The Study And Application On Assay Of Major Nutrients In Amino Acid-based Enteral Nutrition Powder XNX

Enteral nutritions Pharmaceuticals are composed a variety of nutrient. The study to assay the content of major nutrients can provide data for quality control. It can support the declaring, marketing, testing and clinical applications of the enteral nutritions.Based on the composition of major nutrients in XNX powders, such as amino acids, vitamins and trace elements, the relevant methods were established for the determination of quantitation of these nutrients. The work was divided into the following three parts:1. The method for the determination of amino acids in XNX powdersAmino acid-based enteral nutrition powder XNX contains19kinds of essential and non-essential amino acids. According to the structure of different amino acids, two methods were established to determine the contents of amino acids. Using O-phthalaldehyde as a derivatization reagent, the method of pre-column derivatization-high performance liquid chromatography was established to determine18kinds of amino acids. An Agilent C18g column (150×4.6mm,5.0μm) was used with a mobile phase consisting of methanol-sodium acetate (pH5.6,50mmol/L) for gradient elution analysis. The flow rate was1.0mL/min. The column temperature was30℃. The λex of FLD was340nm and the λem was460nm. The eighteen amino acids were firstly ultrasonic extracted by0.1mol/L HC1, then20μL of the sample after derivatization was injected for HPLC analysis. The linear regression coefficients of eighteen amino acids were within the range of0.9988-0.9999. The range of average recoveries was94.12%～113.73%. Both the inter-day RSD and the intra-day RSD were less than10%. The sample solvent was stable in24h. This part of research outcome has already been published in Chinese Journal of New Drugs and Clinical Remedies.Using FMOC-C1as derivatization reagent, the method of pre-column derivatization-high performance liquid chromatography was established to determine proline. An Agilent C18column (150mm×4.6mm,5.0μm) was used with a mobile phase consisting of acetonitrile-sodium acetate (pH4.2,50mmol/L) for gradient elution analysis. The flow rate was1.0mL/min. The column temperature was30℃. The λex of FLD was263nm and the λem was315nm. The proline was ultrasonic extraction by0.1mol/L HC1, then20μL of the sample after derivatization with FMOC-C1for15min at room temperature was injected for HPLC analysis. The linear range of Proline was0.1～1μg·mL-1(r=0.9996). The range of average recoveries was109.42%～112.72%. Both the inter-day RSD and the intra-day RSD were less than7%. The sample solvent was stable in24h. This part of research outcome has already been published in Chinese Journal of Clinical Pharmacy.Two methods above are sensitive, simple, specific, and be able to assay amino acids in XNX powders accurately, avoiding the interferences from other components in pharmaceuticals.2. The method for the determination of vitamins in XNX powdersThe method for determination of vitamin E, vitamin A, vitamin B1, B2, B6, and carnitine were developed in this paper.The method for determination of vitamin E:A Diamonsil C18column (150×4.6mm,5.0μm) was used with a mobile phase consisting of methanol-acetonitrile (95/5, v/v). The flow rate was1.0mL/min. The column temperature was30℃. The λex of FLD was292nm and the λem was340nm. The vitamin E was firstly extracted by95%ethanol, then20μL of the sample after hydrolysis by potassium hydroxide was injected for HPLC analysis. The linear range of vitamin E was0.3～12μg·mL-(r=0.9997). The range of average recoveries was99.83%～108.14%. Both the inter-day RSD and the intra-day RSD were less than9%. The sample solvent was stable in8h. This part of research outcome has already been published in Chinese Journal of Clinical Pharmacy.The method for determination of vitamin A:A Diamonsil C18column (150×4.6mm,5.0μm) was used with a mobile phase consisting of methanol-acetonitrile (95/5, v/v). The flow rate was1.0mL/min. The column temperature was30℃. The λ of VWD was325nm. The vitamin A was firstly ultrasonic extracted by methanol, then20μL of the sample was injected for HPLC analysis. The linear range of vitamin A was32.5-650ng/mL (r=0.999). The range of average recoveries was95.09-96.91%. Both the inter-day RSD and the intra-day RSD were less than8%.The method for determination of vitamin B1&B2:A Diamonsil C18column (150×4.6mm,5.0μm) was used with a mobile phase consisting of methanol-5mM sodium heptanesulfonate, containing0.5%acetic acid and0.1%triethylamine (30/70, v/v). The flow rate was1.0mL/min. The column temperature was30℃. The λ of VWD was260nm. The vitamin B2&B2were firstly ultrasonic extracted by deionized water, then20μL of the sample was injected for HPLC analysis. The linear range of vitamin B1&B2was0.5-4μg/mL (r=0.999&0.991, respectly). The range of average recoveries was98.34-107.68%. Both the inter-day RSD and the intra-day RSD were less than9%.The method for determination of vitamin B6:A Diamonsil Cig column (150×4.6mm,5.0μm) was used with a mobile phase consisting of methanol-5mM sodium heptanesulfonate, containing0.5%acetic acid and0.1%triethylamine (28/72, v/v). The flow rate was1.0mL/min. The column temperature was30℃. The λex of FLD was295nm and the λem was397nm. The vitamin B6was firstly ultrasonic extracted by deionized water, then20μL of the sample was injected for HPLC analysis. The linear range of vitamin B6was0.1-1μg/mL (r=0.9999). The range of average recoveries was98.31-101.0%. Both the inter-day RSD and the intra-day RSD were less than4%.The method for determination of vitamin carnitine:The enzymatic method for the determination of carnitine in XNX powders was established. The carnitine was firstly ultrasonic extracted by deionized water, then determined the absorbance values before and after enzyme reaction by UV spectrophotometer. The linear range of carnitine was4-16μg/mL (r=0.9995). The range of average recoveries was85.84-106.82%.The methods above are sensitive, simple, specific, and be able to assay vitamin E, vitamin A, vitamin B1, B2, B6, and carnitine in XNX powders accurately, avoiding the interferences from other components in pharmaceuticals.3. The method for the determination of trace elements in XNX powdersThe method for determination of metal ions and iodide were developed in this paper.The method for determination of metal ions:The content of copper, iron and zinc in XNX powders were determined by atomic absorption spectrophotometry. The linear regression coefficients of copper, iron and zinc were within the range of0.9928-0.9999. The precision and recoveries of copper, iron and zinc were good. The method is able to determine copper, iron, zinc in XNX powders accurately and quickly.The method for determination of iodide:The method of ion-selective electrode for determination of iodide in XNX powders was established. The XNX powders were damaged at high temperatures, and then the iodide were extracted which was determined by potentiometric method. The linear range of iodide was0.07-0.2ug/mL (r=0.9970). The range of average recoveries was96.5%. The inter-day RSD was less than3%. The method is accurate, sensitive and be able to determine of iodide in XNX powders quickly.