High-performance liquid chromatography quantitative determination

Typically, quantitative protein determination is done on the one hand by colorimetric or nephelometric methods, on the other hand for more difficult analytical problems by more sophisticated techniques such as high performance liquid chromatography (HPLC), gel-electrophoresis and immunoassay. However, these methods are tedious, time-consuming and expensive. 

Chromatography is a separation process employed for the separation of mixtures of substances. It is widely used for the identification of the components of mixtures, but as explained in Chapters 8 and 9, it is often possible to use the procedure to make quantitative determinations, particularly when using Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC). 

High performance liquid chromatography is used for the separation and quantitative analysis of a wide variety of mixtures, especially those in which the components are insufficiently volatile and/or thermally stable to be separated by gas chromatography. This is illustrated by the following method which may be used for the quantitative determination of aspirin and caffeine in the common analgesic tablets, using phenacetin as internal standard where APC tablets are available the phenacetin can also be determined by this procedure. 

Milbemectin consists of two active ingredients, M.A3 and M.A4. Milbemectin is extracted from plant materials and soils with methanol-water (7 3, v/v). After centrifugation, the extracts obtained are diluted to volume with the extraction solvent in a volumetric flask. Aliquots of the extracts are transferred on to a previously conditioned Cl8 solid-phase extraction (SPE) column. Milbemectin is eluted with methanol after washing the column with aqueous methanol. The eluate is evaporated to dryness and the residual milbemectin is converted to fluorescent anhydride derivatives after treatment with trifluoroacetic anhydride in 0.5 M triethylamine in benzene solution. The anhydride derivatives of M.A3 and M.A4 possess fluorescent sensitivity. The derivatized samples are dissolved in methanol and injected into a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector for quantitative determination. 

Fan used a high performance liquid chromatographic method for the qualitative and quantitative analysis of miconazole [59], Miconazole sample was dissolved in methanol and determined by high performance liquid chromatography using methanol-water (75 25) as the mobile phase and ultraviolet detection at 214 nm, the recovery was more than 99.4% and the accuracy was satisfactory for the qualitative and quantitative analysis. 

High performance liquid chromatography is used to determine the purity of calcitriol, and to separate it from related compounds. Using a 10 micron silica column of 25 cm length, and a mobile phase of spectroquality heptane ethyl acetate. methanol (50 50 1) at a flow rate of 1.7 ml/ minute, separation and quantitation are achieved. p-Dimethyl-aminobenzaldehyde may be used as an internal standard to compensate for variations in injection technique and instrumental conditions. With a 254 nm ultraviolet absorbance detector, 0.01 ug of calcitriol may be detected (3). 

Quantitative determination of ginsenosides by high performance liquid chromatography-tandem mass spectrometry. Phytochem. Anal. 12, 320-326. 

Li, Q. C., Harkey, M. R., Henderson, G. L., Gershwin, M. E., Stern, J. S., and Hackman, R. M. (2001b). Quantitative determination of ginsenosides by high-performance liquid chromatography-tandem mass spectrometry. Phytochem. Anal. 12, 320-326. 

Boonen G, Beck MA, Haberlein H. (1997). Contribution to the quantitative and enantioselective determination of kavalactones by high-performance liquid chromatography on ChiraSpher NT material. J Chromatogr B Biomed Sci Appl. 702(1-2) 240-44. 

Aboul-Enein, H.Y. and Serignese, V., Quantitative determination of clenbuterol enantiomers in human plasma by high-performance liquid chromatography using the macrocyclic antibiotic chiral stationary phase teicoplanin, Biomed. Chromatogr., 13, 520, 1999. 

Foglia JP, Sorisio D, Kirshner MA, Mulsant BH, Perel JM. 1995. Quantitative determination of perphenazine and its metabolites in plasma by high-performance liquid chromatography and coulometric detection. J Chromatogr B Biomed Appl 668(2) 291-297. 

KoUroser M, Schober C. 2002. Direct-injection high performance liquid chromatography ion trap mass spectrometry for the quantitative determination of olanzapine, clozapine and N-desmethylclozapine in human plasma. Rapid Com-mun Mass Spectrom 16(13) 1266-1272. 

High-performance liquid chromatography (HPLC), followed by GC/MS, has been used to fractionate and then quantitate the aliphatic and aromatic hydrocarbons present in liquid fuel precursors in order to determine the fuel potential of the compounds. Kerosene had the advantage of not requiring any sample preparation. Other light fuel oils may require the use of methylene chloride as a solvent prior to HPLC analysis (Lamey et al. 1991). The sensitivity, precision, and recovery of this method were not reported. 

In bioanalysis, High-Performance Liquid Chromatography (HPLC) is the analytical technique most frequently used. Often, extended sample preparation is required to make a biological sample (the matrix) suitable for HPLC-analysis. The compound of interest, the analyte, has to be isolated from the matrix as selective and quantitative as possible. The quality of the sample preparation largely determines the quality of the total analysis procedure. In a survey Majors [2] showed that approximately 30% of an error generated during sample analysis was due to sample preparation, which indicates the need for error reduction and quality improvement in sample preparation. 

A method for the determination of personal exposure to benzidine-based dyes has been developed. This procedure involved the reduction of benzidine-based dye filter samples to free benzidine with neutral buffered sodium hydrosulfite solution. The benzidine-containing reduction solution was then analyzed by high performance liquid chromatography. The reduction was found to be quantitative by visible-spectrum analysis. This reduction and analysis method was evaluated with four benzidine-based dyes over the range from 12 to 300 micrograms per sample. Precision for the reduction and analysis of the four dyes falls within % coefficient of variation. This method can differentiate between benzidine-and benzidine congener-based dyes. Results are reported in terms of free benzidine. 

The availability of stable isotope-labeled PA makes an accurate quantitative determination of this imino acid possible. A short high-performance liquid chromatography (HPLC) run prior to the mass spectrometer inlet will result in a discrete peak of PA. For the definitive diagnosis of AASA dehydrogenase deficiency, a simultaneous determination of AASA would be preferred. The absence of a commercially available labeled standard leaves this analysis in the experimental stage. 

Suormala TM, Baumgartner ER, Bausch J, Holick W, Wick H (1988) Quantitative determination of biocytin in urine of patients with biotinidase deficiency using high-performance liquid chromatography (HPLC). Clin Chim Acta 177 253-270... 

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