27


HPLC Conditions: Flow Rate: 1.0 or 0.2 mL/min


Detection: UV @ 220 nm or MS/MS Temperature: Ambient.


Injection Volume: 5 - 20 µL (depending on analyte response)


Sample Concentration: 500 µg/mL (UV); 200 ng/mL (MS/MS) Columns: LuxTM


Cellulose-1 5 µm 250 x 4.6mmor 3 µm150 x 2.0mm


Cellulose tris (3,5- dimethylphenylcarbamate) LuxTM


Cellulose-2 5 µm 250 x 4.6mmor 3 µm150 x 2.0mm


Cellulose tris (3-chloro-4-methylphenylcarbamate)


LuxTM Amylose-2 5 µm250 x 4.6mm


Amylose tris (5-chloro-2-methylphenylcarbamate)


Mobile Phase for basic or neutral compounds:


NH4 NH4


HCO3 HCO3


; NH4Ac; or NH4 Ac + 0.1%DEA; + 0.1%DEA with CH3CN orMeOH


Mobile Phase for acidic and neutral compounds:


0.1 % HAc with CH3 CN or MeOH


(DEA = Diethylamine; HAc = Acetic acid; CH3CN = Acetonitrile; MeOH= Methanol)


Compound Cellulose-1 Cellulose-2 Compound Cellulose-1 Cellulose-2 Labetalol Nifenalol


Benfluorex


Promethazine Bupropion


Trimipramine Metolazone Clenbutero


Resolution Baseline resolution


X X X


Partial X X √ X


X √ X X


√ X √


Felodipine Ketamine Verapamil Nicardipine


Partial Dichloroisoproterenol Diltiazem


Nimodipine Sulpiride


Summary of Results Cellulose-1 2


X


Partial √ X X X X X


Cellulose-2 6


Partial resolution 21 Total separations on both phases Baseline resolution - 8 (50 %) √ : Baseline resolution: Rs > 1.5


Partial resolution: 0.8 < Rs < 1.5 X : No separation: Rs < 0.8 Table 1: Enantioresolution of 18 Racemates on Cellulose-1 and -2 in RP


√ √ X X √ X X X


Results and Discussion Chiral LC(UV) and LC/MS/MS Applications Three polysaccharide-based CSPs were explored in the reversed phase (RP) elution mode for over 200 compounds of pharmaceutical interest, in mobile phases made of ammonium bicarbonate with acetonitrile or methanol as organic modifier. Figures 1 and 4 demonstrate representative chiral separations on Lux™ CSPs. Most compounds were eluted in less than 15 min with baseline resolution in mobile phases of various eluting strength. The results show that Cellusose-1 was most successful in separating benzodizepines and ß-blockers, Cellusose-2 in separating imidazoles, and Amylose-2 in separating antihistamines and imidazoles. Comparing success rates in resolving racemates in RP, NP and PO elution modes revealed that RP elution mode using mobile phases compatible with MS/MS detection showed the highest potential for providing baseline resolution (Figure 2) and is suitable for MS/MS detection.


Effect of Mobile Phase Additives The enantioresolution on Lux™ CSPs was evaluated in both CH3COONH4/CH3CN and NH4HCO3/CH3CN mobile phases (Figure 5). In general NH4HCO3 provided similar or occasionally superior resolution to CH3COONH4 as mobile phase additive. The addition of DEA to the mobile phase can improve resolution for very basic compounds (e.g. ß-blockers and tricyclic antidepressants), but it severely suppresses analyte response in ESI+ MS/MS even at levels as low as 0.025 % (Figure 5). However, DEA or acidic additives do not affect the enantioresolution of benzodiazepines, imidazoles or neutral stereoisomers. For all these compounds baseline separation can be achieved without DEA or acidic additives.


Effect of Organic Modifier Decreasing the eluting strength of the mobile phase will increase retention and resolution as shown for nifenalol in Figure 5 and trimipramine and ketamine in Figure 3. However, once enantiomers elute later than 10 minutes with only partial resolution, baseline separation can be rarely achieved by further decreasing the mobile phase strength. In our study, acetonitrile provided more successful RP chiral separations than methanol on Lux™ CSPs.


Figure 2: Success Rates of Lux™Phases in RP for over 200 Racemates (Rs>1.5) – Various Conditions


Complementary Enantioselectivity in RP Mode 16 basic or neutral compounds not separated in NP and PO were screened in RP on Lux™Cellulose-1 and Lux™


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