Mobile phase deuterated

Separation of five compounds (DL, 6-OH-DL, 3-OH-DL, 7V-OH-DL, and 1-pyridine-/V-oxide-DL) was achieved using an Alliance HPLC system (Waters Corp., Milford, CA) equipped with a 2690 model pump, an autoinjector, a Polaris Cl8-A guard column (Varian Inc., Lake Forest, CA), and a Luna Phenyl-Hexyl analytical column (Phenomenex, Inc., Torrance, CA) maintained at 40°C. For robust characterization of each isomeric compound, an online HDX LC-MS method was developed. The composition of regular and deuterated mobile phases is summarized below ... 

Compound m/z Regular Mobile Phase Deuterated Mobile Phase Mass Increase Number of Labile Hydrogens in Molecule... 

Figure 9.4. LC-APCI-MS spectra of DL, 6-OH-DL, 3-OH-DL, N-OH-DL, and 1-pyridine-N-oxide-DL generated using a TSQ Quantum triple-quadrupole mass spectrometer and deuterated mobile phase.

Microderivatization or chemical modification of analytical samples can be useful in enhancing the ion response, and hence the LC-MS sensitivity, for any particular analyte. In the case of alkaloids, which are already capable of ready ionization, chemical modification can be more valuable in elucidating structures by providing tandem MS evidence for positions of substitution in the parent molecule. Such derivatization or chemical modification for HPLC-MS can include H/D exchange by using deuterated mobile phases, Jones oxidation of aliphatic hydroxyls, selective acetylation of hydroxyl and amine groups, and N-oxide reduction [19,20]. 

To avoid the expensive use of deuterated solvents for H/D exchange experiments, Tolonen et al. [21] have described the postcolumn infusion of D2O to facilitate the LC-MS detection and identification of labile protons in a column eluant. Whilst acknowledging the potential limitations with respect to a reduced level of exchange, and hence sensitivity, compared to the use of deuterated mobile-phase solvents, they optimized the column effluent flow rate (via a splitting connector) with the infused D2O flow rate to enable the very useful determination of up to four labile protons. The method was exemplified by the differentiation of hydroxylated metabolites of the alkaloidal drugs imipramine and omeprazole (Figure 13.5) from the N-oxide and sulfone metabolites, respectively [21]. This was a differentiation that could not be achieved by high-resolution mass measurements. 

Figure 8-53. Mass spectra of 5-aminoindazole in deuterated mobile phase.

LC-NMR One information-rich spectral technique that is more suited to the liquid mobile phase of HPLC than to the vapor phase of GC is NMR. LC-NMR has been implemented, but it has significant limitations. To obtain interpretable spectra of unknowns, concentrations in the measurement cell must be higher than with other detectors. The cell must be smaller than the usual NMR tube, so for any but the very highest concentrations of analytes, FT-NMR acquisition is preferred, with each eluted peak being retained in the measurement cell by a stopped-flow procedure similar to that employed to increase sensitivity in GC-IR (Section 12.8.2). Expensive deuterated mobile phase solvents are required for proton NMR, which mandates the use of low mobile phase volume flow columns narrow bore or even capillary HPLC. LC-NMR is expensive to implement and not readily available from commercial vendors at this time. 

Both MS and NMR coupling to HPLC have been employed for the analysis of p-carotene isomers and determination of lutein and zeaxanthin isomers in spinach, sweet com, and in retina. Capillary high performance hquid chromatography with stop flow connected to NMR (600 MHz) was used for stracture elucidation of all-trans deoxylutein 11 and its isomers.Efforts are in progress to eliminate the remaining major drawbacks such as obligatory use of deuterated solvents in the mobile phase, poor sensitivity, and low throughput of HPLC-NMR analyses. 

NMR/MS has also been hyphenated to LC-SPE and LC-DAD modules. Sample enrichment and exchange of the HPLC mobile phase with an NMR suitable solvent is advantageous. LC-SPH-NMR/MS gains up to a factor of four in LC-NMR S/N for a single injection. No deuterated solvents are needed for separation and trapping. Optimisation of the separation procedure is less critical than for HPLC-UV. 

The second group of recently developed ionic liquids is often referred to as task specific ionic liquids in the literature [15]. These ionic liquids are designed and optimised for the best performance in high-value-added applications. Functionalised [16], fluorinated [17], deuterated [18] and chiral ionic liquids [19] are expected to play a future role as special solvents for sophisticated synthetic applications, analytical tools (stationary or mobile phases for chromatography, matrixes for MS etc.), sensors and special electrolytes. 

The amount of futile acetylation observed for this compound in the rat, at ca. 7-10% for parent compound and metabolites, was less than that seen for phenacetin and similar to that found for paracetamol itself. The bulk of the radio-label was rapidly excreted in urine as practolol itself (albeit with 7-10% reacetylation) and the remainder as either the ring-hydroxylated metabolite or its glucuronide conjugate. As in previous examples, deuterated methanol was used in the mobile phase rather than methanol in order to be able to more easily observe the acetyl resonances of practolol and related compounds. 

In principle, it would be possible to use fully deuterated solvents for HPLC-NMR coupling, and then solvent suppression techniques would be unnecessary however, due to the high costs of these solvents, only the use of D2O is economically acceptable. Solutions to this problem can be seen in the development of hyphenating capillary HPLC to NMR spectroscopy, and there, because of the reduced solvent consumption, the use of fully deuterated solvents would be reasonable. However, this approach is difficult and the development is still an on-going process (see Chapter 7.3 below). From the viewpoint of NMR spectroscopy, there could be a completely new situation, when separation techniques could be found which use proton-free solvents as the mobile phases. 

Another aspect affecting the solvent choice in the case of aH NMR measurements is that deuterated solvents are the most convenient option.53 In principle, the deuterated version of regular LC mobile phases could be used, as deuteration has been shown to have only a minor effect on the partition,52 and they were applied in the studied outlined above. 

Another mobile phase modification technique is termed Hydrogen/Deuterium (H/D) exchange, in which deuterium oxide and/or other deuterated... 

To confirm the tentative assignments of the degradation products and to further elucidate the structures of Deg-4, Deg-5, and Deg-6, HPLC/NMR analyses were performed. Conditions were as described for HPLC/UV and MS, with the exception that NMR-grade acetonitrile and deuterated water were used as mobile-phase components. The injection volume was additionally increased to 25 ml to place 5-50 pig of each degradation product on column. 

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