Colla was hydrolyzed for 3 h at 120°C using 6 mol/L hydrochloric acid, and the amino acids were determined by RP-HPLC after being derived with phenyl isothiocyanate. HPLC was performed on an Eclipse AAA column (75 mm × 4.6 mm, 3.5 μm) with acetonitrile-110 mmol/L ammonium acetate solution (1:99) as mobile phase A and acetic acid-acetonitrile-water (0.05:80:20) as mobile phase B in the gradient mode at a flow rate of 0.6 mL/min. The column temperature was 42°C, and the detection wavelength was set at 254 nm. Results: The 13 amino acids were separated within 36 min with a good linearity (r > 0.9977) in the range of the test concentration. The average recoveries (n = 3) were 95.2%-104.4% and the RSD values were less than 5.0%. Conclusion: The method is simple, accurate, and repeatable, which could be available for the quality control of Asini Corii Colla. It may also serve as a good reference for the determination of amino acids in other pharmaceuticals.