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September 2009
an significant amount of the undesired D-T4 (1.7%) but also significant amounts of both enantiomers of T3 amounts (0.6% and 0.2% of L- and D-T3).
Recently, Cinchona carbamate-type CSPs have been implemented as stereoselective separation tools for themonitoring of fluorescence-labeled D-amino acids in various mammalian tissues, employing a sophisticated 2D column switchingmethod [25]
. The operational scheme is given in Figure 5.
Figure 4. Enantioselective HPLC monitoring of impurities in a levothyroxin (L-T4) formulation. Experimental conditions: Column, Chiralpak QN-AX (150 mm × 4 mm ID); mobile phase, acetonitrile-50 mM ammonium acetate (60:40, v/v) (pHa 4.5); flow rate, 0.7 mLmin−1; UV detection, 240 nm; temperature, 25 C. Reproduced from [25] with permission.
The sensitive detection and quantification of D-amino acids in biological samples are currently of considerable interest because of their relevance as potential disease biomarkers. After extraction fromtissue samples and fluorescence labeling the amino acid derivatives were first chromatographed in an “achiral dimension” using a RP18 narrow bore column to achieve the chemoselective resolution of the four hydrophobic racemic amino acid derivatives of interest (Val, allo-Ile, Ile, Leu). Fractions of each peak were collected in amultiloop trapping device and then transferred sequentially into the second “chiral” chromatographic dimension, presented by either a ChiralpakQN-AX column or a ChiralpakQD-AX column, to achieve the separation of the enantiomers. The incorporation of both pseudoenantiomeric cinchona carbamate-type CSPs allowed reversal of enantiomer elution orders on demand, facilitating the peak integration of theminor enantiomers and thus the quality of the quantification. The 2D-method was fully validated for the concentration range between 0.005 and 0.5 pmol for D-amino acids and 0.05–5 pmol for L-amino acids, providing excellent performance characteristics. LODs and LOQs were reported to be as low as 3 fmol and 5 fmol, making thismethod to one of themost sensitive analysismethod for amino acid enantiomers currently available for mammalian tissue samples.
2.3.2. Capillary Electrophoresis
Figure 5. Enantioselective monitoring of hydrophobic D-amino acids as NBD-derivatives (obtained by precolumn derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole NBD-F) in rat tissue using an online 2-dimensional HPLC system combining RP18 and chiral anion-exchanger columns with fluorescence detection. Experimental conditions: (b) first dimension:M1: THF-TFA-
H2O (25:0.05:75; v/v); flow rate, 75 µL min−1; C1: Capcellpak C18MG II (150 mm × 1.0 mm ID, 40 C); (c) second dimension:M2: acetonitrile–methanol (50:50; v/v) containing 10 mM citric acid; flow rate, 1.5 mLmin−1; C2: Chiralpak QN-AX (150 mm × 4.0 mm ID, 40◦C); (d) same as (c) but C2: Chiralpak QD-AX (150 mm × 4.0 mm ID, 40◦C). Fluorescence detection, λex 470 nm,
λem 530 nm. Legend:M1 andM2, mobile phase 1 and 2; C1 and C2, column 1 and 2; DG, degasser; P, pump; I, injector; CO, column oven; D, detector; R, integrator;W, waste;ML, multiloop trapping device; CS, column selection valve. Reproduced from [28] with permission.
purity of these compounds aremandatory as the individual thyroid hormones display very different biological activity spectra.While the L-enantiomer stimulates themetabolic rate and regulates growth and the development in infants and is used for the treatment of thyroid disorders, the D-enantiomer has seen applications as antihyperlipidemic agent.
Simultaneous separation of the enantiomers of T4 and T3 could be achieved using a hydro-organicmobile phase conditions. This robustmethod was subsequently employed tomonitor the quality of levothyroxin in tablets. A typical chromatogramis depicted in Figure 4. It could be shown that the investigated formulation contained not only
Cinchona carbamate-type selectors have been shown to be highly efficient chiral background electrolyte (BGE) additives for the enantiomer separation of chiral acids via capillary electrophoresis (CE) [29]
. The
enantiomer separation by CE involves stereoselective ion-pair formation of oppositely charged cationic selector and anionic solutes, which leads to a difference of netmigration velocities of both enantiomers in the electric field. Under acidic conditions a favorable countercurrent-likemigration scenario of free (anodic direction) and complexed solute species (cathodic migration of ion-pairs with EOF) can be obtained, facilitating enantiomer separation. Ion-pairCEwith cinchona carbamate-type chiral
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