Acids nonvolatile, HPLC analysis

Sen and Kubacki (54) have reviewed the derivatization techniques for the GC determination of nonvolatile NOC in foods. These include trimethylsilylation (for acidic as well as hy-droxylated NOC), esterification with CH2N2 or BF3/CH3OH, and O-methylation using CH3I/ NaH. Only those applied to HPLC analysis are briefly discussed here. 

Nonvolatile Nitrosamines In Tobacco. A method which we developed several years ago for the analysis of tobacco-specific nitrosamines (TSNA 31) involves extraction of tobacco with buffered ascorbic acid TpH 4.5) followed by partition with ethyl acetate, chromatographic clean-up on silica gel, and analysis by HPLC-TEA (Figure 9). Results obtained with this method for a large spectrum of tobacco products (Table IV), strongly support the concept that the levels of nitrate and alkaloids, and especially the methods for curing and fermentation, determine the yields of TSNA in tobacco products. Recent and as yet preliminary data from snuff analyses indicate that aerobic bacteria play a role in the formation of TSNA during air curing and fermentation. 

The extent of oxidation can also be assessed by the analysis of oxidized fatty acids by spectroscopic means such as IR and NMR techniques (102). Moreover, GC-MS for volatile profile analysis (103) and HPLC for determination of DNPH derivatives of nonvolatile higher carbonyl compounds (62) provide qualitative... 

The stable nonvolatile intermediate phenylthiocar-bamoyl derivatives are formed in basic media and can be analyzed directly by reverse-phase high-performance liquid chromatography (RP-HPLC). Their cyclization into hydantoins requires acid catalysis. This mode of derivatization is a very important supplement to the Edman s method of N-terminated sequencing of polypeptides. Before GC analysis, any hydantoins can be converted into N-trifluoroacetyl or enol-O-trimethylsilyl derivatives, which increases the selectivity of their determination in complex matrices. 

FIGURE 2 N-terminal sequence analysis of protein samples purified by HPLC. Samples recovered in volatile buffers are directly subjected to sequence analysis following application to the sequencer filter disk. Samples recovered in nonvolatile buffers are desalted by precipitation and applied on the sequencer filter disk after reconstitution in volatile solvents. Dilute sample solutions are desalted on ProSpin units by centrifugation and subsequent washing of the excised protein containing PVDF membrane disks. The disks are either sequenced in a standard or a specialized reaction cartridge (Blott cartridge). Insert shows the ProSpin Unit. AAA, amino acid analysis for protein quantitation. 

However, RP-HPLC is challenging for the separation of polar compounds that are poorly retained on the hydrophobic stationary phase. This limits the application of RP-HPLC for the analysis of nonvolatile flavor compounds. Many taste-active compounds, especially those with reported savory (umami = glutamate-like) taste are of a hydrophilic nature. Hydrophilic di- to tetrapeptides consisting of high molar ratios of acidic and other hydrophilic amino acids such as Glu-Asp, Glu-Glu, Thr-Glu, Asp-... 

In cases where the pH range is not limited by the needs of the separation, it is common practice to utilize acidic solvents for the analysis of protonated species with positive ion ESI-MS. Indeed, a number of investigators have reported that the response of weakly basic species to analysis with ESI is optimal at low pH. " The most commonly used electrospray solvents consist of small amounts (0.1-0.5%) of volatile acids such as acetic or formic acid dissolved in methanol/water or acetonitrile/water. Nonvolatile acids such as hydrochloric acid are to be avoided because they cause signal suppression." Signal suppression can also result when a volatile acid is added to the solution in too great an amount, so the concentration added should be only enough to optimize protonation of the analyte and, in the case of HPLC/ESI-MS, to facilitate successful HPLC separation. 

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