Ion-pair reversed-phase HPLC with

The activity is found in erythrocytes, platelets, and lymphocytes, and determination of its value aids in diagnosis of some blood disorders. In this assay, which can readily be used for purine and pyrimidine 5 - and 3 -nucleotidase activities, the nucleoside monophosphate (the substrate) was separated from the nucleoside (the product) using ion-pair reversed-phase HPLC with a mobile phase of 5% methanol-5 mAf potassium dihydrogen phosphate 0.25 mAf 1-decanesulfonic acid was also added to the mobile phase. The elution was carried out at room temperature and the eluent monitored at 254 nm. 

HPLC determination This technique has been used extensively for determining only one vitamer or both vitamers together. Simultaneous determination is difficult due to the differences in basicity and polarity of both vitamers and the interference problems. After water-phase extraction and sometimes cleanup steps as deproteinization, nicotinic acid and/or nicotinamide are analyzed by ion pair reversed-phase HPLC with UV detection. Most methods use different ion pair reagents for each vitamer, but a single ion pair reagent for nicotinamide and nicotinic acid can be also employed. 

Ion-pair reverse phase HPLC. Aqueous samples confaining pyridoxine as infernal standard and 0.5% heptafluorobutyric acid as ion-pairing agent (IP) were injected in an RP-HPLC system with a Varian ODS-80TM column and eluted with 0.15% heptafluorobutyric acid in 24% methanol. Pyridinoline cross-link fluorescence was defecfed af Xgx 295 nm and Xem 400 nm (Bank ef al., 1997). 

Dai, G., Wei, X., Liu, Z., Liu, S., Marcucci, G., and Chan, K. K., Characterization and quantification of Bcl-2 antisense G3139 and metabolites in plasma and urine by ion-pair reversed phase HPLC coupled with electrospray ion-trap mass spectrometry. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences 825(2), 201-213, 2005. 

Determining Class III caramel in food products is more difficult. Semiquantitative results have been achieved with some food products by ion-pair reversed-phase HPLC.201... 

Figure 1.4 Separation of substrates and products of an adenosine kinase reaction on ion-paired reversed-phase HPLC. The separation was carried out on a prepacked Ctg (/xBondapak) column with a mobile phase of 65 mAf potassium phosphate (pH 3.7) containing 1 mAf tetrabutylammonium phosphate and 5% methanol. The column was eluted isocratically, and the detection was at 254 nm. Four relative elution positions (elution times) arc shown.

An interesting and useful variant of reversed-phase HPLC is called ion-paired reversed-phase HPLC. In such a system the analytical columns are packed with the same material, but a compound such as tetrabutylammonium is added to the mobile phase. The separation of ATP and adenosine on such a system is shown in Figure 2.15. A comparison of this profile to that shown for the same compounds in Figure 2.14 immediately highlights the change in the elution sequence. Whereas without ion pairing, the order is ATP followed by adenosine, with ion pairing the order is adenosine followed by ATP. 

The assay, which measured only the amount of AD formed, used ion-paired, reversed-phase HPLC chromatography. The separation was carried out on a C18 (Nucleosil) column with a mobile phase of 0.1 M sodium phosphate buffer (pH 2.3-3.5) containing 5 mM sodium pentanesulfonate as the counterion to form ion pairs with the catecholamines, and 0.5% (v/v) acetonitrile. The separation of NA from AD is shown in Figure 9.14. 

The assay involves the isolation of the pyrrole by ion-paired, reversed-phase HPLC (Hypersil-SAS) with a mobile phase of methanol and water (45 155, v/v) and 0.005 mol/L 1-heptanesulfonic acid (PIC B-7). The radioactive product was detected by scintillation counting. The separation obtained when the compounds were in distilled water is shown in Figure 9.57a. However, when the analysis was carried out on freeze-dried samples, the salts present led to the results shown in Figure 9.57b. 

The assay involves the separation of ALA from PBG by ion-paired, reversed, phase HPLC (Hypersil SAS) with a mobile phase of methanol-water (22 78, v/v) and PIC B-7 (0.005 M 1-heptanesulfonic acid) adjusted to pH 3.5. An internal standard of 2-methyl-3-carbomethoxy-4-(3-propionic add)pyrrole was used. All three compounds were readily separated in 6 minutes (Fig. 9.58). Detection was at 240 nm. 

In the assay of Jahngen and Rossomando (1984), AMP and IMP were separated by means of ion-paired reverse-phased HPLC on a Q8 (/uBondapak) column with a mobile phase of 65 mM potassium phosphate (pH 3.6), 1 mM tetra-n-butylammonium phosphate, and 4% methanol. The column was eluted isocratically, and the eluent was monitored at 254 nm. When the formycin analogs were used, detection was at 295 nm at this wavelength there was no... 

The separation of product from substrate was accomplished using ion-paired, reversed-phase HPLC on a C18 (/xBondapak) column with a mobile phase of 65 mM potassium phosphate and 1 mM tetrabutylammonium phosphate adjusted to pH 3.6 with phosphoric acid and 1.5% acetonitrile. The column was eluted isocratically and monitored at 254 nm. The separations obtained are shown in Figure 9.97. 

Figure 9.97 Separation of adenine nucleotides and adenosine by ion-paired, reversed-phase HPLC. Standards of AMP, adenosine, ADP, ATP, and cAMP (approximately 2 nmol of each) in Tris-HCl (pH 7.4) were injected onto a Cis /xBondapak reversed-phase column (300 mm X 7.8 mm) and eluted with 65 mM KH2P04 (pH 3.6), 1 vaM tetrabutylammonium phosphate, and 2% acetonitrile. The flow rate was 2 mL/min, and detection was at 254 nm. (From Rossomando, 1987.)...

For the HPLC assay, IMP, sAMP, GTP, GDP, and AMP are separated by ion-paired, reversed-phase HPLC (Qg) with a mobile phase of 65 mM potassium phosphate (pH 4.4), 1 mM tetrabutylammonium sulfate, and 10% methanol. Detection was at 254 nm. A representative chromatogram is shown in Figure 9.112. 

All compounds, including substrates and both primary and secondary reaction products, were separated by ion-paired, reversed-phase HPLC (LiChro-sorb RP-18) with a mobile phase of 9.4% 2-propanol containing tetrabutylam-monium hydroxide at several pH values adjusted with phosphoric acid. The separations obtained are shown in Figure 9.145. The column was eluted isocrat-ically and monitored by UV at 254 nm and by liquid scintillation counting. 

The vast majority of separations described for the leukotrienes have used isocratic reversed phase conditions with an ODS stationary phase, although a single report exists which used ion-pair reversed phase chromatography with gradient conditions. Examples of these separations will not be discussed, together with some of the problems associated with HPLC of the leukotrienes. 

Thiamine (vitamin Bj) occurs in foods in free and bound forms, the free form predominates in cereals and plants, whereas the pyrophosphate ester is the main form in animal products. Acid hydrolysis is required to release thiamine from the food matrix. Enzymatic hydrolysis is then needed to convert phosphate esters to thiamine. Prior to CE analysis it is necessary to clean up samples by using ethanol to precipitate protein and by passing through an ion-exchange resin. Thiamine has been determined in meat and milk samples using MEKC with ultraviolet (UV) detection at 254 nm, obtaining comparable sensitivity to that achieved by HPLC using an ion-pair reversed-phase column with postcolumn derivat-ization and fluorescence detection. 

Fig. 4 Ion-pair reversed-phase HPLC-MS/MS chromatogram using heptafluoro-butyric acid (HFBA) as the ion-pair reagent in the mobile phase and a Zorbax SB-Aq column. The standard mixture of alkyl phosphonic adds with a concentration of 10 p-g/ml each was detected using MRM 1) MPA (mh %.8 78.7) 2) EMPA (miz 125 96.8) 3) EDMAPA (miz 154.2 126) 4) iPrMPA (m z 139.1 %.8) 5) PinMPA (m z 181.3 96.8) 6) DiPrMPA (m z 181.3 139.1). See the text for further chromatographic details and the MS/MS conditions used.

The determination of silica and phosphorus as mol5djdenum heteropolyacids by ion-pair reversed-phase HPLC is described. The chromatographic behavior of molybdenum heteropolyacids of silicon and phosphorus in the form of its association with tetrabutylam-monium bromide on a reversed-phase column C18 is investigated using ion-pair reversed-phase HPLC (UV detection at 310 nm). Optimal conditions for the separation of the polyacids for the determination of silicon and phosphorus in water were foimd. The calibration curve has a linear behavior in the concentration range of 0.01-0.1 pg/mL silicon and 0.02—0.15 pg/mL phosphorus. The detection limit of silicon and phosphorus is (1.4 0.3)10 and (6.7 1.2)10 pg/mL, respectively. 

Gregory and Feldstein (94) developed an ion-paired, reversed-phase HPLC method for individual Be vitamers extracted with sulphosalicylic acid from different foods. Using a ternary solvent program, elution of nutritionally active Be vitamers from the analytical column was complete within 30 min. PLP was determined as its hydroxysulfonate derivative, following postcolumn introduction of a buffered solution of sodium bisulfite. This method was found suitable for vitamin Be analysis in foods of both plant and animal origin. Recoveries for PLP and PL from pork loin were <90% it was suggested that these vitamers were not completely released from muscle proteins, even in the presence of 5% sulfo-salicylic acid. 

FIGURE 28.5 Typical chromatogram for separation of five nucleotides by ion-pair reversed-phase HPLC method. (1) cytidine 5 -monophosphate (CMP) (2) uridine 5 -monophosphate (UMP) (3) guanosine 5 -monophosphate (GMP) (4) inosine 5 -mono-phosphate (IMP) (5) adenosine 5 -monophosphate (AMP). The concentration of each nucleotide injected onto the column was 15 mg L h (Reprinted from Food Chem., 7A, Ferreira, I. M. P. L. V. O. et al., The determination and distribution of nucleotides in dairy products using HPLC and diode-array detection, 239-244, Copyright 2001, with permission from Elsevier.)... 

For chromium speciation with ICP-DRC-MS analysis, ammonia was selected as the reaction gas. Airborne Cr(VI) was separated from Cr(III) using a silica gel determination of ultratrace quantities of Cr(VI) was possible using preconcentration via complexation and ICP-DRC-MS. For aqueous samples, ion pair reversed phase HPLC was applied. ... 

AdoMet and AdoHcy are separated and measured as their etheno derivatives using reverse-phase HPLC with isocratic elution in separate runs, using ion-pairing for AdoMet. In both runs the column is flushed with 100% acetonitrile followed by reequilibration with the mobile phase between sample injections. 

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