Application Of HPLC Detection To Pharmaceutical And Pesticide Samples

At present, high performance liquid chromatography (HPLC) has mostly been used to enhance the selectivity of analytical methods, because of its high efficiency for separation of analyses from various interfering substances. One of the major research areas about HPLC involves the development of sensitive detection methods. Significantly more sensitive detection can be realized by HPLC when the analyses are subjected to the using of chemiluminescence (CL) detection. The chemiluminescence (CL) detection system combined with HPLC separation method can offer excellent analytical selectivity and sensitivity. No doubt, chemiluminescence linked to HPLC will be widely applied in environmental science, biological science, pharmaceutical science and clinical science.The thesis consists of two parts. One is a review related to the application of HPLC with post-column chemiluminescence detection to pharmaceutical samples. Emphasis is taken on the development in the recent ten years. The other is research reports which is composed of 4 components as follows. 1) Determination of thiamin and ascobic acid by reversed-phase highperformance liquid chromatography with chemiluminescence detection;2) Derermination of pyridoxine by reversed-phase HPLC with chemiluminescence detection;3) Determination of vitamin B2, B6, B12, C and folic acid by ion-pair Reverse-Phase HPLC;4) Application of RP-HPLC for the detection of methyl-parathion and parathion residues in vegetables. 1. Determination of Thiamin and Ascobic Acid by Reversed-phase HPLC with Chemiluminescence Detection A novel high performance liquid chromatograph chromatography (HPLC) -Chemiluminescence (CL) detection method for simultaneous determination of thiamin (Vbi) and ascorbic acid (Vc) based on the chemiluminescence reaction of Vbi and Vc with Luminol-FeK3(CN)6-KBr system was developed. The proposed method has been applied to the determination of VBI and Vc in a pharmaceutical and honey preparation. This HPLC-CL method showed good linearity when it was evaluated simultaneously on Vbi and Vc and the working range for the system was 5.0x10'8~8.0xl0-5 g/mL and 1.0xl(r9~5.0xl0-6 g/mL with a limit of 8.0x1 (T9 g/mL and 5.0x10"11 g/mL respectively. The relative standered deviations (RSD) ( n=ll ) were 0.8% for Vbi, and 2.9% for Vc with a regression equation of the form y= 53.237x+1.2735and^5.734xl0Vl.266.2. Determination of Pyridoxine by Reversed-Phase High Performance Liquid Chromatography with Chemiluminescence DetectionA novel high performance liquid chromatograph chromatography (HPLC) -Chemiluminescence (CL) detection method for simultaneous determination of Pyridoxine (Vb6) based on the chemiluminescence strength reaction of Vb6 with Luminol- KIO4 system was developed. The proposed method has been applied to the detennination of Vb6 in drinks. This HPLC-CL method showed good linearity when it was evaluated simultaneously on Vb6 and the working range for the system was S.OxlO'^.OxlO"4 g/mL with a limit of 2.0xl0'8 g/mL respectively. The relative standered deviations (RSD) (n=5) were 2.8% for VB6, with a regression equation of the form.y=954.472 x+1.4883. Determination of Vitamin B2, B6, B12, C and Folic Acid by ion-pair Reverse Phase HPLCAn accurate and reliable ion-pair reverse phase HPLC method was established for the determination of VB2, VB6, VBi2, Vc and folic acid. These vitamins can be separated on HypersilC18(ODS2,5um,4.6nimxl50mm,)column by using V(methanol): V(0.5% acetic acid)=30:70, (add 2.5mmol pentane sodium mercuric thiocyana) as mobile phase with UV detector at a wavelength of 254nm. The standard curve was linear:2.0x 10"7~8.0x 10-6g/mL(VB2),2.0x 10-6~5.0x loVmL^ae^.Ox 10"7~8.0x loV mL(VBi2),5.0xl0-7~8.0xl0-5g/mL(Vc)and 2.0xl0"*~5.0xl0"5g/mL (folic acid).Thedetection limit were 6xl0"9g/mL,6><10"8 g/mL,7><10'9 g/mL,lxlO'9g/mL and 6xlO'8 g/mL for VB2,Vb6,Vbi2,Vc and folic acid. The method has been successfully applied to the determination of tablet of complex Vitamin and honey on the market, which proved to be simple and rapid.4. Application of RP-HPLC for the Detection of Methyl-parathion and Parathion Residues in vegetablesA rapid method has been developed for the determination of methyl-parathion and parathion residues in vegetables by the reversed-phase high performance liquid chromatography. This approach allows the simultaneous determination of parathion and methyl-parathion, without dedious sample treatments. Hypersil Cis column (4.6 mm x 300 mm, 5 um) with CH3OH: H2O (60:40, V/V) solution as mobile phase, ultraviolet detection wavelength was at 270 nm. The calibration graphs was linear in the concentration range of 0.04 ~ 10.00 ug/mL, 0.002 ~ 10.00 ug/mL for methyl-parathion and parathion, respectively. The limits of detection were 0.036 Hg/mL for methyl-parathion and 0.00043 ug/mL for parathion (S / N = 3). The average recovery of methyl-parathion and parathion were 91.8%~94.8% with RSD from 1.06% to 2.41%. This method is simple and reliable.