Study On The Determination Of Lactoferrin By High Performance Capillary Electrophoresis

The methods for the accurate assay of the minor lactoferrin in milk products and the trace natamycin in jam by high performance capillary electrophorsis (HPCE) and high performance liquid chromatography (HPLC), respectively, were investigated in detail. The thesis consists of the following four sections:(1) A new method for the quantitative determination of lactoferrin (Lf) by HPCE under acid separation conditions was established. The separation was carried out in an uncoated capillary with 100μm i.d. and total length of 37 cm. The detection wavelength was set at 214 nm. The running buffer was composed of 35mmol/L phosphoric acid and 14 g/L poly (ethylene oxide) (PEO) (pH 2.50, adjusted with 1 mol/L CsOH). The addition of PEO in the running buffer not only suppressed analyte adsorption but also increased the resolution significantly. The samples were simply extracted by 50mmol/L sodium dihydrogen phosphate (pH 2.20, adjusted with 1 mol/L H₃PO₄). A linear relationship was established for the lactoferrin concentration in the range from 50 mg/L to 300 mg/L with a linear correlation cofficient (r=0.9986). The limit of detection (LOD) and quantitation (LOQ) were 10 mg/L(S/N=3) and 50 mg/L (S/N=10), respectively. Since the sample must be diluted 1:10 with water before injection. The sensitivity was not high enough for the determination of the minor Lf in real samples. The method was appropriate to analyze tablets and raw drugs with high Lf contents.(2) A new method for the quantitative determination of the Lf by HPCE under basic separation conditions was established. The electrophoretic separation was carried out in an uncoated capillary with 100μm i.d. and total length of 77 cm (70 cm to the detector) with a separation voltage of 30 kV. The running buffer was composed of 100mmol/L boric acid, 10mmol/L sodium dodecyl sulfate (SDS) and 8g/L hydroxyethylcellulose (HEC) (pH 8.80, adjusted with 1 mol/L CsOH). The sample buffer contained 15mmol/L boric acid and 20 mmol/L SDS (pH 8.60, adjusted with 0.25 mol/L LiOH). A linear relationship was established for the Lf concentration in the range from 10 mg/L to 1000 mg/L with a linear correlation cofficient (r=0.9993). The LOD and LOQ were 3 mg/L (S/N=3) and 10 mg/L (S/N=10), respectively. Since the minor Lf could not be efficiently separated from the major whey proteins in milk products under the present separation conditions, the newly established method was suitale for the determination of Lf in tablets, raw drugs and milk products with simple matrix.(3) A new method for the accurate assay of the minor bovine lactoferrin (Lf) in infant formula by HPCE under basic separation conditions was established. The electrophoretic separation was carried out in an uncoated capillary with 50μm i.d. and total length of 57 cm (50 cm to the detector) maintained at 25℃under 30 kV. The running buffer were composed of 300 mmol/L boric acid, 20 mmol/L SDS and 8 g/L HEC (pH 8.80, adjusted with 1 mol/L CsOH). The commercially available Lf-fortified samples were simply extracted by 50 mmol/L acetic acid and then directly injected after centrifugation. The linear range of the method was from 50 to 800 mg/L. The LOD and LOQ were 15 mg/L (S/N=3) and 50 mg/L (S/N=10). The method was successfully applied for the determination of Lf in bovine colostrum products and achieved satisfactory results.(4) To establish an analytical method for the determination of natamycin in jam by reversed-phase HPLC. Natamycin was extracted from jam by methanol-water-acetic acid (20:80:10, v/v/v). The sample was separated at room temperature on a Hypersil ODS₂ C₁₈ column (200 mm×4.0 mm i.d., 5.0μm) with methanol-0.75% phosphoric acid (75:25, v/v) as mobile phase at a flow rate of 1.00 mL/min. Natamycin was detected and quantified by UV absorbance at 304 nm. The linear responses covered the range from 0.5 to 100 mg/kg (r=0.9999) for natamycin. The detection limits (S/N = 3) of natamycin in tomato jam and strawberry jam sample were shown to be 0.06 mg/kg and 0.05 mg/kg. The recoveries were 93.14 % - 98.24 % with relative standard deviations between 0.72 % and 1.49 %. The method is simple and accurate. It can be used for the routine determination of natamycin in some jams.