Trifluoroacetic acid, reversed-phase HPLC

Fig. 3 Resolution of the six major iso-a-acids in beer into five bands by reverse-phase HPLC. Mobile phase acetonitrile water methanol 0.2 M magnesium acetate (aq) formic acid trifluoroacetic acid (840 490 480 24 3.6 1.2 v/v/v/v/v/v). Column NovaPak C18 5 /rm cartridge (100 X 8 mm i.d.) under radial compression. Mobile phase flow rate = 2 ml min-1. (Chromatogram redrawn from original).

Rezk et al. [74] developed and validated a reversed-phase HPLC assay method for the simultaneous quantitative determination of omeprazole and its three metabolites in human plasma. The method provides excellent chromatographic resolution and peak shape for the four components and the internal standard within a 17-min run time. The simple extraction method results in a clean baseline and relatively high extraction efficiency. The method was validated over the range of 2-2000 ng/ml. The resolution and analysis for the four analytes omeprazole, hydroxyome-prazole, omeprazole sulfone, and omeprazole sulfide and the internal standard utilized a Zorbax C18 (15 cm x 3 mm, 5 /im) with a Zorbax C18 (12.5 cm x 4.6 mm) guard column. The mobile phase consisted of two components. Mobile phase A was 22 mM phosphate monobasic, adjusted to a pH of 6 with diluted sodium hydroxide. This solution was filtered through a 0.45-/im membrane filter, then mixed as 900 ml buffer to 100 ml methanol. Mobile phase B was composed of 100 ml of the phosphate buffer as mobile phase A, mixed with 800 ml of acetonitrile, 100 ml of methanol, and 100 /A of trifluoroacetic acid with an initial flow-rate of 0.55 ml/min and detection at 302 nm. 

For pharmaceutical compounds, LC-MS has found extremely wide acceptance due to the low-level detection that can be achieved, in addition to the selectivity and specificity that are attained by using HPLC in conjunction with MS detection. LC-MS is also convenient because of its compatibility with reversed-phase HPLC mobile phases. Volatile mobile phase additives such as trifluoroacetic acid, formic acid, and ammonium hydroxide are very common and can be utilized not only to aid in the chromatographic separation but also to influence the ionization state of the molecule (i.e., acid modifiers to protonate [M + H]+1, and basic modifiers to deprotonate [M — H] ). This requirement may require a modification of the potency method if phosphate was utilized however, it is not viewed as a major drawback. 

The eluents most commonly used in peptide purification by reversed phase HPLC are water and acetonitrile. These are often buffered with trifluoroacetic acid (0.1% v/v, TFA), ammonium acetate (0.05-0.1 mol/dm3 at pH 4-8) or phosphate (0.05-0.1 mol/dm3 sodium or potassium salt at pH 2-8). In addition, polymeric reversed phase media also performs well at high pH and is often buffered with ammonium hydroxide or ammonium bicarbonate (0.05-0.1 mol/dm3 at pH 8-9). 

After renaturation, the majority of the recombinant chemokines are easily quantified by UV spectroscopy, although there are examples of chemokines such as NAP-2, which do not possess an aromatic amino acid that serve as chromophores. In this case, quantification is achieved by comparison of the peak height on reverse phase HPLC analysis to that of a known concentration of another chemokine. They can then be lyophilized after a change of buffer into a trifluoroacetic acid or acetic acid solution, which facilitates their storage as lyophilized powders. It is important that they are redissolved in water, before dilution into buffer or medium. Their handling is easy and rapid, as they are instantly soluble at concentrations as high as 1 mM if necessary, in aqueous solutions. 

Reverse phase HPLC buffers Buffer A 0.1% trifluoroacetic acid. Buffer B Buffer A containing 90% acetonitrile. 

The resulting active fraction was further processed in five steps of high performance liquid chromatography (HPLC) reverse phase HPLC on Develosil 5Co (Nomura Kagaku) with 20-25% acetonitrile in 0.08% trifluoroacetic acid (TEA) (step 12), reverse phase HPLC on Hi-Pore RP-304 (Bio-Rad) with 20-40% acetonitrile in 0.08% TEA (step 13), reverse phase HPLC on Hi-Pore RP-304 with 20-40% acetonitrile in 0.1% heptafluorobutyric acid (HEBA), cation exchange HPLC on TSKgel (Toyo Soda) with... 

The chemical scheme for C-terminal sequencing is shown in Figure 2. The first step involves treatment of the peptide or protein sample with diisopropylethylamine in order to convert the C-terminal carboxylic acid into a carboxylate salt. Derivatization of the C-terminal amino acid to a thiohydantoin is accomplished with diphenylisothiocyanatidate (liquid phase) and pyridine (gas phase). The peptide is then extensively washed with ethyl acetate and acetonitrile to remove reaction by-products. The peptide is then treated briefly with gas phase trifluoroacetic acid, followed by water vapor in case the C-terminal residue is a proline (this treatment has no effect on residues which are not proline). The derivatized amino acid is then specifically cleaved with sodium or potassium trimethylsilanolate to generate a shortened peptide or protein which is ready for continued sequencing. In the case of a C-terminal proline which was already removed by water vapor, the silanolate treatment merely converts the C-terminal carboxylic acid group on the shortened peptide to a carboxylate. The thiohydantoin amino acid is then quantitated and identified by reverse-phase HPLC. 

Reversed phase HPLC was performed using fused silica columns and solvent delivery systems developed and built in our laboratory (5,6). All chromatographies were carried out on Vydac S itm C18 RP support with or without an SDS removal precolumn. SDS removal resin was obtained from Poly-LC Solvent A was 0.1 % trifluoroacetic acid (TFA) in water and solvent B was 0.07 % TFA, 90 % acetonitrile in water. Water was obtained from a Milli-Q system. Samples were eluted with a gradient from 2% to 92 % solvent B in 45 minutes unless otherwise noted As a standard, cytochrome C digested with Lys-C (CCKCD) was used The separation of the peptides was carried out with or without SDS. 

Reversed-phase HPLC was performed on a SynChropak RP-8 (SynChrom, Inc., Linden, IN). The gradient was from 19% to 34% acetonitrile, at 1%/min., in 0.1% trifluoroacetic acid. This method resolves the correctly refolded rSLPI from the unfolded form. 

For HPLC-MS/MS assays, the mobile phase is an important consideration. For reversed-phase HPLC systems (the most common), mobile phase A is water plus one or more modifiers while mobile phase B is usually either acetonitrile or methanol with one or more modifiers. Modifiers have to be volatile for HPLC-MS/MS assays. Typical modifiers are acetic acid, formic acid, and ammonium acetate. Formic acid is so popular that it is now available as a premixed HPLC solvent (0.1% in water or acetonitrile). Most other modifiers are not recommended. Triethylamine (TEA) and trifluoroacetic acid (TFA) are a problem as they are known to cause ion-suppression problems. For some special HPLC-MS/MS assays that need ion-pairing reagents, hexylamine and heptafluorobutyric acid have been found to be successful [51-53], Gao et al. [54] recently evaluated a series of ion-pairing reagents in terms of their suitability for an HPLC-MS/MS assay. 

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