In Part I, work was done to develop a novel gradient chromatofocusing liquid chromatography-tandem mass spectrometry (MS/MS) technique for the determination of ionic compounds in biological matrix. The analysis of ionic compounds poses a great challenge to current LC-MS techniques, due to the fact that there is little or no retention. These ionic compounds are eluted at almost the same time as biological matrix, which is normally directed to waste prior to connecting to the mass spectrometer. A review of current LC-MS techniques for ionic compounds is given in Chapter 1. They all have advantages and disadvantages.
To retain and quantify ionic compounds in biological matrix, gradient chromatofocusing LC-MS was investigated and improved in this dissertation. In gradient chromatofocusing, a linear pH gradient is generated on an ion-exchange column by the gradient mixing of an acidic buffer and a basic buffer. Gradient chromatofocusing LC-MS was successfully applied in the quantification of choline and arginine in whole blood. This is the first report of the use of cation-exchange HPLC columns in gradient chromatofocusing work. It is also the first chromatofocusing technique employing on-line MS/MS analysis.
Improvement of the above technique was made by elimination of signal enhancement solution. Detection limit has been improved around 100 times compared to previous work. Peak shape and peak tailing has also been greatly improved. The concentrations of choline in 60 whole blood samples (26 were diagnosed with acute coronary syndrome [ACS] and 34 were diagnosed with non-ACS) were quantified. The concentrations of choline in the 34 ACS whole blood samples are 61% higher with a p value equaling 0.0013, supporting the conclusion made by Danne et. al that choline level was elevated in the whole blood of ACS patients. The concentrations of arginine in 44 human serum samples (23 ACS patients and 21 non-ACS patients) were quantitatively determined. The p value is 0.12, meaning that there is no significant difference of the serum arginine levels in these two groups of patients.
In Part II, identification of a caspase 3 enzyme cleavage site of a recombinant sodium/hydrogen exchange protein (cNHE1) was done by HPLC-MS. cNHE1 has been shown to protect renal tubular epithelial cells against apoptosis, and thus its degradation by caspase 3 has implications in progressive chronic kidney disease. The identity and molecular weight of cNHE1 has been confirmed. The molecular weight of cNHE1 has been checked by LC-MS and MALDI-TOF. After In vitro caspase 3 digestion of cNHE1, three peptides were recovered. The molecular weight of these three peptides were measured by LC-MS. One peptide (peptide 2) with M.W. 8760.6 matched very well to part of cNHE1 sequence: DDKD↓RWGSPLVDLLAVKKKQETKRSINEEIHTQFLDHLL TGIEDICGHYGHHHWKDKLNRFNKKYVKKCLIAGERSKE↓PQL. No other part of cNHE1 sequence matched this molecular weight.