Polymeric sulfated amino acid surfactants: a class of versatile chiral selectors for micellar electrokinetic chromatography (MEKC) and MEKC-MS

Abstract

In this work, three amino acid-derived (l-leucinol, l-isoleucinol, l-valinol) sulfated chiral surfactants are synthesized and polymerized. These chiral sulfated surfactants are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation, and partial specific volume. For the first time the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy. The polysodium N-undecenoyl-l-leucine sulfate shows distinct tubular structure, while polysodium N-undecenoyl-l-valine sulfate also shows tubular morphology but without any distinct order of the tubes. On the other hand, polysodium N-undecenoyl-l-isoleucine sulfate (poly-l-SUCILS) displays random distribution of coiled/curved filaments with heavy association of tightly and loosely bound water. All three polymeric sulfated surfactants are compared for enantioseparation of a broad range of structurally diverse racemic compounds at very acidic, neutral, and basic pH conditions in micellar electrokinetic chromatography (MEKC). A small combinatorial library of 10 structurally related phenylethylamines (PEAs) is investigated for chiral separation under acidic and moderately acidic to neutral pH conditions using an experimental design. In contrast to neutral pH conditions, at acidic pH, significantly enhanced chiral resolution is obtained for class I and class II PEAs due to the compact structure of polymeric sulfated surfactants. It is observed that the presence of a hydroxy group on the benzene ring of PEAs resulted in deterioration of enantioseparation. A sensitive MEKC-mass spectrometry (MS) method is developed for one of the PEAs (e.g., (+/-)-pseudoephedrine) in human urine. Very low limit of detection (LOD) is obtained at pH 2.0 (LOD 325 ng/mL), which is approximately 16 times better compared to pH 8.0 (LOD 5.2 microg/mL). Another broad range of chiral analytes (beta-blockers, phenoxypropionic acid, benzoin derivatives, PTH-amino acids, benzodiazepinones) studied also provided improved chiral separation at low pH compared to high-pH conditions. Among the three polymeric sulfated surfactants, poly-l-SUCILS with two chiral centers on the polymer head group provided overall higher enantioresolution for the investigated acidic, basic, and neutral compounds. This work clearly demonstrates for the first time the superiority of chiral separation and sensitive MS detection at low pH over conventional high-pH chiral separation and detection employing anionic chiral polymeric surfactants in MEKC and MEKC-MS.

Figure 1

Synthesis of the N-undecenoxy carbonyl-L-amino acid sulfated surfactants and their polymers.

Figure 2

Intermediate magnification cryo-etch-HRSEM of (A) poly-L-SUCLS imaged at 10000 x, scale bar = 1.00 µm, (B) poly-L-SUCILS imaged at 20000, scale bar = 500 nm and (C) poly-L-SUCVS imaged at 15000 x, scale bar = 667 nm. For poly-L-SUCLS, poly-LSUCILS and poly-L-SUCVS, images were taken at 5 mg/mL, 6-min etch-time and −115 °C. Blue asterisk (*) represents the remnant patches of nonsublimed ice; red color and green color arrows point the loosely and tightly bound water around the nanorods, respectively.

Figure 3

Comparison of 20 mM poly-L-SUCLS, poly-L-SUCILS and poly-L-SUCVS for dependence of electroosmotic velocity (V_eof, methanol), micelle migration velocity (V_app, dodecanophenone) and micelle electrophoretic velocity (V_ep, calculated) on pH. MEKC conditions: 25 mM NH₄OAc / 25 mM TEA, 25 °C, pressure injection: 50 mbar for 15s, ±20 kV applied for separations, UV detection at 214 nm.

Figure 4

Chemical structures of the racemic compounds studied.

Figure 5

Comparison of (A) poly-L-SUCAAS for enantioseparation of class I PEA (0.17 mg/mL in 14:86, MeOH/H₂O) at low pH under optimum conditions (see Table 3). MEKC conditions: 25 mM TEA/H₃PO₄, pressure injection 5 mbar for 1s, −20 kV, UV detection at 200 nm, (B) poly-L-SUCAAS for enantioseparation of class I PEA (0.25 mg/mL in MeOH/H₂O) at moderately acidic to neutral pH under optimum conditions (see Table 3). MEKC conditions are same as Figure 5A except 20 °C, pressure injection of 40 mbar for 2s.

Figure 6

Comparison of (A) poly-L-SUCAAS for enantioseparation of class II PEA at low pH under optimum conditions (see Table 4). Other conditions are same as Figure 5(A), (B) poly-L-SUCAAS for enantioseparation of class II PEA at moderately acidic to neutral pH under optimum conditions (see Table 4). Other conditions are same as Figure 5(B) except pressure injection of 25 mbar for 2s.

Figure 7

Comparison of (A) poly-L-SUCAAS for enantioseparation of class III PEA at low pH under optimum conditions (see Table 5). Other conditions are same as Figure 5(A). (B) poly-L-SUCAAS for enantioseparation of class III PEA at moderately acidic to neutral pH under optimum conditions (see Table 5). Other conditions are same as Figure 5(B) except pressure injection of 25 mbar for 1s.

Figure 8

Electrochromatogram comparing simultaneous MEKC separation and MS detection of class III PEA (0.17 mg/mL in 14:86 MeOH/H₂O) using 25 mM poly-L-SUCLS without (A), and with (B) valeric acid in the sheath liquid. Conditions: (A) 15 mM NH₄OAc / 15 mM TEA, + 20 % (v/v) ACN, 20 °C; injection, 15 mbar for 2 sec, pH 2.0 and −15 kV, 70 cm, 50 µm (I.D.), sheath liquid: 5 mM NH₄OAc in MeOH/H2O (80:20, v/v), 0.5 mL/min. Spray chamber: drying gas flow 6 L/min, nebulizer pressure 4 psi, drying gas temp, 250 °C, V_cap 3000 V, fragmentor, 72 V. ESI SIM positive ions (3 ions) monitored as group SIM at m/z 166, 166, 152, (B) same as Figure 8(A) except sheath liquid is 1% (v/v) valeric acid in MeOH/H2O (80:20, v/v).

Figure 9

Analysis of human urine spiked with (±)-psuedoephedrine enantiomers. The electropherogram [positive SIM 166 and 168 (m/z)] of human urine spiked with (±)-pseudoephedrine (2,2’) and (−)-phenylephrine (1) as IS at low pH of 2.0 (A) and high pH of 8.0 (B). Conditions are same as Figure 8(B), except 35 mM poly-L-SUCLS, sheath liquid flow rate 7.5 µL/min, pH 8.00 and +15 kV. Conditions in (B) are same as (A) except pH 2.00 and −15 kV. The insets on the right and left of Figure 9(A) and 9(B) show the enhanced region for (±)-pseudoephedrine at the LOD and calibration curves for psuedoephedrine enantiomers, respectively.

Figure 10

Comparison of (A) 35 mM poly-L-SUCAAS for enantioseparation of (±)-atenolol (0.25 mg/mL in 25:75, MeOH/H₂O) and 15 mM poly-L-SUCAAS for enantioseparation of (±)-metoprolol (0.25 mg/mL in 25:75, MeOH/H₂O). MEKC conditions: pH 2.0, 25 mM NaH₂PO₄ + 25 mM CH₃COONa + H₃PO₄, 25 °C, pressure injection 5 mbar for 1s, −20 kV applied for separations, UV detection at 220 nm, (B) 25 mM poly-L-SUCAAS for enantioseparation of (±)-atenolol and (±)-metoprolol (0.25 mg/mL in MeOH/H₂O). MEKC conditions: pH 8.0, 25 mM NH₄OAc / 25 mM TEA, 25 °C, pressure injection of 5 mbar for 1s, +20 kV applied for separations; UV detection at 220 nm.

Figure 11

Comparison of enantioseparation of ±-2-(2-chlorophenoxy)propanoic acid (±-2-PPA, 0.5 mg/mL in 50:50, MeOH/H₂O) using (A) 50 mM poly-L-SUCAAS, pH 2.0. MEKC conditions are same as Figure10(A) except, 15 °C, pressure injection 50 mbar for 1s, UV detection at 200 nm, (B) 25 mM poly-L-SUCAAS, pH 8.0. MEKC conditions are same as Figure 10(B) except, pressure injection of 50 mbar for 1s, UV detection at 200 nm.

Figure 12

Comparison of simultaneous enantioseparation of four benzoin derivatives (0.33 mg/mL in 33:66, MeOH/H₂O) using (A) 50 mM poly-L-SUCAAS, pH 3.0. MEKC conditions are same as 10(A) except, 20 °C, pressure injection 50 mbar for 1s, UV detection at 200 nm, (B) 25 mM poly-L-SUCAAS, pH 8.0. MEKC conditions are same as Figure 10(B) except, 20 °C, pressure injection of 50 mbar for 1s, UV detection at 200 nm.

Figure 13

Comparison of simultaneous enantioseparation of three PTH-amino acids (0.17 mg/mL in 50:50 MeOH/H₂O) using (A) 15 mM poly-L-SUCAAS, pH 3.0. MEKC conditions are same as Figure 10(A) except, 20 °C, pressure injection of 50 mbar for 1s, UV detection at 269 nm, (B) 15 mM poly-L-SUCAAS, pH 8.0. MEKC conditions are same as Figure 10(B) except, pressure injection 50 mbar for 1s, UV detection at 269 nm.

Figure 14

Comparison of simultaneous enantioseparation of three benzodiazepines (0.17 mg/mL in 50:50, MeOH/H₂O) using (A) 10 mM poly-L-SUCAAS, pH 3.0. MEKC conditions are same as 10(A) except 15 % (v/v) ACN was used in buffer, 17 °C, pressure injection of 25 mbar for 1s, UV detection at 200 nm, (B) 10 mM poly-L-SUCAAS, pH 8.0, 10 %ACN. MEKC conditions are same as 10(B) except, 17 °C, pressure injection 25 mbar for 1s.